Evaluation of the antioxidant effect of a phytocomplex addition in clean label pork salami enriched in n-3 PUFA.

Control and n-3 PUFA enriched raw material was used to manufacture clean label and conventional salami; the former were added with a phytocomplex having iron chelating, DPPH, and FRAP activity, obtained from olive vegetation water, oregano, green tea and blueberry leaves, and with acerola powder. Salami were dried at 3 ± 1 °C until an established decrease in the combined pH and aw values, while only the conventional ones underwent a standard process. In the cold dried salami pH changes, aw and weight decrease were delayed; the phytocomplex contributed to lower the pH, and to prevent lipid and protein oxidation, despite the n-3 PUFA enrichment and heme iron release due to nitrite removal. TBARS and protein carbonyls were the highest in the nitrite-added salami undergoing cold and standard drying, respectively. The oxidation marker MDA tended to increase in the simulated digests of salami n-3 PUFA enriched or subjected to cold drying.

Group detection of DON and its modified forms by an ELISA kit.

Deoxynivalenol (DON) and its modified forms (3-, and 15-acetyl-DON, DON-3-glucoside) are commonly analysed by chromatographic methods. Indeed, coupled with proper extraction and clean-up, LC-MS represents the best approach for multi-mycotoxin measurements. On the other hand, immunochemistry-based methods are possibly able to detect a family of structurally related compounds, although the determination of single contributions is not possible so far. However, ELISA methods often lead to an apparent overestimation of the mycotoxins content because modified forms and matrix components can potentially cross-react with the antibodies (designed for the parent toxin). Several data about the possible cross-reactivity of commercial DON-detecting ELISA kit are reported in the literature so far. Data are commonly obtained in buffer solutions or in matrix-matched solutions, but comparison of a set of naturally incurred samples has never been reported. In the present work the accuracy of a commercial DON-detecting ELISA kit was evaluated on naturally incurred soft wheat (n = 15) and maize (n = 15), taking into account the matrix effect. Recovery was calculated considering the DON concentration found by LC-MS/MS and the total DON concentration, expressed as the sum of DON and its modified forms found by LC-MS/MS. The obtained data clearly show that, when 3-modified forms of DON occur in the sample, the ELISA kit does actually detect them, thus returning an apparent overestimation if only DON content is considered. When the ELISA recovery is calculated on the total DON content, the accuracy of the analysis increases and the variability decreases. According to our data, the ELISA kit seems to be a promising group detection tool for the accurate evaluation of DON and its modified forms, expressed as sum of DON, DON-3Glc and 3Ac-DON, for soft wheat and maize samples.

Oleoyl and linoleoyl esters of fumonisin B1 are differently produced by Fusarium verticillioides on maize and rice based media.

Fatty acid esters of fumonisins, namely oleoyl- and linoleoyl esters of fumonisin B1 (EFB1OA and EFB1LA, respectively), are modified forms of fumonisins whose formation and occurrence have been reported so far in naturally infected maize and in artificially inoculated rice. There is a lack of knowledge about the mechanism of formation, mainly in relation to the role played by the substrate. Therefore, in this work we studied the dynamics of accumulation of the toxin and its esters, together with their precursor, in maize and rice based media inoculated with different strains of F. verticillioides and incubated at 25 °C for 7-45 days. The production pattern of FB1 and its modified forms was significantly influenced by growth media, reaching a higher concentration in cornmeal compared to rice based medium. Similarly, cornmeal was more supportive for the conversion of FB1 by considering the esterification rate, with a prevalence of linoleoyl esters compared to oleoyl esters resembling the OA/LA rate in both media. The conversion of FB1 into fatty acid esters was also shown as strain-related. Results, thus, strongly support the hypothesis that fatty acid esters of FB1 are produced by the fungus itself at a late stage of growth, or at a certain point of FB1 accumulation in the medium, using fatty acids from the substrate.

A simple and reliable liquid chromatography-tandem mass spectrometry method for the determination of aflatoxin M1 in milk.

A new chromatographic method is proposed for the analysis of aflatoxin M(1) in milk. The method is based on liquid-liquid extraction followed by LC-MS/MS analysis. Liquid-liquid extraction (LLE) is performed on the defatted milk plus sodium chloride by using ethyl acetate as an extraction solvent. Accuracy and precision were evaluated at the LOQ (15 ng kg(-1)) spiked sample as well as with three other different naturally contaminated reference materials. The mean overall recovery (n = 24) was 95% with a confidence interval of 1.9% and a CV% of 4.5%. The performance of the proposed method was compared with that of the Official ISO Method based on the use of immunoaffinity chromatography columns (IAC): LLE protocol could be considered a valid alternative to the LC-IAC. In general it showed better accuracy with lower data dispersion. Moreover, the sample preparation is very simple and straightforward, potentially being applicable as a high-throughput method which, on account of its simplicity and low cost, may be applied to the analysis of a large number of samples in the occasion of outbreaks of large-scale contamination.

Volatile fingerprinting of chestnut flours from traditional Emilia Romagna (Italy) cultivars.

The volatile profile of nine monocultivar chestnut flours, obtained from fruits grown in Italy (Parma province), was characterised by a head-space solid-phase microextraction (HS-SPME) coupled with GC-MS technique. The volatile fraction was composed of 44 main compounds belonging to different classes, mainly aldehydes, ketones, alcohols, furans and terpenes. Aldehydes, in particular hexanal, are the most abundant components. In order to better understand the origin of the different volatile compounds during the drying and milling processes, samples of fresh fruit were also analysed by the same technique and the data obtained were statistically and critically compared in order to get a picture of the volatile evolution in chestnut from fresh fruit to flour. Finally, the nine monocultivar flours were chemometrically classified on the basis of the main odour descriptors associated with the volatile fingerprinting.

Cheese peptidomics: a detailed study on the evolution of the oligopeptide fraction in Parmigiano-Reggiano cheese from curd to 24 months of aging.

In this work, we performed a detailed evaluation of the evolution of the oligopeptide fractions in samples of Parmigiano-Reggiano cheese from the curd up to 24 mo of aging. The samples were taken from wheels produced the same day, in the same factory, from the same milk, during the same caseification process, thus simplifying the natural variability of a whey-based starter fermentation. This unique and homogeneous sampling plan, never reported before in the literature, provided a detailed study of the peptides produced by enzymatic events during Parmigiano-Reggiano aging. Given the large dimensions of the 35-kg wheels of Parmigiano-Reggiano, samples were taken from both the internal and external parts of the cheese, to evidence eventual differences in the oligopeptide composition of the different parts. Fifty-seven peptides were considered, being among the most abundant during at least one of the periods of ripening considered, and their semiquantification indicated that the peptide fraction of Parmigiano-Reggiano cheese constantly evolves during the aging period. Five trends in its evolution were outlined, which could be clearly correlated to the enzymatic activities present in the cheese, making it possible to discriminate cheeses according to their aging time. Several known bioactive peptides were also found to be present in Parmigiano-Reggiano cheese samples, and for the first time, the age at which they are most abundant has been identified. Aged cheeses have been shown to be dominated by nonproteolytic aminoacyl derivatives, a new class of peptide-like molecules recently reported. Finally, the changing peptide pattern may be related to the changing enzymatic activities occurring inside the cheeses during the aging period, which, in turn, are also related to the microbiological composition.

Hyphenated chromatographic techniques for structural characterization and determination of masked mycotoxins.

Mycotoxins are secondary metabolites produced by fungi that can contaminate a wide range of food and feed commodities and that are harmful to humans for their poisonous and toxic effects. An increasing amount of data have been accumulated in the last years, showing that mycotoxins may also occur in modified forms originating by plant, fungi or animal metabolism or by food processing. In particular, this modified forms may be produced via conjugation with sugars or other biological components (masked mycotoxins) or may occur as non extractable forms on account of strong interaction, association or binding with macromolecules in the food matrix (bound or hidden mycotoxins). Analytical methods have been set up in order to check for the occurrence of these forms and to evaluate their amount, in order to obtain reliable data for toxicity and exposure studies. In this paper hyphenated chromatographic methods for the determination and structural characterization of masked mycotoxins are reviewed, with a particular emphasis on liquid chromatography-(tandem) mass spectrometry as the most effective approach for their determination.

Brand-dependent volatile fingerprinting of Italian wines from Valpolicella.

Head-space solid-phase microextraction (HS-SPME) coupled to gas-chromatography-mass spectrometry was developed and applied to obtain the volatile aromatic fingerprints of three typical Italian wines, Valpolicella, Amarone and Recioto, all produced in the restricted geographical area of Valpolicella (Veneto, Italy) with the same grape cultivars within the regulations of a rigid disciplinary of production. Differences between the three typologies are mainly linked to the different withering times to which grapes are subjected before vinification, which strongly influences the concentration and the development of volatile aroma compounds. A total of 22 different wines (7 Valpolicella, 10 Amarone and 5 Recioto) were characterised in terms of aromatic volatile profile with the aim to distinguish the different products and to evaluate the possibility to differentiate the same product from different brands. For the chemometric evaluation of the data one-way analysis of variance (ANOVA), principal component analysis (PCA) and hierarchical cluster analysis (HCA) were tested. All the chemometric tools employed allow to differentiate between the three products. More intriguing is the ability of the chemometric approach to differentiate between the same product (Amarone, Recioto) from different winery, thus showing the potential of this approach to characterize the brand-dependent typicality of wines, which is usually related to subtle technological differences which nevertheless have strong influences on the organoleptic characteristics of the products.

Fate of Fusarium mycotoxins in the cereal product supply chain: the deoxynivalenol (DON) case within industrial bread-making technology.

Fusarium mycotoxins are a relevant problem in the cereal supply chain at a worldwide level, with wheat, maize and barley being the main contaminated crops. Mould growth can happen in the pre-harvest phase and also during transport and storage due to ineffective drying conditions. Among Fusarium toxins, deoxynivalenol (DON) is considered the most important contaminant in wheat due to its widespread occurrence. In the last years the European Food Safety Authority (EFSA) and the European Commission have frequently expressed opinions on Fusarium toxins, setting limits, regulations and guidelines in order to reduce their levels in raw materials and food commodities. In particular, European legislation (Reg. 1881/2006) sets the maximum limit for DON in flour and bread as 750 and 500 microg kg(-1) respectively. Relatively few studies have taken into account the loss of trichothecenes during processing, focusing on how processing factors may influence their degradation. In particular, the description of DON behaviour during bread-making is very difficult, since complex physico-chemical modifications occur during the transformation of the raw ingredients into the final product. In the present study, we studied how DON concentration may be influenced by modifying bread-making parameters, with a special emphasis on the fermentation and baking stages, starting from a naturally contaminated flour at both pilot and industrial scales. Exploiting the power of a Design of Experiments (DoE) approach to consider the great complexity of the studied system, the obtained model shows satisfying goodness-of-fit and prediction, suggesting that the baking step (time/temperature ranges) is crucial for minimizing native DON level in bread.

Complexation of the mycotoxin zearalenone with ß-cyclodextrin: Study of the interaction and first promising applications.

This work reports the study of the interactions between native and substituted ß-cyclodextrins and zearalenone and its derivatives α- and ß-zearelonol. The data obtained by fluorescence and NMR experiments suggested that zearalenone, α- and ß-zearalenol and cyclodextrins give rise to host-guest complexation, with the inclusion of the phenolic moiety inside the cyclodextrin cavity. The high stability of these complexes induces a high fluorescence enhancement upon complexation. These results have been successfully applied to the spectrofluorimetric determination of zearalenone in maize raw samples, without any chromatographic separation.

A new validated HPLC-FLD method for detecting ochratoxin A in dry-cured meat and in blue cheese.

In the present study, a fast and sensitive method for the quantification of ochratoxin A in two lipidicproteic food matrices has been developed. In particular, the sample preparation procedure has been optimized for dry-cured meat products and blue cheeses and tested for several validation parameters (LOD, LOQ, recovery, repeatability and within-laboratory precision). The procedure has been then applied to several dry-cured meat products and blue cheeses from the market.Ochratoxin A has been occasionally found in dry-cured and smoked ham from the market and the contamination occurred both in the outer and in the inner part of the products. Concerning the blue cheese, the occurrence of ochratoxin A is reported for the first time: OTA was occasionally found at low levels (0.1-3 µg/kg) in commercial samples of Roquefort from France and Gorgonzola from Italy, opening a new issue for risk assessment and quality control.

An innovative LC/MS approach applied to the determination of zeralenone in maize: Alternate Isotope-coded Derivatization Assay (AIDA).

Zearalenone is a mycotoxin mainly produced by severalFusarium species, which are known to colonize grains in temperate climates. The purpose of the study is to provide a reliable isotope dilution method for the quantification of this mycotoxin. A derivative of the analyte to be used as standard is obtained by reaction with acetic anhydride, which is available in two pure isotopic forms, a protonated ("light") and a hexadeuterated ("heavy"). The derivatized standards are added to the matrix split intwo parts. Then, the derivatization procedure is repeated on both matrices derivatizing the part containing the "heavy" labelled standard with the "light" acetic anhydride and the part containing the "light" labelled standard with the "heavy" acetic anhydride. Both extracted mixtures are analyzed by LC/MS, monitoring the "light" and the "heavy" labelled analytes and using the former as standard for the latter in one case and viceversa in the other case. The method allowed to obtain very good results, without the need of IAC purification.

Simultaneous detection of type A and type B trichothecenes in cereals by liquid chromatography-electrospray ionization mass spectrometry using NaCl as cationization agent.

A LC/MS method for the simultaneous determination of both type A and type B trichothecenes by using an electrospray ionization (ESI) interface in the positive ionization mode with a single quadrupole analyzer is described. In order to enhance the ionization of both groups of trichothecenes, the sodium ion was used as cationization agent by adding sodium chloride to the eluent. All LC/MS parameters were optimized. The newly developed LC/ESI-MS method was applied to the analysis of a wheat reference material and cereal-based foods and feeds.

Simultaneous liquid chromatography-fluorescence analysis of type A and type B trichothecenes as fluorescent derivatives via reaction with coumarin-3-carbonyl chloride.

A method for the simultaneous LC-fluorescence detection (FLD) determination of eight trichothecenes A and B by pre-column derivatization with coumarin-3-carbonyl chloride, a highly fluorescent fluorophore, has been developed. The reaction conditions (temperature, reaction time, reactant ratios) were optimized to give a reproducible quantitative conversion. All derivatives were characterized by LC-MS. The chromatographic parameters were optimized (column, eluent) to give a very good separation of three type A (diacetoxyscirpenol, T-2 toxin, HT-2 toxin) and five type B trichothecenes [deoxynivalenol (DON), nivalenol, fusarenon-X, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol]. The best conditions were obtained on a narrow-bore C18 column with a water-methanol gradient. The detection limits (S/N = 3:1) in grain samples, with an injected volume of 5 microl, were 0.2-1 ng/g for all trichothecenes. These values are more than one order of magnitude lower than those of other LC-FLD and LC-MS methods and are similar to those obtained by GC-MS. The calibration curves were linear between 100 and 2500 ng/g. The method was successfully applied to the analysis of a certified wheat reference material, after solvent extraction and clean-up on a Mycosep column, obtaining a good recovery (89% for DON) and a high accuracy (z-score value: 0.67).

Reversed-phase liquid chromatographic method for the determination of ochratoxin A in wine.

In this paper, we propose a new, rapid, highly sensitive and reproducible RP-HPLC-FLD method for the detection of ochratoxin A (OTA) in wine, by directly injecting the liquid in the chromatographic system without any extraction or clean-up. An alkaline mobile phase (NH4Cl:CH-CN 85:15 (v/v), 20 mM, pH 9.8) was used to obtain a distinct fluorescence enhancement. This improvement allows to reach, without an immunoaffinity clean-up or concentration, a detection limit of 0.05 ng/ml, which is similar to those commonly obtained after immunoaffinity purification and acidic elution. The method was statistically validated and directly applied to a series of wine samples.

Design and synthesis of fluorescent beta-cyclodextrins for the enantioselective sensing of alpha-amino acids.

Fluorescent monofunctionalized beta-cyclodextrins bearing a copper(II) binding side arm and a dansyl group (CD-NH-AA-CH(2)CH(2)NH-DNS) were designed as enantioselective sensors for unmodified alpha-amino acids. The side arm was derived from amino acid synthons (AA = L- and D-phenylalanine (1 and 2), L- and D-phenylglycine (3 and 4), L-proline (5), and L-cyclohexylglycine (6)) and was chosen in order to contain an amide, an amine, and a sulphonamide group. Enantioselectivity was evaluated by addition of copper(II) complexes of D- or L-valine and D- or L-proline. Chiral discrimination in the fluorescence response was observed in all cases, due to a ligand exchange process. The best conditions for these experiments were found to be the use of an excess (10:1) of the copper complex. The cyclodextrin 4 containing a D-phenylglycine unit was found to be poorly enantioselective, as found for 2, suggesting that the best design can be obtained by using L-amino acids. All L-amino acid containing cyclodextrins showed good enantioselectivities, some of which were higher than those already reported for 1. Other analytes related to amino acids were studied using cyclodextrins 1 and 3. Enantiomers of alpha,alpha-disubstituted amino acids, N-methylamino acids, and amino acid amides were found to be discriminated, while beta-phenylalanine and other molecules bearing a poor anchoring group at the alpha-carbon gave poor enantioselectivity. On the basis of the present data a model for the recognition process, based on the formation of ternary diastereomeric complexes, is proposed.

Enantiomeric separation of hydroxy acids and carboxylic acids by diamino-beta-cyclodextrins (AB,AC,AD) in capillary electrophoresis.

Selectively modified 6,6'-dideoxy-6,6'-L-diamino-beta-cyclodextrins (AB, AC, AD) were successfully used as chiral selectors for the enantiomeric separation of hydroxy acids and carboxylic acids (in particular, phenoxyalkanoic acid herbicides) in capillary electrophoresis (CE). Chiral separations were obtained at a low selector concentration (1 mM) with good enantioselectivity and resolution factors. Separations were optimized as a function of pH. The different position of the charged groups on the upper rim greatly influenced the separation, accounting for electrostatic interactions between the protonated amino groups of the cyclodextrins (CDs) and the carboxylate of the selectands. The best enantiomeric separation of hydroxy acids was obtained with the AC regioisomer, whereas carboxylic acids were well resolved only by the AB regioisomer. A recognition model is proposed, based on two-dimensional nuclear magnetic resonance (2-D NMR) experiments, in which the orientation of the guest in the inclusion complex is determined by the electrostatic interactions between the selectand and the CD upper rim.

Chiral separation of amino acids by copper(II) complexes of tetradentate diaminodiamido-type ligands added to the eluent in reversed-phase high-performance liquid chromatography: a ligand exchange mechanism.

In this paper we report a study on the mechanism of the enantiomeric separation of unmodified D,L-amino acids in RP-HPLC by copper(II) complexes of two tetradentate diaminodiamido ligands, (S,S)-N,N'-bis(phenylalanyl)ethanediamine (PheNN-2) and (S,S)-N,N'-bis(methylphenylalanyl)ethanediamine (Me2PheNN-2), added to the eluent. The aim is to investigate whether and how a copper(II) complex with no free equatorial positions can perform chiral discrimination of bidentate analytes such as unmodified amino acids. The problem is approached in a systematic way by: (a) varying the different chromatographic parameters (pH, selector concentration, eluent polarity); (b) performing chiral separation with the selector adsorbed on the stationary phase; (c) studying the ternary complex formation of these ligands with D- and L-amino acids in solution by glass electrode potentiometry and electrospray ionization MS. All the experimental data are consistent with a mechanism of chiral recognition, based on ligand exchange, which involves as selectors the species [Cu2L2H(-2)]2+ and [CuLH(-2)] and proceeds by displacement of two binding sites from the equatorial positions, giving rise to the ternary species [CuLA]+ and [CuLH(-1) A]. The most important factor responsible for chiral discrimination seems to be the affinity of the diastereomeric ternary complexes for the stationary phase since no enantioselectivity is observed in solution.

Incidence of tail structure distortions associated with dysplasia of the fibrous sheath in human spermatozoa.

Dysplasia of the fibrous sheath (DFS) is an anomaly found in spermatozoa of severe asthenozoospermic patients. Marked hypertrophy and hyperplasia of the fibrous sheath is the common characteristic. Immunocytochemistry allowed us to visualize the distortions and incidence of tail structure abnormalities associated with this phenotype in six patients; four with a complete form and two with an incomplete form of this pathology previously diagnosed and studied by electron microscopy. Microtubules and fibrous sheaths were studied using monoclonal antibodies against alpha-acetylated tubulin and anti-FSC1 (the major protein component of the fibrous sheath). Mitochondrial sheaths were visualized using the mitochondrion-specific vital dye MitoTracker green FM(TM). Phase contrast and fluorescent microscopy of semen samples showed large numbers of spermatozoa with short, rigid, thick and irregular tails. As expected, anomalous and completely distorted fibrous sheaths, severe alterations of the axonemal microtubules and different patterns of mitochondrial sheath configurations were found. While ultrastructural studies of thin sections allow an in-depth knowledge of the internal organization of the sperm tail, fluorescence labelling of selected sperm components affords a unique view of the whole flagellum including topographical relationships of various organelles. The combination of these different approaches is essential for a comprehensive understanding of this particular pathology.