The risks associated with the presence of hidden allergens in the food chain have raised the need for fast, sensitive, and reliable methods to trace food allergens in different commodities. We highlight advances and future trends in biosensor systems applied to food-allergen management. We discuss critical aspects of biosensor development with particular emphasis on integrating nanotechnology.

Food allergy (FA) is a relatively new food safety issue still deemed to be on the rise in the recent years and with an increasing number of food allergens identified and a growing number of consumers suffering from FA. No actual cure currently exists for food allergy and therefore food allergic consumers can only manage their condition by carefully avoiding products which contain the allergenic food(s). In order to address this effectively, food allergic consumers absolutely rely on the availability, accuracy and reliability of information provided on foods they intend to buy, resulting in the urgent need for specific labeling legislation. On this regard, the European Union (EU) is very restrictive, with the last Directive 2007/68/EC requiring the mandatory labeling of a total of 14 allergenic ingredients, whenever used and irrespective of the amounts, on the respective food label with few exemption cases also listed in the same Directive [1]. This regulatory framework is intended to provide consumers with information about allergens when they are introduced in foods as ingredients; however, allergens may also inadvertently contaminate the food when manufacturing processes and control measures are not adequate to prevent cross-contact between allergen-containing and allergen-free foods then becoming "hidden allergens". This last represents the major threat for allergic consumers and for food manufacturers, the latter being responsible for the safety of food products brought onto the market.In the last two decades, mass spectrometry has played a pivotal role in proteomic research being the election method for protein identification in complex mixtures. Such MS approach has been implemented in the food allergen field proving to be a reliable confirmatory tool overcoming the objective limitations of antibody-based kits commercially available, accounted by epitope masking phenomena or epitope modification occurring upon application of thermal treatments [2]. Additional advantages rely on the possibility to run multiple-allergen analysis in one shot, quantitative analysis, structural protein elucidation, characterization of protein modifications and epitope mapping. In the last five year, our Institute actively contributed to such research field thanks to the acquisition of last-generation high resolution mass spectrometers (HR-MS) based on OrbitrapTM technology with very high selectivity and sensitivity achieved in the food allergen detection methods developed [3-5].In the present communication, different examples of our achievements will be presented and critically discussed, highlighting the relevance and benefit of the accurate mass detection of allergens markers for the fulfillment of specific objectives. Features and analytical performances of different HR-MS platforms were investigated and will be thoroughly discussed along with their strengths and weaknesses applied to the multi-allergen screening in different food matrices.

Laccases (LCs) are multicopper oxidases that find application as versatile biocatalystsfor the green bioremediation of environmental pollutants and xenobiotics. In this study weelucidate the degrading activity of Lac2 pure enzyme form Pleurotus pulmonarius towards aflatoxinB1 (AFB1) and M1 (AFM1). LC enzyme was purified using three chromatographic steps andidentified as Lac2 through zymogram and LC-MS/MS. The degradation assays were performedin vitro at 25 °C for 72 h in buffer solution. AFB1 degradation by Lac2 direct oxidationwas 23%. Toxin degradation was also investigated in the presence of three redox mediators,(2,20-azino-bis-[3-ethylbenzothiazoline-6-sulfonic acid]) (ABTS) and two naturally-occurring phenols,acetosyringone (AS) and syringaldehyde (SA). The direct effect of the enzyme and the mediatedaction of Lac2 with redox mediators univocally proved the correlation between Lac2 activity andaflatoxins degradation. The degradation of AFB1 was enhanced by the addition of all mediators at10 mM, with AS being the most effective (90% of degradation). AFM1 was completely degraded byLac2 with all mediators at 10 mM. The novelty of this study relies on the identification of a pureenzyme as capable of degrading AFB1 and, for the first time, AFM1, and on the evidence that themechanism of an effective degradation occurs via the mediation of natural phenolic compounds.These results opened new perspective for Lac2 application in the food and feed supply chains as abiotransforming agent of AFB1 and AFM1

There is an increasing demand for animal-derived products in the developing countries. This posesmajor concerns for the sustainable production of safe and nutritious food. Consequently, to address these needsalternative sustainable sources of valuable dietary proteins are sought for.Scope and approach: In this review, we discuss alternative protein sources for human food consumption such asnovel foods derived from other animal sources like insects. Before these novel foods can enter the market place,their safety for consumers should be demonstrated. We herein provide an overview of the legislative frameworkcurrently in place across Europe, the key elements required for allergenicity assessment of novel foods, the toolsat disposal for allergenicity prediction and the most advanced technologies available for food allergen detectionand characterization.Key finding and conclusions: Effective characterization of potential protein-based allergenic hazards in novel foodingredients is essential to support effective risk assessment. Development of a cost-effective, validated tool box toallow improved hazard characterization for allergenicity risk assessment is needed. Although novel methodologies, such as mass spectrometry, have great potential for allergen characterization and allergen detection indifferent food contributing to reduce the risk for allergic consumers, some work is still required for methodvalidation and the creation of protein sequence databases for proteomic analysis.

The aim of this work was to check the efficacy of bovine lactoferrin (BLF) and its pepsin-digested hydrolysate (LFH) to control spoilage bacteria contaminating the governing liquid of high moisture (HM) Mozzarella cheese during cold storage. These natural substances resulted effective when tested in vitro against five potential spoilage bacteria contaminating cold-stored HM Mozzarella cheese. Among six LFH fractions, only the fraction containing lactoferricins, mainly represented by LfcinB?????, resulted effective against Escherichia coli K12 at the same extent of the whole pepsin-digested hydrolysate. LFH tested throughout seven days for its antimicrobial activity against the main bacterial groups growing in cold-stored commercial HM Mozzarella cheese samples delayed significantly the growth of pseudomonads and coliforms in comparison with the un-treated samples. This is the first report providing a direct evidence of the ability of LFH to inhibit the growth of cheese spoilage bacteria.

Le pigmentazioni anomale di mozzarelle sono state associate a ceppi psicrotrofi di Pseudomonas fluorescens presenti a basse concentrazioni nel liquido di governo già nelle prime fasi di frigoconservazione di tale formaggio fresco. Il mancato controllo di questi batteri alterativi ne causa la crescita determinando il ritiro dal mercato dei prodotti alterati. Scopo di questo lavoro è stato valutare l'efficacia antimicrobica in vitro ed in mozzarella dell'idrolizzato peptico di lattoferrina bovina (HLF) contro P. fluorescens 84095 isolato da mozzarelle blu. I risultati hanno mostrato che la crescita in vitro a 4?C del ceppo è stata inibita da 50 mg/mL di HLF. La stessa concentrazione, applicata a mozzarelle inoculate, ha determinato una significativa riduzione delle conte di 84095 rispetto a quelle registrate in campioni senza HLF fino al 14 giorno di frigoconservazione. Il trattamento ha inoltre bloccato lo sviluppo della colorazione blu. Le analisi LC-MS hanno confermato la presenza della leucoindigoidina, forma ridotta ed incolore del pigmento blu indigoidina solo nei campioni di mozzarella inoculati non trattati con HLF. Questo lavoro, pertanto, suggerisce l'applicazione di un idrolizzato antimicrobico di una proteina del latte, efficace per prevenire la colorazione blu, senza modificare la composizione della mozzarella. Parole chiave: peptidi antimicrobici, shelf-life, controllo alterazioni

The constant increase in seafood consumption worldwide has led to a parallel growth of the incidence of products obtained by aquaculture on the market, but also of the fraudulent commercialization of farmed products as wild-type ones. A careful characterization of the lipid component of seafood products based on chromatography-mass spectrometry techniques has been reported as a promising approach to reliably differentiate farmed from wild-type products. In this context, a fast method based on Direct Analysis in Real Time (DART) coupled to High Resolution Mass Spectrometry (HRMS) based on a single stage Orbitrap mass analyzer, integrated by Principal Component Analysis (PCA), was developed in the present study and applied to scout for spectral features useful to discriminate wild-type from farmed salmon of Salmo salar species. In particular, normalized intensities obtained for the 30 most intense signals (all referred to fatty acids, FA) detected in negative ion DART-HRMS spectra of the lipid extracts of salmon fillets [26 wild-type from Canada, 74 farmed from Canada (25), Norway (25) and Chile (24)] were considered as the variables for PCA. The scatterplot referred to the first two principal components showed a clear distinction between wild-type and farmed salmon, which gathered as a unique cluster, despite the remarkable differences in their geographical origin. In accordance with previous studies based on more complex and time-demanding analytical approaches, three saturated (14:0, 16:0 and 18:0) FA, along with unsaturated ones having 20 or 22 carbon atoms, were found as the main discriminating variables for wild-type salmons, whereas FA with compositions 18:1, 18:2, 18:3 and several oxidized forms arising from them were found to have a significantly higher incidence in farmed salmon. The method was further validated by Discriminant Analysis (DA) performed on the same dataset used for PCA integrated by data obtained from 6 commercial samples, putatively referred to farmed Norwegian salmon. Results showed that 100% of the latter were correctly classified as farmed type. Relative abundances of DART-HRMS signals related to specific FA appear then very promising for the differentiation of wild-type salmon from farmed ones, a very relevant issue in the context of consumers' protection from seafood frauds.

Ingestion of food is considered a major route of exposure to many contaminants including mycotoxins. The amount of mycotoxin resisting to the digestion process and potentially absorbable by the systemic circulation is only a smaller part of that ingested. In vitro digestion models turn useful for evaluating mycotoxins bioaccessibility during the intestinal transit and can be intended as a valuable tool for the assessment of mycotoxin bioavailability in food. In this paper we describe a study aimed at investigating toxicity of in vitro gastro-duodenal digests of mycotoxin contaminated bread collected along the digestion time-course. Toxicity tests were carried out on a sensitive RPMI lymphoid B cell line chosen as the most suitable lineage to assess toxicity retained by gastro-duodenal digests. In parallel, a chemical quantification of T-2 and HT-2 toxins contaminating the bread digests was accomplished during the gastric and duodenal transit. The digestive fluids undergoing chemical and toxicological analysis were collected at the beginning and end of gastric phase, and after completion of the duodenal phase. Results proved that a correlation between HT-2 content and toxicity did exist although a more persistent toxic activity was displayed in the later stage of the duodenal phase. This persistent toxicity might be explained by the co-occurrence of unknown HT-2-related conjugates or metabolites formed during digestion.

Nella presente comunicazione saranno descritte le potentizlità dell'accoppiamento della sorgente DART interfacciata alla spettrometria di massa ad alta risoluzione applicate ai diversi settori in campo alimentare per studi di tracciabilità e sicurezza.

ScopePeanut allergy is one of the most frequent allergies especially affecting developed countries. In order to reduce the risk of eliciting undesired reactions, a number of technological approaches have been devised to inhibit/remove allergens in order to deliver a hypoallergenic food. In the present work we investigated alternative strategies based on thermal treatments like autoclaving to decrease peanut immunogenicity.MethodsRaw and autoclaved peanuts were extracted, separated on SDS-PAGE and further submitted to immunblot analysis using sera of allergic patients. Each individual extract was further analysed by ELISA in order to estimate the residual immunoreactivity of the processed peanuts. The most resistant allergenic proteins displayed in the gel were finally identified by LC-HRMS analyses.ResultsA progressive reduction in the intensity of the major allergenic bands was highlighted in autoclaved samples; such behaviour was even more evidenced with a total disappearance of the major allergenic proteins when samples were preliminary exposed to hydration. These data also confirmed results obtained by ELISA analysis. Raw and treated peanut material were finally submitted to Western blot analysis in order to assess the residual immunogenicity of the treated peanuts. ConclusionHydrating peanut seeds prior to autoclaving increased the efficacy of the thermal treatment contributing to the disappearance of the main allergenic proteins and reducing significantly the final immunoreactivity, as assessed by ELISA tests and immunoblot analysis.

T-2 and HT-2 toxins are Fusarium mycotoxins frequently occurring in cereal-based products with a toxic effectascertained on different biological systems. Despite other mycotoxins, to date bioaccessibility of T-2 and HT-2toxins has never been investigated. In order to provide insights on T-2 and HT-2 stability and bioaccessibilityalong digestion in the upper intestinal tract, differently contaminated bread samples produced at laboratoryscale were submitted to in vitro digestion experiments. Two different contaminated bread models were preparedwith this aim. One employed naturally contaminated wheat flour for production of naturally contaminated bread,whereas in the other model dough was fortified with both mycotoxins during bread preparation before undergoing the leavening and baking process. Gastro-duodenal fluids were collected at different time-points along simulated digestion and mycotoxin content determined by LC-High Resolution-Mass Spectrometry. Our data report that HT-2 content in the beginning of the gastric phase was not significantly different from what was recorded at the end of the duodenal phase, although an apparent decrease of the signal was displayed in the early duodenal phase in both bread models tested. By contrast, the consistent drop of approximately 50% observed for T-2 when the duodenal digestion started remained constant until the digestion reached completion. In conclusion both approaches provided similar results thus considering incurred food models valid alternatives to undertake such investigations. Finally, bioaccessibility of T2 and HT2 was calculated in both food models tested with a higher bioaccessibility found in artificially contaminated bread samples. Industrial Relevance: Food is very complex both in composition and structure; therefore, generic realistic models that can mimic this complexity are required. Such models would greatly facilitate evaluation of the impact of changing composition or processing conditions on nutrition and safety. The primary scope of this work (part of the EU funded DREAM project) is to integrate experimental and mathematical approaches elsewhere optimised to develop a cereal-based food model realistic enough to be used by the industry and sufficiently versatile to be used as predictive tool of food behaviour. This is would be also applicable to study the release of contaminants from food such as mycotoxins. In general, food parameters may affect mycotoxins bioavailability from foods. A number of mycotoxins are currently regulated in foodstuffs by the European Commission and as a consequence there is growing interest and concern of the public health authorities for the presence of mycotoxins in human food. Due to the different chemical structure and stability, the fate of mycotoxins during food processing and the relevant bioaccessibility upon digestion process vary considerably depending on the different food preparations. Limited data are currently available on the fate

Durum wheat is naturally more susceptible to Fusarium graminerum infection in comparison to common wheat. The improvement of durum wheat resistance against F. graminearum is a challenge due to the lack of resistance sources in its gene pool. FHB-resistance factors were introduced in durum wheat by generating recombinant inbred lines (RILs), obtained by crossing the hexaploid resistant accession 02-5B-318 with the susceptible durum wheat cv. Saragolla. In this work we explored the possible contribution of cell wall (CW) in RILs with improved FHB resistance. We thoroughly studied CW components, mycotoxins content and the expression of related genes in different RILs selected for their extremely high and low resistance to FHB. Differences were found in resistant and susceptible lines in the degree of pectin methylesterification and in deoxynivalenol (DON) accumulation after fungal infection. Genes involved in biochemical modification of CW structure (WheatPme-1, Glu-1) and mycotoxins accumulation (ns-LTP-1) were analyzed as putative candidates for FHB resistance. Our results indicate that durum wheat plants with cell wall structure and gene response acquired from common wheat displayed an increased resistance to FHB.

Owing to its extensive use in human diet, wheat is among the most common causes of food-related allergies and intolerances. Gluten proteins and particularly the gliadin fractions represent the main factor triggering celiac disease. Given the extremely high structural heterogeneity of gliadins, generated by amino acid insertions, deletions and substitutions, the physico-chemical properties of gliadins can vary significantly among wheat genotypes (species, cultivars and breeding lines) influencing in parallel the immunoreactive properties and the susceptibility to enzymatic treatment [1]. Therefore, the structural characterization and the correlation with relevant toxicity, by tracking the fate upon gastrointestinal digestion of wheat-based commodities [2], gains significance to deepen the knowledge at the molecular level of the immunological pathway and to identify naturally low toxic wheat species and/or efficient detoxification technologies.Recent development in proteomics have contributed to give insights in this field, although the analytical capabilities of the proteomic approach are challenged by the complexity of the wheat seed proteome and particularly of the gluten protein fraction. Limited database entries available, complexity arising from sets of homologue proteins, large occurrence of repeated motifs, very low number of basic residues for tryptic hydrolysis represent drawbacks that complicate the comprehensive proteomic cataloguing of the gluten proteins. These challenging issues can only be addressed by the use of integrated, up-to-date analytical approaches, which together constitute the platform of modern food proteomics, and where a pivotal role is played by mass spectrometry.

The improvement of a surface plasmon resonance (SPR)-based immunoassay for the detection of traces of egg-based fining agents in red wines is herein described. The latter represents an extension of a previously developed direct assay targeted to the detection of ovalbumin (OVA) as marker of the presence of egg white powder residues, a typical fining agent utilized by the winery industry. In this paper, a suitable pre-treatment procedure was optimized for the sensitive detection of OVA at sub-ppm levels in red wines fortified with egg-white powder, by using an immunoassay proved to be reliable for both white and roseé wines. A red wine from Chianti grapes selected as reference matrix was artificially contaminated with egg-white powder before undergoing different sample purification. Several purification strategies were investigated and tested in order to challenge the limit of detection (LOD) obtained with the methods currently in use for egg allergen detection. Finally, the optimized two-step pre-treatment, combining polyvinylpolypyrrolidone-based purification and size exclusion chromatography, enabled to achieve an LOD in red wine as low as 0.2 ?g/mL. The optimized SPR-based method met the method performance criteria issued by the International Organization of Vine and Wine (OIV) concerning the minimum sensitivity required for the analyses of potentially allergenic fining agent proteins in wines, confirming the biosensor as promising tool to monitor the residual contamination level of fined red wines.

in questa relazione saranno isllutrati i vari approcci utilizzait per ridurre l'allergenicità degli alimenti. Sarà inoltre presentato un caso studio su trattamenticombinati applicati a nocciole al fine di ridurne il potere allergenico per i consumatori allergici. Inoltre saranno presentati alcuni risultati emersi dallo studio nato in collaborazione fra ISPA-CNR di Bari e l'unità di Allergologia del Policlinico di Bari sotto la guida del prof. Macchia.

The identification of wheat genotypes with low toxicity could represent a valid alternative for the prevention of wheat intolerance onset. Over the last years, great efforts have been devoted to develop effective gluten detoxification strategies mostly based on enzymatic strategies, which, however, involve a simultaneous detrimental alteration of the technological properties. In this frame, obtaining low-gluten wheat products without affecting their rheological properties is still a challenging issue.In this contribution, we presented an integrated approach encompassing both proteomic characterization and grains yield/quality evaluation for the identification of durum wheat genotypes combining potential lower toxicity/immunogenicity with satisfactory rheological properties. A preliminary profiling of gluten proteins was accomplished by immunoassay-based quantification and liquid chromatography coupled to UV detection focusing on the gliadin fraction as main responsible for immunoreactivity in celiac disease patients. In addition, complementary information about productivity-related traits and quali-quantitative characteristics were collected, such as grain protein content, grain yield per spike, dry gluten and gluten index. The whole pool of data was statistically evaluated confirming that durum wheat breeding programs accomplished in the last 25 years improved the pasta-making quality (gluten strength), without causing an increment of toxic epitopes towards CD patients. Tracking the fate of gluten proteins upon in-vitro simulated gastroduodenal digestion experiments and in-silico assessing the risk of toxicity according to the most recent guidance provided by EFSA1, confirmed such statement. The selected genotypes boasting medium and strong gluten strength, all presented a significantly lower number of toxic epitopes compared to commercial semolina. In perspective, such genotypes could represent an innovative alternative for preventive and therapeutic wheat based foods in genetically predisposed individuals who may develop CD after prolonged wheat or gluten consumption.

Food allergies are a serious health concern with increasing worldwide prevalence. Food legislation in place in several countries, requires detailed declaration of allergens in foods implying capability of methodologies to reliably trace food allergens. However, detecting and quantifying food allergens remains a challenge. Current common methods for food allergen analysis utilize antibody-based assays although some drawbacks are encountered such as matrix/processing effects and epitope masking especially when dealing with complex and processed foods. Therefore, sensitive, reliable, robust, fast, reproducible, and standardized methods are necessary for improved allergen analysis and reduce the risk of allergen contamination. In the last decade, mass spectrometry (MS) techniques have been developed and applied with success to food allergen detection. This review compares different aspects of food allergen quantification using advanced MS techniques including multiple reaction monitoring. The latter provides low limits of quantification for multiple allergens in complex food matrices, while being robust and reproducible. (C) 2018 Elsevier B.V. All rights reserved.

Mass spectrometry (MS) represents an essential tool in proteomics studies, in the last years also exploited for monitoring allergens contamination in food products. Milk and egg are renowned allergens often used as fining agents to promote clarification of wines, therefore any residual amount in the endproducts could represent a menace for allergic individuals. In view of this, it is of paramount importance to have at disposal sensitive analytical methods able to detect traces of milk and egg allergens in food. In this work we describe the upgrade and the optimization of an analytical workflow based on the use of a pre-enrichment column coupled with HPLC separation and MS/MS detection for the selective and sensitive detection of milk and egg allergens in white wine. Two different sample pre-treatments based on the use of mass cut-off filters or size exclusion cartridges were evaluated and compared, before tryptic digestion and LC-SRM-MS/MS analysis of the resulting peptides mixture. The devised UF based method coupled with peptide on-line pre-enrichment enabled to reach the lowest LODs down at 0.036 ug/mL and 0.05 ug/mL for egg and milk allergens respectively, proving to be the most sensitive strategy for monitoring allergens contamination in wine.

Allergen detection and quantification is an essential part of allergen management as practiced by food manufacturers. Recently, protein MS methods (in particular, multiple reaction monitoring experiments) have begun to be adopted by the allergen detection community to provide an alternative technique to ELISA and PCR methods. MS analysis of proteins in foods provides additional challenges to the analyst, both in terms of experimental design and methodology: (1) choice of analyte, including multiplexing to simultaneously detect several biologically relevant molecules able to trigger allergic reactions; (2) choice of processing stable peptide markers for different target analytes that should be placed in publicly available databases; (3) markers allowing quantification (e.g., through standard addition or isotopically labeled peptide standards); (4) optimization of protease digestion protocols to ensure reproducible and robust method development; and (5) effective validation of methods and harmonization of results through the use of naturally incurred reference materials spanning several types of food matrix.

A sensitive and accurate method employing a single stage high resolution mass spectrometer equippedwith a high-energy collision-dissociation cell (HCD) for the simultaneous determination of deoxynivalenol(DON), T-2 toxin (T-2) and HT-2 toxin (HT-2) in a processed bread model food has been developed.Two sample pre-treatment routes for the extraction of these mycotoxins were investigated, based onMycosep® column clean up or QuEChERS-like procedure, respectively. The former approach suffered lessfrom matrix effects and allowed to achieve in bread samples LODs of 7, 12 and 17 ng/g for T-2, HT-2and DON, respectively, with 0.5 ppm mass accuracy. Two acquisition modes, full scan MS and all ionfragmentation, exploiting the fragmentation features offered by an HCD chamber and integrated withinthe Orbitrap analyser, were compared for quantitative purposes. The method was applied to investigatethe degradation of these mycotoxins during bread processing using a bread model food. Most T-2hydrolyzed to HT-2 during dough preparation, and about 20-30% of HT-2 and DON was degraded duringbread baking.

A Mass Spectrometry ImmunoAssay (MSIA) specifically designed for the detection of egg allergens in wines is described. This approach is based on an immunoaffinity enrichment procedure combined with targeted MS/MS detection of selected egg peptide markers. Polyclonal antibodies raised against native ovalbumin, chosen as the target protein tracing for egg powder, were immobilized onto low backpressure monolithic MSIA customized disposable tips. Ovalbumin-free wine samples were fortified with standard protein at different concentration levels in the low µg/mL range. A simple protocol was devised consisting in a 1:4 dilution of the wine sample with a basic solution for pH adjustment, followed by a semi-automated purification/enrichment step on customized MSIA disposable tips fitted on a multichannel electronic pipette. Among the main figures of merit LOD and LOQ values as low as 0.01 and 0.03 ?g/mL, respectively, of 18 % should be noticed. Noteworthy, the developed assay outperformed current MS-based methods for the detection of allergenic protein in wine matrices, thanks to the immunoaffinity enrichment. In addition compared to other immunoassays, the present approach boasts the unquestionable advantage to provide an unambiguous identification of the target protein, by simultaneous detection of three unique peptide markers each giving three specific MS/MS transitions.

A method using the combination of size exclusion-solid phase extraction and ultrafiltration, followed by trypticdigestion and analysis of the protein digest by liquid chromatography-electrospray ionization-3D ion trap-mass spectrometry (LC-ESI-3D IT-MS), was developed for the detection and quantification of caseinate traces potentially resulting from fining processes in white wines. In particular, several tryptic peptides generated from the main proteins constituting caseinate (?-, ?S1-, and ?S2-casein-caseins) were used as markers of its presence in the wine matrices; among them, the ?-casein peptide GPFPIIV was found to be the best marker for quantification purposes. Method linearity and sensitivity were assessed on a series of Italian commercial white wines, first checked for the absence of any peptide signal attributable to caseins introduced during their production and subsequently spiked with increasing concentrations of caseinate, to provide samples for matrix-matched calibrations. Limits of detection ranging between 0.09 and 0.29 mg/L (S/N = 3), according to the wine, were achieved using a 10 mL sample volume and the MS signal of GPFPIIV as the response related to the caseinate concentration. Such levels are comparable or even lower than the one (0.25 mg/L) recently adopted as a threshold by European Union legislation concerning the indication of milk- and egg-derived fining agents on wine labels, that is, the most restrictive one among those currently proposed in the world.

Food allergy is nowadays regarded as a problem of public-health relevance, the main concern being the unintentional exposure of allergic consumers to the offending ingredient through allergen-containing food. Rapid diagnostic tools are increasingly being requested by food companies to verify the efficiency of their management schemes for food safety. Although no specific reference analytical method for the determination of fining agent proteins has been prescribed, the international Organization of Vine and Wine (OIV) resolution 427-2010 modified by the OIV/COMEX 502-2012 set up the analytical requirements to be fulfilled by methods under development. In particular, ELISA methods must comply with the detection limits and the quantification limits of <=0.25 and 0.5 mg/L, respectively.In the present communication, the development of a surface plasmon resonance (SPR)-based biosensor tailored to the fast detection of egg related fining allergens in wines is described. Ovalbumin (OVA) was chosen as target protein to be monitored due to its highest abundance in the egg white (EW) powder, a typical fining agent used by the winery industry to promote wine clarification. A direct assay was designed, basing on the use of polyclonal anti-OVA antibody as bio-specific receptor. After the fine tuning of all parameters able to influence the final response, the assay was tested in a direct assay for OVA in commercial wines artificially contaminated with EW powder. The devised assay allowed to trace, in a short analysis time and with a minimal sample pre-treatment, the presence of egg allergens at the lowest concentration comprised between 0.03 and 0.2 ?g/mL [1].

Recent technological advances in instrumentation and sample preparation have raised the attention towards MS-based targeted protein immunoassays as attractive alternative to classical ELISAs [1]. The Mass Spectrometry ImmunoAssay (MSIA(TM)) approach utilizes the MSIA D.A.R.T.'s technology which comprises a molecular trapping micro-column contained within a pipette housing. A capture antibody is immobilized onto the micro-column and the antigen containing sample is applied by repetitive pipetting cycles. After washing and elution steps, the target antigen can be either directly injected into mass spectrometers or subjected to enzymatic digestion for peptide markers detection. The combination of, immunoaffinity capture and mass spectrometry provide complementary advantages: the first enables a specific binding of the target molecule from a complex matrix using the immobilized antibody with its consequent selective enrichment on the micro-column and the prospective to fully automate the procedure; MS instead provides an unambiguous detection, as antigen signals are observed at characteristic m/z values in the mass spectrum, overcoming common issues of immunoassays, such as false positives.So far, MSIA technology has found mainly applications in clinical fields where the complexity of biofluids, such as blood plasma and serum, impairs the sensitivity, robustness and throughput of routine MS-based protein detection [2 and reference thereof].In the present investigation, MSIA approach was applied to the food safety field. Notably, the applicability of a MSIA based workflow was tested for the detection of residual egg proteins in wine. Egg-derived products, in various commercial preparations, are commonly utilized in winemaking thanks to the ability to interact and promote precipitation of wine polyphenols and other undesirable compounds [3]. However, any residues of egg white proteins remaining in wine could represent a risk for allergic consumers. In this scenario, the development of analytical approaches for the detection of egg proteins might open new perspectives for the producers, that might spot the real risk associated to certain procedures where allergens are likely to remain as residues. Till now, no official method for the determination of fining agent proteins is prescribed.Here, the feasibility of the MSIA approach for the detection of such residues in white wines was investigated, combining the semi-automated immunoaffinity enrichment with selective reaction monitoring (SRM) detection of specific peptide markers. The micro-columns in the MSIA D.A.R.T's were chemically modified to bind a polyclonal antibody commercially available to specifically recognize native ovalbumin (OVA), the most abundant protein in the egg based fining agent typically used for wine clarification. As a proof of concept, the MSIA D.A.R.T's were employed with the Finnpipette(TM) Novus i Multichannel Electronic Pipette which processes up to 12 samples simultane

Direct analysis in real time-high resolution mass spectrometry (DART-HRMS) was applied to the detection of lipid species in the lipid extracts of farmed salmon samples collected from a local retailer and analyzed right after the purchase and after storage for 4 and 6days under refrigerated conditions. The recognition of type and composition of lipids detected in DART-HRMS spectra was performed by using the relevant accurate m/z data (accuracy better than 5ppm) as input for a search on the LipidMaps database. As a result, several fatty acids (FA), either saturated or mono-/poly-unsaturated, and triacylglycerols (TAG) were recognized in the three types of samples from the corresponding negative and positive ion DART-HRMS spectra, respectively. Following, spectral intensities were exploited to monitor the evolution of selected FA and TAG during the refrigeration of salmon meat. In particular, after 4days of refrigeration, a statistically significant increase was recorded for FA with side chain compositions 18:2, 18:1, 20:5, and 22:6 despite a significant decrease found for TAG with overall side chain compositions 50:4, 52:5, 52:4, and 52:3 after the same time. These evolutions were consistent with a general model already proposed for the effect of low temperature treatments on seafood, implying the action of endogenous lipases, with consequent increase of the free FA amount and decrease in glycerophospholipids and triglycerides contents. The described results indicate DART-HRMS as a promising MS-based rapid tool for the assessment of fish, or other seafood, freshness.

Introduction Food authenticity aims to guarantee product safety, transparency and protection of consumers' health. In order to safeguard food authenticity, a variety of analytical techniques have been investigated some of which are routinely used. If on one hand some methodologies typically based on MS detection revealed successful for the identification of selected molecules as suitable markers for species identification, on the other hand the untargeted methods utilizing MS detection appear not deeply investigated in this specific field. In the present communication, a non targeted approach based on DART ionization coupled to High Resolution Mass Spectrometry was described and applied for the discrimination of two types of salmons -farmed versus wild type-, belonging to the Salmo salar species and collected in Canada. In particular, the apolarfractions were analysed and the final data were further subjected to multivariate statistical analysis to highlight eventual differences between bothdata-set. ExperimentalDifferent fish homogenates were prepared from 26 canadian wild type salmons (CND-WT) and25 canadian farmed salmons (CND-F). Aliquots of 2.5 g of each fish homogenate were added with 5 mL of refrigerated methanol and 5 mL of refrigerated chloroform and stirred for 15 min at R.T.After decantation for 2 hours at 4°Cthe lower apolar fraction was withdrawn and first evaporated under nitrogen flow and then resuspended in an equal volume of isopropanol before HRMS analysis. Analysis were performed by DART ionization (IonSense) coupled to HRMS using an Exactive(TM) (Thermo Fisher Scientific) system in negative ion mode by setting the gas temperature 150°C. The total peak lists of all ions generated from each analysis was converted in .csv format and processed for the multivariate statistical analysis by using the free online software MetaboAnalyst 3.0. ResultsAfter duly inspecting the HRMS spectra referred to the analysis of the apolar fraction, it appears that the ion m/z 281.247 is the most intense in CND-F samples despite the ion m/z 255.232 instead dominating the CND-WT spectra. By entering the m/z values into the LipidMaps database, the ion species were likely attributed to thestearic acid (m/z 281.247) and palmitic acid (m/z 255.232) suggesting their possible role as potential biomarkers indicator of the type of capture. By applying the multivariate statistical analysis e.g. the Principal Component Analysis(PCA) on the list of ionsa good separation between the two fish groups (farmed vs wild type) was foundwith PC1 and PC2 that explained respectively 68.5% and 23.3% of the total variance.ConclusionsIon differences between the CND-F and CND-WT samples werehighlightedby applying DART-HRMS analysis followed by multivariate statistical analysis on the whole dataset. In particular, PCA resultsshowed a good separation of both salmon groups. In future, a supervisioned approach might be envisaged to further improve such separation between b

Saranno illustrate nella Lezione Magistrale i diversi approcci metodologici sviluppati e volti alla riduzione di allergenicità di alimenti

Several buffer compositions were compared for their efficiency in protein extraction from both raw and roasted peanut and hazelnut samples, the final goal being to understand the modification of protein solubility upon roasting and maximize the extraction yield. Denaturant conditions provided by urea-TBS buffer resulted in satisfactory extraction yields for both peanut and hazelnut samples, before and after the thermal treatment. In addition, different varieties of peanuts and hazelnuts were characterized to highlight the extent of variability in the protein profile accounted by the varietal factor and eventual differential resistance among cultivars to protein modification induced by the thermal processing. The protein profile was characterized by gel electrophoresis, and specific bands were analyzed by micro-HPLC-MS/MS coupled to software-based protein identification. No significant difference was observed for the investigated hazelnut cultivars, namely, Campana, Romana, and Georgia, whereas interesting features were presented for the peanut varieties Virginia, Zambia, and China. In particular, Zambia variety lacked two bands of approximately 36 and 24 kDa that were visible in Virginia and China varieties, which could suggest a lower allergenic potential of this particular variety which deserves to be further investigated before drawing final conclusions.

La contaminazione da micotossine rappresenta una delle maggiori problematiche nella gestione del rischiodella sicurezza e della salubrità dei prodotti alimentari. I cereali -come frumento, orzo e mais -e i prodotti derivati, risultano frequentemente contaminati dai tricoteceni quali deossinivalenolo (DON) e dalle tossine T-2 e HT-2 come risultato dell'accrescimento di specie fungine Fusarium su piante o granaglie. È stato ampiamente dimostrato che il processo di trasformazione degli alimenti può notevolmente influire sulla stabilità delle micotossine, alterandone i contenuti nei prodotti finali. Nel presente lavoro è stato valutato l'effetto della panificazione sulla stabilità dei tricoteceni DON, T-2 e HT-2 in panetti prodotti a partire da farine naturalmentecontaminate dalle tre micotossine. Insieme alle forme native, sono state analizzate anche alcune forme glicosilate ovvero DON-3 Glucoside, T-2 Glucoside e HT-2 Glucoside che, al pari delle micotossine native,potrebbero costituire un rischio per la salute umana. I risultati mostrano che i livelli finali di DON aumentanoin seguito alla panificazione, mentre quelli del DON-3 Glucoside appaiono dimezzati. Per le tossine T-2e HT-2 si osserva in generale una variabile suscettibilità alla cottura.

Deoxynivalenol, T-2 and HT-2 toxins are mycotoxins frequently occurring in cereals and cereal-based products along with their conjugated forms. In this paper, we provide insights into the fate of deoxynivalenol, T-2 and HT-2 toxins and their glucoside derivatives during bread making, using naturally contaminated wheat flour. High-resolution mass spectrometry was used to assess the extent of degradation of the three mycotoxins during bread baking and to identify some glucoside conjugates, namely deoxynivalenol, T-2 and HT-2 mono-glucosides, detected both in the flour and in the respective breads. Our findings show deoxynivalenol's levels markedly increased upon baking, whereas those of HT-2 and T-2 toxins were decreased in the final bread with special regard to the T-2 toxin.

The capability of a capillary LC-Q-TOF mass spectrometer system as a qualitative tool for theidentification and confirmation of milk allergens in thermally processed food was investigated. Milkpowder incurred cookies were produced in-house and chosen as the model food matrix. Tounequivocally assess the presence of milk allergens, samples testing positive to ELISA were analysed bya capillary LC-Q-TOF MS/MS method in order to identify specific peptides that can be used as markersfor milk allergens. Results show that a-S1 casein was the protein identified with the highest score(in 100 mgg1milk powder incurred cookies) and its identity was confirmed by detection of the peptidesm/z 692.86 and 634.34 and their specific MS/MS ions providing a fingerprint for a-S1 casein. Besidesthat, other milk proteins were highlighted by performing database searching. The proteomic MS-basedmethod employing a capillary LC-Q-TOF system proved to be a valuable tool to carry out qualitativeand confirmative analysis to trace contamination of milk allergens in processed food matrices.

Almond is consumed worldwide and renowned as a valuable healthy food. Despite this, it is also a potent source of allergenic proteins that can trigger several mild to life-threatening immunoreactions. Food processing proved to alter biochemical characteristics of proteins, thus affecting the respective allergenicity. In this paper, we investigated the effect of autoclaving, preceded or not by a hydration step, on the biochemical and immunological properties of almond proteins. Any variation in the stability and immunoreactivity of almond proteins extracted from the treated materials were evaluated by total protein quantification, Enzyme Linked Immunosorbent Assay (ELISA), and protein profiling by electrophoresis-based separation (SDS-PAGE). The sole autoclaving applied was found to weakly affect almond protein stability, despite what was observed when hydration preceded autoclaving, which resulted in a loss of approximately 70% of total protein content compared to untreated samples, and a remarkable reduction of the final immunoreactivity. The final SDS-PAGE protein pattern recorded for hydrated and autoclaved almonds disclosed significant changes. In addition, the same samples were further submitted to human-simulated gastro-intestinal (GI) digestion to evaluate potential changes induced by these processing methods on allergen digestibility. Digestion products were identified by High Pressure Liquid Chromatography-High Resolution Tandem Mass Spectrometry (HPLC-HRMS/MS) analysis followed by software-based data mining, and complementary information was provided by analyzing the proteolytic fragments lower than 6 kDa in size. The autoclave-based treatment was found not to alter the allergen digestibility, whereas an increased susceptibility to proteolytic action of digestive enzymes was observed in almonds subjected to autoclaving of prehydrated almond kernels. Finally, the residual immunoreactivity of the GI-resistant peptides was in-silico investigated by bioinformatic tools. Results obtained confirm that by adopting both approaches, no epitopes associated with known allergens survived, thus demonstrating the potential effectiveness of these treatments to reduce almond allergenicity.

Mass spectrometry (MS) has played a pivotal role in proteomic research being the election method for protein identification in complex mixtures. In the last decade, the MS-based proteomic approaches have demonstrated to be a valuable confirmatory tool for allergen contamination management [1]. Our research group, actively contributed to such field developing label-free quantitative methods for the multiple allergen detection in several food matrices based on both low and high-resolution mass spectrometers [2-8]. Thanks to the advances provided by last-generation high resolution mass spectrometers (HR-MS) based on OrbitrapTM technology very high selectivity and sensitivity were achieved by the developed methods.In the present communication, performance provided by a hybrid quadrupole-OrbitrapTM MS platform will be presented. In particular, different acquisition modes were compared: Full-MS acquisition, targeted-Selected Ion Monitoring with data-dependent fragmentation (t-SIM/dd2) and Parallel Reaction Monitoring (PRM). The different acquisition modes were tested towards the detection of specific peptide markers arising from five different allergenic ingredients (milk, egg, soy, hazelnut, peanut) in home-made incurred cookies, selected as model processed matrix. In order to challenge the HR-MS platform, the sample pretreatment was kept as simply as possible, limited to a 30 min protein extraction followed by quick purification based on size exclusion chromatography by disposable cartridges. The three acquisition modes were independently optimized and compared in term of sensitivity, by means of ad-hoc calibration curves. In addition, performances provided by such hybrid HR-MS platform were compared with an optimized HPLC-ESI-SRM method we recently developed based on linear ion trap MS spectrometer [7] for the same kind of processed food matrix.

Peanut represents one of the most harmful allergenic foods capable of triggering severe and sometimes lethal reactions in allergic consumers upon ingestion of even small amounts. Several proteins capable of inducing allergic reactions that have been recognised by patients' IgE antibodies have been identified from this nut source. Methods mainly based on ELISA assays have been developed in order to detect peanuts in several food commodities. In addition LC-MS/MS methods based on different mass analysers have also been devised for tracing peanut contamination in different foods achieving low limits of detection. The applicability of a benchtop high-resolution Exactive(TM) mass spectrometer has never been investigated for the rapid screening of peanut contamination in complex food matrices like mixtures of nuts. We report in this paper the design of suitable peanut markers and the development of an high-resolution Orbitrap(TM) mass spectrometer-based method for peanut detection in a mixture of nuts species. With this aim, different types of samples were prepared: (1) nuts-based powder made up of a mixture of hazelnuts, pistachios, almonds and walnuts; and (2) nuts powder fortified with peanuts. Different levels of fortifications were produced and the applicability of the method was tested. Finally, a subset of six peptides fulfilling specific analytical requirements was chosen to check the suitability of the method tailored to the detection of peanuts in nuts-based products, and two of them, peptides VYD and WLG, were selected as quantitative markers. The method proved to be a suitable screening tool to assess the presence of traces of peanuts in other tree nuts with a limit of detection as low as 4 µg of peanuts proteins or 26 µg of peanuts in 1 g of matrix.

Deoxynivalenol, T-2 and HT-2 toxins are trichothecene mycotoxins frequently occurring in cereals and cereal based products. Due to the toxic effects they can exert on human health, the study of the influence of food processing on the final mycotoxin content becomes a crucial point for the estimation of the risk. In addition, investigation needs to be extended also to modified mycotoxins, that still possess a toxic potential and which could co-occur along with their native forms in raw material and derived food. In thispresentation a previously developed HighResolution mass spectrometry based method has been applied to quantify the presence of DON, T-2 and HT-2 both in naturally contaminated wheat flours and in their respective bread samples in order to investigate the fate of these mycotoxins through baking. On the other hand the same method was also applied for the identification of DON, T-2 and HT-2 glucoside conjugates in both matrices. Our findings show that unlike DON which shows a significant increase upon baking, HT-2 and T-2 levels decreasedin bread with a more remarked drop displayed by T-2. On the other hand a parallel decrease of DON-glucoside onjugate (DON-3Glu) was recorded after baking. Significant levels of T-2 and HT-2 mono-glucosides were also identified and detected both in the wheat flour and bread although a more careful estimation on the final levels cannot be made due to the lack of a standard. This represents an important issue worthy to be deepened for the implications that some conjugated mycotoxins can have on human health, in order to avoid underestimation of the risk.

A method based on capillary liquid chromatography combined with electrospray ionization-tandem mass spectrometry (CapLC-ESI-MS-MS) for the detection and identification of casein deriving peptides in fined white wine is described. This is the first step towards the development of a liquid chromatography mass spectrometric method for the detection/identification of markers of potentially allergenic milk proteins used as wine fining agents. The method demonstrated to be capable of detecting some peptides arising from alpha and beta casein (with the relative aminoacidic sequences elucidated) in extracts of white wine fined with casein at 100 and 1000 mu g/mL. This MS based approach appears to be a useful tool for screening purposes as well as a confirmatory tool for the unequivocal identification of caseins in ELISA positive samples.

The hazard of hidden allergens in the food chain has generated the need for sensitive and reliable methods tracing food allergens in different commodities. The most recent methods employing immunochemical and DNA recognition for food allergen detection are reviewed and compared to mass spectrometric methodologies. Major issues such as the influence of food matrix and food processing on the extraction/detection of food allergens are also tackled In order to produce trustful results there is urgent need for reliable methods underpinning quality assurance scheme. Elements including the use of reference materials, method validation and proficiency testing scheme in food allergen analysis are discussed.

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 60-100mugingred/g allergenic ingredient/matrix in incurred cookies.

Milk allergens are common allergens occurring in foods, therefore raising concern in allergic consumers.Enzyme-linked immunosorbent assay (ELISA) is, to date, the method of choice for the detection of foodallergens by the food industry although, the performance of ELISA might be compromised when severefood processing techniques are applied to allergen-containing foods. In this paper we investigated theinfluence of baking time on the detection of milk allergens by using commercial ELISA kits. Baked cookieswere chosen as a model food system and experiments were set up to study the impact of spiking a matrixfood either before, or after the baking process. Results revealed clear analytical differences between bothspiking methods, which stress the importance of choosing appropriate spiking methodologies for methodvalidation purposes. Finally, since the narrow dynamic range of quantification of ELISA implies that dilutionof samples is required, the impact of sample dilution on the quantitative results was investigated. Allparameters investigated were shown to impact milk allergen detection by means of ELISA.

Soy is an important component of the human diet thanks to its nutritional value and high protein content; however, it also represents a risk for allergenic consumers due to its potential to trigger adverse reactions in sensitized individuals. The putative correlation between immunoreactivity and resistance to the human gastrointestinal (GI) digestion has drawn attention to investigating soybean proteins digestibility. In this work, we provided further insights into this field by performing in vitro simulated GI digestion experiments directly on ground soybean seeds, to provide more realistic results obtained from the digestion of the whole food matrix. Soybean digestion products were analyzed by SDS-PAGE followed by untargeted HPLC-MS/MS analysis and the final data were software treated to enable protein/peptide identification. The latter allowed monitoring the proteolytic degradation of the main soybean proteins during the gastric and duodenal phases. In particular, beta-conglycinin and trypsin inhibitors showed the highest resistance to the combined activity of GI enzymes, showing a partial degradation at the end of the duodenal phase as ascertained by the strong electrophoretic bands displayed at 50 kDa and 20 kDa, respectively. Glycinin subunits also presented, even if to a lower extent, resistance to the complete proteolytic degradation, as demonstrated by polypeptide fragments with molecular weight lower than 20 kDa displayed in the gel at the end of duodenal digestion. In addition, by bioinformatics analysis it was demonstrated that the GI resistant fragments of the allergenic proteins, beta-conglycinin and glycinin, retained in their primary structure linear epitopes potentially able to trigger an immunoreaction when exposed to the intestinal mucosa. Moreover, such resistant peptides also presented a structural homology with epitope sequences recognized in other legume species, presenting a potential risk of adverse cross-reaction for a larger category of allergic consumers.

IntroductionEffective enzymatic gluten detoxification strategies have been developed in the last years; however, obtaining low-gluten wheat products without impairing the rheological properties remains a challenging issue [1]. In this contribution, we presented an integrated analytical approach for the identification of wheat genotypes with reduced toxicity and satisfactory rheological properties [2].MethodsA comprehensive characterization of durum wheat genotypes was performed including grain quality traits (productivity-related and quali-quantitative characteristics) and proteomic profiling (R5-ELISA, HPLC-UV analysis). The data were evaluated statistically, and a selected list of candidates was subjected to in-vitro gastroduodenal digestion [3] and discovery MS/MS analysis for in-silico toxicity risk assessment [4].Results38 accessions of Triticum turgidum sp., including both wild and cultivated ones, were investigated. A preliminary profiling of gluten proteins was accomplished on the whole collection focusing on the gliadin fraction as main responsible for immunoreactivity in celiac disease (CD) patients. Complementary information about grain protein content, grain yield per spike, dry gluten and gluten index were collected. Cluster analysis was performed on original variables supporting the proper selection of five genotypes featuring medium and strong gluten strength, together with R5-reactivity and gliadin content lower than commercial semolina. Finally, the fate of gluten proteins was evaluated upon in-vitro simulated gastroduodenal digestion experiments carried out on raw wheat flours. The in-silico toxicity evaluation assessed a significantly lower number of toxic epitopes than commercial semolina.ConclusionsThe integrated approach confirmed that durum wheat breeding programs accomplished in the last decades improved the gluten strength, without causing an increment of toxic epitopes. Even if none of the selected genotypes can be considered safe for CD patients, a lower toxicity level could be envisaged and, in perspective, they may represent an innovative solution in genetically predisposed individuals who may develop CD after prolonged gluten consumption.

In this communication, we investigated the feasibility to develop an SPR based method tailored to the detection of egg residues in wines, the final goal being the elimination of matrix-effect on the analytical response. Two model wines matrices were selected, subjected to various purification procedures, and compared pair-wise with the standard curve both in terms of specific analytical responses and calibration curve slopes. Different statistical tools were used for significant comparisons.

Deoxynivalenol (DON) and its main conjugate derivative, DON-3 glucoside (DON-3G), are Fusarium mycotoxins frequently occurring in cereal and cereal-based products destined to human consumption. In this paper, we investigated the stability of DON and DON-3G along the gastric and duodenal-jejunal tract (GI) by submitting a bread food naturally contaminated with both mycotoxins to in vitro digestion experiments. Gastro-duodenal digestion fluids were collected at different time-points along the whole digestion process (gastric and duodenal phases) and mycotoxin content was determined by LC/High Resolution Mass Spectrometry. Our findings show a rather stable behaviour displayed by DON during gastric digestion, whereas a 43% DON decrease was recorded during the passage from the gastric to the duodenal compartment. Ultimately, the apparent and actual bioaccessibility was calculated for DON with values ranging between 34 and 57%, respectively. Data obtained for DON-3G showed no significant differences between the beginning and the end of the gastric phase, whereas a remarked increase of this conjugated mycotoxin was highlighted during the passage in the duodenal compartment. Our results suggest that a conversion of DON in DON-3G is likely to occur during gastro-duodenal digestion of contaminated bread.

Food allergen research has considerably expanded its field of interest in recent years probably due to the increasing incidence of food allergies throughout the population. According to the last legislation issued on this issue, there is a current trend to develop reliable methods tailored to the detection of food allergens for routine-like applications. Lately, MS-based methodologies have attracted the interest of researchers owing to the great potentials offered by this technology, the ability to perform multi-allergen screening in food products. The MS approach is currently being adopted by the allergen detection community, proving to be a valid alternative to ELISA and PCR methods. MS methodologies applied to food allergen detection are herein presented.

In the present investigation, an LC-MS method for sensitive multiplex detection of five allergenic ingredients in a processed food matrix is presented. Cookie was chosen as complex food model and was incurred with egg, milk, soy, hazelnuts and peanuts before baking. Extraction, purification and pre-concentration protocols were applied to ground cookie basing on protocols described elsewhere. Specific instrumental features of a dual cell linear ion trap MS instrument were exploited to identify suitable peptide markers for each allergen and to deliver a sensitive multiplex SRM-based method for the simultaneous detection of common allergenic ingredients which might contaminate such a commodity.

Ambient ionization techniques have been demonstrated challenging and straightforward strategies for food fingerprinting or food-authenticity assessment. Among them, direct analysis in real time (DART) is an ambient ionization technique that coupled with various MS analyzers demonstrated to be successful for both target and non target analysis of several complex foods [1, 2]. Fish is an important source of nutrients in the human diet and salmon, in particular, is considered a valuable source of "healthy" fatty acids thanks to the presence of long chain omega-3 fatty acids eicosapenteanoic acid and docosahexaenoic acid. However, factors such as the living conditions of the salmons whether farmed or caught or the non appropriate storage conditions applied during food circulation or dispatch to the local retailers might affect considerably the quality and the final composition of the lipid fraction, also compromising the availability of some beneficial compounds.In this presentation the coupling between DART and a mono-stage Orbitrap(TM) based High Resolution Mass Spectrometer has been for the first time exploited for rapid lipid profiling of Salmo salar stored for several days under refrigerated conditions. First DART and HRMS parameters were duly tuned in order to achieve a rapid and reliable analysis in the shortest time. Afterwards, samples were extracted according to a general protocol and further analysed by DART-HRMS in order to highlight significant changes in the lipid profile over time. Thanks to the high mass resolving power offered by the instrument in use, providing a mass accuracy better than 5 ppm, the composition of some lipids could be also inferred by entering the elemental composition predicted by the XCalibur(TM) software into the Lipid Maps database.The method DART-HRMS applied to the analysis of salmon under different storage conditions highlighted significant changes appearing along the lipid profile over the explored time. Results showed that prolonging storage of the product for a period longer than 3 days produced a remarked increase in the levels of specific fatty acids and a consequent decrease in the triacylglicerol levels. In perspective these findings open towards the possibility to employ such strategy to monitor salmon freshness through the analysis of the lipid profile. Acknowledgments: This work has been supported by the European project FOODINTEGRITY (FP7-KBBE-2013-single-stage, No 613688).

An overview about the methodologies devepoed over the last years for mutliple allergen detection are in this communication discussed and presented into details.

The protection of allergic consumers have become an important health issue especially in industrialized countries. To protect allergic people, labelling rules in European Directives 2003/89/EC and 2006/142/EC establish that consumers must be provided with comprehensive ingredient listing information to allow them to easily identify ingredients they need to avoid. Such labeling requirements were extended in 2014 to unpackaged food including catering outlets, deli counters, bakeries and sandwich sellers. At the moment, food manufacturers can use an advisory label (such as "May contain ...") on packaged and unpackaged foods to inform consumers about the potential presence of allergens due to cross-contamination and/or clean down efficiencies during food production. Currently, allergen detection in contaminated food is mainly based on immunochemistry techniques (ELISA) although this method could be affected by cross-reactivity and/or by possible epitope masking caused by food processing. As consequence, an alternative method for confirmatory analysis is required. Mass spectrometry is a non-immunological method for high-specificity and high-sensitivity detection of allergens in foods. Several mass spectrometry-based methods have been developed in last years for the single or multi-allergen detection on different matrices (wine and bakery products). Although many efforts have been done till now, some aspects remain to be investigated mainly related to the efficacy of the methods used for the protein extraction from complex matrices and the effects that food process could have on protein modification and aggregation probably altering the efficacy of protein/peptide detection. A review of recent researches in this field and the last results obtained by our Institute will be presented in this communication.

There is a raising demand for sensitive and high throughput MS based methods for screening purposesespecially tailored to the detection of allergen contaminants in different food commodities. A challengingissue is represented by complex food matrices where the antibody-based kits commercially availablemight encounter objective limitations consequently to epitope masking phenomena due to a multitudeof interfering compounds arising from the matrix. The performance of a method duly optimized forthe extraction and simultaneous detection of soy, egg and milk allergens in a cookie food matrix by microHPLC-ESI-MS/MS, is herein reported. Thanks to the innovative configuration and the versatility shown by the dual cell linear ion trap MS used, the most intense and reliable peptide markers were first identified by untargeted survey experiment, and subsequently employed to design an ad hoc multitarget SRM method, based on the most intense transitions recorded for each selected precursor peptide. A sample extraction and purification protocol was optimized also including an additional step based on sonication, which resulted in a considerable improvement in the detection of milk allergen peptides. Data Dependent TM Acquisition scheme allowed to fill out a tentative list of potential peptide markers, which were further filtered upon fulfilling specific requirements. A total of eleven peptides were monitored simultaneously for confirmation purposes of each allergenic contaminant and the two most sensitive peptide markers/protein were selected in order to retrieve quantitative information. Relevant LODs were found to range from 0.1 g/g for milk to 0.3 g/g for egg and 2 g/g for soy.

A method based on High-Resolution Mass Spectrometry was developed for the simultaneousdetermination 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 theanalogues of the best peptide markers identified for ? S1-casein and ovalbumin. 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. Six-point calibrations were performed by adding caseinate and egg-white powder in the concentration rangebetween 0.25 and 10 ug/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 FVAPFPEVFGK for caseinate. Limits of detection (LODs) ranged from 0.4 to 1.1 ug/mL. 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.

To date it is well known that there is not a unique best analytical technique that alone is capable of answering the question whether a food is authentic or not, nor there is the best mathematical classifier to correctly interpret the results assigning one sample to one or another population. A holistic and multidisciplinary approach benefitting from the knowledge and the skills/expertise acquired by different researches is therefore necessary to tackle this task. In this regard, it is fundamental to know in details the product, the different variables involved, the raw materials and the production processes. It is important to have an in thorough knowledge of the storage conditions of the food along the process chain, and to combine all the available information into an experimental design that should consider all the possible sources of variation.The collection of all these preliminary information represents the starting point to develop tailor-made projects, specifically designed on the objective of the study.In this regard, we need:o representative sampling, with respect to the objective to be achieved;o robust analytical results;o appropriate data interpretation (method, theoretical classification and real classification) based on mathematical classifiers able to distinguish / classify according to the pre-established purpose;o validation protocols. All these issues will be schematized and concisely presented in the present communication, aiming at establishing a universal protocol to be intended as guideline by food industries, suppliers, control laboratories, etc.. to help developing non targeted based methodologies. All authors thank Food Integrity Project, Università del Piemonte Orientale, CNR-ISPA, Innovative Solutions, ICETA, Coop Italia, Thermo Fisher Scientific and Mérieux NutriSciences for the opportunity to participate in this Research, of utmost importance for the dissemination of analytical services designed to verify the full transparency of final food products in terms of Authenticity and Integrity.

Salmon is one of the most valuable and beneficial fish sold worldwide, also thanks to the enormous benefits on human health related to its consumption. The increasing demand of this product on the market has led, on the one hand, to the depletion of the wild type species in the oceans, on the other hand, to the intensification of farming practices in aquaculture systems. In the last years, the increase of food fraud and adulterant substitutions with serious consequences on the consumers and the economic systems, has solicited the development of new analytical and fast methods able to detect illegal manipulation or food mislabeling. Non targeted methods have recently emerged as an alternative and rapid strategy to confirm food authenticity. Despite the classical targeted methods mainly aimed at detecting specific indicators, untargeted analysis represents a novel tool able to collect a multitude of data, not correlated to known parameters, that are further processed via advanced statistical tools. In the present communication, an untargeted workflow based on High Resolution Mass Spectrometry analysis was applied to the protein fraction of farmed and wild type salmon extracts (belonging to Salmo salar species) aimimng at assessing its capability to discriminate between both salmon groups. The protein mixture extracted by using an urea based buffer, was further enzymatically cleaved by using trypsin and then submitted to a cleanup step before LC-HRMS analysis. MS/MS experiments were performed on a triple TOF-MS in DIA analysis by applying the SWATH mode and data evaluation was further accomplished by using a chemometric software. A preliminary analysis (PCA) was carried out on all peptides detected after data processing and the peak area values were used to highlight any eventual discrimination between both salmon groups. The resulting score plot referred to the PCA using all peptides already demonstrated a clear separation between wild type and farmed salmons. In addition, an evaluation on the extracted ion chromatograms (XICs) relative to the most abundant peptides in wild or farmed salmons showed that some peptides displayed a different abundance, in terms of intensities, in farmed or wild salmons, suggesting the possibility to identify a list of candidate peptide markers capable of discriminating farmed from wild salmons. In conclusion, the application of untargeted LC-HRMS/MS approach, integrated by multivariate statistical analysis, appears to be a promising tool for the discrimination of farmed and wild type salmons.

Food allergen research has made giant steps in the last years thanks to the features offered by the latest technology of mass analyzers placed on the market allowing multiplex sensitive detection of proteins. Potentials and features of two mass analyzers namely a linear ion trap capable of performing a data dependent or selected reaction monitoring analysis and an OrbitrapTM stand-alone MS enabling a broadband fragmentation without mass selection at highest mass resolving power are herein described and applied to the multiplex screening of allergens in a type of wine chosen as a reference matrix. Quantitative and confirmative capabilities of both platforms were assessed on the specific case study, the multiple detection of egg and milk-related proteins, typically employed in white wines as fining agents. Commercial bioinformatic tools used for a quick allergen identification will be also discussed.

Authenticity and traceability of food products are of primary importance at all levels of the production process, from raw materials to finished products. Authentication is also a key aspect for accurate labeling of food, which is required to help consumers in selecting appropriate types of food products. With the aim of guaranteeing the authenticity of foods, various methodological approaches have been devised over the past years, mainly based on either targeted or untargeted analyses. In this review, a brief overview of current analytical methods tailored to authenticity studies, with special regard to fishery products, is provided. Focus is placed on untargeted methods that are attracting the interest of the analytical community thanks to their rapidity and high throughput; such methods enable a fast collection of "fingerprinting signals" referred to each authentic food, subsequently stored into large database for the construction of specific information repositories. In the present case, methods capable of detecting fish adulteration/substitution and involving sensory, physicochemical, DNA-based, chromatographic, and spectroscopic measurements, combined with chemometric tools, are illustrated and commented on.

Peanut allergy is a typical IgE mediated immune disease and has become a major health concern worldwide. Even a low intake of peanuts or peanut containing foods can cause severe and sometimes fatal allergic reactions in sensitive individuals. Differences in the preparation of peanuts before consumption could contribute to whether an individual will eventually display an allergic reaction. Several thermal and non thermal treatments might account for this change in allergenicity due to an alteration of the allergenic proteins therein contained. Such paper will report and review the different strategies aimed to reduce peanuts allergenicity.

Stability to proteolytic degradation in the digestive tract is considered a general feature shared by most food allergens. Current digestibility models exclusively utilize purified allergen proteins, neglecting the relevant effects of matrix that occur for foodstuff systems. In the present study, we investigated digestion stability of the major peanut allergens directly in the natural matrix using an in vitro static model that simulates the gastrointestinal digestion including the oral, gastric, duodenal and intestinal (brush border membrane enzymes) phases. Immunogenicity was evaluated by Western Blot using N=8 pooled sera of peanut allergic pediatric subjects. Immunoreactive, large-sized and fragments of Ara h 2, Ara h 6 and Ara h 3 survived hydrolysis as assessed by SDS-PAGE. Smaller resistant peptides mainly arising from Ara h 3 and also Ara h 1 were detected and further identified by LC-high resolution-MS/MS. RP-HPLC purification followed by dot-blot analysis and MS/MS-based identification demonstrated that stable IgE-binding peptides derived from Ara h 3. These results provide a more realistic picture of the potentially allergenic determinants of peanuts that survived the human digestion, taking into account the role of the food matrix, which may significantly affect gastrointestinal breakdown of peanut allergens. (C) 2017 Elsevier Ltd. All rights reserved.

The aim of this work was to check the efficacy of bovine lactoferrin hydrolyzed by pepsin (LFH) to prevent blue discoloration of Mozzarella cheese delaying the growth of the related spoilage bacteria. Among 64 Pseudomonas fluorescens strains, isolated from 105 Mozzarella samples, only ten developed blue discoloration in cold-stored Mozzarella cheese slices. When Mozzarella cheese samples from dairy were treated with LFH and inoculated with a selected P.fluorescens strain, no pigmentation and changes in casein profiles were found up to 14 days of cold storage. In addition, starting from day 5, the count of P.fluorescens spoiling strain was steadily ca. one log cycle lower than that of LFH-free samples. ESI-Orbitrap-based mass spectrometry analyses allowed to reveal the pigment leucoindigoidine only in the blue LFH-free cheese samples indicating that this compound could be considered a chemical marker of this alteration. For the first time, an innovative mild approach, based on the antimicrobial activity of milk protein hydrolysates, for counteracting blue Mozzarella event and controlling psychrotrophic pigmenting pseudomonads, is here reported. © 2014 Elsevier Ltd.

The development of a surface plasmon resonance (SPR)-based biosensor tailored to the fast detection of egg-related fining allergens in wines is herein described. Ovalbumin (OVA) was chosen as the target protein to be monitored due to its highest abundance in the egg white powder, a typical fining agent used by the winery industry to promote wine clarification. A direct assay was designed, basing on the use of polyclonal anti-OVA antibody as bio-specific receptor. With the aim of optimizing the assay conditions, different parameters able to influence the final biosensor response were carefully investigated (i.e., pH, ionic strength, and additional surfactant concentration). After the fine tuning of these parameters, the assay was tested in the direct analysis of OVA in commercial wines artificially contaminated with egg white powder at different concentration levels in order to assess the reliability of the biosensor in detecting traces of OVA in complex matrices. The devised assay allowed to trace, in a short analysis time and with a minimal sample pre-treatment required, the presence of egg allergens at the lowest concentration comprised between 0.03 and 0.2 ?g/mL. Finally, the response provided by the developed biosensor was correlated with an established liquid chromatography mass spectrometry (LC-MS) method developed in our laboratories, and performances of both approaches were assessed for the fast monitoring of egg allergen contamination in fined wines.,[Figure not available: see fulltext.]

Bacillus subtilis TR50, a gram-positive endospore-forming bacteria isolated from a cured sausage, proved to be source of several antimicrobial compounds against food-borne pathogenic bacteria (Caputo et al., 2011). The wide chemical variability shown by these metabolites and their related antimicrobial efficacy depend on both the specific strain producer and on nutritional/environmental growth conditions (Baruzzi et al., 2011;Dhouha and Ellouze-Chaabouni, 2011).The difficulty for reliably identifying the antimicrobial compounds released by strains hampers their application in many fields such as controlling food microbial spoilage or fighting the emergence of antibiotic resistance to human pathogens. In general, identification of these antimicrobial compounds such as bacteriocins and lipopeptides in culture filtrates is accomplished by MALDI-TOF analysis or filtrate fractionation followed by HPLC separation and ESI -MS/MS analysis (Pabel et al., 2003; Kim et al., 2004; Caputo et al., 2011).In this work we describe a LC/MS method for the rapid screening of antimicrobial compounds produced by Bacillus strain TR50. By using HPLC separation and High Resolution MS analysis on a benchtop Orbitrap(TM)-based mass spectrometer equipped with a collision chamber we identified sixteen compounds belonging to three family of lipopeptides in the cell-free supernatant directly.This approach is herein described and proposed as a high throughput tool for the rapid identification of antimicrobial and antifungal compounds in microbial cultures.

Several Bacillus strains, typically isolated from different food sources, represent renowned producers of a multitude of low and high molecular weight compounds, including lipopeptides and macrolactones, with an importance for their antimicrobial activity. The high homology shared by many of these compounds also occurring as closely related isoforms poses a challenge in their prompt detection.Identification and structural elucidation is generally achieved by matrix-assisted laser desorption/ionization (MALDI) or liquid chromatography (LC) coupled to mass spectrometry (MS) after a pre-fractionation and/or purification step of the extract. In this paper we report the application of a method based on LC separation and high-resolution Orbitrap(TM)-based MS for the rapid screening of raw filtrate of the strain Bacillus subtilis TR50 endowed with antimicrobial activity, without requiring any sample pre-treatment.Upon direct analysis of the cell-free filtrate of Bacillus subtilis TR50 by high-resolution mass spectrometry (HRMS), different compounds families, that proved to exert a remarked antimicrobial activity against several foodborne pathogens, can be readily displayed along the chromatographic run. Among them, three different classes were identified and characterized belonging to the iturin, fengycin and surfactin groups. The high resolving power and accurate mass accuracy provided by the HRMS system in use ensured an enhanced selectivity compared to other mass spectrometers. In addition, after activation of the HCD cell, the HR-MS/MS spectra can provide insights in the structural elucidation of several compounds.The acquisition of HRMS spectra of raw filtrates of subtilis strains allows untargeted analysis of the major classes of compounds produced to be performed, thus facilitating identification of other unknown bioactive molecules after retrospective analysis. These features make this approach a fast tool applicable to the rapid screening and further identification of antimicrobial compounds released by Bacillus strains in raw filtrates

The global interest in saving food resources is leading to recycle wasted-food materials to extract useful nutrients. In dairy industry, the recycling of whey proteins determines their utilization in the healthy-addressed foods, which, however, can cause immunological responses in allergic subjects. In this work, a whey protein concentrate (WPC) was alternatively hydrolyzed with pepsin, papain, trypsin and rennin in order to attenuate or abolish the ?-lactoglobulin (BLG) antigenicity. The electrophoretic profiles of both pepsin and papain WPC hydrolysates proved the disappearance of the BLG band, even though a slight antigenicity was still found by ELISA. Pepsin hydrolysates, filtered through a 10-kDa cut-off membrane, did not produce immunological response. A deeper investigation carried out on pepsin digested and ultrafiltered samples by LC-MS/MS showed the disappearance of the immunoreactive BLG-fragment IVTQMKGLDIQKVAGTW. The remaining peptides, partially overlapped to major IgE binding epitopes, were not able to give immunoreactivity response. The combined WPC pepsin digestion with ultrafiltration confirmed to be a user-friendly strategy to reduce markedly the WPC antigenicity. The improvement of this two-steps process could be used to produce novel hypoallergenic infant food formulas.

Reliable methods are needed for detection ofallergenic milk proteins in complex food matrixes.The feasibility of an LC/high-resolution MS methodfor the analysis of milk proteins in a thermallyprocessed model food (incurred cookies) and inwhite wine spiked, respectively, with milk powderand caseinate is described. Detection of milkproteins was based on the identification of uniquepeptides in the tryptic digests of cookie/wineextracts using an RP-HPLC separation coupled toan Exactive(TM) nonhybrid mass spectrometer usingOrbitrap technology. The extremely high massaccuracy and resolution provided by the Orbitrapanalyzer allowed a fast preliminary identification offour previously proposed peptide markers ofcaseins using only accurate values of the m/z oftheir ions. No interference was observed, despitethe complexity of the analyzed matrixes. Moreover,the availability of a high- energy, collisionallyactivated dissociation cell integrated in the massspectrometer enabled acquisition of peptideMS/MS-like spectra through post-sourcefragmentation. Confirmation of peptide markeridentity could then be achieved by a comparisonbetween experimental and predicted product ions.The described method shows the great potential ofOrbitrap MS as a reliable technique in the field ofprotein allergen detection once the peptidemarkers are identified.

La resistenza delle proteine allergeniche della soia in seguito a digestione gastro-duodenale simulata sarà discussa nella presente comunicazione unitamente all'uso di software di bioinformatica per valutare l'allergenicità residua.

Over the last years, great efforts have been devoted to develop effective gluten detoxification strategies with a consequent detrimental alteration of the technological properties as well. Obtaining low-gluten products without affecting the rheological properties of wheat could still be considered a new challenge to face.In this investigation, we presented a comprehensive characterization of durum wheat genotypes aimed at identifying low gluten ones, which combine the potential lower toxicity/immunogenicity with conserved yield and rheological properties to encompass the perspective usability for bread or pasta making. A preliminary profiling of gluten proteins was accomplished by immunoassay-based quantification and liquid chromatography coupled to UV detection, focusing on the gliadin fraction as main responsible for immunoreactivity in celiac disease patients. In addition, data on grain protein content, grain yield per spike, dry gluten and gluten index were collected in order to provide complementary information about productivity-related traits and quali-quantitative characteristics related to wheat nutritional value and its technological properties. The whole pool of data was statistically evaluated driving to the selection of a preferred list of candidate low-toxicity genotypes that were subjected to in-vitro simulated gastroduodenal digestion and untargeted HR-MS/MS peptide identification. Finally, an in-silico risk assessment of potential toxicity for celiac disease patients was performed according to the most recent guidance provided by EFSA.

Contamination of food products by allergens represents a matter of concern especially for allergic consumers due to the risk of triggering an immunological reaction upon ingestion of allergen-containing foods [1]. Due to the widespread extent of such pathology, and in order to protect the health of sensitive consumers, specific legislation has been issued in different countries on the proper labeling of a restricted list of food allergens whenever added to food [2]. Besides their intentional incorporation into the commodity, a risk of accidental contamination is likely to exist. In this case allergens are defined hidden as they have not been declared on the product label and might unexpectedly reach the end products through several routes [3]. Different analytical methods have been developed in the last years for monitoring food allergen contamination along the food chain. Recently, mass spectrometry (MS) methods [1], have been considered a promising analytical strategy for f ood allergens detection thanks to the advances made in this technology that enables to overcome several restrictions of antibody-based methods, such as ELISA. Among them the risk of false positives, especially when applied to complex or processed food matrices that might cause epitope modification or masking, and the limitations in multiplexing. In the present investigation, a sensitive LC-MS method tailored to the multiplex detection of several allergenic ingredients in a processed food matrix will be described. Cookie was chosen as complex and processed food model, incurred with egg, milk, soy, hazelnut and peanut allergens. Starting from our previous investigation [4], extraction, purification and enzymatic digestion conditions were duly optimized in order to design a relatively fast and easy sample handling procedure which allowed to considerably reduce the time requested for sample preparation before LC-MS analysis. Features of two different MS instrumental set-up, also exploiting High Resolution Mass Spectrometric detection, to monitor 5 allergenic foods in complex foods will be presented. An automated on line pre-enrichment procedure onto a trap-column followed by chromatographic separation was designed enabling the pre-enrichment and partial purification of the candidate markers with challenging LODs obtained.

Allergenic ingredients in pre-packaged foods are regulated by EU legislation mandating their inclusion on labels. In order to protect allergic consumers, sensitive analytical methods are required for detect allergen traces in different food products. As a follow-up to our previous investigations, an optimized, sensitive ,label-free LC-MS/MS method for multiplex detection of five allergenic ingredients in a processed food matrix is proposed. A cookie base was chosen as a complex food matrix and home-made cookies incurred with whole egg, skimmed milk, soy flour, ground hazelnut and ground peanut were prepared at laboratory scale. In order to improve the analytical workflow both protein extraction and purification protocols were optimized in order to achieve a sensitive streamlined SRM based analytical method for allergens detection in incurred cookies. The effect of baking on the detection of selected markers was also investigated .

Saranno descritte nella presente comunicazione le procedure sviluppate per la riduzione di allergenicità in arachidi trattati e non.

A method based on LC-ESI-high-resolution (HR)-MS analysis, using a single-stage Orbitrap mass spectrometer, was developed for the quantification of casein allergens potentially present in white wines as a result of fining by caseinate. The method consists of (1) extraction from the matrix by ultrafiltration, (2) digestion with trypsin and (3) detection/quantification of residual caseins, obtained by monitoring the LC-MS response of representative tryptic peptides (peak areas in extracted-ion chromatograms). Method linearity was assessed first on caseinatesolutions prepared either in water or in wine matrix (the ultrafiltration residue of a protein-free white wine). Limits of detection (LOD) ranged from 0.1 to 0.3 mg ml1 (S/N ¼ 3) in water, and between 0.15 and 0.7 mg ml1 inwine matrix, depending on the selected peptide. Method repeatability and reproducibility, measured as responsevariability (standard deviation) due to LC-MS analysis alone and to both enzymatic digestion and LC-MSanalysis, were assessed on caseinate standard solutions in water and ranged from 5 to 12% and from 8 to 20%,respectively. A higher variability was usually observed for the peptide marker response in the case of matrixmatched samples, the only exception being peptide GPFPIIV from -casein, the marker also providing thehighest sensitivity. The method was finally applied to a casein-free white wine ('Greco di Tufo') fined withcaseinate at different concentrations, after discarding the precipitate due to casein-wine components aggregation. Minimum detectable added caseinate concentrations (i.e. those corresponding to responses with S/N ¼ 3) were estimated between 39 and 51 mg ml1 , according to the peptide marker chosen. These limits are compatible with caseinate concentrations typically adopted for wine-fining purposes. Moreover, a cross-check with the calibration performed in wine matrix led to an estimation of the concentration of dissolved caseinate to be in the low ng ml1 range.

Fusarium is a genus frequently contaminating different agricultural products and is also a renown producer of Trichothecenes1. This group of mycotoxins can be further subdivided into subgroups with T-2/HT-2 toxins and deoxynivalenol being the main representatives of the type A and B respectively. In general Trichothecenes are responsible for a wide variety of toxic effects in humans and animals. Within the type class A, in particular, T-2 and HT-2 toxins represent the most acutely toxic compounds2 and have been lately reviewed by EFSA3. In human health risk assessment, ingestion of contaminated food is considered the major route of human exposure to these compounds although only a smaller amount will be further available for absorption during its transit along the gut4. In vitro digestion models have been used to evaluate mycotoxins bioaccessibility during the intestinal transit. As a result, the bioaccessibility can be calculated representing a measure for the assessment of mycotoxin bioavailability in food. Some investigations have been already carried out in the past by our group in order to assess T-2 and HT-2 bioaccessibility in bread chosen as reference food model5 In this note we describe a study aimed to investigate toxicity of gastro-duodenal digests of mycotoxins contaminated bread samples collected along the digestion time-course, assayed on RPMI lymphoid B cell line, as a sensitive cellular model. In parallel, a chemical quantification of T-2 and HT-2 toxins contaminating the bread digests was accomplished along the gastric and duodenal phase. The digestive fluids undergoing chemical and toxicological analysis, were collected at the beginning and end of the gastric phase, and after completion of the duodenal phase Results proved that a correlation between HT-2 content and toxicity did exist in digestive fluids although a more persistent toxic activity was noticed in the later stage of the duodenal phase.

Saffron is a valuable and highly appreciated spice derived from the dried red stigmas of the flowers of the cultivated plant Crocus sativus L. It is commonly used as a coloring and flavouring agent in food preparation. In addition, it is a good source of flavonoids, proteins, sugars, vitamins, amino acids, mineral matter, gums, and other chemical compounds that make it a health promoting spice. To produce saffron spice, harvested stigmas are submitted to a mild food processing peculiar of the region of production thus it could be considered a traditional product, which aroma and chemical composition strictly depend on the geographic location of production. Due to its high costs of production, saffron is one of the most expensive spice commercialized across the world, and often susceptible of adulteration. Different plant-derived adulterants have been discovered, and the most frequently involve cut and/or dyed Carthamus tinctorius L. petals and Curcuma longa L. powdered rhizomes (Kanti et al., 2011). Several analytical methods have been reported in literature for the detection of plant adulterants in saffron samples based on different techniques (e.g. Maggi et al., 2011, Zalacain et al., 2005, Tarantilis et al., 2004, Zougagh et al., 2005) although official methods (ISO 3632-1; ISO 3632-2) exist. Very recently, high-performance liquid chromatography coupled to high resolution mass spectrometry (HRMS) was successfully proposed for saffron authentication/traceability according to the geographical origin based on untargeted metabolic fingerprinting (Rubert et al., 2016). In this work, we investigated two HRMS based approaches one using ESI ionization coupled to LC separation and the other based on DART ionization before HRMS detection to assess saffron authenticity by an untargeted metabolic approach. Pure saffron samples and saffron spiked with different amounts of Carthamus and Curcuma adulterants were extracted with a mix of ethanol and water to extract the majority of metabolites and final MS fingerprints obtained were used for authenticity assessment and/or adulteration detection. The produced spectra were then processed via the commercial software Compound Discoverer v.2.1 SP1 (Thermo Fisher Scientific). The detection and grouping of the unknown compounds by setting a mass accuracy <= 5ppm was further accomplished, together with a preliminary statistical analysis of the integrated peak areas. As grouping factor "Type of adulterant" (Carthamus and Curcuma) was set and data were further pretreated by aligning the extracted chromatograms on the respective retention time. In order to filter the compound list to the species most suitable in discriminating pure saffron from the adulterated one, Volcano plots combining the statistical significance of the identified compounds and magnitude of change in the extracted peak areas, were investigated. Edited compounds list constrained by means of p-value thresholds was then subjected to statistical evalua

In the present contribution, the potential of LC-HR-MS coupled to software based data handling for fish authenticity and traceability will be presented. Salmon salar discrimination based on geographic origin was accomplished by untargeted LC-HR-MS analysis on hybrid quadrupole/OrbitrapTM-based mass spectrometer and by raw data mining with commercial software for small molecules identification. Salmon filets were extracted by methanol/chloroform mixtures and both the polar and non-polar fractions were analysed for a comprehensive compound characterization and differential analysis. In particular, conventional chromatographic separation was accomplished with a relatively short linear gradient with full scan HR-MS acquisitions performed in both positive and negative modes. The gathered spectra were processed via the commercial software Compound Discoverer v.2.0 (Thermo Fisher Scientific) in order to detect and group the unknown compounds with an accuracy <= 2ppm and achieve a preliminary statistical analysis of the integrated peak areas. The study was carried out by setting as grouping factor the geographic origin (Canada, Norway and Chile) and data were pretreated by means of the retention time alignment of extracted chromatograms and background subtraction. Volcano plots combining the statistical significance of the identified compounds and magnitude of change in the extracted peak areas were investigated in order to filter the compound list to the main species most suitable in discriminating the different salmon groups. Edited compounds list constrained by means of p-value thresholds was subjected within the same workflow to further statistical evaluation by PCA in order to simplify the multivariate system and highlight the intrinsic trend of the analyzed samples in the reduced projected space. The PCA performed on polar fraction of the salmon extracts allowed to distinguish clearly three data groups belonging to the three geographic origins (Canada, Norway and Chile).

Authentication of food is a burning topic and the development of rapid and reliable analytical strategies to authenticate the origin of food has become a priority at global level to combat food frauds. Food authentication is typically attained by applying the classical targeted approach where a certain analyte defined by characteristic parameters is further confirmed by reference standards. The non targeted approach offering the advantage of rapidity of the analysis without requiring any knowledge about the composition of the food sample to be analysed, has emerged in the last years gaining increasing attention and has been taken up by European research projects such as Food Integrity as objective of the WP18. Such approach allows to capture the highest number of information also referred to as features that are strictly correlated to the whole food matrix analysed. The comparison of food fingerprints to an authentic sample set will enable through the use of multivariate statistical models, detection of food sample adulteration or misdescription. However the bottleneck of such approach relies on the extensive data treatment required before submitting the pre-processed matrix to statistical analysis.In this work we present the optimization of a workflow based on the coupling between a Direct Analysis in Real Time (DART)ambient pressure source and a single cell Orbitrap(TM) based mass spectrometer applied to the non targeted analysis of foods to track the geographical origin and/or food integrity/authenticity.A typical workflow describing the main steps of a DART-HRMS analysis such as optimization of instrumental settings, sample preparation and post-acquisition data treatment to make the final data suitable for further statistical evaluation, will be presented along with the most critical steps. The pre-processing should include noise filtering, background subtraction, mass shift correction, mass alignment, spectra normalization, etc. Finally, an averaged spectrum representative of a certain food is typically generated from the analytical platform in use, that can be further converted into a format compatible for the subsequent statistical analysis. The duly optimization of all pre-processing steps on the acquired MS data is fundamental to produce reliable data prone to be submitted to multivariate statistic to generate trustful results. A case study reporting a typical DART-HRMS based workflow herein optimized will be shown applied to different food samples for food authenticity studies.