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.

Microwave-Assisted Extraction of Bioactive Compounds from Lentil Wastes: Antioxidant Activity Evaluation and Metabolomic Characterization.

The recovery of industrial by-products is part of the zero-waste circular economy. Lentil seed coats are generally considered to be a waste by-product. However, this low-value by-product is rich in bioactive compounds and may be considered an eco-friendly source of health-promoting phytochemicals. For the first time, a sustainable microwave-assisted extraction technique was applied, and a solvent screening was carried out to enhance the bioactive compound content and the antioxidant activity of green and red lentil hull extracts. With respect to green lentil hull extracts that were obtained with different solvents, the aqueous extract of the red lentil seed coats showed the highest total phenolic and total flavonoid content (TPC = 28.3 ± 0.1 mg GAE/g dry weight, TFC = 1.89 ± 0.01 mg CE/100 mg dry weight, respectively), as well as the highest antioxidant activity, both in terms of the free radical scavenging activity (ABTS, 39.06 ± 0.73 mg TE/g dry weight; DPPH, IC50 = 0.39 µg/mL) and the protection of the neuroblastoma cell line (SH-SY5Y, IC50 = 10.1 ± 0.6 µg/mL), the latter of which has never been investigated so far. Furthermore, a metabolite discovery analysis was for the first time performed on the aqueous extracts of both cultivars using an HPLC separation which was coupled with an Orbitrap-based high-Resolution Mass Spectrometry technique.

In house validation of a high resolution mass spectrometry Orbitrap-based method for multiple allergen detection in a processed model food.

In recent years, mass spectrometry (MS) has been establishing its role in the development of analytical methods for multiple allergen detection, but most analyses are being carried out on low-resolution mass spectrometers such as triple quadrupole or ion traps. In this investigation, performance provided by a high resolution (HR) hybrid quadrupole-Orbitrap™ MS platform for the multiple allergens detection in processed food matrix is presented. In particular, three different acquisition modes were compared: full-MS, targeted-selected ion monitoring with data-dependent fragmentation (t-SIM/dd2), and parallel reaction monitoring. In order to challenge the HR-MS platform, the sample preparation was kept as simple as possible, limited to a 30-min ultrasound-aided protein extraction followed by clean-up with disposable size exclusion cartridges. Selected peptide markers tracing for five allergenic ingredients namely skim milk, whole egg, soy flour, ground hazelnut, and ground peanut were monitored in home-made cookies chosen as model processed matrix. Timed t-SIM/dd2 was found the best choice as a good compromise between sensitivity and accuracy, accomplishing the detection of 17 peptides originating from the five allergens in the same run. The optimized method was validated in-house through the evaluation of matrix and processing effects, recoveries, and precision. The selected quantitative markers for each allergenic ingredient provided quantification of 60-100 µgingred/g allergenic ingredient/matrix in incurred cookies.

Standardization of a Mass Spectrometry-Based Workflow for Food Allergen Quantification.

In this chapter, the analytical workflow typically used for the development and validation of an analytical method tailored to food allergen detection and quantification is presented. The main steps defining the workflow are herein described and commented with specific notes about the critical issues that can be faced and common solutions to be adopted. References to guidelines and/or recommendation available from official bodies, as well as main papers from international consortia operating on the specific research field, are also reported, whenever possible. As such, this chapter may represent a practical guide to drive method development in the standardization of analytical methods for food allergen detection.

Multi-allergen quantification of fining-related egg and milk proteins in white wines by high-resolution mass spectrometry.

RATIONALE: A method based on High-Resolution Mass Spectrometry was developed for the simultaneous determination of fining agents containing potentially allergenic milk (casein) and egg-white (lysozyme and ovalbumin) proteins, added to commercial white wines at sub-ppm levels. Selected tryptic peptides were used as quantitative markers. An evaluation of protein digestion yields was also performed by implementing the (15)N-valine-labelled analogues of the best peptide markers identified for αS1 -casein and ovalbumin. METHODS: The method was based on the combination of ultrafiltration (UF) of protein-containing wines, tryptic digestion of the dialyzed wine extracts and liquid chromatography/high resolution mass spectrometry (LC/HRMS) analysis of tryptic digests. Peptides providing the most intense electrospray ionization (ESI)-MS response were chosen as quantitative markers of the proteins under investigation. RESULTS: Six-point calibrations were performed by adding caseinate and egg-white powder in the concentration range between 0.25 and 10 µg/mL, to an allergen-free white wine. The following three peptide markers, LTEWTSSNVMEER, GGLEPINFQTAADQAR and ELINSWVESQTNGIIR, were highlighted as best markers for ovalbumin, while GTDVQAWIR and NTDGSTDYGILQINSR for lysozyme and YLGYLEQLLR, GPFPIIV and FFVAPFPEVFGK for caseinate. Limits of detection (LODs) ranged from 0.4 to 1.1 µg/mL. CONCLUSIONS: The developed method is suited for assessing the contemporary presence of allergenic milk and egg proteins characterizing egg white and caseinate, fining agents typically employed for wine clarification. The LODs of the method enable the detection of sub-ppm concentrations of residual fining agents, that could represent a potential risk for allergic consumers.

Multi-Target Detection of Nuts and Peanuts as Hidden Allergens in Bakery Products through Bottom-Up Proteomics and High-Resolution Mass Spectrometry.

Due to the growing global incidence of allergy to nuts and peanuts, the need for better protection of consumers sensitive to those products is constantly increasing. The best strategy to defend them against adverse immunological reactions still remains the total removal of those products from their diet. However, nuts and peanuts traces can also be hidden in other food products, especially processed ones, such as bakery products, because of cross-contamination occurring during production. Precautionary labelling is often adopted by producers to warn allergic consumers, usually without any evaluation of the actual risk, which would require a careful quantification of nuts/peanuts traces. In this paper, the development of a multi-target method based on liquid chromatography-tandem high resolution mass spectrometry (LC-MS, MS/MS), able to detect traces of five nuts species (almonds, hazelnuts, walnuts, cashews and pistachios) and of peanuts in an in-house incurred bakery product (cookie) through a single analysis is described. Specifically, allergenic proteins of the six ingredients were used as the analytical targets, and the LC-MS responses of selected peptides resulting from their tryptic digestion, after extraction from the bakery product matrix, were exploited for quantification, following a bottom-up approach typical of proteomics. As a result, nuts/peanuts could be detected/quantified down to mg·kg-1 levels in the model cookie, thus opening interesting perspectives for the quantification of hidden nuts/peanuts in bakery products and, consequently, for a more rational use of precautionary labelling.

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.

Reactivity to allergenic food contaminants: A study on products on the market.

BACKGROUND: The frequency and severity of reactions in food-allergic consumers exposed to unintentional food allergen contamination during production is unknown. To warn allergic consumers, it has been suggested for pre-packaged foods to be precautionary labelled when the food allergen contamination may exceed the amount to which 1%-5% of the population could react (ED01-ED05). ED01 for hazelnut and milk have been estimated at 0.1 and 0.2 mg, respectively, by the Voluntary Incidental Trace Allergen Labelling (VITAL) initiative. The respective reference doses recommended by the FAO/WHO Codex consultation are 3 and 2 mg. We evaluated the reactivity to potential traces of milk and hazelnut allergens in allergen-free pre-packaged products by children affected by severe allergies to milk and hazelnuts. METHODS: Oral Food Challenges with commercially available hazelnut-free wafer biscuits and milk-free chocolate pralines were administered to patients with severe food allergies to hazelnut and cow's milk, respectively. Contamination levels of milk or hazelnut allergens were measured using chromatographic separation interfaced with triple quadrupole mass spectrometry. RESULTS: No hazelnut allergic patient showed allergic reactions to exposure to biscuits, nor any milk allergic patient displayed allergic reactions to the dark chocolate praline. While no hazelnut trace was detected in biscuits, the praline was found to be contaminated by milk at concentrations ranging between 8 and 35 mg total protein/kg food. In our dose model, these amounts exceeded 1.5-10 times the VITAL ED01 and reached the threshold suggested by the FAO/WHO Codex consultation. CONCLUSIONS: Upon the consumption of food products available on the market, many patients with severe food allergies tolerate significantly higher doses of allergen than reference doses indicated in the VITAL system used for precautionary allergen labelling. These doses support the safety of the FAO/WHO recommended reference doses.

Tree Nuts and Peanuts as a Source of Beneficial Compounds and a Threat for Allergic Consumers: Overview on Methods for Their Detection in Complex Food Products.

Consumption of tree nuts and peanuts has considerably increased over the last decades due to their nutritional composition and the content of beneficial compounds. On the other hand, such widespread consumption worldwide has also generated a growing incidence of allergy in the sensitive population. Allergy to nuts and peanuts represents a global relevant problem, especially due to the risk of the ingestion of hidden allergens as a result of cross-contamination between production lines at industrial level occurring during food manufacturing. The present review provides insights on peanuts, almonds, and four nut allergens-namely hazelnuts, walnuts, cashew, and pistachios-that are likely to cross-contaminate different food commodities. The paper aims at covering both the biochemical aspect linked to the identified allergenic proteins for each allergen category and the different methodological approaches developed for allergens detection and identification. Attention has been also paid to mass spectrometry methods and to current efforts of the scientific community to identify a harmonized approach for allergens quantification through the detection of allergen markers.

In Vivo and In Vitro Assessment and Proteomic Analysis of the Effectiveness of Physical Treatments in Reducing Allergenicity of Hazelnut Proteins.

Hazelnut is a widespread nut species, especially present in Europe, that can be consumed raw or roasted thanks to its pleasant taste and nutritional properties. In addition to renowned beneficial properties hazelnuts contain several proteins capable of inducing food allergy in sensitized individuals, including Cor a 2 (a profilin), Cor a 8 (a lipid transfer protein), Cor a 9 (an 11S seed storage globulin, legumin-like), and Cor a 11 (a 7S seed storage globulin, vicilin-like). In the present paper we investigated the effectiveness of autoclave-based treatments in decreasing the allergic potential of hazelnut as assessed by submitting the treated material to an in vivo skin prick test and an in vitro immunoblot analysis, with sera of allergic individuals exposed to the treated food material. This preliminary analysis showed that autoclave treatment preceded by hydration and/or followed by drying seems to be a promising approach and appears to be effective in reducing the allergenicity of hazelnuts in most patients, probably due to the denaturation of most major and minor allergenic proteins. This work opens up the opportunity to produce hypoallergenic hazelnut derivatives that can be tolerated by allergic subjects.

Milk Ingredients in Meat Products: Can Autoclaving and In Vitro Gastroduodenal Digestion Mitigate Their IgE-Binding Capacity?

The food industry commonly uses milk ingredients as technological aids in an uncounted number of products. On the other hand, milk contains allergenic proteins causing adverse allergic reactions in sensitized/allergic individuals. This work intends to evaluate the effect of autoclaving and in vitro digestion on the allergenicity of milk proteins incurred in meat products. Protein profiles of raw and autoclaved sausages without and with the addition of 10% of milk protein concentrates were analyzed by gel electrophoresis and liquid chromatography-mass spectrometry. Additionally, residual IgE-reactivity was evaluated by immunoblot analysis using pooled sera of cow's-milk-allergic individuals followed by bioinformatic analysis. Results showed that autoclaving led to an increase in protein fragmentation (higher number of short peptides) and consequently to a higher digestion rate, that was found to be more pronounced in ß-casein. The IgE-binding capacity of milk proteins seems to be reduced after autoclaving prior to digestion, with a residual reactivity in caseins, but was eliminated following digestion. This study highlights the importance of autoclaving as a processing strategy to produce hypoallergenic formulas.

New insight into microbial degradation of mycotoxins during anaerobic digestion.

Anaerobic digestion represents an interesting approach to produce biogas from organic waste materials contaminated by mycotoxins. In this study a shotgun metagenomic analysis of lab-scale bioreactors fed with mycotoxin-contaminated silage has been carried out to characterize the evolution of microbial community under the operating conditions and the key enzymatic activities responsible for mycotoxin degradation. The study was conducted at two different level of contamination for fumonisins and aflatoxin B1. After 15 days biogas production was not influenced by the presence of mycotoxins. Metagenomic analysis revealed that a high contamination rate of mycotoxins interfere with microbial diversity. Degradation of mycotoxins accounted in about 54% for aflatoxin B1 and 60% for fumonisins. The degradation activity of fumonisins resulted in the presence of partially hydrolyzed forms in both tested contamination levels. Accordingly, metagenomic functional analysis revealed the presence of two new carboxylesterase genes belonging to D. bacterium and P. bacterium putatively involved in fumonisin degradation.

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.

Effect of polymer additives on heat transport in turbulent thermal convection.

In this Letter, we explore the possible effects of polymer additives on heat transport in turbulent thermal convective flows. Using both direct numerical simulations and shell-model calculations, we show that polymer additives can significantly enhance the heat transport in homogeneous turbulent thermal convection, which mimics the bulk of turbulent Rayleigh-Bénard convection. We also discuss the implication of our results for turbulent Rayleigh-Bénard convection, in which there are boundary layers in addition to the central bulk.

Food allergens: Classification, molecular properties, characterization, and detection in food sources.

Food allergy is a large and growing public health problem in many areas of the world. The prevalence of food allergy has increased in the last decades in a very significant way in many world regions, particularly in developed countries. In that respect, the research field of food allergy has experienced an extensive growth and very relevant progress has been made in recent years regarding the characterization of food allergens, the study of their immunological properties, and their detection in food sources. Furthermore, food labeling policies have also been improved decidedly in recent years. For that immense progress made, it is about time to review the latest progress in the field of food allergy. In this review, we intend to carry out an extensive and profound overview regarding the latest scientific advances and knowledge in the field of food allergen detection, characterization, and in the study of the effects of food processing on the physico-chemical properties of food allergens. The advances in food labeling policies, and methodologies for the characterization of food allergens are also thoroughly reviewed in the present overview.

Prototype Gluten-Free Breads from Processed Durum Wheat: Use of Monovarietal Flours and Implications for Gluten Detoxification Strategies.

In this investigation, we reported the production of prototype breads from the processed flours of three specific Triticum turgidum wheat genotypes that were selected in our previous investigation for their potential low toxic/immunogenic activity for celiac disease (CD) patients. The flours were subjected to sourdough fermentation with a mixture of selected Lactobacillus strains, and in presence of fungal endoproteases. The breads were characterized by R5 competitive enzyme linked immunosorbent assay in order to quantify the residual gluten, and the differential efficacy in gluten degradation was assessed. In particular, two of them were classified as gluten-free (<20 ppm) and very low-gluten content (<100 ppm) breads, respectively, whereas the third monovarietal prototype retained a gluten content that was well above the safety threshold prescribed for direct consumption by CD patients. In order to investigate such a genotype-dependent efficiency of the detoxification method applied, an advanced proteomic characterization by high-resolution tandem mass spectrometry was performed. Notably, to the best of our knowledge, this is the first proteomic investigation which benefitted, for protein identification, from the full sequencing of the Triticum turgidum ssp. durum genome. The differences of the proteins' primary structures affecting their susceptibility to hydrolysis were investigated. As a confirmation of the previous immunoassay-based results, two out of the three breads made with the processed flours presented an exhaustive degradation of the epitopic sequences that are relevant for CD immune stimulatory activity. The list of the detected epitopes was analyzed and critically discussed in light of their susceptibility to the detoxification strategy applied. Finally, in-vitro experiments of human gastroduodenal digestion were carried out in order to assess, in-silico, the toxicity risk of the prototype breads under investigation for direct consumption by CD patients. This approach allowed us to confirm the total degradation of the epitopic sequences upon gastro-duodenal digestion.

Understanding the Fate of Almond (Prunus dulcis (Mill.) D.A. Webb) Oleosomes during Simulated Digestion.

BACKGROUND: Almond kernels contain phytochemicals with positive health effects in relation to heart disease, diabetes and obesity. Several studies have previously highlighted that almond cell wall encapsulation during digestion and particle size are factors associated with these benefits. In the present study, we have characterized almond oleosomes, natural oil droplets abundant in plants, and we have investigated their integrity during simulated gastrointestinal digestion. METHODS: Oleosomes were visualized on the almond seed surface by imaging mass spectrometry analysis, and then characterized in terms of droplet size distribution by dynamic light scattering and protein profile by liquid chromatography high-resolution tandem mass spectrometry analysis. RESULTS: The almond oleosomes' distribution remained monomodal after in vitro mastication, whereas gastric and duodenal digestion led to a bimodal distribution, albeit characterized mainly by a prevalent population with a droplet size decrease related to a rearrangement of the protein profile. Oleosins, structural proteins found in plant oil bodies, persisted unchanged during simulated mastication, with the appearance of new prunin isoforms after gastric and duodenal digestion. CONCLUSIONS: The rearrangement of the protein profile could limit lipid bioaccessibility. The data improve our understanding of the behavior of almond lipids during gastrointestinal digestion, and may have implications for energy intake and satiety imparted by almonds.

Validation of a MS Based Proteomics Method for Milk and Egg Quantification in Cookies at the Lowest VITAL Levels: An Alternative to the Use of Precautionary Labeling.

The prevalence of food allergy has increased over the last decades and consequently the food labeling policies have improved over the time in different countries to regulate allergen presence in foods. In particular, Reg 1169 in EU mandates the labelling of 14 allergens whenever intentionally added to foods, but the inadvertent contamination by allergens still remains an uncovered topic. In order to warn consumers on the risk of cross-contamination occurring in certain categories of foods, a precautionary allergen labelling system has been put in place by food industries on a voluntary basis. In order to reduce the overuse of precautionary allergen labelling (PAL), reference doses and action limits have been proposed by the Voluntary Incidental Trace Allergen Labelling VITAL project representing a guide in this jeopardizing scenario. Development of sensitive and reliable mass spectrometry methods are therefore of paramount importance in this regard to check the contamination levels in foods. In this paper we describe the development of a time-managed multiple reaction monitoring (MRM) method based on a triple quadrupole platform for milk and egg quantification in processed food. The method was in house validated and allowed to achieve levels of proteins lower than 0.2 mg of total milk and egg proteins, respectively, in cookies, challenging the doses recommended by VITAL. The method was finally applied to cookies labeled as milk and egg-free. This method could represent, in perspective, a promising tool to be implemented along the food chain to detect even tiny amounts of allergens contaminating food commodities.

Emerging Allergens in Goji Berry Superfruit: The Identification of New IgE Binding Proteins towards Allergic Patients' Sera.

The goji berry (Lycium barbarum L.) (GB) is gaining increasing attention with high consumption worldwide due to its exceptional nutritional value and medicinal benefits displayed in humans. Beyond their beneficial properties, GBs contain renowned allergenic proteins, and therefore deserve inclusion among the allergenic foods capable of inducing allergic reactions in sensitive consumers. GB allergy has been frequently linked to the panallergen lipid transfer protein (LTP), especially across the population of the Mediterranean area. Methods: In this study, we investigated the protein profile of GBs focusing on the most reactive proteins against immunoglobulins E (IgE) of allergic patients' sera, as ascertained by immunoblot experiments. The protein spots displaying a clear reaction were excised, in-gel digested, and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) followed by data searching against a restricted database for a reliable protein identification. Results: According to our data, three main spots were identified in GB extract as IgE binding proteins after immunoblot analysis. Some major proteins were identified and the three proteins that provided the highest reactivity were putatively attributed to vicilin and legumin proteins followed by a protein matching with 11S globulin belonging to the cupin superfamily. Finally, the whole GB protein extract was also submitted to bottom-up proteomics followed by a software-based database (DB) screening and a more exhaustive list of GB proteins was compiled.