**1. Introduction**

Terrestrial gastropods, commonly named land snails, constitute a niche food product traditionally appreciated by many European countries, especially France and Italy. The use of land snails as food is still steadily growing, and 26,000 tons of snails were imported from Africa and countries in the Middle East [1]. *Cornu aspersum*, *Eobania vermiculata,* and *Theba pisana* are the land snail species most consumed in Italy [2]. Land snails are consumed in different ways all over the world, but the principal cooking procedures recognized are roasting and boiling, according to the traditions of the countries. According to Milinsk et al. [3], there is a correlation between land snails' diet and their nutritional values. Recently, increasing attention was paid to the fatty acid composition, due to nutritional and health-related aspects [4–8]. However, few studies are available about the fatty acid (FA) profile in

land snails [3,9,10] and, as far as we know, no data have been reported regarding the fatty acid profile of *T. pisana*. Snails are commonly consumed in di fferent ways after boiling due to the risk posed by the possible presence of potentially pathogenic microorganisms [2]. The cooking temperature can influence the nutritional aspect of mollusks [11]. At present, there are too few studies about the influence of heat processing (such as boiling) on the nutritional composition of land snails.

In this context, the present work aimed at evaluating the fatty acids content of wild *C. apsersum*, *T. pisana,* and *E. vermiculata* samples collected in Sicily (Southern Italy). Furthermore, the e ffect of boiling on the fatty acid composition was evaluated to have a comprehensive nutritional evaluation of this product after processing.

#### **2. Materials and Methods**

#### *2.1. Reagents and Standards*

All chemicals, solvents, and reagents employed were of analytical grade (≥99.9%). Acetone, hexane, diatomaceous earth, sodium sulfide nonahydrate, methanol, and hydrochloric acid were purchased from Sigma-Aldrich (Amsterdam, Holland). All of the gas used for gas chromatography (GC) analysis was pure (≥99.9995%). Water used for the separation of fatty acid methyl esters (FAMEs) phase was bidistilled in Milli-Q ® Integral 5 (Merck KGaA, Darmstadt, Germany). FA standards were purchased from Sigma-Aldrich (Amsterdam, Holland). The 10,000 mg/<sup>L</sup> standards were prepared by diluting 100 mg of a pure standard solution with 10 ml of n-hexane. A mixture of FA standards was used for the identification of each peak.

#### *2.2. Sample Collection and Preparation*

A total of 128 samples of *C. aspersum*, 400 samples of *T. pisana,* and 162 samples of *E. vermiculata*, were collected from Palermo provinces (Sicily, Southern Italy) in 2018 during July for *C. aspersum*, August for *T. pisana*, and September for *E. vermiculata* to have the maximum assimilation e fficiency according to the literature [12–14]. The shell of the snail samples was removed and only the meat was considered for the chemical analysis. The meat of the snail samples was grouped into three pools according to the species, then homogenized by a vertical mixer B-400 (Büchi, Flawil, Switzerland) and stored at −10 ◦C for 24 h to prevent a decrease in fatty acid content during the storage period [15]. The FA content of each sample pool was determined both raw and after cooking at 100 ◦C with boiled water for 30 s. The entire procedure of analysis is shown in Figure 1.

**Figure 1.** Scheme of the cooking process of the land snails samples collected.

*2.3. Extraction of Fatty Acids and Gas Chromatography with a Flame Ionization Detector (GC-FID) Analysis*

An amount of 10 ± 0.1 g of each pool of samples was placed in a glass of polypropylene and mixed with diatomaceous earth (Sigma-Aldrich, Amsterdam, Holland). The mixture was transferred in an accelerated solvent extraction (ASE) ASE 200 cell (Thermo Fisher, Waltham, Massachusetts, USA). The ASE operating conditions were set up as follows: 20 mL of hexane/acetone, 70:30; extraction temperature 120 ◦C for 6 min with a pressure of 120 pound per square (PSI).

The extract was filtered (size 240 nm) and dehydrated in rotavapor (Büchi, Flawil, Switzerland) at +40 ◦C. For the preparation of FAME, 100 mg of the oil extracted was trans-esterified in a pyrex tube by using 2 mL of HCl/MeOH (2:98 v/v) to obtain the fatty acid methyl esters (FAMEs). The solution was mixed in a vortex for 1 min and put in the oven at 120 ◦C for 1 h. After cooling, 2 mL of bidistilled water and 1 mL of hexane were added, and the mixture was centrifuged at 300 rpm for 1 min. Approximately 1 mL of the upper n-hexane phase was transferred in a vial and injected in gas chromatography (GC) with a flame ionization detector (FID).

Each pool of samples was examined in triplicate by GC-FID analysis. The analysis was carried out by a Trace GC/ULTRA HP 5890 GC + 7673 A/S (Thermo Fisher, Waltham, Massachusetts, USA); a Famexax column (30m × 0.25 mm i.d. × 0.25 μm df) was used for the separation. A flame ionization detector (FID) and ChromQuest 4.2.1 software (Thermo Fisher Scientific, Waltham, Massachusetts, USA)were used for the qualification and quantification of the analytes. The injector port and the detector temperatures were 220 ◦C and 230 ◦C, respectively. The split ratio was 1:20. The flow rates of compressed air and hydrogen were 350 mL min−<sup>1</sup> and 35 mL min–1, respectively. The carrier gas was helium (1.5 mL min−1). The oven temperature was programmed at a rate of 6.0 ◦C min−<sup>1</sup> from 130 to 225 ◦C, held for 15 s.

Individual FAME was identified by comparison with the chromatographic behavior of authentic standards by the formula:

$$TR = TR\_{st} \pm 0.5\tag{1}$$

where *TR* is the determined retention time (min), and *TR st* is the retention time for each FA standard. The relative percentages of the fatty acids were also determined. Quantitation of individual FAs is thus based on the comparison of their peak areas (Ai), and the peak area of a suitable standard. The relative percentages of fatty acids (C) were determined by the formula:

$$C = \frac{A}{\sum A} 100\tag{2}$$

#### *2.4. Validation of the GC-FID Method*

The repeatability mean and standard deviation of the analytical procedure were all calculated according to Taverniers et al. [16]. Separated FA standards were used to calculate the mean retention times (RTs) in the FID detector. The precision of the quantitative method was checked by the repeatability test, based on ten series of experiments [17]. The area of each peak was measured and corrected manually. The relative percentage of the fatty acids was also determined by comparing their peak areas.

#### *2.5. Data Collection and Statistical Analysis*

The data were expressed as g/100g FA in fat extracted and grouped according to species and treatment (raw *T. pisana, C. aspersum,* and *E. vermiculata* and cooked *T. pisana*, *C. aspersum,* and *E. vermiculata*). The variation of fatty acids after heat treatment was calculated as follow:

$$Fa\_{\%} = 100 - \left(\frac{Fa\_b}{Fa\_{raw}}\right) 100\tag{3}$$

where *Fa*% is the variation (expressed as a percentage), *Fab* and *Faraw* are the fatty acid content in boiled and raw samples, respectively (expressed as mg/100 g).

Before calculating the principal component analysis (PCA) model, the erucic acid variable was removed from statistical analysis because its presence was found only in raw *T. pisana* samples. All of the variables were pre-treated by Pareto scaling [18,19], in order to have a compromise between highlighting the contribution of the most abundant analytes and keeping at the same time the information brought by the less abundant ones. The PCA model was calculated using the software PLS-Toolbox ver. 8.6 (Eigenvector Research Inc., Wenatchee, WA, USA), running in the MATLAB environment (ver. 9.3, The Mathworks Inc., Natick, MA, USA).
