## 2 Materials and Methods
### Physico-Chemical and Microbiological Analyses  
For the determination of the physico-chemical analysis, the Bottarga samples were finely ground and analyzed for water activity (W\({}_{\text{a}}\)) using an electric hygrometer (Thermoconstanter model, Novasina, Germany). pH was measured by a pH meter (Thermo Fisher Scientific 0250A0, Milan, Italy, pH/mV/relative mV/temperature meter, model 250A). The AOAC methods [17] were used for the analysis of carbohydrates (method 974.06), total fats (method 948.15), and proteins (method 981.10). For the determination of sodium, calcium potassium, and phosphorus (method 985.01), the samples underwent a digestion process with a microwave system [18].  
Finally, cholesterol was determined according to [PERSON] et al. [19] using an enzymatic kit (Boehringer Mannheim/R-Biopharm, Milan, Italy).  
For the microbiological analysis, 25 g of bottarga sample was homogenized in a 225 mL sterile Ringer's solution for 3 min in a Stomacher Lab Blender 80 (PBI, Milan, Italy). Aliquots (1 mL) were 10-fold diluted in Ringer's solution and plated/inoculated on specific media used to quantify different microbial groups. Both mesophilic (MMC) and psychrophilic (PMC) microbial counts were enumerated on plate count agar (Oxoid, Milan, Italy) at 30 \({}^{\circ}\)C and 5 \({}^{\circ}\)C for 72 h, respectively; yeasts and molds were counted on YPDA that consisted of 1% \(w/v\) yeast extract, 2% \(w/v\) dextrose, 2% \(w/v\) peptone, 1.5% \(w/v\) agar (all ingredients were purchased from Oxoid), and with 100 mg mL\({}^{-1}\) of chloramphenicol (Merk, Darmstadt, Germany) at a pH of 4.5 after incubating at 25 \({}^{\circ}\)C for 48-96 h; enterobacteria in VRBGA (Oxoid) and subsequent incubation at 37 \({}^{\circ}\)C for 48 h; fecal coliforms were enumerated in brilliant-green bile broth (Oxoid) with Durham bell after 48 h of incubation at 44 \({}^{\circ}\)C; and presumptive \(E\). \(coli\) was counted by indole testing: a subculture (0.1 mL) of fecal coliforms with positive BGBB tubes was transferred to tryptone water to perform the  
Figure 2: Map of Sardinia Island with area in box where bottarga samples have been collected.  
indole production test with Kovac's reagent (Sigma-Aldrich, Darmstadt, Germany) after 37 \({}^{\circ}\)C for 24 h (MPN method).  
Staphylococci in Baird-Parker agar (Oxoid) supplemented with egg yolk tellurite emulsion (Oxoid) were incubated at 37 \({}^{\circ}\)C for 48 h; presumptive colonies of coagulse-positive staphylococci were assayed for coagulase activity using the staphylase test (Oxoid, Italy); spores of sulphite-reducing clostridia (MPN method) were placed in DRCM broth (Oxoid) after heat treatment (80 \({}^{\circ}\)C for 10 min) of the samples and incubated at 37 \({}^{\circ}\)C for 48 h in anaerobic conditions.  
The results of microbiological analysis were expressed as CFU/g or MPN/g. Three replicates (one sample from three different trays) and two repetitions of each analysis were carried out.  
The _Listeria_ spp. was determined, involving two pre-enrichment phases. Briefly, 25 g of sample was first diluted in 225 mL of Fraser Broth (Oxoid) and enriched with Fraser Supplement SR166M and placed to incubate at 30 \({}^{\circ}\)C for 21-24 (1 st enrichment). Subsequently, 1 mL of the primary enrichment was transferred into 10 mL of buffered listeria enrichment broth base (BLEBB) enriched with the SR141E supplement and incubated at 30 \({}^{\circ}\)C for 21-24 min (2 nd enrichment).  
In case of blackening of the broth culture, a smear is carried out on Palcam Agar (Microbiol, Cagliari, Italy) differential and selective media after incubation at 30 \({}^{\circ}\)C for 48-72 h. The results for presumptive _Listeria_ spp. were expressed as present (+)/absent (-) in 25 g.