**3. Results**

Five calibration curves were prepared using standard references of the selected elements. Table 1 reports the wavelength, slit width, limit of detection (LOD), limit of quantification (LOQ), the equation of the calibration curve, the linear range, and the regression coefficient for each element. The regression coefficients obtained were greater than 0.99, except for that calculated for determination of Zn, which was found to be higher than 0.94 (Table 1). The limits of detection (LOD) and the limits of quantification (LOQ) were respectively calculated to be three times and 10 times the signal of the blank.


**Table 1.** Analytical parameters of atomic absorption spectrometer analysis.

\* LOD, limit of detection; LOQ, limit of quantification; LR, linear range; R2 regression coefficient.

According to Italian LARN (Livelli di Assunzione di Riferimento di Nutrienti ed energia—Reference Intake Levels of Nutrients and Energy) a standard portion of ripened cheese is 50 g. Considering the high nutritional value of 40-month ripened PR, a portion could be quantified as 30 g. Therefore, Table 2 reports the results expressed as average value and standard deviation (SD) of the selected trace element concentrations in 100 g and in a portion of 50 g for 24-month ripened PR, and in a portion of 30 g for 40-month ripened PR.


**Table 2.** Concentration of trace elements in Parmigiano Reggiano cheese at different months of ripening, expressed per 100 g and per consuming portion (30 g and 50 g).

\* (n = 25, ±SD); different letters indicate statistically significant differences (*p* < 0.05) between four groups (24 summer: cheese aged 24 months obtained from summer milk; 40 summer: cheese aged 40 months, summer milk; 24 winter: cheese aged 24 months, winter milk; 40 winter: cheese aged 40 months, winter milk).

Average chromium concentration in cheese samples obtained with summer milk and aged 24 months was found to be 13.93 ± 4.80 μg/100 g, while in cheese samples obtained with winter milk aged 24 months, the concentration was 14.61 ± 3.44 μg/100 g. For samples aged 40 months, Cr was present at higher concentrations equal to 25.34 ± 2.59 μg/100 g for summer milk samples and 26.19 ± 4.12 μg/100 g for winter milk samples. Tukey's multiple comparisons test (Table 2) showed a statistically significant difference between samples aged 24 months and samples aged 40 months.

Average manganese concentration in cheese samples obtained with summer milk and aged 24 months resulted to be 15.96 ± 4.60 μg/100 g, while in cheese samples obtained with winter milk aged 24 months, the concentration was 7.64 ± 7.11 μg/100 g. For samples aged 40 months, Mn was present at higher concentrations equal to 40.24 ± 16.09 μg/100 g for summer milk samples and 24.96 ± 11.95 μg/100g for winter milk samples. Comparison tests showed a statistically significant difference between all groups (Table 2).

Average selenium concentration found in cheese aged 24 months from summer milk was 25.27 ± 5.82 μg/100 g, and from winter milk was 25.82 ± 2.97 μg/100 g. In cheese aged 40 months the Se concentration was equal to 37.34 ± 6.86 μg/100 g from summer milk samples, and 34.22 ± 4.26 μg/100 g from winter milk samples. Here too, the statistical analysis determined statistically significant differences between the samples aged at 24 months and samples aged at 40 months.

Average zinc concentration found in cheese aged 24 months made from summer milk was 3196 ± 1095 μg/100 g, and from winter milk it was 1752 ± 627 mg/100 g. Contrastingly, in cheese aged 40 months the Zn concentration was equal to 3952 ± 1330 μg/100 g in the summer milk samples, while it came to 2478 ± 772 μg/100 g from winter milk samples. Statistically significant differences were found in Zn concentration between groups: cheese aged 24 months, summer milk vs. cheese aged 40 months, summer milk; cheese aged 24 months, summer milk vs. cheese aged 24 months, winter milk; cheese aged 40 months, summer milk vs. cheese aged 24 months, winter milk; cheese aged 40 months, summer milk vs. cheese aged 40 months, winter milk. No statistical differences were found between the groups: cheese aged 24 months, summer milk vs. 40 months, winter milk; cheese aged 24 months, winter milk vs. cheese aged 40 months, winter milk. (Table 2).
