The objective of this study was to investigate <em>Clostridium botulinum</em> growth and toxin production in the industrially manufactured Italian Parma ham. The study focuses on the Parma ham production phase identified as maximum risk to <em>C. botulinum</em> proliferation, <em>i.e.</em> the transition from cold phase (salting and resting) to a phase carried out at temperature between 15 and 23°C (drying). A preliminary in vitro test was carried out in order to verify the capability of 6 <em>C. botulinum</em> strains (1 type A, 4 type B, and 1 type E strains) to grow in conditions of temperature, pH and NaCl concentration comparable to those of the beginning stage of ham drying. Five <em>C. botulinum</em> strains grew at 20°C and pH 6, four strains produced toxin when inoculated at a concentration equal to 10<sup>3</sup> cfu/mL at NaCl concentration of 4%, while when the inoculum concentration was 10 cfu/mL, NaCl concentration of 3% resulted the toxin-genesis limiting factor. An experimental contamination with a mixture of the 5 <em>C. botulinum</em> strains selected by the preliminary in vitro test was performed on 9 thighs inoculated at the end of the resting phase. The study was designed to evaluate the potential growth and toxin production in extremely favourable conditions for the bacterium. Type B proteolytic <em>C. botulinum</em> toxin was produced after 14 days of incubation at 20°C in 2 thighs characterised by high weight, low number of days of resting and anomalous physiochemical characteristics [one for very low NaCl concentration (1.59%), the other for elevated pH (6.27) and both for high water activity values (>0.970)]. The results of this research confirm that the cold resting step is a critical phase in the production process of Parma ham for the investigated hazard. Based on the present study, the long resting phase adopted in the manufacturing of Parma ham is proven effective to prevent the growth of <em>C. botulinum</em>, an event which could not otherwise be excluded if the hams were processed under less stringent technological conditions.
Embryonic Stem Cell-Derived Neurons Grown on Multi-Electrode Arrays as a Novel In vitro Bioassay for the Detection of Clostridium botulinum Neurotoxins