Comparison of the freezing point, chloramines-T, and HPLC methods for determination of the extent of lactose hydrolysis in milk

Presently there are several methods for determination of the extent of lactose hydrolysis in milk, some of which, despite high precision and reliability, are not used extensively due to their high cost. The objective of this study was to compare effectiveness 3 of such methods, namely, HPLC, freezing point measurement, and chloramines-T, with a view to finding a suitable and less expensive method. The lactose content of skimmed milk samples was measured, followed by treatment for 4 hours with β-galactosidase to hydrolyze lactose and then determining the extent of hydrolysis using the HPLC, freezing point, and chloramines-T methods. The β-galactosidase exerted its maximum effect during the first 2 hours, resulting in reduction of the lactose content to less than 0.5% of its original value. In the HPLC method, the percentage of relative standard deviation (%RSD) for the milk samples after 2 hours treatment with the enzyme was more than 40% and the reproducibility was not desirable. With regard to the freezing-point method, after a 2-hour decreasing trend of the freezing point the rate of decrease slowed down. The freezing point decreased about -0.274 ºC after 4 hours, such that for every 1% hydrolysis of lactose the freezing point decreased by 0.0028 ºC. Furthermore, no linear relationship was observed between the freezing point and the rate of lactose hydrolysis at rates higher than 88%. Finally, in the chloramines-T method, it was shown that with increasing lactose hydrolysis, the consumption of sodium thiosulfate decreased, the trend slowing down after 2 hours. On the whole, in the preliminary stages of hydrolysis (the first 60%) the amounts of lactose determined by the 3 methods was almost similar, but after that the chloramines-T method gave figures slightly different from those given by the other 2 methods. Since the correlation between the HPLC and the freezing point methods was very high (r2 =0.9976), it can be concluded that the freezing point can be recommended as an appropriate, simple, precise and rapid method for the assessment of lactose concentration, as well as determining its hydrolysis extent, in enzyme-treated milk. Furthermore, the chloramine-T method can be said to be suitable for determination of lactose in untreated milks.