[33]. For each batch, the viability was expressed as the percentage of
[33]. For every batch, the viability was expressed because the percentage of living animals around the whole batch. 2.2. Abundance of Vibrio spp. and Pathogenic Vibrios (WPs 1, two, and three) The abundance of Vibrio spp. was checked on thiosulfate-citrate-bile salts-sucrose (TCBS) agar (Oxoid, Milan, Italy) NaCl three (final) by the spread plate system at 20 C for 3 days, following an internal protocol [34]. The sample units (100 individuals) have been ready in accordance with a slight modification on the ISO 6887-3:2017/Amd.1:2020 strategy. Briefly, gastropods had been rinsed with sterilized seawater, then their shells were cut aseptically to receive 10 g from the entire physique. Each and every unit was added to 90 mL of saline remedy NaCl three and homogenized using a rotary blender at medium speed. From this first dilution, additional ten-fold serial dilutions have been prepared with saline option NaCl three . From each and every homogenate and its ten-fold serial dilutions, one hundred have been spread plated around the agar plates and incubated at 20 C. The outcomes had been expressed as log10 Colony Forming Units (CFU) g-1 . V. parahaemolyticus, V. cholerae and V. vulnificus were investigated using the homogenate of each sample unit, from which 100 had been spread plated on agar plate of CHROMagarTM Vibrio (CAV) (PBI International, Milan, Italy), then incubated at 37 C for 24 h. Suspected colonies (V. parahaemolyticus mauve, V. vulnificus/V. cholerae green-blue to turquoise blue, V. alginolyticus colorless) were submitted to biochemical screening and genotyped by Polymerase Chain Reaction (PCR). Briefly, soon after a biochemical screening to ascertain the characteristics in the genus (pleomorphic Gram-negative rods, oxidase-positive, Fmoc-Gly-Gly-OH Formula capable to lessen nitrate, glucose-fermenting, sensitive towards the vibriostatic O129/150), suspected strains had been genotyped by PCR as previously described [35] to test their respective species-specific and pathogenic gene markers. For V. parahaemolyticus toxR, tdh and trh; for V. cholerae toxR, ctx and stn/sto; for V. vulnificus only the species-specific gene marker vvhA, for the reason that, in line with the Food and Agricultural Organization on the United Nations and the World Health Organization [36], all V. vulnificus strains could be prudently deemed virulent. The oligonucleotide primers along with the PCR conditions are reported in Supplementary Materials Table S1. 2.three. Biogenic Amines (WP2) BAs had been quantified by an HPLC method employing a UV detector following derivatization by Dansyl chloride. Analyses have been performed by an external service (FoodMicroTeam s.r.l., Academic Spin-off of the University of Florence, Italy), and samples had been supplied frozen (-20 C). Contemplating the results for each and every batch, the sum of histamine, putrescine, cadaverine, and tyramine quantities have been calculated and expressed as BAI (Biogenic Amines Index) as outlined by -Irofulven Purity Veciana-Nogu et al. [37]. two.four. Abundance of IPB (WP3) IPB load was determined in T. mutabilis by Most Probable Quantity process, indicated by Leit and Rios [38] because the most dependable method for the quantitative evaluation on the indole constructive microflora. 3 series of 3 tubes containing five mL of tryptone broth 1 were ready. 1 series was inoculated with 0.5 mL with the homogenate (dilution 1:ten), and the remaining two with 0.five mL of two additional ten-fold serial dilutions each. Following incubation at 20 C for three days, Kovac’s reagent was added to every single tube. The reagent builds with indole a cherry-red complicated, permitting to confirm the enzymatic activity of tryp.