**4. Discussion**

In this study, the concentrations of four healthy trace elements were determined in a large number of PR PDO samples with two different degrees of ripening, 24 and 40 months, taking into account different milk production seasons (summer and winter) and the percentage distribution of wheels produced among provinces of the geographical area of origin.

Trace and major elements are included in milk and cheese in a colloidal or aqueous phase depending on their type. The colloidal phase consists of proteins, caseins, organized as micelles, which during the coagulation process include the fat globules forming the curd. The aqueous phase consists of whey, which includes the soluble protein fraction, as well as monomers, small polymers, and the majority of the sugars. In the colloidal phase of milk, the casein micelles are made up of casein submicelles, cross-linked together by

calcium and phosphorus, in the form of colloidal calcium phosphate (CCP). Depending on the dominant phase, colloidal or soluble, in which major minerals and trace elements can be found in milk, these can wind up in either whey or curd during the cheesemaking process. During the cheesemaking process, in the case of coagulation via lactic acid, the micelles are destabilized by acidic conditions (pH values near the isoelectric point of casein) which cause the loss of saline components including Ca and P, leading to dissociation in submicelles, which are then regrouped due to hydrophobic interaction, but not in micellar form, with Ca and P solubilized in the aqueous phase. In the case of rennet coagulation, the micelles are destabilized by rennet enzymes that cut k-casein, leading to the aggregation of many different micelles thanks to hydrophobic bonds. In this case the micelle structure is maintained, with Ca, P, and many other minerals and trace elements present in the colloidal phase (CCP). The minerals not associated with CCP (i.e., Na, and K), are almost completely lost in the whey phase, unlike the CCP minerals, such as S, Mg, and Zn, that are largely retained in curd as structural components [22]. Different factors (pH, temperature, and salinity) characterize the different cheesemaking processes, and thus play a key role in the colloidal-soluble form equilibrium of minerals and trace elements. Lactic acid fermentation leads to a decrease in milk pH, which throws off the equilibrium of the soluble form, causing a progressive solubilization of the casein-bounded minerals, losing them into the whey [23].

On the other hand, the higher temperatures promote casein-bound mineral forms. The different conditions of different cheesemaking processes lead to a variation in the concentrations of minerals and trace elements across the different kinds of cheese, despite having the same ripening period [22]. According to the literature data, the concentration of minerals with a predominantly colloidal form decreases in semi-cooked cheeses, compared to uncooked cheeses, and to lactic acid coagulation cheeses [22].

PR coagulation involves both lactic acid and rennet coagulation, with the latter being predominant. Thus, PR should be characterized by a higher concentration of minerals and trace elements that are mainly found in the colloidal casein-bound form, such as Ca, P, Zn, Se, and Cu, compared to other types of cheese, due to the predominant rennet coagulation and the long ripening process.

In addition to the cheesemaking process, cheese ripening is an important factor that influences the chemical composition of cheese, being a complex process that involves physical, chemical, and microbiological modifications, including the diffusion of salt from external to inner parts and consequent aqueous phase loss, gradual lactose loss mainly through lactic bacteria fermentation, and lactic acid neutralization leading to a pH increase. In the case of PR cheese, lactose is fully processed within 12 h post-production [24]. During the cheese ripening, minerals are progressively concentrated into the colloidal phase, due to the loss of the aqueous phase and to pH changes [25]. In addition, the basic pH environment promotes the retention of minerals in the colloidal phase. The water content of a 12-month ripened PR is 30%, decreasing to 28% in a 40-month cheese [26].

The results of our investigation show statistically significant differences between the trace element mean concentrations in 40 month-ripened PR and in 24 month-ripened PR, registered for all elements. The milk's season of origin, however, does not influence the concentrations of the studied trace elements, with the exception of the concentration of Mn and Zn in 40 and 24 month-ripened PR, which were found to have lower concentrations in cheese obtained from winter milk. This result is due to several concurrent causes, especially humidity loss and pH changes.

At the end of 1990, Gambelli et al. [14] published a paper on minerals and trace minerals in Italian dairy products, giving a healthy connotation to trace elements for the first time. These were previously considered to be toxicological components, such as heavy metals. The research group used ion exchange liquid chromatography with suppressed conductivity for the determination of the major minerals (Na, K, Mg, and Ca) and instrumental neutron activation analysis for the determination of the trace elements (Co, Cr, Fe, Rb, Se, and Zn). The results identified two subgroups within cheeses, the

stirred curd group and the hard group, both being foods with high levels of nutritionally important trace elements (i.e., Se, Zn, Fe, and Co). In particular, the hard cheese analyzed, Grana Padano (1–2 years ripened cheese samples), yielded the following concentrations: Zn 4.50 + 0.00 mg/100 g, Se 10.00 + 1.03 μg/100 g, Cr 9.90 + 2.00 μg/100 g [13], in which the concentration of Zn is similar to that found in PR, while the concentrations of Se and Cr are lower in Grana Padano than in PR according to the results of the present study.

More recently, Manuelian et al. [4] published a paper regarding major and trace elements, fatty acid composition and cholesterol content of different types of PDO cheese, including PR. Mineral concentration was measured by inductively coupled plasma optical emission spectrometry. In PR ripened for a period ranging from 12 to 24 months, Zn concentration was 33.93 ± 2.30 mg/g, and Se concentration was 0.91 ± 0.13 μg/g [4]. This concentration of Zn is similar to that found in the present investigation, and so, our results are in line with those of Manuelian and colleagues. On the contrary, the concentration of Se found by Manuelian et al. is about four times higher than the one we found, and is reported by CREA Italian Council (Consiglio per la Ricerca in agricoltura e L'analisi dell' Economia Agraria—Council for Research in Agriculture and the Analysis of Agricultural Economics) [27].

The selenium concentration reported by Manuelian actually seems to be very high, as a portion of PR alone (50 g) would provide about 45 μg, satisfying more than 82% of the Dietary Reference Values for selenium. In any case, the concentration of Se found in PR in this investigation, compared to other long-ripened cheeses, could be ascribed to the specific product PDO regulation. It requires a rationing of dairy cows based on the use of fodder from the production area of PR, and at least 50% of the dry matter of said fodder must consist of hays. Interestingly, an agronomical and geological study published in 2007 has shown that the cheese production area corresponds to a one of the three geographically separate soils richer in selenium within the Italian peninsula (Figure 1) [28]. It is well established that the selenium content of soil affects the amounts of selenium in the plants that animals eat. Nevertheless, the selenium concentration in soil has a smaller effect on trace element levels in animal products than in plant-based foods, owing to the homeostatic mechanisms present in animals and their effect on the maintenance of selenium tissue concentrations.

**Figure 1.** PR production area and selenium content data in Italian soil (modified from Spadoni et al., 2007) [28].

At the level of the European Union, as previously reported, the Regulation (EC) 1924/2006 harmonizes the laws of the Member States concerning nutrition and health claims. Generally speaking, a detailed chemical characterization of a food might be a useful

approach for producers and consumers to communicate and understand the evidence-based health properties of a food.

Regarding the daily nutrient reference intakes values (NRVs) for adults, reported in annex 13 of the Regulation (EU) 1169/2011, a 30 g portion of 24-month PR could satisfy about 14% of the NRV for Se, and 11% for Cr. A 30 g portion of 40-month PR could satisfy more than 19% for both Se and Cr, making PR ripened for 40 months a confirmable source of Se and Cr, as it contains much more than the required 15% of the nutrient reference values per portion and thus easily per 100 g of food product. As far as Mn and Zn are concerned, long-aged PR contributes only a very small of the NRV for Mn. However, regarding Zn, our results showed a significant contribution towards NRV levels but only at the 100 g level, too much for a single portion consumption.

These findings allow claims to be made for PR for the health properties ascribed to the food sources of selenium and chromium, according to the Regulation (EU) 432/2012 concerning health claims (Figure 2).

**Figure 2.** Health claim for selenium and chromium, according to the Regulation (EU) 432/2012.

The most studied property of selenium is its ability to protect DNA, proteins, and lipids from oxidative damage, through its role as a cofactor for antioxidant enzymes, such as gluthation peroxidase [29]. Selenium plays a key role in both thyroid function, through the selenoproteins involved in deiodination of thyroid hormones, and in the immune system, being able to stimulate the proliferation of T cells, increase the activity of natural killer cells and the response to antigen stimulation. In addition, selenium is an important factor in spermatogenesis, as the selenoproteins of the sperm mithocondrial capsule exert structural and enzymatic functions being responsible for the motility and structural integrity of the sperm tail [30]. Finally, a deficiency in selenium, shown in patients receiving total parenteral nutrition lacking selenium, results in impairment of hair and nails, with the clinical manifestation of white nail beds, pseudoalbinism, alopecia and thin hair, which disappeared after the administration of selenium [31]. A correct intake of selenium is considered important for the maintenance of normal hair and nails.

If a correct selenium intake leads to the above reported health benefits, a selenium intake much higher than the NRV induces acute selenium toxicity, which can cause severe gastrointestinal and neurological symptoms, acute respiratory distress syndrome, myocardial infarction and, in rare cases, death, as has been observed in cases of misformulated over-the-counter products containing excessive amounts of selenium. In 2008, the US National Institute of Health (NHI) reported that 201 people experienced severe adverse reactions from taking a dietary supplement containing 200 times the labelled amount [32].

The fixed selenium upper intake level (UL) from food and supplements in the adult population is 400 μg. However, a recently published observational cohort study in a diabetes-free Italian population found that a daily quantity of Selenium equal or higher to 80 μg/day is positively associated with hospitalization for type 2 diabetes [33]. In this regard, as a selenium source, long-aged PR ripened for 40 months is a useful option for nutritionists looking to give the correct daily amount of this trace element to the general healthy population, without resorting to food supplements which remain the best option for selenium deficient subjects.

As far as chromium is concerned, this occurs in nature in the forms of trivalent and hexavalent chromium. This former form is found in various foods, including cheese, reaching higher concentrations in bivalve mollusks and Brazil nuts [19]. The latter form of chromium may be found in foods as a toxic contaminant released by the tools used in the production process (e.g., certain types of stainless steel) [34]. Trivalent chromium, taken with the diet, has positive health effects in the body [35]. Adverse reactions related to chromium deficiency have been highlighted in patients undergoing parenteral nutrition for extended periods without supplementation of this element, resulting in impaired glucose tolerance and an altered metabolism [36]. In particular, trivalent chromium is linked with an increase in insulin action and an improvement in glucose tolerance in type 2 diabetes [37]. In addition to the glucose metabolism, chromium also influences nitrogen and lipid metabolism, as it inhibits the hepatic enzyme HMG-CoA reductase and lowers LDL cholesterol levels [38]. Numerous investigations have demonstrated a relationship between the consumption of dairy products and a reduction in the occurrence of diabetes. This property of dairy products, especially PR, is generally ascribed to the protein content and the type of fats. Further studies should be conducted to identify the role of chromium in the protective effects of PR against diabetes and metabolic syndrome.

Regarding the safety of trivalent chromium, it is safe to the point that the Food and Nutrition Board of the USA National Academies of Sciences, Engineering, and Medicine has stated that no adverse effects have been linked to high intakes of chromium from food or supplements, and so it did not establish a UL for chromium [34]. In fact, no case of toxicity is recorded by the intake of trivalent chromium with food, and the only negative effects recorded are in isolated case reports of misformulated chromium supplements, which might cause weight loss, anemia, thrombocytopenia, liver dysfunction, renal failure, rhabdomyolysis, dermatitis, and hypoglycemia [39,40].
