Alkaline Phosphatase Test for Pasteurized Milk

Alkaline Phosphatase Test for Pasteurized Milk

Pasteurisation is an essential process in the production of milk which is safe and free from pathogens. Alkaline Phosphatase is an enzyme which is naturally present in milk, but is destroyed at a temperature just near to the pasteurization temperature. Alkaline Phosphatase test is used to indicate whether milk has been adequately pasteurised or whether it has been contaminated with raw milk after pasteurisation. This test is based on the principle that the alkaline phosphatase enzyme in raw milk liberates phenol from a disodium para-nitro phenyl phosphate and forms a yellow coloured complex at alkaline pH (Scharer, 1943). The intensity of yellow colour produced is proportional to the activity of the enzyme. The colour intensity is measured by direct comparison with standard colour discs in a Lovibond comparator. The test is not applicable to sour milk and milk preserved with chemical preservatives.

Apparatus required

1.    Water-Bath -maintained at 37±l⁰C, thermostatically controlled.

2.    Comparator - with special discs of standard colour glasses calibrated in µg p-nitrophenol per ml milk, and 2 x 25 mm cells.

3.    Test Tubes - of size 16 x 1.50 mm and rubber stoppers to fit.

4.    Pipettes - 1, 5, and 10 ml.

5.    Filter Paper - Whatman No. 2 or equivalent.

6.    Litmus Paper


1.    Sodium Carbonate-Bicarbonate Buffer - Dissolve 3.5 g of anhydrous sodium carbonate and 1.5 g of sodium bicarbonate in one litre of distilled water.

2.    Buffer Substrate - Dissolve 1.5 g of disodium p-nitrophenyl phosphate in one litre of sodium carbonate-bicarbonate buffer. This solution is stable if stored in a refrigerator at 4°C or less for one month but a colour control test should be carried out on such stored solutions



1.    Pipette 5 ml of buffer substrate into a clean, dry test tube followed by 1 ml of the milk to be tested. Stopper the tube, mix by inversion and place in the water-bath

2.    At the same time place in the water-bath a control tube containing 5 ml of the buffer substrate and 1 ml of boiled milk of the same kind as that under test that is pasteurized homogenized, low fat.

3.    After 2 hours, remove the tubes from the bath, invert each and read the colour developed using the comparator and special disc, the tube containing the boiled milk control being placed on the left of the stand and the tube containing the sample under test on the right. Record readings which lie between two standard colour discs by adding a plus (+) or minus (-) sign to the figure of the nearest standard.

NOTE - If artificial light is needed when taking these readings, an approved ‘day light’ source of illumination must be used.




1.    IS:8479 (Part-1)-1977, method for determination of Phosphatase activity in milk and milk products