Study on the Effect of Heavy Metal Contamination of Milk on the Coagulation Process

Overview

This controlled experiment quantified how lead, cadmium and copper distribute between the curd (casein-rich) and whey fractions when milk from five species is coagulated. Metals were added at two levels: the EU regulatory maximum level (ML) for milk, and ten times that level (10 x ML). Both enzymatic coagulation and acid coagulation at pH 4.6 were compared. All five raw milks had lead (Pb), cadmium (Cd) and Cu below the limit of quantification before spiking, confirming they served as valid blanks.

The key finding is that casein-rich curd retains the majority of all three metals under both coagulation methods and across all five species. This is mechanistically attributed to casein micelles carrying negatively charged phosphoserine clusters and carboxylate residues that act as coordination sites for divalent metal ions.

Key numbers

Curd retention of metals at the EU ML level (Table 2):

• Overall range at ML: curd retention 77.56% to 96.64% across all species and metals.
• At 10 x ML, curd retention decreased for all metals: Pb in donkey milk fell to 74.96% (acid) and 72.56% (enzymatic); buffalo Cd maintained the highest curd retention.
• Species ranking: buffalo and cow milk retained metals more strongly in curd than donkey milk. Donkey milk consistently showed lower retention, attributed to lower casein content and different micellar structure.
• Coagulation method (enzymatic vs acid) did not show a uniform directional effect; its influence was condition-dependent and statistically significant only in interaction with species and metal type.

Methods (brief)

Milk from each species was spiked with certified standard solutions of Pb(NO3)2, Cd(NO3)2 and Cu(NO3)2 added simultaneously to 50 mL aliquots, pH readjusted, then equilibrated 1 h before coagulation. Enzymatic coagulation used mesophilic starter and rennet at 32 degrees Celsius for 1 h; acid coagulation used 1% acetic acid to pH 4.6 at 32 degrees Celsius. Curd and whey were separated by centrifugation (4000 rpm, 20 min, 4 degrees Celsius) and digested by microwave-assisted acid digestion (HNO3/H2O2). Lead and cadmium were measured by graphite furnace AAS; copper by flame AAS. CRM: ERM-BD151 skimmed milk powder (Pb 0.207 mg/kg, Cd 0.106 mg/kg, Cu 5.00 mg/kg). Recovery 90-110%.

Evidence fitness

This is a controlled spiking experiment, not an occurrence study. The metal concentrations used correspond to the EU ML (Pb 0.020 mg/kg, Cd 0.010 mg/kg, Cu 2.0 mg/kg) and 10x ML. The findings describe metal behavior during processing rather than the contamination levels of casein as an ingredient. The practical implication for the casein page is mechanistic: casein-rich dairy fractions (curd, cheese, casein powder) tend to concentrate lead, cadmium and copper from milk above the level found in whey.

Related evidence

• Casein
• Milk and Dairy
• Lead
• Cadmium

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