This method describes the determination of phosphatase activity in:
dried high fat milk or high fat milk powder,
dried whole milk or whole milk powder,
dried partly skimmed milk or partly skimmed-milk powder,
dried skimmed milk or skimmed-milk powder.
The phosphatase activity of dried milks is a measure of the quantity of active alkaline phosphatase present in the product. It is expressed as the quantity of p-nitrophenol in micrograms liberated by 1 ml of the reconstituted sample, under the conditions described.
The reconstituted sample is diluted with a buffer substrate at pH 10,2 and incubated at a temperature of 37 oC for two hours. Any alkaline phosphatase present in the sample will, under these circumstances, liberate p-nitrophenol from added disodium p-nitrophenyl phosphate. The p-nitrophenol liberated is determined by direct comparison with standard colour glasses in a simple comparator using reflected light.
Dissolve 3,5 g of anhydrous sodium carbonate and 1,5 g of sodium bicarbonate in water and dilute to 1 000 ml in a volumetric flask with water.
Dissolve 1,5 g of disodium p-nitrophenylphosphate in sodium carbonate-bicarbonate buffer (4.1) and dilute to 1 000 ml in a volumetric flask with buffer (4.1).
This solution is stable if stored in a refrigerator (≤ 4 oC) for one month but a colour control test should be carried out on such stored solutions — see 6, precaution number 3.
Dissolve 30,0 g of zinc sulphate (ZnSO4) in water and dilute to 100 ml in a volumetric flask with water.
Dissolve 17,2 g of potassium hexacyanoferrate (II) trihydrate (K4Fe(CN)6.3H20) and dilute to 100 ml in a volumetric flask with water.
Precautions:
After use, test tubes must be emptied, rinsed in water, washed in hot water containing an al kaline detergent, followed by thorough rinsing in clean hot tap water. Finally, they must be rinsed in water and dried before use.
Pipettes must be thoroughly rinsed in clean cold tap water immediately after use, followed by rinsing in water and dried before use.
The test tube stoppers must be thoroughly rinsed in hot tap water immediately after use, followed by boiling for two minutes in water.
The buffer substrate solution (4.2) should remain stable for at least one month if stored in a refrigerator at 4 oC or less. Any instability is denoted by the formation of a yellow colour. Whilst the test is always read against a boiled product control containing the same buffer substrate solution, it is recommended that the solution should not be used if it gives a colour reading in excess of 10 μg when read in a 25 mm cell in the comparator using distilled water in the other 25 mm cell.
Use a separate pipette for each sample and avoid contaminating the pipette with saliva.
The test must not be exposed to direct sunlight at any time.
Dissolve 10 g of the powder in 90 ml of water. The temperature for dissolving the powder must not exceed 35 oC.
The direct reading obtained under 6.2.4 is recorded as μg p-nitrophenol per ml sample or per ml of reconstituted sample.
The difference between the results of two determinations carried out simultaneously or in rapid succession on the same sample, by the same analyst, under the same conditions, shall not exceed 2 μg of p-nitrophenol liberated by 1 ml of reconstituted milk.