ALKALINE

PHOSPHATASE TEST
• Alkaline Phosphatase (ALP) is an enzyme naturally present in all raw milks, which is used
as an indicator of proper milk pasteurization.
• Complete pasteurization will inactivate the enzyme to below levels which are detectable by
conventional methods.
• Because the heat stability of ALP is greater than that of pathogens which may be present in
milk, the enzyme serves as an indicator of product safety.
 PRINCIPLE
 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
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 .
Materials Required
 Test tubes
 Pipettes (one and 10 ml capacity)
 Volumetric flask (100 ml and 500 ml capacity)
 Water bath at 37ºC
 Buffer solution is prepared by mixing 0.75g anhydrous sodium carbonate and l.75g Sodium
bicarbonate in 500 ml distilled water
 Buffer-substrate solution is prepared by placing 0.l5 g of di-sodium para-
nitrophenylphosphate (the substrate) into a clean 100 ml measuring cylinder.
 Note: All glassware must be rinsed, cleaned, demineralized in dichromate solution and boiled
in water for 30 min. Store this buffer and substrate solution in a refrigerator and protected
against light. It should not be used after one week. Prepare a fresh stock.
PROCEDURE
1) Pipette 5 ml buffer-substrate solution into a test tube, cover with suitable stopper and warm
in the water bath at 37ºC.
2) Add to the test tube 1ml of the milk to be tested, stopper and mix well and place in water
bath at 37ºC.
3) Prepare a blank sample from boiled milk of the same type as that undergoing the test.
4) Incubate both the test samples and the blank sample at 37ºC for 2hrs.
5) After incubation, remove the tubes and mix them thoroughly.
6) Place one sample against the blank in a Lovibond comparator" All purposes using
“A.P.T.W”. disc and rotate the disc until the color of the test sample is matched and read the
disc number.