Simona L. Bavaro

High-resolution Orbitrap™-based mass spectrometry for rapid detection of peanuts in nuts.

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™ 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™ 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. Graphical Abstract

Fungal and bacterial metabolites associated with natural contamination of locally processed rice (Oryza sativa L.) in Nigeria

This study reports the fungal and bacterial metabolites associated with natural contamination of 38 composite samples of locally processed rice from five agro-ecological zones of Nigeria (AEZs). The samples were evaluated for the presence of microbial metabolites by LC-MS/MS. Among the identified metabolites, 63 fungal and 5 bacterial metabolites were measured at varying concentrations and occurrence levels. Fusarium toxins had the highest incidence of 79%, but occurred in low amounts with fumonisin B1 (FB1) having the highest percentage incidence of 39.5% and a mean of 18.5 µg/kg. Among the Aspergillus toxins, aflatoxins (AFs) occurred in 36.9% of the rice samples, with aflatoxin B1 (AFB1) having the highest occurrence level of 18.4% and a mean value of 5 µg/kg. About 12 metabolites had incidence levels > 50%, including beauvericin (BEA) and tryptophol, which had occurrence levels of 100%. Among the emerging toxins under evaluation by international organisations such as the European Food Safety Authority (EFSA) and the Food and Agriculture Organisation of the United Nations (FAO), citrinin, sterigmatocystin (STER) and beauvericin were detected with maximum values of 207, 125 and 131 μg/kg, respectively. This paper also reports the first documented evidence of the contamination of Nigerian rice by bacterial and Alternaria metabolites, nivalenol, kojic acid, STER, moniliformin, fusaric acid, fumonisin B3, citrinin, 3-nitropropionic acid, andrastin A, cytochalasins, emodin and physicon.

Application of the adverse outcome pathway (AOP) concept to structure the available in vivo and in vitro mechanistic data for allergic sensitization to food proteins

BackgroundThe introduction of whole new foods in a population may lead to sensitization and food allergy. This constitutes a potential public health problem and a challenge to risk assessors and managers as the existing understanding of the pathophysiological processes and the currently available biological tools for prediction of the risk for food allergy development and the severity of the reaction are not sufficient. There is a substantial body of in vivo and in vitro data describing molecular and cellular events potentially involved in food sensitization. However, these events have not been organized in a sequence of related events that is plausible to result in sensitization, and useful to challenge current hypotheses. The aim of this manuscript was to collect and structure the current mechanistic understanding of sensitization induction to food proteins by applying the concept of adverse outcome pathway (AOP).Main bodyThe proposed AOP for food sensitization is based on information on molecular and cellular mechanisms and pathways evidenced to be involved in sensitization by food and food proteins and uses the AOPs for chemical skin sensitization and respiratory sensitization induction as templates. Available mechanistic data on protein respiratory sensitization were included to fill out gaps in the understanding of how proteins may affect cells, cell–cell interactions and tissue homeostasis. Analysis revealed several key events (KE) and biomarkers that may have potential use in testing and assessment of proteins for their sensitizing potential.ConclusionThe application of the AOP concept to structure mechanistic in vivo and in vitro knowledge has made it possible to identify a number of methods, each addressing a specific KE, that provide information about the food allergenic potential of new proteins. When applied in the context of an integrated strategy these methods may reduce, if not replace, current animal testing approaches. The proposed AOP will be shared at the www.aopwiki.org platform to expand the mechanistic data, improve the confidence in each of the proposed KE and key event relations (KERs), and allow for the identification of new, or refinement of established KE and KERs.

Isolation, Characterization, and Selection of Molds Associated to Fermented Black Table Olives

Table olives are one of the most important fermented food in the Mediterranean countries. Apart from lactic acid bacteria and yeasts that mainly conduct the olive fermentation, molds can develop on the brine surface, and can have either deleterious or useful effects on this process. From the food safety point of view, occurring molds could also produce mycotoxins, so, it is important to monitor and control them. In this respect, identification of molds associated to two Italian and two Greek fermented black table olives cultivars, was carried out. Sixty strains were isolated and molecularly identified as Penicillium crustosum (21), P. roqueforti (29), P. paneum (1), P. expansum (6), P. polonicum (2), P. commune (1). A group of 20 selected isolates was subjected to technological (beta-glucosidase, cellulolytic, ligninolytic, pectolytic, and xylanolytic activities; proteolytic enzymes) and safety (biogenic amines and secondary metabolites, including mycotoxins) characterization. Combining both technological (presence of desired and absence of undesired enzymatic activities) and safety aspects (no or low production of biogenic amines and regulated mycotoxins), it was possible to select six strains with biotechnological interest. These are putative candidates for future studies as autochthonous co-starters with yeasts and lactic acid bacteria for black table olive production.

Investigation of Heat and Pressure Treatments on Almond Protein Stability and Immunoreactivity after Simulated Human Digestion

Almond is worldwide consumed and renowned as a valuable healthy food. In spite of this, 14 it is also a potent source of allergenic proteins able to trigger several mild to life-threatening 15 immunoreactions. Food processing proved to alter biochemical characteristics of proteins, thus 16 affecting the respective allergenicity. In this paper we investigated the effect of autoclaving, preceded 17 or not by a hydration step, on the biochemical and immunological properties of almond proteins. 18 Any variation in the stability and immunoreactivity of almond proteins extracted from the treated 19 materials, were evaluated by total protein quantification, ELISA assay and protein profiling by 20 electrophoresis-based separation (SDS-PAGE). The autoclaving alone was found to weakly affect 21 almond proteins stability, despite what observed for the combination of hydration and autoclaving, 22 which resulted in a loss of approximately 70% of total protein content compared to untreated sample, 23 and in a final negligible immunoreactivity, as well. The final SDS-PAGE protein pattern recorded for 24 almonds hydrated and autoclaved disclosed significant changes. In addition, the same samples were 25 further submitted to in vitro simulated gastro-duodenal (GI) digestion to evaluate potential changes 26 induced by these processing on allergens digestibility. Digestion products were identified by HPLC27 HRMS/MS analysis followed by software-based data mining, and complementary information were 28 provided by analyzing the proteolytic fragments lower that 6 kDa in size. The autoclave based 29 treatment was found not to alter the allergens digestibility, whereas an increased susceptibility to 30 proteolytic action of digestive enzymes was observed in almonds subjected to the combination of 31 prehydration and autoclaving. Finally, the residual immunoreactivity of the GI resistant peptides 32 was investigated in-silico by bioinformatic tools, confirming that by following both approaches, no 33 epitopes survived the almond digestion, thus demonstrating the potential effectiveness of these 34 treatments to reduce almond allergenicity. 35

Effects of the Varietal Diversity and the Thermal Treatment on the Protein Profile of Peanuts and Hazelnuts

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.

Fungal isolates and metabolites in locally processed rice from five agro-ecological zones of Nigeria

ABSTRACT This study reports the distribution of fungal isolates and fungal metabolites that naturally contaminate locally processed rice from five agro-ecological zones of Nigeria. The fungal species were isolated by the dilution plate technique and identified by appropriate diagnostics, while metabolites were determined by a liquid chromatographic tandem mass spectrometric method. Aspergillus and Penicillium species were the predominant isolates found in the rice samples while Fusarium spp. were not isolated. The mean fungal count differed significantly (p < 0.05) across the zones and ranged from 9.98 × 102 cfu g−1 in the Southern Guinea Savannah to 96.97 × 102 cfu g−1 in the Derived Savannah. For 16 fungal metabolites, selected from 63 positively identified fungal metabolites based on their concentration and spread across the zones, an occurrence map was constructed. The Northern Guinea Savannah recorded the highest contamination of fungal metabolites while the Sudan Savannah zone recorded the least.