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Background Goji berries belonging to the Solanaceae family were considered officinal plants because of their health benefits. More recently this fruit has been qualified as superfood for the high nutritional value, low fats and high antioxidant content, therefore its inclusion in the diet is highly recommended. Despite their health-promoting properties, Goji berries represent a threat for allergic consumers. Allergenicity of Goji berries has not been thoroughly studied due to the paucity of investigations performed and the little information available about allergenic proteins. It was reported that nearly 77% of individuals allergic to plant foods reacted towards Goji berries and two bands were recognized by most of allergic individuals putatively attributed to LTPs. Method We report a case of two female patients (mean age 50 ys old) that displayed a severe anaphylactic reaction after ingestion of Goji berries. Conventional in vitro test resulted negative. Prick to prick with fresh Goji berries resulted positive. In order to have more insights in the reactive Goji proteins, we performed a proteomic investigation aiming at identifying the proteins reacting towards allergic patients' sera. Goji berries were homogenized, extracted by adding TrisHCl/NaCl buffer enriched with Urea then purified by size exclusion chromatography and loaded onto SDS-PAGE for electrophorethic separation. Several proteins banding above 100 kDa, in the region of 50-30 kDa and below 25 kDa were displayed long the electrophoretic profile. In order to identify the immunoreactive proteins, immunoblot experiments were accomplished by using allergic sera. Each individual band was excised from the gel, in vitro digested and analyzed by LC-MS/MS followed by software-based protein identification. Online databases (Eudycotiledons and Viridiplantae) were interrogated for searching any match with Goji berries protein sequences (Lycium barbarum). Results Fibrillin proteins and proteins belonging to vicilin family were identified in the reactive bands comprised in the range 30-50Kda, whereas the band (below 25kDa) showing the highest reactivity was attributed to glutelin and legumin proteins. Conclusion These preliminary results could be a useful starting point for future investigation to deepen the knowledge on Goji allergenic proteins and its likely cross reactivity with other proteins of plant-related species.

Goji berries belonging to the Solanaceae family were considered officinal plants in ancient China because of their health benefits. More recently this fruit has been qualified as superfood for the high nutritional value, low fats and high antioxidant content, therefore its inclusion in the diet is highly recommended. Despite their health-promoting properties, Goji berries represent a threat for allergic consumers. Allergenicity of Goji berries has not been thoroughly studied due to the paucity of investigations performed and the little information available about allergenic proteins. It was reported that nearly 77% of individuals allergic to plant foods reacted towards Goji berries and two bands were recognized by most of allergic individuals putatively attributed to LTPs. Cross-reactivity with other proteins belonging to the same botanical family was also demonstrated due to the high homology with other proteins. In this study we report a case of two patients (mean age 50 ys old) that displayed a severe anaphylactic reaction after ingestion of Goji berries. Conventional in vitro test resulted negative. Prick to prick with fresh Goji berries resulted positive. In order to have more insights in the reactive Gojii proteins we performed a proteomic investigation aiming at identifying the proteins reacting towards allergic patients' sera. Gojii berries were homogenized, extracted by adding TrisHCl/NaCl buffer enriched with Urea then purified by size exclusion chromatography and loaded onto SDS-PAGE for electrophorethic separation. Several proteins banding above 100 kDa, in the region of 50-30 kDa and below 25 kDa were displayed long the electrophoretic profile. In order to identify the immunoreactive proteins, immunoblot experiments were accomplished by using allergic sera. Each individual band was excised from the gel, in vitro digested and analyzed by LC-MS/MS followed by software-based protein identification. Online databases (Eudycotiledons and Viridiplantae) were interrogated for searching any match with Gojii berries protein sequences (Lycium barbarum). Finally fibrillin proteins and proteins belonging to vicilin family were identified in the reactive bands comprised in the range 30-50 KDa, whereas the band (below 25kDa) showing the highest reactivity was attributed to glutelin and legumin proteins. These preliminary results could be a useful starting point for future investigation to deepen the knowledge in Gojii allergenic proteins and its likely cross reactivity with other proteins of plant-related species.

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

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.

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

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

A method using the combination of size exclusion-solid phase extraction and ultrafiltration, followed by tryptic digestion 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.

A method based on High-Resolution Mass Spectrometry was developed for the simultaneous determination of fining agents containing potentially allergenic milk (casein) and egg-white (lysozyme and ovalbumin) proteins, added to commercial white wines at sub-ppm levels. Selected tryptic peptides were used as quantitative markers. An evaluation of protein digestion yields was also performed by implementing the analogues 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.

La contaminazione da micotossine rappresenta una delle maggiori problematiche nella gestione del rischio della 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 naturalmente contaminate 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 aumentano in seguito alla panificazione, mentre quelli del DON-3 Glucoside appaiono dimezzati. Per le tossine T-2 e 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.

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.

All of 58 Aspergillus e Penicillium mould strains isolated from dairy products collected in Southern Italy, have been analysed for mycotoxin production ability on synthetic media (CYA and YES) by rapid tests on agar cultures and chromatographic analysis of methanolic extracts. The results showed that the 72% of strains produced cyclopiazonic acid, mycophenolic acid, patulin, penitrem A, PR toxin, roquefortin C, sterigmatocystin. The highest number of strains (42%) produced cyclopiazonic acid, 20% mycophenolic acid and 12% sterigmatocystin. These toxigenic fungi were isolated mainly from ripe cheese samples made with sheep milk. A. versicolor (100%), P. camemberti (75%) and P. commune (56%) resulted the major producers of cyclopiazonic acid. Artemia salina bioassay has been carried out in order to evaluate the toxicity of methanolic extracts; P. commune and P. crustosum have shown a high toxicity probably related to the occurrence of penitrem A. A correlation between the concentration of sterigmatocystin and cyclopiazonic acid to the toxicity of methanolic extracts was found.

A new high-performance liquid (HPLC) chromatographic method is described as cyclopiazonic acid (CPA) determination in fungal cultures on a propylamino-bonded stationary phase with a CH3CN/CH3COONH4 buffer as mobile phase. Retention of CPA on propylamino modified silica under acidic conditions (protonated amino groups and deprotonated CPA) is governed by a mixed ion-exchange-reversed-phase mechanism. In addition to non-polar (hydrophobic) interactions, polar interactions with the surface silanols are also possible and become important as the polarity of the mobile phase decreases. A detection limit of 25 pg of CPA standard is obtained that represents an improvement of more than two orders of magnitude compared to existing HPLC procedures. UV-detector response was linear to 200 ng of CPA. Fungal extracts can be analysed after a simple dilution step with UV diode array detection that provides peak identity/purity assessment. The suitability of the proposed method as a rapid confirmatory test to assess the toxigenic potential of different Aspergillus and Penicillium strains is demonstrated by the analysis of 54 fungal extracts.