Roberto Piro

Raw milk cheeses harbor complex microbial communities. Some of these microorganisms are technologically essential, but undesirable microorganisms can also be present. While most of the microbial dynamics and cross-talking studies involving interaction between food-derived bacteria have been carried out on agar plates in laboratory-controlled conditions, the present study evaluated the modulation of the resident microbiota and the changes of metabolite production directly in ripening raw milk cheese inoculated with Listeria innocua strains. Using a proxy of the pathogenic Listeria monocytogenes, we aimed to establish the key microbiota players and chemical signals that characterize Latteria raw milk cheese over 60 days of ripening time. The microbiota of both the control and Listeria-inoculated cheeses was analyzed using 16S rRNA targeted amplicon sequencing, while direct analysis in real time mass spectrometry (DART-HRMS) was applied to investigate the differences in the metabolic profiles of the cheeses. The diversity analysis showed the same microbial diversity trend in both the control cheese and the inoculated cheese, while the taxonomic analysis highlighted the most representative genera of bacteria in both the control and inoculated cheese: Lactobacillus and Streptococcus. On the other hand, the metabolic fingerprints revealed that the complex interactions between resident microbiota and L. innocua were governed by continuously changing chemical signals. Changes in the amounts of small organic acids, hydroxyl fatty acids, and antimicrobial compounds, including pyroglutamic acid, hydroxy-isocaproic acid, malic acid, phenyllactic acid, and lactic acid, were observed over time in the L. innocua-inoculated cheese. In cheese that was inoculated with L. innocua, Streptococcus was significantly correlated with the volatile compounds carboxylbenzaldheyde and cyclohexanecarboxylic acid, while Lactobacillus was positively correlated with some volatile and flavor compounds (cyclohexanecarboxylic acid, pyroxidal acid, aminobenzoic acid, and vanillic acid). Therefore, we determined the metabolic markers that characterize a raw milk cheese inoculated with L. innocua, the changes in these markers with the ripening time, and the positive correlation of flavor and volatile compounds with the resident microbiota. This multi-omics approach could suggest innovative food safety strategies based on the enhanced management of undesirable microorganisms by means of strain selection in raw matrices and the addition of specific antimicrobial metabolites to prevent the growth of undesirable microorganisms.

The study aimed to assess the seasonal variation in raw milk volatile organic compounds (VOCs) from three indoor feeding systems based on maize silage (n = 31), silages/hay (n = 19) or hay (n = 16). After headspace solid-phase microextraction (HS-SPME), VOC profiles were determined by gas chromatography (GC). Chemical and VOC (log₁₀ transformations of the peak areas) data were submitted to a two-way ANOVA to assess the feeding system (FS) and season (S) effects; an interactive principal component analysis (iPCA) was also performed. The interaction FS × S was never significant. The FS showed the highest (p < 0.05) protein and casein content for hay-milk samples, while it did not affect any VOCs. Winter milk had higher (p < 0.05) proportions of protein, casein, fat and some carboxylic acids, while summer milk was higher (p < 0.05) in urea and 2-pentanol and methyl aldehydes. The iPCA confirmed a seasonal spatial separation. Carboxylic acids might generate from incomplete esterification in the mammary gland and/or milk lipolytic activity, while aldehydes seemed to be correlated with endogenous lipid or amino acid oxidation and/or feed transfer. The outcomes suggested that VOCs could be an operative support to trace raw milk for further mild processing.

Nutritional information provided on food labels can impact healthy dietary decisions of consumers. The accuracy of the information provided is of paramount importance to guide consumers’ food choices and prevent food-related chronic diseases. The present study aimed to verify the veracity of nutritional labels of 103 food products purchased online through well-known e-commerce websites (80 processed and 23 unprocessed items) using near infrared spectroscopy. Among processed food products, surprisingly, 28 food products out of 80 (35%) did not bear nutritional labels. Considering the European tolerances for nutrient values declared on a label, the comparison of experimental values with those reported on the labels showed that more than 74% of the values declared on the label were congruent with the NIR experimental data, whereas 7.5% of the label values were non-compliant with the tolerance limits, and about 11.3% were slightly outside the tolerance limits. Note that 6.6% of the values indicated in the labels did not abide the regulation at all. Finally, 35.8% of the samples showed at least one value outside the tolerance limits. The current study demonstrated the capability of NIR spectroscopy for monitoring the compliance of nutritional labels with EU tolerance limits and guiding the choice of reference methods for further confirmation purposes.

This feasibility study reports the use of direct analysis in real-time high-resolution mass spectrometry (DART-HRMS) in profiling the powders from edible insects, as well as the potential for the identification of different insect species by classification modeling. The basis of this study is the revolution that has occurred in the field of analytical chemistry, with the improved capability of ambient mass spectrometry to authenticate food matrices. In this study, we applied DART-HRMS, coupled with mid-level data fusion and a learning method, to discriminate between Acheta domesticus (house cricket), Tenebrio molitor (yellow mealworm), Locusta migratoria (migratory locust), and Bombyx mori (silk moth). A distinct metabolic fingerprint was observed for each edible insect species, while the Bombyx mori fingerprint was characterized by highly abundant linolenic acid and quinic acid; palmitic and oleic acids are the statistically predominant fatty acids in black soldier fly (Hermetia illucens). Our chemometrics also revealed that the amino acid proline is a discriminant molecule in Tenebrio molitor, whereas palmitic and linoleic acids are the most informative molecular features of the house cricket (Acheta domesticus). Good separation between the four different insect species was achieved, and cross-validation gave 100% correct identification for all training samples. The performance of the random forest classifier was examined on a test set and produced excellent results, in terms of overall accuracy, sensitivity, and specificity. These results demonstrate the reliability of the DART-HRMS as a screening method in a future quality control scenario to detect complete substitution of insect powders.

Aims The efficacy of ambient mass spectrometry to identify and serotype Legionella pneumophila was assessed. To this aim, isolated waterborne colonies were submitted to a rapid extraction method and analysed by direct analysis in real time mass spectrometry (DART-HRMS). Methods and Results The DART-HRMS profiles, coupled with partial least squares discriminant analysis (PLS-DA), were first evaluated for their ability to differentiate Legionella spp. from other bacteria. The resultant classification model achieved an accuracy of 98.1% on validation. Capitalising on these encouraging results, DART-HRMS profiling was explored as an alternative approach for the identification of L. pneumophila sg. 1, L. pneumophila sg. 2-15 and L. non-pneumophila; therefore, a different PLS-DA classifier was built. When tested on a validation set, this second classifier reached an overall accuracy of 95.93%. It identified the harmful L. pneumophila sg. 1 with an impressive specificity (100%) and slightly lower sensitivity (91.7%), and similar performances were reached in the classification of L. pneumophila sg. 2-15 and L. non-pneumophila. Conclusions The results of this study show the DART-HMRS method has good accuracy and it is an effective method for Legionella serogroup profiling. Significance and Impact of Study These preliminary findings could open a new avenue for the rapid identification and quick epidemiologic tracing of L. pneumophila, with a consequent improvement to risk assessment.

Background: Bronchial thermoplasty (BT) is a new therapeutic option for severe refractory asthma not controlled despite high dose inhaled corticosteroids plus long-acting bronchodilators and omalizumab in selected cases. Risk of pulmonary atelectasis after BT in severe asthma has been described in literature, but no details have been reported on the possible mechanisms of the complication. Case presentation: A 49-year-old male with severe uncontrolled asthma was referred to BT. One hour after the first procedure, acute respiratory failure occurred with PaO2/FiO2 < 300. A CT scan showed atelectasis of the right lower and middle lobes. A new bronchoscopy was performed under non-invasive ventilation; the right lower and middle lobe bronchus were occluded by bronchus-shaped plugs, that were very difficult to remove despite repeated saline washings and fragmentation with forceps. The patient had a rapid resolution of respiratory failure. Four weeks later, 6 hours after the second session of BT, severe bronchospasm occurred with respiratory failure. Chest X-Ray showed atelectasis of the left lower lobe, prompting to perform a new flexible bronchoscopy on non-invasive ventilation. The exam showed again a plug occluding the left lower lobar bronchus, removed with forceps and washings. The histological analysis of the plugs demonstrated the massive presence of fibrin with mucus debris, rare Charcot-Leyden crystals, scattered macrophages, neutrophils, eosinophils and bronchial epithelial cells. Conclusion: The originality of our case report is related to the recurrence of bronchial plugging with lobar atelectasis within one and five hours respectively, after two sequential BT procedures. At the histological evaluation the bronchial plugs appeared very different from the typical mucoid asthma plugs, being composed prevalently by fibrin. It can be hypothesized that intense thermal stimulation of the bronchial mucosa may represent a strong boost for inflammation in susceptible patients, with microvascular alteration induced directly by heat or through the release of mediators. Although in severe asthma a risk of atelectasis from the classical asthma mucoid plugs may be expected, the peculiarity of our case resides in the formation of fibrin plugs whose direct correlation with BT should be considered.