The Koyal Group Journals, Milk fraud: Proteins uncover adulteration with cheaper milk

Mixing milk

Food adulteration is an unsavoury fact of modern life. Many foodstuffs are targeted by criminal gangs because of their high value, so mixing them with cheaper alternatives while selling at the price of the pure material leads to higher profits. Pursue this tactic at the national or international scale and there is good money to be made.

One of the more surprising foods that have been targeted is milk. Not the common cows’ milk that we drink in the Western world, but milk from more exotic species likes the yak, buffalo or camel. These types have been mixed with milk from the cow or goat, and goat milk itself has been adulterated with cheaper cow milk. It is difficult to see or taste the difference when milk has been adulterated but scientific methods are available.

In particular, NMR and IR spectroscopy, mass spectrometry, capillary electrophoresis, ELISA and the polymerase chain reaction have been successfully employed. A further technique is 2D gel electrophoresis (2-DE) which has been reported recently for the characterisation of mammalian milk but there has been little work to study the differences in the 2-DE maps of different species.

This situation has now been rectified by Chinese researchers who have examined five types of milk. Jiaqi Wang and colleagues from the Institute of Animal Science at the Chinese Academy of Agricultural Sciences, Beijing, and the Institute of Animal Science and Veterinary Medicine at Anhui Academy of Agricultural Sciences, Hefei, used 2-DE backed up by mass spectrometry to help identify milk adulteration.

Well gel

Milk was collected from buffalos, yaks, camels, goats and cows at various farms in China. Samples of the same species were pooled to provide uniformity and the skimmed milk was prepared for analysis. Samples from one species alone or with up to 5% adulteration by one other type of milk were prepared.

All of the samples were subjected to 2-DE with isoelectric focussing over pH 4-7 followed by separation on polyacrylamide gels. This process separated the proteins across the gel in two dimensions and they were visualised by staining the gels with the dye Coomassie Brilliant Blue G-250. The intensities of the dye on the respective spots are a measure of the amount of protein in that spot.

Each pure milk was subjected three times to 2-DE and master gel maps for each species were created by comparing the 2-DE maps. Then, the researchers looked for proteins that were present in the milk mixtures but absent from the pure milks as a way to detect adulteration and these spots were identified by mass spectrometry.

Animal species identified

At first glance, the protein maps of cow, yak and buffalo milk were similar but those of camel and goat were different, as might be expected. Those differences not only allowed modified mixtures to be detected, but also revealed which type of milk had been added. They key proteins were β-lactoglobulin, α-lactalbumin and αS1-casein. 

For instance, β-lactoglobulin and α-lactalbumin from cow or goat milk mark the adulteration of camel milk. This is supported by the detection of the αS1-caseins from the cow and the goat, which have different molecular masses and isoelectric points from that of the camel, so appear in different positions on the gels.

Yak milk adulteration by cow milk was highlighted by a β-lactoglobulin variant which is absent in the yak but present in cow milk. Similarly, the β-lactoglobulin and α-lactalbumin of goat milk revealed its presence in yak milk. Conversely, goat milk adulteration with yak milk was marked by the presence of yak β-lactoglobulin and α-lactalbumin.

These three proteins from each species were sufficient to unearth the adulteration of one milk by another, down to contamination levels of 0.5%. The gels are reproducible for each species, with the proteins appearing in the same positions so, in theory, it is not necessary to identify the protein spots for successful tests. However, it would be wise to confirm their identities before establishing a lab test.