## 3 Results and Discussion
Such a high content of CLA compounds (rumenic acid and the other CLA isomers) may reflect a high amount of CLA precursors in the milk and its presence can arise from dietary PUFAs biohydrogenation in the ewes' rumen and the \(\Delta\)9-desaturase action on _trans_ vaccine acid in the mammary gland [24], an FA present at high amounts in these Ch\({}_{\text{C}}\) cheeses. During storage, the CLA content remained stable (1.47 g FA/100 g fat at 15 months) (Figure 5). Clinical studies have associated CLA consumption with positive human health effects, such as anti-obesity (reduced body fat mass), reduced incidence of cardiovascular diseases (improvement of the blood lipid profile and a reduction in total cholesterol), and reduced immune disorders (enhanced levels of protective antibodies) [31]. Due to the high content of CLA verified in the cheeses, an Ag+HPLC separation of CLA isomers of Ch\({}_{\text{C}}\) and HPP P1 (the most intense pressure treatment studied) cheeses at 0 months of storage was performed (Figure 6). As observed from Figure 6, similar qualitative and quantitative CLA isomers profiles were registered, demonstrating that HPP had no direct effect on the CLA isomers profile. Studies performed on _Pecorino_ ewes' milk cheese (made with pasteurized milk) showed a similar relative FAs profile with higher percentages of saturated (SFAs = 68.0 % of total FAs) and monounsaturated fatty acids (MUFAs = 22.5 % of total FAs), and consequently, lower percentages of polyunsaturated fatty acids (PUFAs = 4.43 % of total FAs) [29]. Notably, in the present work, a higher relative amount of PUFAs in the EFAMEs fat fraction of _Serra da Estrela_ cheese was quantified relatively to other ewes' milk chees reported in the literature, such as the previously mentioned _Pecorino_, _Roquefort_[29], and _Serra da Estrela_ cheeses with incorporation of dried plants [26]. As previously discussed, the ingestion of PUFAs from animal sources such as cheese can reduce blood cholesterol and reduce the risk of cardiovascular diseases [31]. It is well known that the animals' diet plays a major role in modulating the fatty acids composition of ruminant milk [32]. The _Serra da Estrela_ ewe diet is mainly from pasture and fresh forages in extensive or semi-extensive grazing, but also based on preserved forage according to land availability and climatic conditions. Figure 5: Evolution of the mean composition of the main SFAs, MUFAs, and PUFAs (g FA/100 g of fat) found in the EFAMEs fraction in control (**A**) and HPP cheeses (**B**) 600 MPa/6 min—P1, (**C**) 450 MPa/6 min—P2, and (**D**) 450 MPa/9 min—P3 _Serra da Estrela_ cheeses, during refrigerated storage. Data are expressed as mean (\(n=2\)). Concerning the Ch\({}_{\rm C}\) cheese NEFAMEs fractions (Figure 4 and Table S3), the total content of FAs was 4001 mg FA/100 g fat, which increased to 5793 mg FA/100 g fat over 15 months of storage. The main FA corresponded, once again, to SFAs (3783 mg FA/100 g fat) with pelargonic (C9; 3007 mg FA/100 g fat), butyric (C4; 153.9 mg FA/100 g fat), capric (C10; 148.5 mg FA/100 g fat), and palmitic acids (C16; 118.6 mg FA/100 g fat) as the main NEFAMEs present (Figure 7). In terms of the MU NEFAs, the NEFAME fractions revealed a composition similar to that of EFAMEs fractions, with oleic (C18:1 9c, 108.7 mg FA/100 g fat) and _trans_ vaccine acids (C18:1 11f; 14.7 mg FA/100 g fat) quantified as the main MU NEFAs present. Among the PU NEFA fraction, linoleic (C18:2 9c,12c, 16.25 mg FA/100 g fat) and \(\alpha\)-linolenic acids (\(\alpha\)-C18:3 9c,12c,15c, 14.5 mg FA/100 g fat) were the main PU NEFAs present; these FAs are known as essential FAs since their biosynthesis is not possible in humans or animals. In general, this qualitative profile was maintained throughout storage.