**3. Results**

#### *3.1. Fatty Acid Profiles*

The fatty acid contents of the land snail samples examined are shown in Table 1. Seventeen FAs were found in all of the species examined: Six saturated fatty acids (C14:0, C16:0, C17:0, C18:0, C20:0, C22:0), six monounsaturated (C14:1, C16:1, C17:1, C20:1, C18:1Ñ:9), and six polyunsaturated (C18:2Ñ:6, C18:3Ñ:3, C20:2, C20:4, C20:5, C22:6). Erucic acid (C22:1) was found only in raw *T. pisana* samples.

**Table 1.** Fatty acids contents (mean ± SD; g/100 g FA) in fat extracted from the land snails species examined (*<sup>n</sup>*=3 replicates of each pool of samples). SFA =Saturated fatty acids, MUFA = monounsatured fatty acids, PUFA = polyunsaturated fatty acids.


#### *3.2. Fatty Acids of Raw Samples*

The polyunsaturated fatty acid (PUFA) content of raw *T. pisana*, *C. aspersum,* and *E. vermiculata* was 48.10 g/100 g, 47.22 g/100 g, and 46.56 g/100 g, respectively, representing the most abundant class of fatty acids, followed by monounsaturated fatty acid (MUFA) in *T. pisana* and *E. vermiculata* (31.27 g/100 g and 27.86 g/100 g, respectively) and saturated fatty acid (SFA) in *C. aspersum* (26.97 g/100 g).

The main PUFA components were C18:2ω6 (18.78–22.15 g/100 g), C18:3ω3 (7.64–15.78 g/100 g), and C20:2 (3.98–5.29 g/100 g). Linoleic acid (C18:2 ω6) represents the most abundant PUFA showing a range between 7.64 and 15.78 g/100 g. A high level of eicosapentaenoic acid (C20:5) was determined in *T. pisana* samples (9.85 g/100 g).

TheMUFA profiles obtained for all the species examined consisted of C14:1 (0.23–0.53 g/100 g), C16:1 (0.32–0.5 g/100 g), C17:1 (0.52–1.01 g/100 g), C18:1ω:9 (23.79–28.83 g/100 g), and C20:1 (0.17–0.37 g/100 g). Oleic acid was the main component of all the samples examined, representing 25% of the total fatty acid content. Erucic acid was found only in raw *T. pisana* samples at low concentrations (0.52 g/100 g).

Among the SFA, palmitic acid (C16:0) was the most abundant in all of the samples examined (from 12.63 to 16.02 g/100 g), followed by stearic acid (5.41–7.66 g/100 g). The SFA profiles in all of the species consisted of C14:0 (ranging from 0.73 to 0.81 g/100 g), C16:0 (12.63–16.02 g/100 g), C17:0 (1.02–1.36 g/100 g), C18:0 (5.41–7.72 g/100 g), C20:0 (0.63–0.81 g/100 g), and C22:0 (0.31–0.64 g/100 g).

The raw *T. pisana* samples showed the highest ω3/ω6 ratio (0.58), followed by *E. vermiculata* (0.55) and *C. aspersum* (0.43).

#### *3.3. Fatty Acids after Heat Treatment*

After boiling at +100 ◦C, the FA profile of all of the species examined verified a decrease of PUFA content up to 8.2%. Differently from PUFA, a species-specific modification of MUFA and SFA contents was found.

In particular, *T. pisana* samples showed an increase of SFA content by 22.20%; only C22:0 verified a decrease after boiling (from 0.31 to 0.29 g/100 g).

The MUFA contents decreased by 2.49%. No erucic acid was found after boiling. The PUFA content decreased from 48.10 to 44.19 g/100 g (8.13%), with a significant reduction of C20:5 content (from 9.85 to 1.15 g/100 g).

Regarding *C. aspersum*, the SFA content decreased by 14.68%, showing a reduction of C20:0 from 0.71 to 0.39 g/100 g; only C17:0 showed an increase from 1.13 to 1.35 g/100 g.

The total MUFA content increased by 29%. Palmitoleic acid (C16:1) showed a significant increase from 0.32 to 1.35 g/100 g, followed by C14:1, C17:1, and C30:1.

The *E. vermiculata* samples verified a reduction of SFA content after boiling (8.72%), especially for arachidic acid (C20:0) (from 0.81 to 0.37 g/100 g), followed by C17:0 (1.36–1.04 g/100 g) and C14:0 (0.81–0.63 g/100 g). The behenic acid (C22:0) content increased by 25.81%. The MUFA content verified an increase from 27.86 to 32.24 g/100 g. Palmitoleic acid (C16:1) was the only MUFA that decreased, from 0.40 to 0.37 g/100 g. The heat treatment of the *E. vermiculata* samples determined a decrease of PUFA components of 4.61%. Eicosapentaenoic acid (C22:6) decreased significantly from 1.30 to 0.34 g/100 g. The MUFA fatty acids more compromised after boiling were palmitoleic acid (C16:1) for *E. vermiculata* and *T. pisana* samples and myristoleic acid (C14:1) for *C. aspersum*. Oleic acid (C18:1Ñ9) remained the main component of all the land snails species examined even after boiling. Moreover, no oleic acid content variation was found after boiling for *T. pisana* samples.

Among the PUFA group, eicosapentaenoic acid (C20:5) showed the highest decrease in *T. pisana* and *E. vermiculata* samples after the heat treatment, whereas docosahexaenoic acid (C22:6) decreased up to 70% in *C. aspersum*.

A decrease of the ω3/ω6 ratio was found only for the *T. pisana* samples, whereas the *E. vermiculata* and *C. aspersum* samples increased the ω3/ω6 ratio up to 35%. Finally, a reduction of the PUFA/SFA ratio was found only for *T. pisana* samples.

## *3.4. Multivariate Analysis*

Given the high number of fatty acids examined as variables, principal component analysis (PCA) was used to explore the dataset structure and to obtain more information on the variables that mainly influence sample similarities and differences after heat treatment. The PCA model calculated after Pareto scaling showed that the data group variation is visible in the first two principal components, accounting for 97.82% of total data variance. Figure 2, which reports the PC1 vs. PC2 score plot (Figure 2a), together with the corresponding loading plot, highlights that PC1 alone explains about 94% of data variance. This extremely high value, together with the fact that all the variables have positive loading values along PC1 (Figure 2b), reflect the high positive correlation among the most significant part of the considered variables, so PC1 describes the prevailing trend of the analyzed FAs for the samples examined.

**Figure 2.** PC1 vs. PC2 score (**a**) and loading (**b**) plots of FA contents of the land snail samples examined, according to the species and treatment (raw vs. boiled).

The score plot shows differences related both to species and to heat treatment. PC1 describes a decrease in the number of fatty acids for each species after boiling. The variation is more marked for the *T. pisana* samples, which in general show the highest amount of unsaturated fatty acids (UFAs). In fact, both raw and boiled *T. pisana* samples lie at positive values of PC1, whereas all *E. vermiculata* and *C. aspersum* samples lie at negative values of PC1; this is due to the fact that, notwithstanding the significant decrease of FA content, boiled *T. pisana* still has a UFA content higher than raw *E. vermiculata* and raw *C. aspersum*. These last two samples have essentially the same overall amount of FAs and boiling leads to a more marked decrease for *C. aspersum* than for *E. vermiculata*.

*T. pisana* shows an opposite trend compared to the two other species considering the position along PC2 of the samples before and after boiling. Recalling that the percentage of variance explained by PC2 (3.93%) is much lower than the percentage of variance explained by PC1 (93.89%), PC2 accounts for the differences between the different species in the variations of the FA compositions after boiling, beyond the overall decrease, explained by PC1.

These variations of the FA profile can be examined more in-depth by means of the corresponding loading plot reported in Figure 2b. In this plot, the variables that are far from the origin contributed to the systematic variability explained by the PCA model, whereas variables close to the origin (like, e.g., C17:1, C14:0, C20:2, C20:1, C22:6, etc.) did not show a systematic trend.

The significant variations after boiling for all of the three species examined are related to PUFA, MUFA, oleic acid, SFA, and linoleic acid, which show higher PC1 values. PC2 showed a different variation of the fatty acid contents between the *T. pisana* samples and the other two species. A more considerable variation of eicosapentaenoic (C20:5) acid, MUFA, and oleic acid was found for *T. pisana* samples, whereas *E. vermiculata* and *C. aspersum* showed a more significant variation of SFA, linoleic acid, palmitic acid, and linolenic acid.
