Michele Tomaiuolo

Within the framework of a project financed by the Italian Ministry of Health, eggs of different brands and types (farming method, color, size), both irradiated (0.5–5 kGy) and non-irradiated, were examined by the laboratories of the Istituto Superiore di Sanità (ISS) and the Istituto Zooprofilattico Sperimentale della Puglia e della Basilicata (IZSPB) using Photo-stimulated Luminescence (PSL) and Thermoluminescence (TL) techniques, which identify irradiated foods through the luminescence emitted from certain types of minerals present in the food matrix (biominerals) or in contaminating dust (silicates). The data obtained with both techniques indicate that the content of luminescent minerals in the shells is rather low, probably due to the cleaning of the eggs during production. The PSL technique applied to 144 irradiated samples results in 75% false negatives and 23% unclassifiable intermediates, with only 3 positive samples originating from eggs with markedly different characteristics (small and white). As for TL technique, the silicates extracted from 6 eggshells were generally sufficient for the analysis but provided rather weak TL signals. The ESR technique applied to eggshells produced excellent results for the identification of X-ray treatment. Furthermore, from the tenth day after irradiation onward, the ESR signal showed no time-dependent attenuation. The relationship between signal intensity and irradiation dose was not linear but exponential up to 1 kGy and logarithmic between 1 and 5 kGy.

Ionizing radiation is a non-thermal sanitization technique used in the food field to eliminate bacteria, molds, insects and other microbes, resulting in delayed spoilage and extended shelf life. In this work, mozzarella cheese was irradiated with X-rays at a dose of 3.0 kGy, and irradiation-induced lipid modifications were evaluated through a comprehensive analysis of the mozzarella lipid fingerprint. To this aim, an optimized microwave-assisted extraction method associated with UHPLC-Q-Orbitrap-MS analysis was used for reliable and accurate lipid identification in the controls and in irradiated samples. The outcomes demonstrated that the X-ray dose employed in this investigation did not cause the formation of new lipid molecules. However, lipidomic chemometric modeling, including partial least squares-discriminant analysis, enabled the discrimination of irradiated versus non-irradiated samples and the selection of five ceramides, eight hexosyl ceramides, four sphingomyelins, one phosphatidylethanolamine, one cholesterol ester, ten oxidized triacylglycerols, and one oxidized diacylglycerol as potential markers of treatment. Finally, an artificial neural network was developed to accurately model the entire pattern in omics data in relation to the treatment. This developed analytical workflow allows for expanding knowledge on the effects of this technology and could have interesting applications in food safety traceability and control plans.

Food irradiation is a clean, safe and non-thermal technology applied to destroy pathogenic microorganisms, i.e., Salmonella spp., in hen eggs. Currently, in Europe only the egg white can be irradiated up to 3 kGy, so different control methods are crucial for official inspections to identify illicit treatments. In this work, an analytical method was proposed to determine the radiolytic markers, namely 2–dodecylcyclobutanone (2–DCB) and 2–tetradecylcyclobutanone (2–TCB) in hen egg samples. This method is based on headspace solid phase micro-extraction coupled with gas chromatography/mass spectrometry (HS–SPME/GC–MS). The eggs were treated by an X-ray irradiator at dose levels of 0.5, 1.0 and 3.0 kGy. The preliminary validation showed good selectivity, without matrix interferences in non-irradiated samples. Spiked samples showed linear responses in the range 2.5–25.0 µg kg⁻¹, where 2.5 µg kg⁻¹ was the limit of detection for both analytes. Irradiated samples showed a dose-dependent increase in signal intensity and a constant 2–DCB/2–TCB ratio. The minimum dose level detected was 0.5 kGy for all samples, and the 2–DCB and 2–TCB signals remained stable over one month after irradiation. Not least, white analytical chemistry was used to evaluate the HS–SPME/GC–MS method validation effectiveness, greenness power and economic efficiency, compared to the EN 1785:2003 standard method. The results of this study prove that the HS–SPME/GC–MS method is a reliable green alternative to the official method, which is suitable in food safety control programs.

In this work, Microwave-Assisted Extraction (MAE) was proposed as an alternative and environmentally friendly technique in lipidomics to study the lipid fingerprint of soft cheeses, such as mozzarella. For method development, a first step concerning an evaluation of extraction solvents was carried out via testing three different mixtures, including methanol/ethyl acetate, isopropanol/ethyl acetate, and ethanol/ethyl acetate, at a 1:2 v/v ratio. The latter was chosen as a solvent mixture for subsequent method optimization. MAE conditions, in terms of solvent volume, time, and temperature, were explored to define their effects on extraction capability through a full factorial experimental design. The best compromise to extract more lipids at the same time was obtained with 24 mL g⁻¹ for solvent-to-solid ratio, 65 °C for temperature, and 18 min for time. Lipid analyses were conducted by UHPLC-Q-Orbitrap-MS associated with multivariate statistics. The developed lipidomic workflow allowed for the extraction of over 400 lipids grouped into 18 different subclasses. The results confirmed that MAE is a suitable technique for lipid extraction in the omics approach with high efficiency, even using low-cost and less toxic solvents. Moreover, a comprehensive structure characterization of extracted lipids, in terms of fatty acid composition and regiochemistry, was carried out.

X-ray irradiation is an emerging non-thermal technology that is used as a preservation and sanitization technique to inactivate pathogens and spoilage organisms, increasing the shelf life of products. In this work, two different types of surface-ripened cheeses, Brie and Camembert, produced with cow milk, were treated with X-rays at three dose levels, 2.0, 4.0 and 6.0 kGy, to evaluate the irradiation effects on the volatile profile using a volatolomic approach. The headspace solid-phase microextraction (HS-SPME) technique combined with gas chromatography–mass spectrometry (GC–MS) was used to extract and analyze the volatile fraction from these dairy matrices. The HS-SPME method was optimized by a central composite design in combination with a desirability optimization methodology. The Carboxen/PDMS fiber, 50 °C for extraction temperature and 60 min for time extraction were found to be the best parameter settings and were applied for this investigation. The obtained fingerprints demonstrated that the irradiation-induced changes are dose dependent. The X-ray irradiation produced many new volatiles not found in the non-irradiated samples, but it also varied the amount of some volatiles already present in the control. Specifically, aldehydes and hydrocarbons increased with the irradiation dose, whereas alcohols, carboxylic acids, esters, methyl esters, ketones, lactones and sulfur-containing compounds showed a non-linear dependence on the dose levels; indeed, they increased up to 4.0 kGy, and then decreased slightly at 6.0 kGy. This trend, more evident in the Camembert profile, is probably due to the fact that these compounds are involved in different oxidation mechanisms of lipids and proteins, which were induced by the radiation treatment. In these oxidative chemical changes, the production and degradation processes of the volatiles are competitive, but at higher doses, the decomposition reactions exceed those of formation. A principal component analysis and partial least square discriminant analysis were used to discriminate between the treated and untreated samples. Moreover, this study allowed for the identification of potential markers of X-ray treatment for the two cheeses, confirming this approach as a useful tool for the control of irradiated surface-ripened cheeses.

In this work, an investigation using UHPLC-Q-Orbitrap-MS and multivariate statistics was conducted to obtain the lipid fingerprint of Camembert cheese and to explore its correlated variation with respect to X-ray irradiation treatment. A total of 479 lipids, categorized into 16 different lipid subclasses, were measured. Furthermore, the identification of oxidized lipids was carried out to better understand the possible phenomena of lipid oxidation related to this technological process. The results confirm that the lipidomic approach adopted is effective in implementing the knowledge of the effects of X-ray irradiation on food and evaluating its safety aspects. Furthermore, Partial Least Squares-Discriminant Analysis (PLS-DA) and Linear Discriminant Analysis (LDA) were applied showing high discriminating ability with excellent values of accuracy, specificity and sensitivity. Through the PLS-DA and LDA models, it was possible to select 40 and 24 lipids, respectively, including 3 ceramides (Cer), 1 hexosyl ceramide (HexCer), 1 lysophosphatidylcholine (LPC), 1 lysophosphatidylethanolamine (LPE), 3 phosphatidic acids (PA), 4 phosphatidylcholines (PC), 10 phosphatidylethanolamines (PE), 5 phosphatidylinositols (PI), 2 phosphatidylserines (PS), 3 diacylglycerols (DG) and 9 oxidized triacylglycerols (OxTG) as potential markers of treatment useful in food safety control plans.

This work reports the results of a study carried out to verify the applicability of the CEN EN 13751 method, based on the photo-stimulated luminescence (PSL) technique, for the identification of irradiated fruit. A total of 15 types of fresh and dried fruit not irradiated and irradiated (0.5 and 1 kGy) were tested. Preliminary tests were performed on samples of fresh kiwis, lemons, mangoes, oranges, papayas, pineapples and tangerines, dried dates, dried prunes and raisins. Successively, an inter-comparative test was organized, whereby four Italian official control laboratories analyzed eight matrices, namely, kiwi, orange, dried fig, raisin, hazelnut, peanut, pistachio and walnut. The results indicated considerable differences in the radiation PSL sensitivities of the different types of fruit as well as among the PSL responses obtained from different batches of the same fruit. The best results were obtained with some types of dried fruit, namely, pistachios, peanuts, dried figs and raisins. Irradiated fresh fruit generally gave non-positive results, mainly intermediate and even very close to the negative threshold. Tests performed on kiwi, mango and orange samples indicated that the parts of the fruit peel close to the stem, which are more wrinkled, provide better results.