Ilario Losito

Highly Efficient Gluten Degradation by Lactobacilli and Fungal Proteases during Food Processing: New Perspectives for Celiac Disease

ABSTRACT Presently, the only effective treatment for celiac disease is a life-long gluten-free diet. In this work, we used a new mixture of selected sourdough lactobacilli and fungal proteases to eliminate the toxicity of wheat flour during long-time fermentation. Immunological (R5 antibody-based sandwich and competitive enzyme-linked immunosorbent assay [ELISA] and R5 antibody-based Western blot), two-dimensional electrophoresis, and mass spectrometry (matrix-assisted laser desorption ionization-time of flight, strong-cation-exchange-liquid chromatography/capillary liquid chromatography-electrospray ionization-quadrupole-time of flight [SCX-LC/CapLC-ESI-Q-TOF], and high-pressure liquid chromatography-electrospray ionization-ion trap mass spectrometry) analyses were used to determine the gluten concentration. Assays based on the proliferation of peripheral blood mononuclear cells (PBMCs) and gamma interferon production by PBMCs and intestinal T-cell lines (iTCLs) from 12 celiac disease patients were used to determine the protein toxicity of the pepsin-trypsin digests from fermented wheat dough (sourdough). As determined by R5-based sandwich and competitive ELISAs, the residual concentration of gluten in sourdough was 12 ppm. Albumins, globulins, and gliadins were completely hydrolyzed, while ca. 20% of glutenins persisted. Low-molecular-weight epitopes were not detectable by SCX-LC/CapLC-ESI-Q-TOF mass spectrometry and R5-based Western blot analyses. The kinetics of the hydrolysis of the 33-mer by lactobacilli were highly efficient. All proteins extracted from sourdough activated PBMCs and induced gamma interferon production at levels comparable to the negative control. None of the iTCLs demonstrated immunoreactivity towards pepsin-trypsin digests. Bread making was standardized to show the suitability of the detoxified wheat flour. Food processing by selected sourdough lactobacilli and fungal proteases may be considered an efficient approach to eliminate gluten toxicity.

NEW FINDINGS ON POLYPYRROLE CHEMICAL STRUCTURE BY XPS COUPLED TO CHEMICAL DERIVATIZATION LABELLING

Abstract Polypyrrole electrosynthesised from aqueous solutions has been investigated by XPS, both in the conducting (PPY) and in the so-called overoxidised (PPYox) state. An accurate analysis of high resolution spectra, including cross-checking between the independent fittings of different though related signals (e.g. C1s and O1s), is presented, enlightening novel findings on polypyrrole structure. In particular, it suggested, for the first time, that overoxidation breaks polymer chains, producing new oxygen function on α-C (mainly Cue5fbO). The application of a new chemical derivatization (CD) method to the labelling of this key functional group (Cue5fbO) would confirm this feature.

Identification of allergenic milk proteins markers in fined white wines by capillary liquid chromatography-electrospray ionization-tandem mass spectrometry.

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 microg/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.

Spectroscopic investigation on polymer films obtained by oxidation of o-phenylenediamine on platinum electrodes at different pHs

An investigation on the structure of poly(o-phenylenediamine) n(PPD) films, obtained by electropolymerization of oPD (1,2-diaminobenzene) on platinum at different pH values, was performed by X-ray Photoelectron Spectroscopy (XPS). n XPS could be used as a “bulk” technique for PPD films analysis, due to their extremely low thickness. The presence of different functionalities, like primary/secondary aminic, iminic and, as minor species, oxygenated groups (carbonyl, oximes) was suggested by curve fitting of carbon (C1s) and nitrogen (N1s) XP spectra. The use of chemical derivatization reactions (CD–XPS) confirmed the presence of primary aminic and hydroxy groups, showing that NH2 groups are present in the PPD structure even at low pH values, though their amount increases on increasing the pH of polymerization. n Optical spectroscopy in the visible region nwas also performed on the electrolytic solutions at the end of polymerization, suggesting a higher conjugation of the oPD oligomers at low pH, which indirectly confirms XPS findings on the presence of NH2 groups in the polymer.

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

RATIONALEnA method based on High-Resolution Mass Spectrometry was developed for the simultaneous determination of fining agents containing potentially allergenic milk (casein) and egg-white (lysozyme and ovalbumin) proteins, added to commercial white wines at sub-ppm levels. Selected tryptic peptides were used as quantitative markers. An evaluation of protein digestion yields was also performed by implementing the (15)N-valine-labelled analogues of the best peptide markers identified for αS1 -casein and ovalbumin.nnnMETHODSnThe 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.nnnRESULTSnSix-point calibrations were performed by adding caseinate and egg-white powder in the concentration range between 0.25 and 10 µg/mL, to an allergen-free white wine. The following three peptide markers, LTEWTSSNVMEER, GGLEPINFQTAADQAR and ELINSWVESQTNGIIR, were highlighted as best markers for ovalbumin, while GTDVQAWIR and NTDGSTDYGILQINSR for lysozyme and YLGYLEQLLR, GPFPIIV and FFVAPFPEVFGK for caseinate. Limits of detection (LODs) ranged from 0.4 to 1.1 µg/mL.nnnCONCLUSIONSnThe 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.

Electrosynthesis and analytical characterisation of polypyrrole thin films modified with copper nanoparticles

Copper–polypyrrole (Cu–PPy) composites nhave been synthesised following an all-electrochemical procedure comprising nthe deposition of a thin PPy film and the subsequent pulsed potentiostatic ndeposition of copper from a CuCl2 solution. Surface and bulk characterisation nof the specimens has been carried out using X-ray Photoelectron and Micro-Raman nspectroscopies, respectively. Cyclic voltammetry has been used to investigate nelectroactive species and copper–polypyrrole interactions in the materials. nComposite morphology has been assessed by Scanning and Transmission Electron nMicroscopies. The cluster size depended upon the amount of copper deposited; nfor a copper loading of 44xa0mCxa0cm−2, clusters nwith a mean diameter of 160xa0nm were deposited. These clusters are mainly ncomposed of Cu2O, although chloride species and CuO were found non the surface of the composites. Evidence is provided that Cu–N complexes nwere formed too.

Towards the quantification of residual milk allergens in caseinate-fined white wines using HPLC coupled with single-stage Orbitrap mass spectrometry

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 caseinate solutions 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 0.3u2009µgu2009ml−1 (S/Nu2009=u20093) in water, and between 0.15 and 0.7u2009µgu2009ml−1 in wine matrix, depending on the selected peptide. Method repeatability and reproducibility, measured as response variability (standard deviation) due to LC-MS analysis alone and to both enzymatic digestion and LC-MS analysis, 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 matrix-matched samples, the only exception being peptide GPFPIIV from β-casein, the marker also providing the highest sensitivity. The method was finally applied to a casein-free white wine (‘Greco di Tufo’) fined with caseinate 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/Nu2009=u20093) were estimated between 39 and 51u2009µgu2009ml−1, 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 ngu2009ml−1 range.

Disposable interference-free glucose biosensor based on an electropolymerised poly(pyrrole) permselective film

Abstract Miniaturised disposable amperometric biosensors for glucose determination in serum are described. A commercially available three-electrode system created on a planar corundum ceramic base was used as biosensor substrate and the working Pt electrode was modified by an electrogenerated overoxidised poly(pyrrole) film (PPYox). After the optimisation of the PPy layer synthesis, two different approaches were investigated for glucose oxidase (GOD) immobilisation: enzyme entrapment into the electropolymerised film by an ‘all-electrochemical’ procedure (PPYox/GOD) and gel-entrapment over the PPYox modified electrode by co-crosslinking with glutaraldehyde/bovine serum albumin (PPYox/GOD-gel). A comparison of the sensitivities to glucose, performed by dropping 50xa0μl of the sample solutions onto the relevant electrode systems, showed that both sensors have a linearity range extending up to 10xa0mM, though Pt/PPYox/GOD-gel sensor is more sensitive (168±15 versus 53±7xa0nAm/M). The two sensors showed remarkable anti-interferent selectivity, moreover the PPYox/GOD-gel sensor had also a good stability and could be used for flow injection analysis of glucose, with a linearity range extending up to 25xa0mM.

Electrosynthesised thin polymer films: the role of XPS in the design of application oriented innovative materials

The paper reviews some significant pieces of work carried out in the authors’ laboratory in the course of about three decades and concerned with the development of market oriented devices, which exploit the singular characteristics of electrosynthesised polymers. The strategic role of X-Ray Photoelectron Spectroscopy is underlined not only as a powerful technique for the characterization of these thin films, but mainly as an unvaluable tool to feedback optimization procedures both in film synthesis and modification. Case studies relevant to the development of permselective membranes for biosensors, of biocompatible coatings and active layers for gas sensors have been selected and reviewed. Future trends and prospects of the work in progress are also described.rnrnPrefacernrnDuring electrochemical experiments electrode materials often undergo more or less pronounced modifications of their surface composition, for example by chemical interaction with the media and/or as a consequence of the applied potential. These phenomena, should they be promoted deliberately or completely undesired, can influence the behaviour of the electrode systems particularly when surface species are directly involved in the electrochemical processes.rnrnThis can lead to a lack of molecular specificity in electrochemical measurements so that the knowledge of the chemical composition of the electrode surface becomes of paramount importance. In this context, surface analysis techniques have played, and still play, a key-role in the characterization of surface chemically-modified electrodes.rnrnIn the course of a systematic investigation performed in our laboratory in the period 1960–1975, aimed at the development of oxygen and hydrogen electrodes to be employed in medium temperature fused salt fuel cells, irreversible potentiometric behaviours, largely dependent on the working temperature, and Nernstian behaviours different from those expected on the basis of the overall electrode processes, were observed. These results were rationalised by hypothesizing potential-determining steps involving solid species (metal oxides) present on the electrode surface [1–4]. In the following years, combined potentiometric and XPS studies were performed on those (and others) electrode systems [5–9] and the results obtained well supported the proposed mechanistic models through the detection and identification of solid surface species.rnrnAs an example of this research activity, results obtained on the system (Ni)CO2,O2/CO3= in a (Na,K)NO3 equimolar mixture will be briefly reviewed. Potentiometric measurements performed in the temperature range 507<T<623 K in molten nitrates containing a carbonate ion concentration in the range 10−5<[CO3=]<10−2 mol kg−1 and fluxed with a mixture of CO2 and O2 at variable partial pressures indicated that at the highest tested temperature the system was irreversible and the potential was independent of oxygen concentration. This was interpreted on the basis of the following mechanistic modelrnrnMechanism I:rn(1)2NiO+CO3= ⇔ NiO-O-NiO+CO2 +2e(fast)rnrn(2)Ni2O3 ⇒ 2NiO+1/2 O2(slow)rnrn-----------------------------------------------------rnrn(3)CO3= ⇔ CO2+1/2 O2+2ernWhere reaction 1 represents the potential-determining step and reaction 3 the overall [10,3] electrode process.rnrnOn the other hand a dependence of the potential on O2 (other than CO2) was seen at lower temperatures. At the same time a large irreversibility of the system was still present. To explain this behavior, the presence of a second parallel mechanism, increasingly competitive with the first one as the temperature decreases, was proposed.rnrnMechanism II:rn(4)2NiO+CO3=⇔ NiO–CO2–NiO +1/2 O2 +2e(fast)rnrn(5)NiO–CO2–NiO ⇒ 2NiO +CO2(slow)rnrn------------------------------------------------------------rnrnCO3= ⇔ CO2+1/2 O2+2e(3)rnrnrnAs is apparent, both mechanisms involve, in the potential determining step, solid species present on the electrode surface. Due to the strong oxidising power of nitrate melts, the following reactions can occur when nickel electrodes are immersed in such a solventrnNi +NO3−⇔ NiO +NO2−rnrn2NiO +NO3− ⇔ Ni2O3+NO2−rnrnNi2O3+Ni⇔ 3NiOrnrnrnAn XPS study was carried out on this system in order to verify the reliability of the proposed mechanistic models. Spectra recorded on nickel foils maintained in contact with the melt at two different temperatures and for different lapses of time are reported in Figs. 1 and 2rnDownload high-res image (149KB)rnDownload full-size imagernrnrnrnFig. 1. Typical photoelectron spectra relevant to the nickel 2p3/2 level recorded at the following metal-melt times of contact (in min): (a)0.0, (b)5.0, (c)30.0, (d)90.0, (e)120.0. T=511 K. The arrows define the range in which binding energies of some nickel compounds (NiO, Ni2O3, Ni(OH)2, NiCO3) fall.rnrnrnDownload high-res image (87KB)rnDownload full-size imagernrnrnrnFig. 2. Typical photoelectron spectra relevant to the nickel 2p3/2 level recorded at the following metal-melt times of contact: (a’) 0.0, (b’) 5.0 s, (c’) 5.0 min. T=609 K. The arrows define the range in which binding energies of some nickel compounds (NiO, Ni2O3, Ni(OH)2, NiCO3) fall.rnrn. The presence of oxidised species is clear; Ni2O3 and Ni(OH)2 species seem to be present in the first tract of the experiment, while, for longer periods of contact, NiO becomes the predominant one. This is consistent with the fact that Ni2O3 is the primary species formed on nickel-oxygen surfaces [11,12], since kinetically favoured, and with the observation that NiO is the only known stable “bulk” oxide.rnrnIn order to test for the presence of carbonate species [involved in the proposed mechanism II] XPS spectra were recorded on nickel foils maintained in contact with the melt for different lapses of time, under two different gas atmospheres, both at low (511 K) and high (609 K) temperature. The C1s signals reported in Figs. 3 and 4rnDownload high-res image (82KB)rnDownload full-size imagernrnrnrnFig. 3. Typical photoelectron spectra relevant to C1s region recorded on nickel samples under the following working conditions. Metal-melt times of contact (in min): (a’) 10.0, (b’) 30.0, (c’) 120.0. T=511 K; gas atmosphere: CO2=0.9 atm, O2=0.1atm.rnrnrnDownload high-res image (62KB)rnDownload full-size imagernrnrnrnFig. 4. Typical photoelectron spectra relevant to C1s region recorded on nickel samples under the following working conditions. Metal-melt times of contact (in min): (a’) 10.0, (b’) 30.0, (c’) 120.0. T=609 K; gas atmosphere: CO2=0.9 atm, O2=0.1atm.rnrnclearly show that at low temperature a carbonate peak (ca. 290 eV) is present, whose intensity depends on carbon dioxide partial pressure and on the time of contact between nickel substrate and molten nitrates. At high temperatures, whatever the composition of the gas mixture and the time of immersion of the nickel foils, no carbonate peak could be recorded. As expected, at this temperature the formation of a nickel carbonate species is strongly prevented because of its thermal decomposition.rnrnIn conclusion, XPS investigation unequivocally showed that solid species (carbonate and/or oxides) are formed on the surface of nickel electrode, thus confirming the hypothesis that the large potentiometric irreversibility as well as the marked dependence of the potentiometric behaviour on the temperature were consequences of the electrode chemical corrosion and could be explained by the establishment of two competitive mechanistic models (mechanisms I and II).rnrnAs a more general conclusion, the research activity carried out by our group in the field of fuel cells contributed further evidence of the great potential of XPS in providing definitely the “chemical situations” existing at modified electrode surfaces.

Phospholipidomics of Human Blood Microparticles

The phospholipidome of blood microparticles (MPs) obtained from platelet-rich plasma of healthy individuals was characterized by hydrophilic interaction liquid chromatography (HILIC) coupled to electrospray ionization tandem mass spectrometry (ESI-MS/MS). The HILIC separation, performed on a silica stationary phase using an acetonitrile/methanol gradient, enabled the separation of several phospholipids (PL) classes, viz., phosphatidyl-cholines (PCs), -ethanolamines (PEs), -serines (PSs), -inositoles (PIs), sphyngomielins (SMs), and lyso forms of PCs and PEs. Structural characterization of species belonging to each class was performed by MS/MS measurements, in either positive or negative ion mode. The set of 131 phospholipids (including regioisomers) here identified represents the most comprehensive phospholipidomic characterization reported for human MPs. Although the phospholipidome composition of MPs and platelets, collected from the same donors, was found to be qualitatively the same, quantitative differences were evidenced for lyso-PCs, which appear to be significantly more abundant in MPs.