**4. Discussion**

In the present study, Zn enrichment of ewes' diet did not induce significant changes on milk composition, and this finding is consistent with what has been previously reported in dairy cows [22,23] and dairy goats [8]. According to what was observed in milk, no variations were evidenced in the chemical composition of Pecorino cheese samples, both in relation to dietary treatment and ripening time. The analysis on milk showed instead the capacity of the experimental diet to markedly reduce the number of somatic cells, a datum already observed in dairy cows by Pechová et al. [24] who tested the effect of dietary Zn at a daily dose of 440 mg/animal, therefore by administering a Zn dose similar to that used in the present work but on animals with a much greater body weight. Such phenomenon was justified by assuming an increased Zn supply into the mammary gland with a consequent improvement of the immune response. A precise and exhaustive evaluation of the influence of this parameter on the quality of dairy products is not feasible, however, it is known that somatic cells contribute to proteolysis in milk and cheese because of their tendency to release proteolytic enzymes in the extracellular environment [25].

A finding deserving special attention concerns the Zn content, which is higher in EG milk samples, whereas no variations were highlighted in cheese. The Zn concentration in cheese could be directly related to the presence of caseins, with which the microelement interacts through a complex kinetic that develops in two phases: an initial rapid stage in which about the 70% of Zn interacts with polar amino acids of casein, and a significantly slower second stage in which the equilibrium is reached [26]. Since after the cheese-making both CG cheese and EG cheese showed similar protein concentrations, it could be supposed that in EG samples the Zn excess, not associated with caseins, may have been lost with serum after rennet breakage. In addition to this, it should be also noted that in this study Zn seems to show a moderate capacity for association with caseins, as can be proved by the reduced Zn concentration in cheeses as compared with milk (considering the casein concentration by cheesemaking).

The Zn enrichment of ewes' diet showed effective in inducing variations in the FA profile, both in milk and its derived cheese; in all EG samples, the amount of vaccenic acid (C18:1 *trans*11), rumenic acid (RA) and total PUFA significantly increased. With regard to vaccenic acid, the result is explainable, at least in part, by taking into account the study of Szczechowiak et al. [27], in which the increase in concentration of this compound was justified by the action of bioactive compounds taken through the diet which tend to slow down or totally inhibit the terminal steps of ruminal biohydrogenation, thus avoiding the formation of stearic acid (C18:0). The relevance of vaccenic acid is its role as substrate of the mammary gland stearoyl coenzyme A desaturase (SCD), an endoplasmic reticulum-bound enzyme which is responsible for the catalytic mechanism that gives origin to CLA [28–30]. In this study the RA concentration effectively increased in both milk and cheese obtained from the experimental group. A similar finding was recently reported by Ianni et al. [9] who tested a dietary zinc supplementation in lactating dairy cows. In that case, the phenomenon was partly justified by advancing the hypothesis

of a role of the Zn supplemented diet in promoting the catalytic function of site-2 protease (S2P), a Zn-dependent metalloprotease which contributes to the activation of the sterol response element binding protein (SREBP), a transcription factor responsible for the regulation of several genes encoding for SCD and other lipogenic factors.

RA has been indicated as a factor with a strong antioxidant function which is able to improve the mammary gland functionality by protecting the mammary epithelial cells from lipoperoxidation through the reduction of the reactive oxygen species [31]. For humans, the ruminant products represent the primary dietary source of CLA, which are credited of several health benefits; the most relevant examples concern the modulation of the immune system response [32], and their potential activity in slowing down the progression of di fferent pathological conditions, as in the case of atherosclerosis [33].

Both in milk and its derived cheese, the feeding strategy based on Zn enrichment also induced an overall increase of PUFA concentration, at the expense of SFA. As a direct consequence of this evidence, the atherogenic and thrombogenic indices decreased, testifying a noteworthy improvement of the health functionality of animal productions.

The oxidative damage in cheese was determined by evaluating the amount of thiobarbituric acid reactive substances. Zn has been reported to act as a free radical scavenger in biological systems, with the consequent inhibition of free radical lipid peroxidation [34]. Additionally, in this study, Zn appeared to provide antioxidant protection since at the end of the 90 days of ripening (T90) the TBA values, although rising in both experimental groups, were found to be significantly lower in cheese samples deriving from the dietary Zn supplementation.

The analysis performed with the purpose to characterize the volatile profile of fresh (T1) and ripened (T90) ewes' milk cheese, led to the identification of di fferent families of compounds, the most represented of which are those of free fatty acid (FFA) and ethyl esters. The biochemical mechanism behind the increased concentration of FFA could relate to the degradation of triglycerides by enzymes of both endogenous and microbial origin. Among FFA the hexanoic acid was present at higher concentrations in EG samples after 90 days of ripening. This compound can only originate from lipolysis [35], and the datum concerning its increase in concentration, should have a significant influence in the determination of cheese flavor, due to its association with strong odors, described as cheesy, rancid and sweaty. In addition to this, the increase of hexanoic acid could also derive from the lipolytic action on longer chain acids, decanoic and dodecanoic, which in fact undergo a marked reduction in the EG samples [36]. What has just been described would sugges<sup>t</sup> a condition characterized by an increase in lipolysis during the ripening period. Some authors explain this phenomenon with an increase in the autolysis of bacterial starters, with the consequent release in the extracellular environment of a wide range of enzymes capable to attack and degrade both the protein component and fatty acids, contributing to the development of the organoleptic properties in ripened cheeses [37]. The autolysis process is, in turn, mediated by peptidoglycan hydrolases, named autolysins, that in the presence of certain stimuli degrade the cellular envelope which separates the cytoplasmic compartment from the extracellular matrix [38,39].

The FFAs, in addition, to directly contribute to the cheese flavor, represent the substrate for the biosynthesis of other classes of compounds: ethyl esters, methyl ketones, secondary alcohols, aldehydes, and lactones [36]. With regard to esters, the EG samples analyzed at the end of the ripening period were characterized by higher concentrations of butanoic acid ethyl ester and hexanoic acid ethyl ester. Generally, this class of compounds is responsible for the supply of sweet, fruity, and floral notes in surface ripened cheese [40], and is considered to be decisive in defining the cheese flavor because of the low odor threshold. Among aldehydes, was instead evidenced a marked increase of hexanal after ripening in EG samples; this compound gives what is called "green grass-like" aroma, with lemon, herbal and slightly fruity notes. A strange behavior is associated to alcohols which mainly derive from FFAs catabolism; after the 90 days of ripening, these compounds are not well represented, and their concentration tends to decrease in the EG samples. Specifically, we found a significant reduction of 3-methylbutanol, which is generally found on surface-ripened cheese and is responsible

for alcoholic, fruity, grainy, and solvent-like notes [34]. No variations were evidenced for lactones and ketones, therefore further analysis could be necessary in order to better characterize the biochemical mechanisms that contribute to their biosynthesis.
