**1. Introduction**

Parmigiano Reggiano cheese (PR), an Italian Protected Designation of Origin (PDO) product, is known worldwide due to its sensorial and nutritional characteristics. Furthermore, despite the product category to which it belongs, it is often associated with health, primarily due to its high protein (32.4 g/100 g), calcium (1155.0 mg/100 g), and phosphorus (691.0 mg/100 g) content, with a lower fat content (29.7 g/100 g) compared to other aged cheese, and a natural absence of lactose (less than 1.0 mg/100 g) [1].

According to the specifications currently in vigor, PR is a hard, cooked, slow-ripening cheese produced with raw partially skimmed milk, coming from cows whose diet is mainly composed of feed from its area of origin. The production area includes the territories of the provinces of Bologna to the left of the Reno River, Mantova to the right of the Po River, Modena, Parma, and Reggio Emilia. Moreover, the milk cannot be thermally treated, the use of additives is not allowed, and all milk introduced into the dairy must comply with the product specifications of PR. The ripening time must last for at least 12 months, starting from the molding of the cheese, with an average ripening of 24 months, potentially lasting up to 60 months and beyond [2].

**Citation:** Santarcangelo, C.; Baldi, A.; Ciampaglia, R.; Dacrema, M.; Di Minno, A.; Pizzamiglio, V.; Tenore, G.C.; Daglia, M. Long-Aged Parmigiano Reggiano PDO: Trace Element Determination Targeted to Health. *Foods* **2022**, *11*, 172. https:// doi.org/10.3390/foods11020172

Academic Editor: Fernando Barbosa Júnior

Received: 29 November 2021 Accepted: 23 December 2021 Published: 10 January 2022

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Typically, the composition of PR varies according to the microbiological and chemical composition of the milk, the cheesemaking technology applied, including the natural whey starter, ripening time, and the environmental conditions that arise during these processes [3]. The milk composition in turn depends mainly on species, breed, season, and animal diet [4].

The specific PR cheesemaking procedures and ripening processes, along with the characteristics of the milk due to its territory of origin, strongly determine the physical properties, the chemical composition, and, in turn, the health profile of the final product. In fact, these practices lead to a selective concentration of nutritional and bioactive components, which increase the health value of this product. Due to natural dehydration occurring during the ripening process, the protein and aminoacidic content increases, mineral concentrations change (e.g., the potassium and magnesium concentrations decrease and selenium increases), and lactose decreases in the early hours following the cheese making process and is no longer detectable at 12 months of ripening [3].

There are several research articles that address PR as a source of protein, vitamins, and minerals, especially calcium, with valuable nutritional properties [5–8]. Moreover, PR, as with other fully ripened cheeses, contains other health-related nutrients, such as fats, and minor components including bioactive peptides. The fat fraction of PR contains butyric acid (123.9 mg per 100 g of fat in the outer part of the wheel at 24 months of ripening) [9], which exerts beneficial effects in obesity, inflammation, and neurological disorders, and conjugated linoleic acid (CLA—0.26 g/100 g), which has shown several beneficial activities on cardiocirculatory and immune systems [4]. As far as bioactive peptides are concerned, they are generated by proteolysis, which takes place during the ripening process and in the digestion process in humans. Among the health properties of the bioactive peptides, the inhibition of the angiotensin converting enzyme (ACE) is the most studied, along with its subsequent anti-hypertensive activity. In this regard, some studies have been published which, starting from the in vitro simulated gastrointestinal digestion of PR, demonstrate that different bioactive peptides (i.e., ACE-inhibitors and antimicrobial), are released and are absorbed in the intestine [10,11]. A recently published in silico study found an inhibitory activity of some PR cheese bioactive peptides against enzymes mainly involved in glucose metabolism, suggesting a potential effect on glycemic parameters [12].

As described above, there are investigations present in the literature that identify compounds with high healthy values in PR, but limited data are published on the concentrations of trace elements [4,13]. These elements are present in living tissues in small amounts and are known to solve essential functions for biological performance, primarily acting as cofactor catalysts in enzyme systems, as well as acting as centers for stabilizing structures of enzymes and proteins or binding molecules on the receptor sites of the cell membrane. Among these trace elements, selenium, zinc, and manganese are directly involved in the antioxidant enzymatic systems as cofactors for a number of enzymes [14]. In particular, selenium is a cofactor of 25 selenoproteins (including glutathione peroxidases, thioredoxin reductases, thioredoxin-glutathione reductase, iodothyronine deiodinases, and selenophosphate synthetase) [15,16], and zinc and manganese are cofactors of superoxide [17,18]. Chromium, as a trivalent ion, is an essential trace element, although no symptoms of chromium deficiency have been reported [19,20]. The low-molecular-weight of chromium-binding substance (LMWCr) has been proposed to be the biologically active form of chromium, being able to activate the kinase activity of insulin receptors in a dose dependent manner and increase insulin sensitivity [21].

According to the current European regulations, at present it is only possible to claim health properties of PR based on proteins, calcium, and phosphorus content, due to the limited available data on its chemical composition. Regulation (EC) 1924/2006 provides harmonized legal standards across Member States concerning nutrition and health claims, to guarantee the effective functioning of the market and a high level of consumer protection. It applies to all foods, including cheese. The term "nutrition claim" means "any indication that states, suggests, or implies that a food possesses beneficial nutritional properties due

to the caloric value it provides or does not provide, or to the nutrients or other substances it contains or does not contain". In addition to nutrition claims, this regulation also allows for health claims, as defined by Article 13 (general function claim) and Article 14 (reduction of disease risk claim). Specifically, a "health claim" is "any indication that affirms, suggests, or implies the existence of a relationship between a category of food, a food or one of its components and health, while, a "reduction of disease risk claim" defines claims relating to the reduction of a disease risk as any health claim that states, suggests or implies that the consumption of a food category, food or one of its constituents significantly reduces a risk factor of development of a human disease".

The Regulation (EU) 432/2012 contains a list of health claims permitted for food products, which includes all 222 functional claims currently approved for description of the health properties of a food.

Considering the widespread consumption of PR, especially in Europe, the aim of this investigation is to determine the chromium, selenium, zinc, and manganese concentrations of 24-month and 40-month ripened PR, to improve the knowledge of the concentrations of these compounds and thus of the biological properties associated with the trace element content, allowing the communication of PR healthy properties to European consumers, according to Regulations 1924/2006 and 432/2012.

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

#### *2.1. Sampling and Treatment*

Cheese samples were provided by Consorzio del Formaggio Parmigiano Reggiano. A total of 100 samples of cheese (1 kg each) were randomly taken from dairies, with a proportional and representative number of samples taken from each of the 5 provinces, reflecting their proportion of the total Parmigiano Reggiano cheese wheel production. Samples were distributed according to milk production season: 25 samples of cheese aged for 24 months obtained from summer milk, 25 samples of cheese aged for 24 months obtained from winter milk, 25 samples of cheese aged for 40 months obtained from summer milk and 25 samples of cheese aged for 40 months obtained from winter milk (see detailed samples description in Supplementary Materials n. 1). Samples were chopped with a mixer (DJ3001 Moulinette Compact, Moulinex, Milano, Italy), collected in plastic tubes, and stored at −20 ◦C until analysis.
