In silico evaluation of the potential allergenicity of a fungal biomass from Rhizomucor pusillus for use as a novel food ingredient.

The world's hunger for novel food ingredients drives the development of safe, sustainable, and nutritious novel food products. For foods containing novel proteins, potential allergenicity of the proteins is a key safety consideration. One such product is a fungal biomass obtained from the fermentation of Rhizomucor pusillus. The annotated whole genome sequence of this strain was subjected to sequence homology searches against the AllergenOnline database (sliding 80-amino acid windows and full sequence searches). In a stepwise manner, proteins were designated as potentially allergenic and were further compared to proteins from commonly consumed foods and from humans. From the sliding 80-mer searches, 356 proteins met the conservative >35% Codex Alimentarius threshold, 72 of which shared ≥50% identity over the full sequence. Although matches were identified between R. pusillus proteins and proteins from allergenic food sources, the matches were limited to minor allergens from these sources, and they shared a greater degree of sequence homology with those from commonly consumed foods and human proteins. Based on the in silico analysis and a literature review for the source organism, the risk of allergenic cross-reactivity of R. pusillus is low.

Preparation of Blinded Food Matrixes for Clinical Oral Challenges.

Clinically, oral food challenges have value in the diagnosis and management of food allergy. Oral food challenges are used not only for diagnostic confirmation that ingestion of a specific food elicits an adverse reaction, but also for determining individual threshold doses, tracking the progress toward desensitization during immunotherapy, determining the effect of processing on the allergenicity of a specific food, assessing the allergenicity of an ingredient derived from an allergenic source, and tracking the progress toward development of age-related tolerance to a specific food. To eliminate bias in oral challenges, the food under investigation is masked in a matrix so that it is not sensorially detectable by the patient or the clinical observer. The preparation of oral challenge foods requires care in the selection of the allergenic components, the selection of the components of the matrix, the masking of the allergenic component, and the homogeneity of the allergen in the overall matrix.

Development of a Sequence Searchable Database of Celiac Disease-Associated Peptides and Proteins for Risk Assessment of Novel Food Proteins.

Celiac disease (CeD) is an autoimmune enteropathy induced by prolamin and glutelin proteins in wheat, barley, rye, and triticale recognized by genetically restricted major histocompatibility (MHC) receptors. Patients with CeD must avoid consuming these proteins. Regulators in Europe and the United States expect an evaluation of CeD risks from proteins in genetically modified (GM) crops or novel foods for wheat-related proteins. Our database includes evidence-based causative peptides and proteins and two amino acid sequence comparison tools for CeD risk assessment. Sequence entries are based on the review of published studies of specific gluten-reactive T cell activation or intestinal epithelial toxicity. The initial database in 2012 was updated in 2018 and 2022. The current database holds 1,041 causative peptides and 76 representative proteins. The FASTA sequence comparison of 76 representative CeD proteins provides an insurance for possible unreported epitopes. Validation was conducted using protein homologs from Pooideae and non-Pooideae monocots, dicots, and non-plant proteins. Criteria for minimum percent identity and maximum E-scores are guidelines. Exact matches to any of the 1,041 peptides suggest risks, while FASTA alignment to the 76 CeD proteins suggests possible risks. Matched proteins should be tested further by CeD-specific CD4/8+ T cell assays or in vivo challenges before their use in foods.

Challenges in Gluten Analysis: A Comparison of Four Commercial Sandwich ELISA Kits.

Gluten is composed of prolamin and glutelin proteins from several related grains. Because these proteins are not present in identical ratios in the various grains and because they have some differences in sequence, the ability to accurately quantify the overall amount of gluten in various food matrices to support gluten-free labeling is difficult. Four sandwich ELISAs (the R-Biopharm AG R5 RIDASCREEN®, the Neogen Veratox® R5, the Romer Labs AgraQuant® G12, and the Morinaga Wheat kits) were evaluated for their performance to quantify gluten concentrations in various foods and ingredients. The Morinaga and AgraQuant® G12 tests yielded results comparable to the two R5 kits for most, but not for certain, foods. The results obtained with the Morinaga kit were lower when compared to the other kits for analyzing powders of buckwheat and several grass-based products. All four kits were capable of detecting multiple gluten-containing grain sources including wheat, rye, barley, semolina, triticale, spelt, emmer, einkorn, Kamut™, and club wheat. Users of the ELISA kits should verify the performance in their hands, with matrices that are typical for their specific uses. The variation in results for some food matrices between test methods could result in trade disputes or regulatory disagreements.

Gluten Cross-Contact in Restaurant-Scale Pasta Cooking.

ABSTRACT: Celiac disease and nonceliac gluten sensitivity are provoked by the consumption of gluten from wheat, barley, rye, and related grains. Affected individuals are advised to adhere to gluten-free diets. Recently, gluten-free foods have become a marketing trend with gluten-free options both in packaged foods and in restaurants and food service establishments. Pasta is one of the primary gluten-containing foods in diets in North America and Europe. Gluten-free pasta formulations are commercially available. In restaurants, multiple pasta dishes are often prepared simultaneously in large pots with multiple compartments and shared cooking water. The objective of this study was to determine whether gluten transfer occurs between traditional and gluten-free pasta when cooked simultaneously in the same water. Pasta was boiled in a commercial, four-compartment, 20-qt (18.9-L) cooking pot containing three batches of traditional penne pasta and one batch of gluten-free penne pasta. The amount of pasta (dry weight) was either 52 g (recommended serving size) or 140 g (typical restaurant portion). Five consecutive batches of pasta were boiled, and cooking water and gluten-free pasta were sampled at completion of cooking. Water and gluten-free pasta samples were tested for gluten with the Neogen Veratox for Gliadin enzyme-linked immunosorbent assay kit. Gluten concentrations were low (<20 ppm) in both water and gluten-free pasta samples through five 52-g batches. Gluten concentrations in the 52-g gluten-free pasta samples slowly increased through five batches but were never >20 ppm. During cooking of the 140-g gluten-free pasta samples, the gluten concentrations in the cooking water increased with each batch to >50 and >80 ppm after the fourth and fifth batches, respectively. The gluten concentrations in the 140-g gluten-free pasta samples approached 20 ppm by the fourth batch and reached nearly 40 ppm after the fifth batch. Although gluten transfer does not occur at a high rate, gluten-free pasta should be prepared in a separate cooking vessel in restaurant and food service establishments.

Purification and Initial Characterization of Ara h 7, a Peanut Allergen from the 2S Albumin Protein Family.

2S albumins are important peanut allergens. Within this protein family, Ara h 2 and Ara h 6 have been described in detail, but Ara h 7 has received little attention. We now describe the first purification of Ara h 7 and its characterization. Two Ara h 7 isoforms were purified from peanuts. Mass spectrometry revealed that both the isoforms have a post-translation cleavage, a hydroxyproline modification near the N-terminus, and four disulfide bonds. The secondary structure of both Ara h 7 isoforms is highly comparable to those of Ara h 2 and Ara h 6. Both Ara h 7 isoforms bind IgE, and Ara h 7 is capable of inhibiting the binding between Ara h 2 and IgE, suggesting at least partially cross-reactive IgE epitopes. Ara h 7 was found in all main market types of peanut, at comparable levels. This suggests that Ara h 7 is a relevant allergen from the peanut 2S albumin protein family.

Development of a Sandwich Enzyme-Linked Immunosorbent Assay for Detection and Quantification of Clam Residues in Food Products.

Seafood is a frequent cause of allergic reactions to food globally. The presence of undeclared trace amounts of clam can cause allergic reactions in sensitive individuals. Limited tools are available to test food products for the presence of traces of clam. We report on the development of a sandwich ELISA that can detect and quantify clam protein in food. Antisera against a mix of two commercially important clam species, Atlantic Surf (Spisula solidissima) and ocean quahog (Arctica islandica), were raised in rabbit and sheep. A sandwich ELISA was constructed with this antisera, and sensitivity and specificity were evaluated. Also, model food products spiked with clam protein were analyzed to assess the performance of the ELISA. Comparison was made with a commercially available ELISA for crustacea. The lower limit of quantification of the sandwich ELISA is 2.5 ppm clam protein in food samples, allowing the detection of low amounts of clam that may trigger a reaction in clam allergic patients. The sandwich ELISA was highly specific with cross-reactivity only noted for other molluscan shellfish (mussel and scallop). Clam protein in tomato juice and potato cream soup was detected well with recoveries ranging from 65 to 74% and from 74 to 113%, respectively. However when potato cream soup was retorted, the recover fell to 20%, imposing the risk of underestimating the clam content of a food product. A commercially available crustacean ELISA test was not suitable to detect clam protein. The sandwich ELISA described here is suitable for detection and quantification of clam protein in food products. Care should be taken with food products that have been retorted as the results may be underestimated.

Full range of population Eliciting Dose values for 14 priority allergenic foods and recommendations for use in risk characterization.

Previously, we published selected Eliciting Dose (ED) values (i.e. ED01 and ED05 values) for 14 allergenic foods, predicted to elicit objective allergic symptoms in 1% and 5%, respectively, of the allergic population (Remington et al., 2020). These ED01 and ED05 values were specifically presented and discussed in the context of establishing Reference Doses for allergen management and the calculation of Action Levels for Precautionary Allergen Labeling (PAL). In the current paper, we publish the full range of ED values for these allergenic foods and provide recommendations for their use, specifically in the context of characterizing risks of concentrations of (unintended) allergenic proteins in food products. The data provided in this publication give risk assessors access to full population ED distribution information for 14 priority allergenic foods, based on the largest threshold database worldwide. The ED distributions were established using broad international consensus regarding suitable datapoints and methods for establishing individual patient's NOAELs and LOAELs and state of the art statistical modelling. Access to these ED data enables risk assessors to use this information for state-of-the-art food allergen risk assessment. This paper contributes to a harmonization of food allergen risk assessment and risk management and PAL practices.

Effect of heat treatment on the conformational stability of intact and cleaved forms ofthe peanut allergen Ara h 6 in relation to its IgE-binding potency.

This work reports on theeffect of heat treatment on the protein conformational stabilityof intact and post-translationallycleaved peanut allergen Ara h 6 in relation to IgE-binding. Intact and post-translationallycleaved Ara h 6 are structurally similar and theirstrong resistance to denaturant-inducedunfolding is comparable. Only upon exposure toautoclave conditions the twoforms of Ara h 6 demonstrated susceptibility toirreversible denaturationresulting in a significant decrease in IgE-binding potency. Thisreduction isfor the intact protein more pronounced than for than for the cleaved form. This isattributed to less conformational constrains of the cleaved form comparedtointact, as suggested by the 2-fold lower activation energy for unfoldingfound for the cleavedform. Overall, harsh conditionsare required to denature Ara h 6 and to significantly reduce its IgE-bindingpotency. The cleavedform possesses more resistance to such denaturation than the intactform.

Updated population minimal eliciting dose distributions for use in risk assessment of 14 priority food allergens.

Food allergy and allergen management are important global public health issues. In 2011, the first iteration of our allergen threshold database (ATDB) was established based on individual NOAELs and LOAELs from oral food challenge in roughly 1750 allergic individuals. Population minimal eliciting dose (EDp) distributions based on this dataset were published for 11 allergenic foods in 2014. Systematic data collection has continued (2011-2018) and the dataset now contains over 3400 data points. The current study provides new and updated EDp values for 14 allergenic foods and incorporates a newly developed Stacked Model Averaging statistical method for interval-censored data. ED01 and ED05 values, the doses at which 1%, and respectively 5%, of the respective allergic population would be predicted to experience any objective allergic reaction were determined. The 14 allergenic foods were cashew, celery, egg, fish, hazelnut, lupine, milk, mustard, peanut, sesame, shrimp (for crustacean shellfish), soy, walnut, and wheat. Updated ED01 estimates ranged between 0.03 mg for walnut protein and 26.2 mg for shrimp protein. ED05 estimates ranged between 0.4 mg for mustard protein and 280 mg for shrimp protein. The ED01 and ED05 values presented here are valuable in the risk assessment and subsequent risk management of allergenic foods.

Deriving individual threshold doses from clinical food challenge data for population risk assessment of food allergens.

BACKGROUND: Food allergies are a significant public health issue, and the only effective management option currently available is strict avoidance of all foods containing the allergen. In view of the practical impossibility of limiting risks to zero, quantitative allergen risk assessment and management strategies are needed. OBJECTIVE: We sought to develop appropriate methods for informing population-based risk assessments and risk management programs to benefit all stakeholders but particularly patients with food allergy. METHODS: Individual thresholds for food allergens (maximum tolerable doses and minimum eliciting doses) can ideally be established through double-blind, placebo-controlled food challenges. If double-blind, placebo-controlled food challenge data are not available, data from widely used open food challenges using predefined objective criteria can also provide useful data regarding minimum eliciting doses. For more than 20 years, the Netherlands Organisation for Applied Scientific Research and the Food Allergy Research and Resource Program at the University of Nebraska-Lincoln have been collecting individual maximum tolerable doses and minimum eliciting doses that produce objective symptoms from published and unpublished clinical data to better refine knowledge regarding the sensitivity of the population to food allergens. RESULTS: In this article we provide in-depth insights into the methodology applied by the Netherlands Organisation for Applied Scientific Research and Food Allergy Research and Resource Program to derive individual maximum tolerable doses and minimum eliciting doses for objective symptoms from clinical food challenge data. More than 90 examples for determining individual allergic thresholds are presented. CONCLUSION: With the methodology presented in this article, we aim to stimulate harmonization and transparency in quantitative food allergen risk assessment and risk management programs, encouraging their wider adoption.

Mycoprotein: The Future of Nutritious Nonmeat Protein, a Symposium Review.

Mycoprotein is an alternative, nutritious protein source with a meat-like texture made from Fusarium venenatum, a naturally occurring fungus. Its unique method of production yields a significantly reduced carbon and water footprint relative to beef and chicken. Mycoprotein, sold as Quorn, is consumed in 17 countries, including the United States. In line with current dietary guidelines, mycoprotein is high in protein and fiber, and low in fat, cholesterol, sodium, and sugar. Mycoprotein may help maintain healthy blood cholesterol levels, promote muscle synthesis, control glucose and insulin levels, and increase satiety. It is possible that some susceptible consumers will become sensitized, and subsequently develop a specific allergy. However, a systematic evidence review indicates that incidence of allergic reactions remains exceptionally low. Mycoprotein's nutritional, health, and environmental benefits affirms its role in a healthful diet. Future research that focuses on the long-term clinical benefits of consuming a diet containing mycoprotein is warranted.

Comparison of recovery and immunochemical detection of peanut proteins from differentially roasted peanut flour using ELISA.

The effect of heat on extractability and immunoreactivity of proteins from roasted peanut flours and whole peanuts was evaluated using two general protein assays and six commercial peanut ELISA kits, respectively. The highest amount of protein was recovered from roasted peanuts with all ELISAs, while recovery showed a decrease with increasing levels of roasting of the peanut flours. Only the Morinaga kit showed sufficient sensitivity to detect peanut at low concentrations of the dark roast peanut flours. Both the protein and immunoassays indicated a decrease in protein solubility with roasting. The underestimation by immunoassays is a combination of decreased solubility and heat induced changes in the proteins that are being targeted by the ELISA antibodies. These findings suggest that most commercial ELISA kits may not reliably quantify peanut present in dark roast peanut flours at ≤25 ppm.

Sensitivity analysis to derive a food consumption point estimate for deterministic food allergy risk assessment.

One of the input parameters in food allergy risk assessment is the amount of a given food consumed at an eating occasion. There is no consensus on how to use food consumption data when assessing the risk from unintended allergen presence in food products. A sensitivity analysis was performed to establish the optimal food consumption estimate for a deterministic food allergy risk assessment. Exposure was calculated for consumption percentiles (50th percentile, P50 to maximum) using the iFAAM consumption database in conjunction with an allergen concentration range from 1 to 1000 ppm. The resulting allergen intakes were compared to the allergic population reference doses proposed by Taylor et al. (2014) for 10 major allergenic foods. Optimal consumption percentiles were defined as those which predicted an intake below the relevant reference dose and met the defined acceptable risk level confirmed by probabilistic risk assessments. Analysis showed that, for 99% of the food groups, the P50 consumption met our criteria, while the P75 did so for 100% of the food groups. We suggest that the P75 is the optimal point estimate for use in deterministic food allergy risk assessment. It meets the safety objective and is adequately conservative for a public health context.

Improved extraction of peanut residues from a wheat flour matrix for immunochemical detection.

The efficacy of different buffers in extracting peanut from a solid model food incurred with peanut and subjected to processing was evaluated using two commercial ELISA kits: Veratox® for peanut allergen and peanut ELISA from Morinaga. Average percentage recoveries of peanut from unprocessed samples using the kit supplied buffers were 46 ±â€¯5 and 28 ±â€¯2 with the Veratox and Morinaga kits, respectively. However, Na2CO3, pH 9.6 and PBS containing 1 M GuHCl recovered 65% ±â€¯4% and 77% ±â€¯10% of peanut, respectively from unprocessed samples with the Veratox kit. These two buffers also performed better than the Veratox buffer with fried, high pressure processed, and baked samples. PBS containing SDS and ß-ME, performed significantly better than the Morinaga buffer in recovering peanut from unprocessed, boiled and fried samples. Thus, the use of alternative extraction buffers provides better recovery of peanut residues from a processed solid food matrix.

Environmental Food Exposure: What Is the Risk of Clinical Reactivity From Cross-Contact and What Is the Risk of Sensitization.

For food-allergic individuals, the typical exposure to food proteins happens during ingestion; however, individuals may be exposed to foods in other ways. In addition to ingestion reactions, allergic patients may have reactions from cutaneous or mucosal exposures to food proteins, with the classic example being a peanut-allergic child touching a counter with peanut butter and then rubbing their eyes. Similar to hands, saliva can also act as a carrier for food proteins. Finally, there is a wealth of new research regarding the presence of food proteins in the environment, for example, within household floor dust. This review will focus on (1) cross-contact of food proteins and (2) environmental food protein exposures. Cross-contact occurs when one type of food comes into contact with another type of food resulting in the mixture of proteins. For food allergies, cross-contact is important when an allergen is inadvertently transferred to a food/meal that is thought to not contain that specific allergen. We will discuss the current literature regarding the presence of detectable food proteins in different locations, how and if these proteins are transferred or eliminated, and the clinical implications of exposures to food proteins under these different scenarios.

Purification and Characterization of Naturally Occurring Post-Translationally Cleaved Ara h 6, an Allergen That Contributes Substantially to the Allergenic Potency of Peanut.

The 2S albumin Ara h 6 is one of the most important peanut allergens. A post-translationally cleaved Ara h 6 (pAra h 6) was purified from Virginia type peanuts, and the cleavage site was mapped using high-resolution mass spectrometry. Compared to intact Ara h 6, pAra h 6 lacks a 5-amino acid stretch, resembling amino acids 43-47 (UniProt accession number Q647G9) in the nonstructured loop. Consequently, pAra h 6 consists of two chains: an N-terminal chain of approximately 5 kDa and a C-terminal chain of approximately 9 kDa, held together by disulfide bonds. Intermediate post-translationally cleaved products, in which this stretch is cleaved yet still attached to one of the subunits, are also present. The secondary structure and immunoglobulin E (IgE) binding of pAra h 6 resembles that of intact Ara h 6, indicating that the loss of the nonstructured loop is not critical for maintaining the protein structure. Commercially available monoclonal and polyclonal immunoglobulin G (IgG) antibodies directed to Ara h 6 react with both intact Ara h 6 and pAra h 6, suggesting that the involved epitopes are not located in the area that is post-translationally cleaved. No differences between intact Ara h 6 and pAra h 6 in terms of IgE binding were found, suggesting that the area that is post-translationally cleaved is not involved in IgE epitopes either. For all main cultivars Runner, Virginia, Valencia, and Spanish, intact Ara h 6 and pAra h 6 occur in peanut at similar levels, indicating that pAra h 6 is a consistent and important contributor to the allergenic potency of peanut.