Direct estimation of sialic acid in milk and milk products by fluorimetry and its application in detection of sweet whey adulteration in milk

Sialic acid, being a biologically active compound, is recognised as an important component of milk and milk products. Almost all the sialic acid estimation protocols in milk require prior hydrolysis step to release the bound sialic acid followed by its estimation. The objective of this work was to estimate sialic acid in milk and milk products by fluorimetric assay which does not require a prior hydrolysis step thus decreasing the estimation time. The recovery of added sialic acid in milk was 91.6 to 95.8%. Sialic acid in milk was found to be dependent on cattle breed and was in the range of 1.68-3.93 g/kg (dry matter basis). The assay was further extended to detect adulteration of milk with sweet whey which is based on the detection of glycomacropeptide (GMP) bound sialic acid in adulterated milk. GMP is the C-terminal part of kappa-casein which is released into the whey during cheese making. For detection of adulteration, selective precipitation of GMP was done using trichloroacetic acid (TCA). TCA concentration in milk was first raised to 5% to precipitate milk proteins, especially kappa-casein, followed by raising the TCA concentration to 14% to precipitate out GMP. In the precipitates GMP bound sialic acid was estimated using fluorimetric method and the fluorescence intensity was found to be directly proportional to the level of sweet whey in adulterated milk samples. The method was found to detect the presence of 5% sweet whey in milk.