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

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

Are current analytical methods suitable to verify VITAL® 2.0/3.0 allergen reference doses for EU allergens in foods?

Food allergy affects up to 6% of Europeans. Allergen identification is important for the risk assessment and management of the inadvertent presence of allergens in foods. The VITAL® initiative for voluntary incidental trace allergen labeling suggests protein reference doses, based on clinical reactivity in food challenge studies, at or below which voluntary labelling is unnecessary. Here, we investigated if current analytical methodology could verify the published VITAL® 2.0 doses, that were available during this analysis, in serving sizes between 5 and 500 g. Available data on published and commercial ELISA, PCR and mass spectrometry methods, especially for the detection of peanuts, soy, hazelnut, wheat, cow's milk and hen's egg were reviewed in detail. Limit of detection, quantitative capability, matrix compatibility, and specificity were assessed. Implications by the recently published VITAL® 3.0 doses were also considered. We conclude that available analytical methods are capable of reasonably robust detection of peanut, soy, hazelnut and wheat allergens for levels at or below the VITAL® 2.0 and also 3.0 doses, with some methods even capable of achieving this in a large 500 g serving size. Cow's milk and hen's egg are more problematic, largely due to matrix/processing incompatibility. An unmet need remains for harmonized reporting units, available reference materials, and method ring-trials to enable validation and the provision of comparable measurement results.

Practical approach to develop a multi-group screening method for detection of mycotoxins, pesticides and veterinary drugs in food.

A multi-group screening method to detect residues of veterinary drugs in meat and environmental contaminants in wheat flour has been developed using liquid chromatography coupled to quadrupole-Orbitrap high-resolution mass spectrometry (LC-HRMS). The procedure was tested for over 300 representative compounds (173 veterinary drugs, 122 pesticides and 9 mycotoxins) analysing in parallel negative and positive (spiked) samples according to European validation rules. The Screening Target Concentrations (STCs) were chosen conservatively with respect to the method purposes. Interpretation of results was based on retention time, mass accuracy of precursor and MS2 spectral library. Evaluating the percentage of false negative results, 280 out of the 304 analytes were detectable at the STCs (false compliant rate ≤ 5%). In wheat flours, incurred levels of mycotoxins, deoxynivalenol and 3-acetyldeoxynivalenol, higher than STCs, were frequently found, whereas in meat, the most detected veterinary drugs were antibiotics generally at negligible concentrations (<10 µg kg-1 ). Finally, seven test materials from proficiency test schemes were successfully tested.

Assessment of Enzymatic Improvers in Flours Using LC-MS/MS Detection of Marker Tryptic Peptides.

Enzymatic improvers are enzymes obtained from microbial or fungal cultures, added as technical adjuvants to flour, with the aim of improving the dough characteristics in bakery products. They are used in a low ppm range and, being technical adjuvants, can go undeclared on the label. Many types of enzymatic improvers are present on the market, such as amylases, lipases, proteases, xylanases, glucose oxidases, and others, each with a different function. Analytical methods capable of detecting these enzymes are needed, particularly for bakery companies, in order to monitor the quality of raw materials and to detect any undeclared presence. In the present work, specific peptide markers, obtained by enzymatic digestion, have been used to detect the presence of enzymatic improvers by LC-MS/MS techniques. Promising results were obtained for some enzymes acting on the carbohydrate fraction (glucoamylase, glucose oxidase, xylanase) in which amounts as low as 20 ppm could be identified in blind flour samples. For lipases and proteases the method proved to be very effective in terms of specific identification, even if less sensitive.

The Influence of Processing Parameters on the Mitigation of Deoxynivalenol during Industrial Baking.

Deoxynivalenol (DON), a frequent contaminant of flour, can be partially degraded by baking. It is not clear: (i) How the choice of processing parameter (i.e., ingredients, leavening, and baking conditions) affects DON degradation and thus (ii) how much DON can be degraded during the large-scale industrial production of bakery products. Crackers, biscuits, and bread were produced from naturally contaminated flour using different processing conditions. DON degradation during baking was quantified with the most accurate analytical methodology available for this Fusarium toxin, which is based on liquid chromatography tandem mass spectrometry. Depending on the processing conditions, 0-21%, 4-16%, and 2-5% DON were degraded during the production of crackers, biscuits, and bread, respectively. A higher NaHCO3 concentration, baking time, and baking temperature caused higher DON degradation. NH4HCO3, yeast, vinegar, and sucrose concentration as well as leavening time did not enhance DON degradation. In vitro cell viability assays confirmed that the major degradation product isoDON is considerably less toxic than DON. This proves for the first time that large-scale industrial baking results in partial detoxification of DON, which can be enhanced by process management.

Development of a DNA Barcoding-Like Approach to Detect Mustard Allergens in Wheat Flours.

The spread of food allergens is a topic of global importance due to its impact on public health. National and International regulations ask food producers and manufacturers to declare product compositions on the label, especially in case of processed raw materials. Wheat flour (Triticum aestivum) can be contaminated by a wide range of species belonging to the Brassicaceae in the field or during grain harvests, storage, and processing. Among them, mustards (Brassica nigra, Brassica juncea and Sinapis alba) are well known allergenic species. Often, food quality laboratories adopt an ELISA approach to detect the presence of mustard species. However, this approach shows cross-reactivity with other non-allergenic species such as Brassica napus (rapeseed). In the last few years, DNA barcoding was proposed as a valid identification method, and it is now commonly used in the authentication of food products. This study aims to set up an easy and rapid DNA-based tool to detect mustard allergenic species. DNA barcoding (matK and ITS2) and chromosome markers (A6, B, C1 genome regions) were selected, and specific primers were validated on incurred reference food matrices. The developed test was proven to be able to distinguish mustard from rapeseed and wheat, overcoming cross-reactivity with Brassica napus.

Food wastes from agrifood industry as possible sources of proteins: A detailed molecular view on the composition of the nitrogen fraction, amino acid profile and racemisation degree of 39 food waste streams.

The nitrogen fraction of 39 food waste streams was characterized by Kjeldahl analysis, amino acid analysis, protein analysis and racemization degree, for assessing their potential for further valorization. For every waste streams the specific nitrogen-to-protein conversion factor was calculated, allowing to assess the accurate protein content. The results indicated which streams are most rich in relevant proteins (all wastes of dairy origin, beer yeast, malted barley germs, brewing cake, rapeseed press cake, sea buckthorn spent pulp, leek leaves, parsley waste, pumpkin kernel cake, and mushroom waste), which ones have valuable proteins, but in too little amount, and also which ones are rich in proteins, but of low nutritional value. Specific data also indicated, for every waste stream, its possible use for supplementing specific amino acids. To date, this represents the most complete characterization with homogeneous methodologies of the nitrogen fraction in food waste streams ever reported in the literature and outlines in unprecedented molecular details the potentialities and the limitations of many waste streams to be used as source of proteins.

Untargeted LC-MS based 13C labelling provides a full mass balance of deoxynivalenol and its degradation products formed during baking of crackers, biscuits and bread.

Deoxynivalenol (DON) is considered to be one of the most important contaminants in cereals and food commodities produced thereof. So far it is not clear i) to which extent DON is degraded during baking and ii) if a degradation results in reduced toxicity. We have elucidated the fate of DON during baking of crackers, biscuits and bread, which were produced from fortified dough and processed under pilot plant conditions. Untargeted stable isotope assisted liquid chromatography (LC) high resolution mass spectrometry was used to determine all extractable degradation products. Targeted LC - tandem mass spectrometry based quantification revealed that DON was partially degraded to isoDON (1.3-3.9%), norDON B (0.2-0.9%) and norDON C (0.3-1.2%). A DON degradation of 6% (crackers), 5% (biscuits) and 2% (bread), respectively, was observed. In vitro translation experiments indicate that isoDON is less toxic than DON.

LC-HRMS for Characterizing Durum Wheat Pasta Production Variability and Consumer Overall Liking.

Semolina pasta represents one of the most important dishes in Italian cuisine worldwide. Italy is the leader in its production and, recently, the worldwide diffusion of its production has begun to grow tremendously. The perceived quality of a food product, such as pasta, is a key feature that allows a company to increase and maintain the competitive advantage of a specific brand. The overall flavor perception of the consumer, therefore, has become as important as other key quality factors such as texture and color; thus, the food industry needs to meet consumer expectations and needs the tools to objectively "measure" the quality of food products. Untargeted fingerprinting by means of coupling LC with high-resolution MS (HRMS) has been well received within the analytical community, and different studies exploiting this approach for the characterization of high-value food products have recently been reported in the literature. In the present work, a tentative application of the sensomics approach to cluster analysis of semolina pasta obtained using different production conditions was developed to objectively define target molecules that correlate with consumer overall liking of an industrial standard product. Principal component analysis of chemical and physical testing, GC-MS, LC-HRMS, and sensory data were performed with the aim of identifying the main parameters to discern similarities and differences among samples and clustering them according to these features. The correlation between analytical data and compounds related to sensory data was further investigated, and lastly, a partial least-squares regression model for the prediction of consumer overall liking was reported.

Migration of selected hydrocarbon contaminants into dry semolina and egg pasta packed in direct contact with virgin paperboard and polypropylene film.

Migration of mineral oil saturated hydrocarbons (MOSH), polyolefin oligomeric saturated hydrocarbons (POSH), and polyalphaolefins (PAO from hot melts) into dry semolina and egg pasta packed in direct contact with virgin paperboard or polypropylene (PP) flexible film was studied. Migration was monitored during shelf life (up to 24 months), through storage in a real supermarket (packs kept on shelves), conditions preventing exchange with the surrounding environment (packs wrapped in aluminium foil), and storage in a warehouse (packs inside of the transport box of corrugated board). Semolina pasta packed in virgin paperboard (without hot melts) had a MOSH content lower than 1.0 mg kg(-1). An increasing contamination with PAO belonging to the adhesives used to close the boxes was detected in egg pasta, wrapped in aluminium (1.5 and 5 mg kg(-1) after 3 and 24 months, respectively). An environmental contribution to total hydrocarbon contamination was observed in egg pasta kept on shelves that, after 3 and 24 months, showed levels of PAO/MOSH < C25 around 3 and 10 mg kg(-1), respectively. The migration of POSH from PP film into egg pasta wrapped in aluminium was around 0.6 mg kg(-1) after 3 months of contact and reached 1.7 mg kg(-1) after 24 months of contact. After 9 months of contact, semolina pasta packed in PP film and stored in the transport box showed that some MOSH migrated into the pasta from the board of the transport box (through the plastic film).

Migration of selected hydrocarbon contaminants into dry pasta packaged in direct contact with recycled paperboard.

This paper deals with the migration of selected hydrocarbon contaminants, namely mineral oil hydrocarbons (MOH), diisopropyl naphthalenes (DIPN) and polyalphaolefins (PAO) from adhesives into dry semolina and egg pasta packaged in direct contact with recycled paperboard. Migration was monitored during its shelf life (for up to two years) simulating storage in a supermarket (packs on shelves) and conditions preventing exchange with the surrounding environment (packs wrapped in aluminium foil). Migration from the secondary packaging (transport boxes of corrugated board) was also studied for semolina pasta. After 24 months of exposure, semolina pasta stored on shelves reached 3.2 and 0.6 mg kg(-1) of MOSH and MOAH, respectively, Migration from the adhesives used to close the boxes and from the transport boxes contributed about 30% and 25% of the total contamination, respectively. The highest contamination levels (14.5 and 2.0 mg kg(-1) of MOSH and MOAH, respectively, after 24 months) were found in egg pasta stored on shelves (no adhesives), and seemed due to the highest contribution from the external environment.

Reliable liquid chromatography-mass spectrometry method for investigation of primary aromatic amines migration from food packaging and during industrial curing of multilayer plastic laminates.

Primary aromatic amines (PAAs) can migrate from packaging into food from different sources such as polyurethanic adhesives used for the manufacture of multilayer films, which may contain residual aromatic isocyanates, or recycled paperboard, because of the presence of azo dyes in the printed paper massively used in the recycling process. In the present work, a reliable analytical method, exploiting a conventional high-performance liquid chromatography-(selected ion monitoring)-mass spectrometry system, for PAAs compliance assessment in food contact materials was developed as an effective alternative to the current standard spectrophotometric one, moving in this way from the screening to the accurate and selective quantitation perspective for the analysis of PAAs both in aqueous and acidic food simulants. The main validation parameters were verified achieving very satisfactory results in terms of linearity range, limit of detection (ranging from 0.1 to 1.0 µg kg(-1)) and quantitation (ranging from 0.1 to 3.6 µg kg(-1)), repeatability and accuracy. Suitability of the method was demonstrated for a wide range of commercial samples, chosen among different producers of the most common used food packaging plastic and paperboard categories and then analyzed to assess the risk related to PAAs migration. Finally, the method was also successfully exploited to monitor the evolution of potential PAAs migration during the industrial curing process of multilayer plastic laminates, prior to their release for delivery to the food industry end user.

Antimicrobial activity of poultry bone and meat trimmings hydrolyzates in low-sodium turkey food.

This research was aimed at the evaluation of the antimicrobial activity exerted by poultry protein hydrolyzates derived from industrial leftovers added to minced turkey meat, intended for the production of burgers for human consumption. Hydrolyzates were obtained through enzymatic hydrolysis from poultry bone and meat trimmings, as by-products from the poultry industry. Colony forming unit assays, under both laboratory and industrial conditions, were performed to assess microbial growth. Poultry protein hydrolyzates inhibited microbial growth occurring in semi-finished turkey meat during the normal retention period because of their water holding capacity resulting in a decreased water activity. Overall, the findings demonstrated that poultry protein hydrolyzates could decrease mesophilic, psychrophilic, and thermophilic bacterial growth for the entire product shelf-life. Bacterial growth inhibition obtained in minced turkey meat by addition of poultry protein hydrolyzates (1.5%), hygroscopic amino acids mixture (1.5%) or sodium chloride (1%) was similar. It is suggested that the use of hydrolyzates could allow the reduction of salt content in poultry meat based products leading to the production of low-sodium turkey food still maintaining acceptable sensory characteristics.

Liquid chromatography-full scan-high resolution mass spectrometry-based method towards the comprehensive analysis of migration of primary aromatic amines from food packaging.

European Union legislation has established that plastic food contact materials shall not release primary aromatic amines (PAAs), which are toxic compounds and suspected human carcinogens. As valid alternative to existing methods for PAA determination, which are based on spectrophotometric test or targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) approaches, in this study a LC-Orbitrap-full scan-high resolution mass spectrometry (HRMS) method was devised and validated for the determination of migration levels of 22 PAAs from food contact materials, thus exploiting the specificity of accurate mass measurement. Direct injection of the simulant (acetic acid 3%, w/v) into the LC-MS system after migration, without any pre-treatment step, makes the developed method of great value for rapid screening analysis of a large number of amines. A very fast and efficient separation (<11min) of PAAs was achieved. Detection limits in the 0.06-0.7µgkg(-1) range were calculated for 17 out of 22 of the investigated PAAs, however obtaining values within 5.3µgkg(-1) for the other 5 amines. Good dynamic linear ranges from two to four orders of magnitude (r(2)≥0.990) were obtained and satisfying results were achieved in terms of intra-day (RSDs<10%) and inter-day repeatability (RSDs<17%). Trueness values in the 70±1-131±5% range proved reliability of the developed method for PAAs quantification also at very low concentration levels. Finally, the method was successfully applied to a range of different real plastic multilayer food packaging materials, noticing in all cases levels below the established limits of detection.

Antioxidant capacity of water soluble extracts from Parmigiano-Reggiano cheese.

In this work the antioxidant capacity of water soluble extracts of Parmigiano-Reggiano cheese (Water Soluble Extracts - WSEs) at different aging time was studied, by measuring their radical scavenging capacity with a standard ABTS assay. The WSEs were also fractionated by semi-preparative HPLC-UV and for each fraction the antioxidant capacity and the molecular composition was determined by LC/ESI-MS, in order to identify the most active antioxidant compounds. The antioxidant capacity was also determined after simulated in vitro gastrointestinal digestion of WSEs. The data indicated that antioxidant capacity in WSE from Parmigiano-Reggiano cheese, quite unaffected by ripening time and gastrointestinal digestion, is mostly due to free amino acids, mainly tyrosine, methionine and tryptophan, and only in minimal part to antioxidant peptides.

Identification and quantification of different species in animal fibres by LC/ESI-MS analysis of keratin-derived proteolytic peptides.

In the present paper, a proteomic method for species determination in fibres has been developed. Keratin was extracted from yak, wool and cashmere fibres and digested by trypsin, providing peptide mixtures that were analyzed by liquid chromatography coupled with electrospray mass spectrometry (LC/ESI-MS) in order to identify peptidic species-specific markers able to differentiate the fibres. Several suitable peptide markers were identified and validated in different fibres of different origin and having undergone different technological treatments, showing 100% specificity and 100% selectivity. Most of the peptide markers were also identified by means of high-resolution mass spectrometry, confirming the origin from species-specific keratin sequences. Some peptides were also used for the quantification of the different species in mixed fibres by LC/ESI-MS. Validation experiments and blind tests confirmed their ability to act as very specific quantitative and qualitative markers. The method here developed is a valid complement to the standard benchmark methods for fibre identification and quantification and will be very useful for assessing the authenticity of textile products.

Microbial origin of non proteolytic aminoacyl derivatives in long ripened cheeses.

Cheese ripening involves a complex series of biochemical events that contribute to the development of each cheese characteristic taste, aroma and texture. Proteolysis, which has been the subject of active research in the last decade, is the most complex of these biochemical events. However, also aminoacyl derivates of non-proteolytic origin (γ-glutamyl-amino acids and lactoyl-amino acids) with interesting sensory properties have been identified in cheeses. In the present work, an enzymatic activity producing γ-glutamyl-phenylalanine in Parmigiano-Reggiano water soluble extracts was observed. It was hypothesized that γ-glutamyl-amino acids and lactoyl-amino acids could be originated by enzymes of bacterial origin. In order to confirm this hypothesis, Lactobacillus helveticus and Lactobacillus rhamnosus were chosen as representative of starter and non starter microbiota of Parmigiano Reggiano cheese. They were used as model bacteria, in the presence of suitable precursors, to verify their ability to produce γ-glutamyl-phenylalanine and lactoyl-phenylalanine. The eventual abilities of these strains were tested both during growth and after cell lyses. While γ-glutamyl-phenylalanine was produced only by lysed cells, lactoyl-phenylalanine was produced either by growing or lysed cells in different amount depending on the species, the cells condition and the time of incubation.

Tolerability of a fully maturated cheese in cow's milk allergic children: biochemical, immunochemical, and clinical aspects.

BACKGROUND: From patients' reports and our preliminary observations, a fully maturated cheese (Parmigiano-Reggiano; PR) seems to be well tolerated by a subset of cow's milk (CM) allergic patients. OBJECTIVE AND METHODS: To biochemically and immunologically characterize PR samples at different maturation stage and to verify PR tolerability in CM allergic children. Seventy patients, with suspected CM allergy, were enrolled. IgE to CM, α-lactalbumin (ALA), ß-lactoglobulin (BLG) and caseins (CAS) were tested using ImmunoCAP, ISAC103 and skin prick test. Patients underwent a double-blind, placebo-controlled food challenge with CM, and an open food challenge with 36 months-maturated PR. Extracts obtained from PR samples were biochemically analyzed in order to determine protein and peptide contents. Pepsin and trypsin-chymotrypsin-pepsin simulated digestions were applied to PR extracts. Each PR extract was investigated by IgE Single Point Highest Inhibition Achievable assay (SPHIAa). The efficiency analysis was carried out using CM and PR oral challenges as gold standards. RESULTS: The IgE binding to milk allergens was 100% inhibited by almost all PR preparations; the only difference was for CAS, mainly α(S1)-CAS. Sixteen patients sensitized to CM tolerated both CM and PR; 29 patients tolerated PR only; 21 patients, reacted to both CM and PR, whereas 4 patients reactive to CM refused to ingest PR. ROC analysis showed that the absence of IgE to BLG measured by ISAC could be a good marker of PR tolerance. The SPHIAa using digested PR preparations showed a marked effect on IgE binding to CAS and almost none on ALA and BLG. CONCLUSIONS: 58% of patients clinically reactive to CM tolerated fully maturated PR. The preliminary digestion of CAS induced by PR maturation process, facilitating a further loss of allergenic reactivity during gut digestion, might explain the tolerance. This hypothesis seems to work when no IgE sensitization to ISAC BLG is detected.