3.1. Caciocavallo Cheese Characteristic and Fatty Acid Profile
The chemical composition of Caciocavallo cheese is reported in
Table 3. Concisely, the range values determined on eighteen cheese samples were: DM 59.7–68.1%; protein 44.6–48.4%; fat 39.9–45.3%; ash 6.2–8.8%; NaCl 2.31–5.64%, respectively. These results showed similar values to those observed by other authors [
31,
32,
42].
Statistical analysis showed significant differences between the cheeses produced by farms for ash and NaCl. In particular, the NaCl content (% DM) was higher (
p < 0.001) in cheeses made by farms F (5.64) > B_org (5.15) > (4.76) than by farms C (2.70) > D (2.56) > A_org (2.81). As expected, a similar trend was registered for ash (% DM) showing higher values
p < 0.01), in products made in farms B_org (9.40) > (9.10) > (8.93) than C (7.13) > D (7.13) > A_org (6.43). Probably, although all the farms carried out the salting in saturated brine, the different times adopted influenced the NaCl absorptions by the cheeses. However, this result can be related to DM content, even if the latter was not significant. In fact, between individual farms, NaCl was higher in cheeses with a higher DM level, as also observed in previous studies [
42,
60], in contrast to what was found between the various seasons.
Table 4 shows the cheeses’ FA composition in relation to production season and farm. The cheeses were high in palmitic (C16:0) and oleic acids (OA, C18:1c9) and, secondarily, by myristic (C14:0) and stearic acids (C18:0), complying with other studies carried out on the same product [
31,
32]. FAs contents and profile on cheese samples were influenced mainly by season rather than farm.
Statistically, the farm influenced only the content in C15:0 (p < 0.01), in C18:2 n6 (linoleic acid, LA) (p < 0.05), in n6 PUFA (p < 0.05) and n6/n3 (p < 0.05). In particular, LA content was higher (p < 0.05) in farm D (2.14%) than in farms E (1.52%), C (1.49%) and B_org (1.36), and in farm F (1.93%) compared to farm B_org (1.36%). This determined an increase (p < 0.05) of n6 FAs in farms D (2.58%) and F (2.30%) compared to farms C (1.93%), E (1.89%) and B_org (1.84%). Although not significant, the higher n6 content in D and the lower n3 content resulted in a higher n6/n3 ratio (p < 0.05) in D (3.44) compared to A_org (1.77) and C (1.62).
Considering the season, most of the short and medium chain FA were significantly higher in cheeses made in winter and spring. In particular, butyric acid (C6: 0), propionic acid (C8: 0), capric acid (C10: 0) and lauric acid (C12: 0) were higher in spring (2.33%, 1.46%, 3.07% and 3.37% respectively) and in winter (2.28%, 1.39%, 2.80% and 3.05%, respectively) than in summer (1.19%, 1.08%, 2.15% and 2.37, respectively). As observed by Di Grigoli et al. [
42] in the same area, the low levels of these FAs in summer milk are attributable to the reduction of their de novo synthesis in the udder, related to the quantitative and qualitative deterioration of pastures, together with unbalanced feeding integrations provided to cows. Moreover, the inadequate diets should lead to a mobilisation of body fat reserves, resulting in the higher content of OA observed in cheeses produced in winter and summer (
p < 0.01), as also found by Chilliard et al. [
61].
In the Mediterranean environment, spring is typically characterised by high availability of forage in the pastures, reducing feeding integrations, especially when the farms raise autochthonous breeds. In spring, the increased intake of cows grazing fresh forage led to a significant increase, compared to other seasons, in C18:1t11 (trans vaccenic acid, TVA), other C18:1, C18:2c9t11 (rumenic acid, RA), other isomers of conjugated linoleic acid (CLA) and C18:3n3 (α-linolenic acid, ALA), as observed by other authors [
31,
42,
62].
The RA and ALA are very important for their positive effects on human health. The consumption of cheeses naturally enriched with RA-induced positive biochemical changes in atherosclerotic markers [
63], as well as the products containing CLA, determine a reduction of the endocannabinoid anandamide and LDL (cholesterol level) in plasma concentrations of hypercholesterolemic subjects [
64]. The ALA is a fatty acid present in high quantities in fresh forage and transferred in milk and cheese. However, ALA is in part biohydrogenated in the rumen, determining a TVA increase, that in the mammary gland is oxidated by the delta-9 desaturase to RA [
62,
65,
66,
67].
Furthermore, the greater amount of fresh grass, that was probably ingested by the animals in spring, has resulted in a higher content of C22:5 (docosapentaenoic acid, DPA) in cheeses (
p < 0.05), an important long-chain n3 FA which plays a role in reducing blood sugar [
68] and in the prevention and treatment of cardiovascular diseases [
69].
Thus, the greater availability of fresh forage in spring has also increased the content of polyunsaturated fatty acids (PUFA) and n3 PUFA in cheeses (
p < 0.01). On the other hand, the higher LA content of winter and summer cheeses led to an increase in the n6 PUFAs levels in these products. Consequently, the n6/n3 ratio was more favourable in cheeses made in spring than in the other seasons (
p < 0.01), as observed by Altomonte et al. [
70], and always lower than 5 (limit indicated by the FAO/WHO) [
71]. LA and ALA being the precursors of the n6 and n3 series of FAs, respectively, represent the simplest members of each PUFA family and are called essential fatty acids because the body is unable to synthesize them. Numerous studies report the related health benefits of n3 PUFAs and their effects on cardiovascular disease, diabetes, cancer, Alzheimer’s disease, and immune function [
72].
The PCA plot is shown in
Figure 1, reporting each selected variable on the main components with a vector of length proportional to its contribution.
The first two principal components accounted for 67.77% of the total variance, allowing for the discrimination of the cheeses for production season. At the same time, they determined a non-linear separation of the farms within the seasonal groupings. The first main component (39.06% of the total variance), with the contributions of DM, Protein, Ash and n6/n3, had a minor impact in discriminating cheeses based on the production season. Instead, the second principal component (28.71% of the total variance) was more responsible for the separation of cheeses based on the production season, mainly due to the contribution of OA, C16:0, TVA and, to a lesser extent, C18:0, C14:0, RA and C12:0.
3.2. PAH and PCB Congeners, Abundance and Possible Source
The concentration of 16 PAH congeners (
Table S1), 20 PCB congeners (
Table S2), and 9 PBDE congeners were investigated in eighteen cheese samples. PBDE results were for all samples per each congener below the limit of detection, so the data was not reported in the tables.
Referring to each congener of PAH (
Table S1), certain contaminations of naphthalene (range: 0.13–30.49 ng/g) > phenanthrene (range: 0.01–13.01 ng/g) > fluorene (range: 0.11–6.65 ng/g) > acenaphthene (range: 0.01–3.46 ng/g) > pyrene (range: 0.01–1.36 ng/g) > fluoranthene (range: 0.01–1.30 ng/g) and > anthracene (range: 0.01–0.66 ng/g) were found in cheese samples, and the highest concentrations were determined in winter. In general, the low molecular-weight ones (PAH
LMW) were greater than the high molecular-weight ones (PAH
HMW), most of them < LOD; indeed, the ratio between PAH
LMW and PAH
HMW was always greater than 1. The PAHs are generated from pyrolysis and due to incomplete combustion of organic matter [
22,
73] and humans and animals can be exposed to these contaminants through different routes [
74]. The principal ones are food, air, and, more generally, several environmental matrices. Humans are also exposed due to smoking. The PAHs are classified as genotoxic and possibly/probably carcinogenic to humans, and the benzo(a)pyrene (BaP) is the most studied, being classified as a human carcinogenic in Group 1 [
75]. According to the EU Scientific Committee on Food, the BaP and ∑4PAHs (benzo(a)pyrene + benzo(a)anthracene + chrysene + benzo(b)fluoranthene) can be used as a marker for carcinogenic PAHs in food [
48,
49], providing maximum levels only for certain foodstuff categories characterized by high PAH levels, for example, the smoked ones. Indeed, the food can be contaminated directly: by the PAH occurrence in air, soil and water, by industrial emission, and by home food preparation (e.g., heating, drying, smoking, grilling and roasting processes) [
74]. The animals can be exposed to PAHs mainly by the inhalation of particulate matter, intake of dietary food, and contact with any other materials contaminated by PAHs (such as soil, which could be more contaminated than fodder and daily ingested in a percentage from 1 to 30 % by grazing the pasture) [
7]. These pollutants are largely excreted in urine or faeces in a hydroxylated form, due to the metabolization process, but are also absorbed in the body [
76,
77]. After the animal exposure to PAHs, due to their lipophilicity, these can be accumulated in animal adipose tissue and milk relative to the complex mobilisation process of energetic animal resources during the lactation and the gestation periods [
76,
78] and, consequently, transferred in cheeses. In the present study, as described above, the low molecular-weight PAHs (PAH
LMW) in cheeses were greater than the high molecular-weight ones (PAH
HMW) for different reasons. The forage usually presents PAH
LMW > PAH
HMW [
77,
79,
80], and it was the feeding basis for the animals involved in the trial. Indeed, the PAH
LMW tend to adhere to the intracuticular wax of the plants differently than the PAH
HMW, which are found in the epicuticular wax and so more exposed to photodegradation and washing [
78,
81]. Moreover, the PAH
LMW can also be transported over long distances [
78,
82] in the gas form [
78], differently to PAH
HMW, that falls out near the emission site [
83], mainly in condensate form. Consequently, the PAH
HMW are generally observed in pasture forage and soil only when the contaminating source is powerful and consistent and the plants are very close to it [
79,
80]. This study did not investigate the contaminants in pasture and feed, but other authors found an abundant presence of specific PAH
LMW in grasses, such as naphthalene, phenanthrene, fluoranthene, and pyrene [
77,
79,
80], the same congeners found in the investigated cheeses. Few PAH
LMW detected in the present cheeses are transferred from feed and soil to milk at low levels (transfer rate from 0.5 to 8%) because most PAHs are probably bio-transformed and excreted by urines [
77,
78,
84,
85]. For dairy products, maximum levels of PAHs are not imposed, therefore the levels of PAHs found in this study were compared with those detected in previous investigations in unsmoked cheeses, showing a similar trend in the contamination of naphthalene > phenanthrene > fluorene > acenaphthene > pyrene > fluoranthene > and > anthracene, as well as the occurrence of heavy PAHs, most of them lower than LOD [
86,
87,
88].
Polybrominated diphenyl ethers (PBDEs), also investigated in this study, are organic chemicals used as flame retardants in numerous consumer products (for example, home electronics, textiles and items containing polyurethane foam, and so on). Humans can be exposed to PBDEs by different sources, inhalation of house dust, absorption by textiles, and diet. PBDE, due to their lipophilicity, could be bioaccumulated from the environment to different foodstuffs [
12,
13,
89]. In the present study, the cheeses showed PBDEs concentrations lower than the detection limit (0.02 ng/g for each congener), differently from those observed in other investigations carried out in Italy [
89] and worldwide [
90], reporting high concentration of PBDEs in dairy products.
Polychlorobiphenyls were other persistent organic pollutants investigated in the cheese samples. Two classes represent them, non-dioxins such as PCBs (ndl-PCB) and dioxin-like PCBs (dl-PCB), which may be distributed over hundreds of kilometres from any sources of emissions [
78]. In some cases, no differences in contamination were found between fresh forage collected near rural areas and those sampled in industrial sites [
91]. Similar to those observed for PAHs, the gas deposit concerns the most-volatile compounds, namely the least-chlorinated PCBs, while the least-volatile compounds are found mainly in the form of particulate deposit [
78], transferring to animal milk by ingestion of contaminated feed and soil. The PCBs are persistent and could be accumulated in livestock products, differently to PAHs that are largely metabolised [
2,
78]. In animal products, these contaminants can achieve different pollution levels, and dairy products represent a considerable portion of total dietary exposure, considering the human diet composition [
3]. The cheeses sampled in this study showed a presence of dl- PCBs 114 + 118 (range: 0.01–5.41 ng/g;
Table S2), being that PCB 118 is one of the principal congeners found in cow milk [
92,
93,
94], a raw product used to make Caciocavallo Palermitano cheese. A prevalence of ndl-PCB congeners (1.5 × 10
−1–2.34 × 10
1 ng/g) on all PCBs, calculated as a sum of PCB (0.23–24.97 ng/g), was found. Most of the dl-PCBs were lower than the detection limits, and were considered equal to ½ LOD in the human health risk assessment. In particular, PCB 52 (0.01–29.37 ng/g) and PCB 28 (0.20–5.22 ng/g) were found in high amounts in cheeses made in winter (
Table S2), followed by PCBs 138 > 153 > 180 (
Table S2), with a different prevalence of congeners, with respect to those usually observed in cow milk, represented by ndl-PCB 153, 138 and 180 [
92,
93,
94]. However, the prevalence of specific PCB congeners in milk and dairy products is observed [
94] and related to various factors. Different studies showed carry-over rates higher than 80% for dl-PCB in milk, and from 5 to 40% for PCB indicators (PCB28, PCB52, PCB101, PCB138, PCB153 and PCB180) [
2,
7,
78]. The PCBs profile in milk is different according to occurrence in the environment, due to levels of pollution [
95], but also due to the physiological animal states, in a close relationship between contaminant–animal, including the rich ruminants microflora [
3,
7,
96]. PCB 81 and especially PCB 77 seem to show poor transfer to milk, probably due to metabolism or poor absorption [
3,
7], and in the present investigation, they were in low concentrations in cheese (0.05 and 0.01 ng/g, respectively). EFSA [
11] reported that keeping the daily intake unchanged, the calculated transfer rate of PCBs to milk increases with the time exposure and is highest in steady-state conditions. Anyway, the PCBs content in milk is significantly affected not only by the specific carry-over rate of each congener but also by the lactation stages and, in general, by animal conditions related to the energy balance and, consequently, the fat mobilisation [
94]. In general, PCBs 28 and 52 are slowly concentrated in corporal deposits and are low-carried in milk. Their presence tends to increase during lactation, probably for the decrease of the more abundant other congeners that are high-carried and also excreted more rapidly [
94]. Moreover, malignant breast lesions can influence the prevalence of specific PCB congeners in milk, as observed for PCB 28 and 52, often associated with these pathologies [
96,
97].
The concentration of summary of 16 congeners PAH and Benzo(a)pyrene_TEQ (BaPy_TEQ), 20 congeners of PCB including the PCBndl, PCB_TEQ (referred to as tetrachloride-dibenzo-para-dioxin—TCDD) were detected in the cheeses (
Table S3).
The ΣPAHs ranged between 0.47 and 27.72 ng/g wet weight (ww), showing lower concentration than those found in unsmoked Caciocavallo (36.70–248.59 ng/g ww) produced in Campania (ITALY) [
98]. The low occurrence of the most toxic PAHs determined a BaP_TEQ ranging between 8.01 × 10
−3 and 3.50 × 10
−2 ng/g; values were also overestimated, considering that the PAHs concentrations lower than the LOD were considered ½ LOD. The BaPy_TEQ was the parameter used for the human health risk assessment.
The ΣPCB, Σndl-PCB and PCB_TEQ in cheese ranged between 0.23–24.97 ng/g, 1.5 × 10−1–2.34 × 101 ng/g, and 3.32 × 10−4–6.79 × 10−4 ng/g, respectively. The PCB_TEQ was also used for the human health risk assessment.
A better comparison of contaminant concentration among cheese samples and among farms and seasons was based on normalised values at fat grams contained in cheese (
Table S3), reported as ΣPAH*, ΣPCB*, Σndl-PCB* and PCB-TEQ*.
The ΣPAH* ranged between 1.76–105.81 ng/g_fat, and the sampled cheeses most contaminated by PAH were Cheese_63, followed by Cheese_64, both made in winter in two different non-organic farms (C and D, respectively). Indeed, the more contaminated cheese by ΣPAH* was found in farms C > D > F > E > B_org > A_org and produced in winter > spring > summer. These trends were shown in the box plots (
Figure 2 and
Figure 3) and as results of statistical analysis in
Table 5. These results are probably due to the influence of the farm management system (organic versus conventional) and the feeding quality. In particular, farms A_org and B_org were organic and integrated the pasture with hay and concentrate in all seasons, unlike the others adopting this management feeding only in winter. Moreover, among the non-organic farms, only farms D and F supplied the concentrate in the spring season, differently to what was observed in summer, where all non-organic farms gave concentrate to the animals. Due to these results, it can be supposed that the animals of organic farms were probably reared in better environmental conditions and fed in all seasons with concentrate and hay less polluted than those used in non-organic farms. These considerations seem to be supported by the different concentration of pollutants found in cheeses in winter > spring > summer, which is in line with the levels of integration adopted especially for non-organic farms, generally higher in winter than in the other seasons.
These considerations could also explain the similar results obtained for ∑PCB* detected on products made in farms F > E > C > D> A_org > B_org (
Figure 2;
Table 5), in highest amounts in winter, followed by spring and then summer (
Figure 3;
Table 5).
The ΣPCB* and Σndl-PCB* ranged between 0.85–98.60 and 0.53–91.81 ng/g_fat, respectively, showing a higher prevalence of Σndl-PCB* on all ΣPCB*. Σndl-PCB* concentrations were 58.30 and 91.81 ng/g_fat on Cheese_5 and Cheese_6, made in winter by the farms E and F, respectively. These values exceeded the maximum level of 40 ng/g_fat provided for Σndl-PCB* by Commission Regulation (EU) No 1259/2011 [
50].
Moreover, the range of PCB_TEQ* in these cheeses was higher than the sum of PCDD/Fs and dl-PCBs TEQ for “raw milk and dairy products, including butter fat” (lower bound mean: 0.73; upper bound mean: 0.88 pg/g fat) reported from EFSA [
11], as result of a European monitoring plan. These results are probably overestimated because most of the dl-PCB determinations were lower than LOD, and the TEQ calculation for the risk assessment was imposed equal to ½ LOD. The regulation EU No 1259/2011 [
50] also provided a limit for the sum of dioxins and dioxin-like PCBs (WHO-PCDD/F-PCB-TEQ*) equal to 5.5 pg/g_fat and, in this study, the PCB-TEQ* ranged between 1.30–2.39 pg/g_fat, representing the 24–43% of the maximum admissible concentration. In this sense, more investigations in cheese samples could be necessary to detect also the PCDD/Fs for a more appropriate comparison to the limits imposed by regulations.
3.3. Potential Human Health Risk Assessment for Cheese Ingestion
The potential human health risk assessment, calculated following Equations (5) and (7), based on BaP_TEQ, ∑ndl-PCB and PCB_TEQ contaminant concentrations detected in cheese, and referring to the INRAN [
52] database for the individual cheese intake by the Italian population across one year and during a lifetime, was reported in
Table 6.
The CR values for BaP_TEQ (CRPAH), ∑ndl-PCB (CRndl-PCB), and PCB_TEQ (CRdl-PCB_TEQ), ranged between 6.95 × 10−9–3.03 × 10−8, 1.88 × 10−5–3.84 × 10−5, and 1.13 × 10−7–1.76 × 10−5, respectively, showed no-risk (<1 × 10−6) for BaP_TEQ (PAH), and probably risk (>1 × 10−6 and <1 × 10−4) for both PCB_TEQ and ∑ndl-PCB.
The CRPAH showed values two-orders of magnitude less than the threshold of 1E-6, evidencing no potential risk by the ingestion of the sample cheeses.
CR∑ndl-PCB for the cheese samples named Cheese_9, Cheese_3, Cheese_4, Cheese_5, and Cheese_6, showed values greater than 1E-6 (1.31 × 10−6, 3.96 × 10−6, 3.39 × 10−6, 1.25 × 10−5, 1.76 × 10−5, respectively). Cheese_9 was made in the spring season by farm C, while the others in the winter season by farms C, D, E, and F, respectively.
The CRdl-PCB_TEQ calculated for all eighteen samples exceeded an order of magnitude with the threshold of 1 × 10−6 and ranged between 1.88 × 10−5 and 3.84 × 10−5. These results are overestimated and should be considered not worrying because most of the dl-PCB determinations were lower than the detection limit and were considered equal to ½ LOD in the human health risk assessment.
Otherwise, if the CR had been calculated only for consumption of cheeses with PCBs concentration greater than the detection limits, the CR would have ranged between 6.65 × 10
−7–1.88 × 10
−5, involving only nine cheeses: Cheese_5 (1.88 × 10
−5) > Cheese_13 (1.02 × 10
−5) > Cheese_6 (8.82 × 10
−6) > Cheese_8 (8.51 × 10
−6) > Cheese_3 (6.76 × 10
−6) > Cheese_7 (5.13 × 10
−6) > Cheese_14 (4.04 × 10
−6) > Cheese_4 (3.50 × 10
−6) > Cheese_12 (1.21 × 10
−6) (data not reported in
Table 6 and calculated only for the discussions).
The human health assessment for a “non-cancer risk” used as indicators the hazard quotient (HQ) and hazard index (HI), which were calculated following Equations (6) and (7). The HQs calculated for B(a)Py_TEQ (HQB(a)Py_TEQ), ∑ndl-PCB (HQndl-PCB), and PCB_TEQ (HQPCB_TEQ) in all sample cheeses, showed values less than 1 for all samples except for the Cheese_6, in which HQndl-PCB was slightly greater than 1 (1.03). These values evidence a non-cancer risk due to the ingestion of this kind of cheese.
Otherwise, the HI values (sum of HQ) evidence possible acute stress (HI > 1) only for the samples named Cheese_5 and Cheese_6 (1.58, 1.66, respectively) due to the contribution of each pollutant, mainly PCB. Indeed, these cheeses (5 and 6) were made in winter in farms E and F, respectively, registering a larger amount of PCBs (as the sum of ∑ndl-PCB and dl-PCB_TEQ) than the others.
The results on risk assessment suggest that a new and more sensible method should be developed to determine pollutants concentration in cheeses, in particular PCB, often below the LOD when detected by conventional ones.