LEAD AND CADMIUM IN SOME MILK AND MILK-BASED MOZZARELLA CHEESES FROM APULIA, ITALY

Headnote

ABSTRACT

The content of lead and cadmium in milk produced near a rural area subjected to illegal dumping and the transfer of the two metals from milk to Mozzarella cheese were investigated. The concentrations found in milk (from 2.00 to 19. 17 for Pb and from 0.04 to 1.43 mg L^sup -1^ for Cd) did not exceed the maximum limits established by European Community regulations. The cheese-making process was responsible for the transfer of the metals into the cheese but the level of contamination differed, also depending on the dairy, the production technology and the extent of exogenous contamination. On the whole, the contribution that Mozzarella makes to the daily intake of lead and cadmium appears to be toxicologically negligible.

- Key words: cadmium, lead, milk, Mozzarella cheese -

INTRODUCTION

The ingestion of contaminated foodstuffs is one of the possible causes of chronic lead and cadmium poisoning. This type of food contamination is ascribed mainly to environmental pollution caused by uncontrolled human activities. For this reason the periodical monitoring of Pb and Cd levels is planned in the HACCP of food industries, in particular those involved in the production of widely consumed foodstuffs. Among these are milk and cheese, in which, fortunately, very low concentrations are usually detected (KOOPS and WESTERBEEK, 1983; BALDINI et al, 1988; LARSEN and RASMUSSEN, 1991; CABRERA et al, 1995; GARCIA et al., 1999; TRIPATHI et al, 1999; YUZBASI étal, 2003). Nevertheless, episodes of milk contamination have been reported, mainly in association with farms situated very close to highways (BHATIA and CHOUDHRI, 1996, SIMSEK et al, 2000), those that are subjected to environmental contamination from industrial waste, or due to feeding animals with contaminated foodstuffs (BAARS et al, 1990; OKADA et al, 1997).

Pb and Cd in milk are mainly associated with casein, and their distribution is not affected by the heating or freezing processes, with the exception of very strong treatments, such as desiccation (JENG et al, 1994; MATA et al, 1996; MORENO-ROJAS et al , 1999) . As regards the fate of these metals during cheesemaking, the information available in the literature is scarce and generally indicates that the metal concentration increase on going from milk to cheese. MATA et al. (1995) reported having recovered almost all the lead and cadmium that had been experimentally added to milk in the casein fraction obtained by enzymatic coagulation; ZURERA-COSANO et al (1994) reported an increased lead concentration in the curd of Manchego-type cheese when compared to milk; CONI et al (1996) reported that curdling resulted in an increased concentration of the two elements. The evaluation of the transfer of lead and cadmium from milk to cheese can be complicated because of the processing equipment used and the cheesemaking procedure (YUZBASI et al, 2003), which may considerably change in connection with the type of cheese that is produced. The problem is far from being clearly defined, and the tolerance levels in dairy products have not yet been established, with the exception of the lead concentration in raw matter which may be 20 mg/kg^sup -1^ (EUROPEAN UNION COMMISSION, 2001).

In the last decade increasing attention has been given to soil contamination caused by illegal dumping of toxic substances in isolated rural areas, where police surveillance is difficult. This is a particularly serious problem in the areas in which dairy cattle are reared. Episodes of soil contamination by heavy metals due to illegal dumping were recently discovered in a small area within a wide rural zone in Apulia, southern Italy. These episodes raised concerns about the safety of the milk products, which prompted this investigation of the Cd and Pb content in milk from farms located in this area, and about the fate of these metals during the manufacture of Mozzarella cheese.

MATERIALS AND METHODS

Assessment of milk contamination

Milk samples were collected from eighteen farms located in the territory under study: nine farms were in the vicinity of the contaminated zone (less than 10 km, Group A), and the remaining nine farms were far away (more than 50 km, Group B). The milk samples (about 2 L each) were collected directly from the storage tanks and transported in refrigerated bags to the lab, where they were immediately analyzed. Precautions were taken to minimize exogenous contamination.

Production of Mozzarella cheese

Six batches of bulk milk were used to produce Mozzarella cheese using two different technologies, direct acidification (DA) and traditional lactic fermentation (TL), at three different dairy plants: one artisanal (Ad), and the other two industrial (IdA and IdB). The manufacturing protocols and experimental design are reported in Schemes 1 and 2. Two replicates (500 kg each) were performed for each cheesemaking trial.

Lead and cadmium determination

by atomic absorption spectrometry (AAS)

Standard solutions of cadmium and lead were purchased from Sigma Aldrich (St. Louis, MO, USA), nitric acid for AAS analyses and hydrogen peroxide for the mineralization process were purchased from J.T. Backer (Phillipsburg, NJ, USA). The matrix modifier used in the analysis was obtained by dissolving 0. 104 g of Mg(NO)3. 6H2O (Sigma-Aldrich) and 1.21 g of NH4H2PO4 (FIuka, Buchs, SG, Switzerland) in a 100 mL flask with 2% HNO3 . All other reagents were of analytical grade. For the mineralization process, a CEM MDS 2100 (CEM, Matthews, NC, USA) was used. The milk samples (4 g) were accurately weighed in Advanced Composite Vessels (ACV), then 3 mL of 70% HNO3 (purity level: [Pb] < 0.05 ppb, [Cd] < 0.01 ppb) and 3 mL of 30% H2O2 were added. The same procedure was used for the mozzarella samples (1.5 g), but 5 mL of 30% H2O2 were used. Autosampler cups, plastic bottles and glassware were cleaned by soaking them in 1.40 mol L1 HNO3 for 24 h and rinsing five times with Milli-Q water. They were dried and stored in a class 100 laminar flow hood. The samples were then subjected to microwave oven programs adjusted for the purposes of the study. The sample solutions were then diluted with water to a final volume of 20 mL, in order to reduce the HNO3 concentration and to prolong the life of the graphite tube. An AAS Shimadzu model AA6701 (Shimadzu Ltd., Kyoto, Japan), equipped with flame and graphite atomiser and autosampler with wizAArd software for automatic dilution was used. Background correction was carried out with a deuterium lamp. Hallow cathode lamps operating at 8 mA and a spectral band width of 0.5 nm were used to determine cadmium and lead at their primary resonances of 228.8 and 283.3 nm, respectively. Argon at 0. 1 mL min-1 was used as inert gas except during the atomization step, where the flow was stopped. Twenty mL were injected and pyrolytic graphite LVov platforms (Shimadzu) were used. In order to avoid matrix interference, 4 mL of Zeeman matrix modifier (Sigma Aldrich, Cotati, CA, USA) were added to each sample and standard injection.

Statistical analysis

Mean values and standard deviations were calculated and analysed with the JMP package program (S.A.S. Ins., Cary, NC, USA) for a oneway analysis of variance (ANOVA) with the Cd and Pb contents in the milk being the source of variance.

RESULTS AND CONCLUSIONS

Analytical characteristics

The calculation of the detection limits in the sample was carried out according to IUPAC rules (LONG and WINEFORDNER, 1983). Accuracy of the method was checked with recovery assays, by adding known amounts of Pb and Cd to five milk samples and processing the mixtures in the same way as indicated for the experimental samples. Precision was determined by 10 replicate determinations on each of five samples. The results are reported in Table 1.

Assessment of milk contamination

The concentration of lead was higher than that of cadmium in all of the milk samples, in agreement with the data reported in the literature (Table 2). On the average, the lead contents were slightly higher than those commonly found in cow milk (GARCIA et al, 1999; HERMANSEN et al, 2005), but all of the values were below the maximum limit established by EU regulations. The concentration levels were close to the tolerance level in only in two cases (sample 3 from Group A and sample 6 from Group B). The results clearly indicate that cadmium was always present in very low amounts. To date, a maximum limit for this element in milk has not yet been established by law, but the concentrations were similar to those reported in the literature (CABRERA et al, 1995; TOKUSOGLU et al, 2004).

The relationships between milk contamination and the geographical area of origin are reported in Figs. 1 and 2. The statistical analysis did not show any significant difference between the two groups of farms regarding the lead and cadmium levels which demonstrates an absence of a significant impact of soil contamination on milk quality. This finding confirms the results already obtained by other authors (SHARMA et al, 1982; VREMAN et al, 1986; OSKARSSON et al, 1992), who reported that only prolonged and acute contamination of soil and fodder can cause a significant increase in the concentration of heavy metals in milk. Moreover, in this geographical area farmers are resorting less and less to grazing and therefore the cattle have less exposure to this type of pollution.

Pb and Cd during the production

of Mozzarella cheese

As previously observed for milk from the individual farms, the lead content was greater than the cadmium in the six batches of milk used for cheesemaking (Table 3). The extent of the transfer of the metals from milk to cheese can be deduced from the data in Table 4, where the levels of Pb and Cd in the samples of Mozzarella and the corresponding "concentration factor" (cf, metal content in cheese/metal content in milk), are shown. The results confirm that the cheesemaking process has a concentration effect, but the extent of the transfer of the two metals was different. The cf for Cd ranged from 3.41 to 7.74, whereas that for Pb ranged from 2.43 to 35.26. The reasons for the wide variations can be inferred from the data of Table 5. First, the type of plant in which the cheesemaking trials were carried out had a great influence. The concentration factor in the artisanal dairy was always higher than those of the two industrial plants which were similar to each other, As regards the production technology, the lactic fermentation seems to have favoured the transfer of lead but, in the case of the artisanal dairy, the cf was very high and exceeded the Pb values found in the raw materials. This abnormal data can only be explained by some exogenous contamination; the source could not be ascertained in the present study and will be the objective of a future investigation. In view of this, it should be noted that traditional and hand-craft production technology involves a more complex series of operations than industrial technology based on direct acidification. Among these operations, the production and addition of the autochthonous lactic starter (the so-called "sieroinnesto"), the prolonged fermentation in open vats, curd-shredding and hand-stretching, involve the use of several tools and containers which could be a potential source of contamination. Finally, some comments about the contribution that mozzarella makes to the daily intake of the two metals should be made. The average per capita consumption of mozzarella in Italy is 5 kg per year (AMBROSI, 2002; AGRISOLE, 2005); this corresponds to about 100 g per week. In the area under study, these data are greatly underestimated, since mozzarella is consumed almost daily. Nevertheless, considering the Provisional Tolerable Intakes (PTI) of 50 mg kg-1 body weight per week for lead and of 7 mg kg-1 for cadmium (NOEL et al, 2003), the data from this investigation excludes any significant risk for human health. Even if the calculations are made from the highest contents observed (2 17.84 and 13.32 mg kg-1 for Pb and Cd, respectively), a person of average weight would have to eat more than 2040 kg Mozzarella per week in order to approach the PTI values, and much more in the case of the lowest concentrations.

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Paper received May 29, 2009 Accepted September 17, 2009