First Commission Directive of 13 November 1979 laying down Community methods of analysis for testing certain partly or wholly dehydrated preserved milk for human consumption (79/1067/EEC)

METHOD 7: DETERMINATION OF PHOSPHATASE ACTIVITY (MODIFIED SANDERS AND SAGER PROCEDURE)

1. SCOPE AND FIELD OF APPLICATION

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.

2. DEFINITION

The phosphatase activity of dried milks is a measure of the quantity of active alkaline phosphatase present. It is expressed as the quantity of phenol in μg liberated by 1 ml of reconstituted milk, as determined by the procedure described below.

3. PRINCIPLE

The phosphatase activity of dried milks is determined by the ability of the phosphatase to liberate the phenol from disodiumphenylphosphate. The quantity of phenol liberated under prescribed conditions is determined by a spectrophotometric measurement of the colour developed with Gibb's reagent.

4. REAGENTS

4.1. Solution A

Barium borate hydroxide buffer: pH 10,6 ± 0,1 at 20 ^o C.

Dissolve: 25,0 g of barium hydroxide (Ba(OH)2.8H2O) in water and dilute to 500 ml.

Dissolve: 11,0 g of boric acid (H3BO3) in water and dilute to 500 ml.

Warm the two solutions to 50 ^o C and mix.

Shake and cool the mixture to room temperature.

Adjust the pH to 10,6 ± 0,1 with the barium hydroxide solution and filter.

Store the solution in a tightly stoppered container.

Before use, dilute the buffer with an equal quantity of water.

4.2. Solution B:

Colour development buffer.

Dissolve: 6,0 g of sodium metaborate (NaBO2) (or 12,6 g of NaBO2.4H2O) and 20,0 g of sodium chloride (NaCl) in water and dilute to 1 000 ml with water.

4.3. Solution C

Buffer substrate solution.

4.3.1. Dissolve 0,5 g of disodiumphenylphosphate (Na2C6H5PO4.2H2O) in 4,5 ml of Solution B (4.2). Add 2 drops of Solution E (4.5) and allow to stand 30 minutes. Extract the colour with 2,5 ml butanol (4.10). If necessary, repeat the colour extraction. After separation, discard the butanol. This solution can be kept for several days in a refrigerator. Develop and extract the colour once more before use.

4.3.2. Pipette 1 ml of this solution into a 100 ml volumetric flask and make up to volume with Solution A. Prepare the buffer solution immediately before use.

4.4. Solution D

Precipitant.

Dissolve 3,0 g of zinc sulphate (ZnSO4.7H2O) and 0,6 g of copper (II) sulphate (CUSO4.5H2O) in water and make up to 100 ml with water.

4.5. Solution E

Gibb's reagent.

Dissolve 0,040 g of 2,6-dibromoquinone 1,4 — chloroimide (O.C6H2Br2.NCl) in 10 ml of 96 % ethanol. Store the solution in a dark glass bottle kept in a refrigerator. Discard this reagent when it has become discoloured.

4.6. Colour dilution buffer

Dilute 10 ml of Solution B (4.2), colour development buffer, to 100 ml with water.

4.7. Copper sulphate solution

Dissolve 0,05 g of copper (II) sulphate (CUSO4.5H2O) in water and make to 100 ml with water.

4.8. Phenol standard solution

Dissolve 0,2 ± 0,001 g of pure phenol in water and make up to 100 ml in a volumetric flask with water. This solution can be stored for several months in a refrigerator. Dilute 10 ml of this solution to 100 ml with water. This diluted solution contains 200 μg of phenol in 1 ml and can be used for preparing more dilute solutions.

4.9. Boiled distilled water.

4.10. n-Butanol.

5. APPARATUS

5.1. Analytical balance.

5.2. Waterbath, thermostatically controlled at 37 ^oC ± 1 ^oC.

5.3. Spectrophotometer suitable for readings at a wavelength of 610 nm.

5.4. Filter paper (Schleicher and Schull 597, Whatman 42 or equivalent filter paper).

5.5. Waterbath, boiling.

5.6. Aluminium foil.

6. PROCEDURE

Precautions:

1.

Avoid direct exposure to sunlight.

2.

All the glassware, stoppers and removal material should be perfectly clean. It is recommended that they be rinsed and boiled with water or that they be treated with steam.

3.

Avoid using plastic materials (stoppers for example) as they may contain phenols.

4.

Saliva contains phosphatase; contamination by traces of saliva must therefore be carefully avoided.

6.1. Preparation of the sample

6.1.1. Weigh, to within 0.1 g, 10 g of the sample and dissolve in 90 ml of water. The temperature for dissolving the powder shall, under no circumstances, exceed 35 ^oC.

6.2. Determination

6.2.1. Introduce in each of two test tubes 1 ml of reconstituted milk prepared as described in 6.1.1.

6.2.2. Heat one of the tubes in boiling water for two minutes. Cover the tube and the waterbath (5.5) or, for example, a beaker with aluminium foil (5.6) to ensure that the entire tube will be heated. Cool in cold water to room temperature. Use this tube for the blank test. For all subsequent operations treat the two tubes identically.

6.2.3. Add 10 ml of Solution C (4.3.2). Mix and place the tube in the waterbath at 37 ^oC (5.2).

6.2.4. Incubate for 60 minutes in the waterbath shaking periodically.

6.2.5. Transfer the tubes immediately to a boiling waterbath (5.5) and heat for two minutes; cool to room temperature in cold water.

6.2.6. Add 1 ml of Solution D (4.4), mix and filter through a dry filter paper; discard the first filtrates until a clear liquid is obtained.

6.2.7. Put 5 ml of each filtrate into test tubes, add 5 ml of Solution B (4.2) and 0.1 ml of Solution E (4.5). Mix.

6.2.8. Allow the colour to develop at room temperature for 30 minutes away from direct sunlight.

6.2.9. Measure the optical density of the sample solution, against the blank, at the wavelength indicated in 5.3.

6.2.10. Repeat the determination if the optical density of the solution is above that of the standard sample with 20 μg of phenol prepared according to 7.

If this limit is exceeded, dilute a suitable volume of reconstituted milk according to 6.1.1 with a suitable volume of this milk carefully boiled as indicated in 6.2.2 to inactivate the phosphatase present.

7. PREPARATION OF THE STANDARD CURVE

7.1. Pipette into four 100 ml volumetric flasks, 1, 3, 5 and 10 ml of the standard solution diluted according to 4.8 and make up to volume with water; these dilutions contain respectively 2, 6, 10 and 20 μg of phenol per ml.

7.2. Pipette 1 ml of water or 1 ml of each standard solution (7.1) into the test tubes in order to obtain a series of samples containing 0 (blank value obtained using the 1 ml of water) — 2 — 6 — 10 and 20 μg of phenol.

7.3. Pipette successively into each test tube 1 ml of the solution of copper (II) sulphate (4.7), 5 ml of the colour dilution buffer solution (4.6), 3 ml of water and 0.1 ml of Solution E (4.5). Mix.

7.4. Leave the test tubes for 30 minutes at room temperature away from direct sunlight.

7.5. Measure the absorbance of the solutions in each of the tubes, compared to the blank value, at the wavelength indicated in 5.3.

7.6. Prepare the standard curve by plotting the absorbance values against the quantities of phenol in μg as indicated in 7.2.

8. EXPRESSION OF THE RESULTS

8.1. Calculation

8.1.1. Convert the figures as determined under 6.2.9 to μg of phenol, by reference to the standard curve.

8.1.2. Calculate the phosphatase activity expressed as μg of phenol per ml of reconstituted milk according to the following formula:

Phosphatase activity = 2,4 x P

where P = the quantity of phenol in μg according to 8.1.1.

8.1.3. If it was necessary to dilute as indicated under 6.2.10 multiply the result obtained in 8.1.2 by the dilution factor.

8.2. Repeatability

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 phenol liberated by 1 ml of reconstituted milk.