Milk sugars in adulteration tests

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  • Published: Nov 2, 2009
  • Author: Steve Down
  • Channels: HPLC
thumbnail image: Milk sugars in adulteration tests

Soy milk and soy protein were introduced a few years ago as high-protein nutritional alternatives to the animal proteins in bovine and ovine milk and in meat. They have been especially useful in countries where livestock is in relatively short supply and have also found a global market for vegetarians and people with bovine milk allergies.

Soy milk has also been added to bovine milk to stretch out supplies but this has not been extended into general practice because many national authorities are reluctant to legalise it. On the other side of the coin, unscrupulous producers of milk and related products such as cheese and infant formula have diluted bovine milk with soy milk. Their motivation is purely financial, since soy milk is cheaper to come by.

Detection of these practices requires a simple test, which could be used in high throughput mode in regions where milk adulteration abounds. Current methods such as SDS-PAGE and gel filtration chromatography tend to concentrate on the protein contents of the two milk types, aiming to detect particular soy proteins. However, a simpler alternative has been proposed by researchers from the National Dairy Research Institute in Karnal, India,

Rajan Sharma and co-workers focused on the milk carbohydrates rather than the proteins. Lactose is the abundant bovine milk sugar whereas soy milk is dominated by sucrose, the trisaccharide raffinose and the tetrasaccharide stachyose. In initial studies, the team passed a mixture of seven standard compounds over an amino column, well-known to be a good separation medium for sugars. Representative sugars from both milks were chosen. They were eluted with an isocratic mixture of aqueous acetonitrile to a refractive index detector.

The monosaccharides eluted first but glucose and galactose coeluted in one peak after fructose. The disaccharides sucrose and lactose were next, followed by the trisaccharide raffinose and the tetrasaccharide stachyose. Sharma noted that the number of hydroxyl groups appeared to determine the elution order. The retention times ranged from 5.0 to 11.0 minutes.

The peak areas of individual sugars varied linearly with their concentrations over ranges from 1-20 µg/20 µL for raffinose to 100-2000 µg/µL for lactose, permitting their measurement in milks over these ranges.

The procedure was tested on bovine, buffalo and soy milks, as well as soy-bovine and soy-buffalo milk combinations. They were diluted, extracted with acetonitrile in the presence of Carrez I solution (potassium ferrocyanide) and filtered for analysis. The extraction was nearly quantitative.

A comparison of the HPLC profiles confirmed the sugar composition of the milks. Lactose was present as a major peak in bovine milk but was absent in soy milk. Conversely, sucrose, raffinose and stachyose were present in soy milk and absent from bovine milk.

In the milk mixtures, the soy sugars sucrose and raffinose eluted too close to the large lactose peak to be useful as differentiators. Stachyose was also a relatively small peak compared with lactose but it eluted about two minutes after the tail of the broad lactose peak in a clear region of the chromatogram.

The stachyose peak was visible in bovine/buffalo milks adulterated with as little as 5% of soy milk, making it an ideal candidate for detecting the presence of soy milk. There appeared to be no interfering peaks and the chromatographic resolution was considered to be acceptable.

The quantitative extraction, relatively short retention time and ease of use of the whole procedure, along with the potential for automation, make it suitable for the high-throughput detection of soy-adulterated bovine milk. It could find application in quality control labs checking incoming milk for authenticity.

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.



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