# Microbial Risk Assessment of Industrial Ice Cream Marketed in Italy
## 2 Materials and Methods
### Microbiological Analyses  
Samples were kept at room temperature for 10 min to ease the sampling step, paying particular attention to collecting a representative portion (including toppings and decorations) of each ice cream. From each sample, two aliquots of \(\sim\)25 g were aseptically sampled and each placed into a sterile stomacher bag.  
One aliquot was diluted with buffered peptone water (Biolife, Milano, Italy) in a ratio of 1:9 \(w/v\) and homogenized through a stomacher (400 Circulator; International PBI s.p.a., Milano, Italy) for 60 s at 230 rpm. Samples thus prepared were processed for the following bacteriological determinations: (i) enumeration of the aerobic colonies at 30 \({}^{\circ}\)C according to ISO 4833-1:2013 [20], on plates of Plate Count Agar (Biolife, Milano, Italy) incubated at 30 \(\pm\) 1 \({}^{\circ}\)C for 72 \(\pm\) 3 h; (ii) enumeration of _Enterobacteriaceae_, according to ISO 21528-2:2017 [21], on plates of Violet Red Bile Glucose Agar (Biolife, Milano, Italy) incubated at 37 \(\pm\) 1 \({}^{\circ}\)C for 24 \(\pm\) 2 h; (iii) enumeration of coagulase-positive staphylococci according to ISO 6888-1:2018 [22], on plates of Baird Parker Agar Base (Biolife, Milano, Italy) supplemented with Egg Yolk Tellurite Emulsion (Biolife, Milano, Italy) incubated at 37 \(\pm\) 1 \({}^{\circ}\)C for 48 h; (iv) detection of _Salmonella_ spp. according to ISO 6579-1:2017 [23], with enrichment in Mueller Kauffmann Tetrathionate Broth base (Biolife, Milano, Italy) and Rappaport Vassiliadis Soy Broth (Biolife, Milano, Italy), followed by a smear on plates of Chromogenic Salmonella Agar (Biolife, Milano, Italy) and Xylose Lysine Deoxycholate Agar (Biolife, Milano, Italy), both incubated at 37 \(\pm\) 1 \({}^{\circ}\)C for 24 \(\pm\) 3 h; (v) enumeration of _L. monocytogenes_ according to ISO 11290-2:2017 [24] on plates of Agar Listeria according to [PERSON] (Biolife, Milano, Italy) and Listeria Palcam Agar (Biolife, Milano, Italy), both incubated at 37 \(\pm\) 1 \({}^{\circ}\)C for 48 \(\pm\) 2 h.  
The other aliquot was diluted with Fraser Broth Base (Biolife, Milano, Italy) in a ratio of 1:9 \(w/v\) and homogenized through a stomacher (400 Circulator; International PBI s.p.a., Milano, Italy) for 60 s at 230 rpm. Samples thus obtained were processed for the detection of _L. monocytogenes_, according to ISO 11290-1:2017 [25], on plates of Agar Listeria according to Ottaviani and Agosti and Listeria Palcam Agar, both incubated at 37 \(\pm\) 1 \({}^{\circ}\)C for 48 \(\pm\) 2 h.  
The identification of all the isolated _Listeria monocytogenes_ strains was further confirmed with Matrix-Assisted Laser Desorption/Ionization-Time of Flight (MALDI-TOF) Mass Spectrometry (MS) [26]. A Vitek Mass Spectrometer Axima Assurance (bioMerieux, Firenze, Italy) was used with the following settings: positive linear mode, laser frequency of 50 Hz, acceleration voltage of 20 kV and extraction delay time of 200 ns. The mass spectra range was set to detect from 2000 to 20,000 Da. MALDI-TOF MS generated unique mass spectra for each tested colony, which were transferred into the SARAMIS software (Spectral Archive and Microbial Identification System, database version V4.12, software year 2013, bioRxiv, Firenze, Italy) and compared to the database of reference bacteria spectra and super spectra, obtaining identification at the genus and species levels. Only a match of at least 70% was considered reliable.