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Determination of Phosphatase enzyme in Curd by Mullen Phosphatase Test

Determination of Phosphatase enzyme in Curd by Mullen Phosphatase Test 


Phosphatase, an enzyme in milk is destroyed during pasteurization. The test is based on the above principle to judge the efficiency of pasteurization. Any Phosphatase present in milk splits the substrate, p-nitro phenyl phosphate to give p-nitro phenol, which is highly colored in alkaline solution. The test does not apply to sour milk and milk preserved with chemical preservatives.


  1.  A Lovibond Comparator with stand to work in reflected light.
  2. A Lovibond comparator disc APTW or APTW 7.
  3. Two Fused glass cells of 25 mm depth
  4. A water bath or incubator capable of being maintained at 37.5°C ± 0.5°C
  5. 1 ml pipette and 5 ml pipette
  6. 1 liter graduated flask
  7. 100 ml measuring cylinder
  8. Test tubes, nominal size 150/16 mm with rubber stoppers


  1. All glassware must be cleaned before use. Cleaning should be done by soaking in Chromic acid solution prepared by slowly adding 4 volumes of concentrated H2SO4 to 5 volumes of 8% Potassium Dichromate. After cleaning in Chromic acid, glassware’s must be rinsed in warm water and distilled water and finally dried. Glassware used for the test must not be used for any other purpose and must be kept apart from other apparatus in the laboratory.
  2. Test must not be carried out in direct sunlight.
  3. A fresh pipette must be used for each sample of milk. Pipettes must not be contaminated with saliva.
  4. The sample of milk should be examined as soon as possible after arrival at the laboratory. If not examined immediately it must be kept at a temperature between 3°C and 5°C until examined. The sample must be brought to room temperature immediately before being tested.


All reagents should be of analytical grade.

  1. Buffer solution – 1.5 g of sodium bicarbonate and 3.5 g of anhydrous sodium carbonate dissolved in water and made upto one liter. Store in a refrigerator and discard after 1 month
  2. Disodium p-nitro phenyl phosphate. The solid substrate must be kept in the refrigerator.
  3. Buffer-substrate solution – Weigh accurately 0.15 g of substrate (disodium p-nitrophenylphosphate) into a 100 ml measuring cylinder and make upto 100 ml with buffer solution. The solution should be stored in refrigerator and protected from light. The solution should give a reading of less than the standard marked 10 on comparator disc APTW or APTW 7 when viewed through a 25 mm cell (distilled water is used as a blank). The solution must be discarded after one week.


  1. Into a test tube pipette 5 ml of buffer substrate solution, stopper and bring the temperature to 37°C.
  2. Add 1 ml of test milk to it, shake and replace stopper, incubate at 37°C for 2 hrs. Incubate one blank prepared from boiled milk of the same type as that undergoing the test with each series of sample.
  3. Remove the tubes after 2 hrs. and the content should be well mixed. Place the boiled milk blank on left hand side of the comparator stand and test sample on the right.
  4. Take reading in reflected light by revolving the disc until the test sample is matched.
  5. Record readings falling between two standards by affixing a plus or minus sign to the figure for the nearest standard.


The test is considered satisfactory if it gives a reading of 10 µg or less of p-nitro phenyl per ml of milk. Properly pasteurized milk gives no discernible color.

(Ref: – F.A.O Manuals of Food Quality control 14/8 page 23)


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