In vitro application of biocontrol agents to improve the safety and quality of grapevine products

Pathogenic fungi are responsible for vine diseases affecting the grapevine yield and the organoleptic quality of the final wine products. Using of biocontrol agents can represent a sustainable alternative to the use of synthetic fungicides whose intense use can have negative effects on the ecosystem and cause increase resistant pathogen population to synthetic agents. The principal aim of my PhD thesis was the isolation and characterization of new yeast strains and Bacillus subtilis SV108 as biocontrol agent and the comprehension of the mechanism of their antimicrobial action. Accordingly, twenty wild yeast and one selected bacterium isolated among 62 samples, isolated from different Italian and Malaysian regions and molecularly identified, were evaluated in a preliminary screening test on agar. Results showed the highest effects on inhibiting mycelial growth by Starmerella bacillaris FE08.05, Metschnikowia pulcherrima GP8 and Hanseniaspora uvarum GM19. On the other side, Bacillus subtilis SV108 showed the ability of inhibit the mycelial growth of selected fungi by producing antimicrobial compounds on Malt Extract Broth medium recovered by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and identified by electrospray ionization (ESI) tandem mass spectrometer Triple TOF 5600. Moreover, in order to analyze the volatile fraction of compounds, the quantitative analysis of the VOCs profiles was performed by GC/MS/SPME. The analysis highlighted the presence of isoamyl and phenylethyl alcohols and an overall higher presence of low-chain fatty acids and volatile ethyl esters. All the data collected suggest that the tested yeasts, found among the epiphytic microbiota associated with grape berries, can be potentially effective for the biological control of pathogenic moulds. On the other hand, the proteomic study conducted on B. subtilis SV108 revealed that there are two cyclic antifungal peptides which can explain the antimicrobial effect of Bacillus subtilis SV108 acting as biocontrol agent against fungal pathogens in grapevine.

Study of the urinary biomarkers of synthetic cannabinoid receptor agonists (SCRAs) for the forensic unequivocal proof of consumption: medico-legal implications for drug abuse prevention

Introduction. Synthetic cannabinoid receptor agonists (SCRAs) represent the widest group of New Psychoactive Substances (NPS) and, around 2021-2022, new compounds emerged on the market. The aims of the present research were to identify suitable urinary markers of Cumyl-CB-MEGACLONE, Cumyl-NB-MEGACLONE, Cumyl-NB-MINACA, 5F-EDMB-PICA, EDMB-PINACA and ADB-HEXINACA, to present data on their prevalence and to adapt the methodology from the University of Freiburg to the University of Bologna. Materials and methods. Human phase-I metabolites detected in 46 authentic urine samples were confirmed in vitro with pooled human liver microsomes (pHLM) assays, analyzed by liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-qToF-MS). Prevalence data were obtained from urines collected for abstinence control programs. The method to study SCRAs metabolism in use at the University of Freiburg was adapted to the local facilities, tested in vitro with 5F-EDMB-PICA and applied to the study of ADB-HEXINACA metabolism. Results. Metabolites built by mono, di- and tri-hydroxylation were recommended as specific urinary biomarkers to monitor the consumption of SCRAs bearing a cumyl moiety. Monohydroxylated and defluorinated metabolites were suitable proof of 5F-EDMB-PICA consumption. Products of monohydroxylation and amide or ester hydrolysis, coupled to monohydroxylation or ketone formation, were recognized as specific markers for EDMB-PINACA and ADB-HEXINACA. The LC-qToF-MS method was successfully adapted to the University of Bologna, as tested with 5F-EDMB-PICA in vitro metabolites. Prevalence data showed that 5F-EDMB-PINACA and EDMB-PINACA were more prevalent than ADB-HEXINACA, but for a limited period. Conclusion. Due to undetectability of parent compounds in urines and to shared metabolites among structurally related compounds, the identification of specific urinary biomarkers as unequivocal proofs of SCRAs consumption remains challenging for forensic laboratories. Urinary biomarkers are necessary to monitor SCRAs abuse and prevalence data could help in establishing tailored strategies to prevent their spreading, highlighting the role for legal medicine as a service to public health.