Council Decision of 14 November 1992 laying down methods for the analysis and testing of heat-treated milk for direct human consumption (92/608/EEC) (c. 608)

ANEX II U.K.

II. DETERMINATION OF FAT CONTENT U.K.

1. SCOPE AND FIELD OF APPLICATION U.K.

This procedure specifies the reference method for the determination of the fat content of raw milk and of whole milk, partly skimmed milk and skimmed milk.

2. DEFINITION U.K.

Fat content of milk: all the material as determined by the specified method. It is expressed as a percentage by mass.

3. PRINCIPLE U.K.

An ammoniacal ethanolic solution of a test portion is extracted with diethyl ether and light petroleum, the solvents removed by distillation or evaporation, and the mass of the extracted material soluble in light petroleum, is determined. (The procedure is usually known as the Rose-Gottlieb method).

4. REAGENTS U.K.

All reagents shall be of recognized analytical quality and shall leave no appreciable residue when taken through a blank test.

To test the quality of the reagents, carry out a determination as specified in 6.3. Use an empty flask, beaker or metal dish (5.8.) for weighing, prepared as specified in 6.4. as a tare (to allow correction for the effects on the weighing result, caused by changes in the atmospheric conditions). If the residue, corrected for the apparent change of the mass of the tare is larger than 2,5 mg, determine the residue or the solvents separately by evaporating 100 ml diethyl ether (4.4.) and 100 ml light petroleum (4.5.) respectively. Also use a tare for the weighing. When the residue is larger than 2,5 mg, cleanse the solvent by means of distillation or replace the solvent.

4.1. Ammonia solution, containing approximately 25 % (m/m) of NH₃. A more concentrated ammonia solution may also be used (see 6.5.1. and A.1.5.1.). U.K.

4.2. Ethanol, at least 94% (v/v). Ethanol denatured by methanol may be used provided it is certain that the results of the determination are not affected. U.K.

4.3. Congo red or Cresol red solution U.K.

Dissolve 1 g of Congo red or Cresol red in water and dilute to 100 ml.

Note: The use of this solution, which allows the interface between the solvent and aqueous layers to be seen more clearly, is optional (see 6.5.2.). Other aqueous colour solutions may be used provided that they do not affect the result of the determination. U.K.

4.4. Diethylether, free from peroxides not containing more than 2 mg/kg of antioxidants, and meeting the requirements of the blank test (6.3.). U.K.

4.5. Light petroleum, having any boiling range between 30 and 60 ^oC. U.K.

4.6 Mixed solvent, prepared shortly before use by mixing equal volumes of diethyl ether (4.4.) and light petroleum (4.5.). U.K.

5. APPARATUS AND GLASSWARE U.K.

Warning: Since the determination involves the use of volatile flammable solvents, any electrical apparatus employed shall comply with legislation relating to the use of such solvents. U.K.

Usual laboratory equipment and, in particular:

5.1. Analytical balance U.K.

5.2. Centrifuge, in which the fat-extraction flasks or tubes (5.6.) can be spun at a rotational frequency of 500 to 600 rev min^-1 to produce a gravitational field of 80 to 90 g at the outer end of the flasks or tubes. U.K.

Note: The use of a centrifuge is optional (6.5.5.). U.K.

5.3. Distillation or evaporation apparatus, to permit the solvents and ethanol to be distilled from the flasks or to be evaporated from beakers and dishes (sec 6.5.12. and 6.5.15.) at a temperature not exceeding 100 ^oC. U.K.

5.4. Oven, electrically heated, with ventilation port(s) fully open, capable of being controlled at a temperature of 102 ± 2 ^oC throughout the working space. The oven shall be fitted with a suitable thermometer. U.K.

5.5. Water bath, capable of being maintained at a temperature of 35 -40 ^oC. U.K.

5.6. Mojonnier-type fat extraction flasks U.K.

Note: It is also possible to use fat-extraction tubes with siphon or wash-bottle fittings, but the procedure is then different and is specified in the Appendix. U.K.

The flasks (or tubes) shall be provided with ground-glass or good quality bark corks or other stoppers unaffected by the reagents used. Rark corks shall be extracted with the diethyl ether (4.4.) kept in water at 60 ^oC or more for at least 15 minutes, and shall then be allowed to cool in water so that they are saturated when used.

5.7. Rack, to hold the fat-extraction flasks (or tubes) (see 5.6.). U.K.

5.8. Wash bottle, suitable for use with the mixed solvent (4.6.). A plastic wash bottle shall not be used. U.K.

5.9. Fat-collecting vessels, for example boiling flasks (flat-bottom), or Erlenmeyer flasks of capacity 125—250 ml or metal dishes. If metal dishes are used, they shall preferably be of stainless steel, shall be flat-bottomed, preferably with a spout, and shall have a diameter of 80 to 100 mm and a height of approximately 50 mm. U.K.

5.10. Boiling aids, fat-free, of non-porous porcelain or silicon carbide or glass beads (optional in the case of metal dishes). U.K.

5.11. Measuring cylinders, of capacities 5 and 25 ml. U.K.

5.12. Pipettes, graduated, of capacity 10 ml. U.K.

5.13. Tongs, made of metal, suitable for holding flasks, beakers or dishes. U.K.

6. PROCEDURE U.K.

Note: The alternative procedure using fat-extraction tubes with siphon or wash-bottle fittings (see the note to 5.6.) is described in the Appendix. U.K.

6.1. Preparations of the test sample U.K.

Adjust the temperature of the laboratory sample to approximately 35—40 ^oC for 15 minutes, by means of a water bath is necessary. Mix the sample thoroughly, but gently, by repeatedly inverting the sample bottle without causing frothing or churning, and cool quickly to approximately 20 ^oC.

6.2. Test portion U.K.

Mix the test sample (6.1.) by gently inverting the bottle three or four times and immediately weigh, to the nearest 1 mg, 10 to 11 g of the test sample, directly or by difference, into one of the extraction flasks (5.6.).

The test portion shall be delivered as completely as possible into the lower (small) bulb of the extraction flasks.

6.3. Blank test U.K.

Carry out the blank test simultaneously with the determination using the same procedure and same reagents, but replacing the test portion by 10 to 11 ml of water.

The change in apparent mass of the fat collecting vessel, corrected for apparent change in mass of the control vessel, should not be greater than 2,5 mg.

6.4. Preparation of fat-collecting vessel U.K.

Dry a vessel (5.9.) together with a few boiling aids (5.10.) to promote gentle boiling during the subsequent removal of solvent in the oven (5.4.) for one hour. Allow the vessel to cool (not in a desiccater but protected from dust) to the temperature of the weighing room (for glass vessels allow at least one hour, for metal dishes allow at least 30 minutes). Taking care to avoid temperature variations, use tongs to place the vessel on the balance and weigh to the nearest 0,1 mg.

6.5. Determination U.K.

6.5.1. Add 2 ml of the ammonia solution (4.1.) or an equivalent volume of a more concentrated ammonia solution and mix thoroughly with the test portion in the small bulb of the flask. After the addition of the ammonia, carry out the determination without delay. U.K.

6.5.2. Add 10 ml of the ethanol (4.2.) and mix gently but thoroughly by allowing the contents of the flask to flow backwards and forwards between the two bulbs; avoid bringing the liquid too near to the neck of the flask. If desired, add two drops of the Congo red or Cresol red solution (4.3.). U.K.

6.5.3. Add 25 ml of diethyl ether (4.4.), close the flask with a cork saturated with water or with a stopper wetted with water (see 5.6.), and shake the flask vigorously, but not excessively (in order to avoid the formation of persistant emulsions), for one minute with the flask in a horizontal position and the small bulb extending upwards. Periodically allow the liquid in the large bulb to run into the small bulb. If necessary, cool the flask in running water, then carefully remove the cork or stopper and rinse it and the neck of the flask with a little of the mixed solvent (4.6.) using the wash bottle (5.8.) so that the rinsings run into the flask. U.K.

6.5.4. Add 25 ml of light petroleum (4.5.), close the flask with the rewetted cork or stopper (rewet by dipping in water), and shake the flask gently for 30 seconds as described in 6.5.3. U.K.

6.5.5. Centrifuge the closed flask for one to five minutes at a rotational frequency of 500 to 600 rev min^-1 (5.2.). If a centrifuge is not available (see note to 5.2.) allow the closed flask to stand in the rack (5.7.) for at least 30 minutes until the supernatant layer is clear and distinctly separated from the aqueous layer. If necessary, cool the flask in running water. U.K.

6.5.6. Carefully remove the cork or stopper and rinse it and the inside of the neck of the flask with a little of the mixed solvent (4.6.) so that the rinsings run into the flask. U.K.

If the interface is below the bottom of the neck of the flask, raise it slightly above this level by gently adding water down the side of the flask to facilitate decantation of solvent.

6.5.7. Holding the extraction flask by the small bulb, carefully decant as much as possible of the supernatant layer into the prepared fat-collecting vessel (6.4.) containing a few boiling aids (5.10.) in the case of flasks (optional with metal dishes), avoiding decantation of any of the aqueous layer. U.K.

6.5.8. Rinse the outside of the neck of the extraction flask with a little of the mixed solvent (4.6.), collecting the rinsings in the fat-collecting vessel and taking care that the mixed solvent does not spread over the outside of the extraction flask. U.K.

If desired, the solvent or part of the solvent may be removed from the vessel by distillation or evaporation as described in 6.5.12.

6.5.9. Add 5 ml of the ethanol (4.2.) to the contents of the extraction flask, using the ethanol to rinse the inside of the neck of the flask and mix as decribed in 6.5.2. U.K.

6.5.10. Carry out a second extraction by repeating the operations, described in 6.5.3. to 6.5.8. inclusive, but using only 15 ml of diethyl ether (4.4.) and 15 ml of light petroleum (4.5.); use the ether to rinse the inside of the neck of the extraction flask. If necessary, raise the interface to the middle of the stem of the flask to enable the final decantation of solvent to be as complete as possible. U.K.

6.5.11. Carry out a third extraction by further repeating the operations described in 6.5.3. to 6.5.8. inclusive, but using only 15 ml of diethyl ether (4.4.) and 15 ml of light petroleum (4.5.); use the ether to rinse the inside of the neck of the extraction flask. If necessary, raise the interface to the middle of the neck of the flask to enable the final decantation of solvent to be as complete as possible. U.K.

The third extraction can be omitted for skimmed milk.

6.5.12. Remove the solvents (including ethanol) as completely as possible from the flask by distillation, or from the beaker or dish by evaporation (5.3.), rinsing the inside of the neck of the flask with a little of the mixed solvent (4.6.) before commencing the distillation. U.K.

6.5.13. Heat the fat-collecting vessel (with the flask placed on its side to allow solvent vapour to escape) for one hour in the oven (5.4.). Remove the fat-collecting vessel from the oven, allow to cool (not in a desiccator, but protected from dust) to the temperature of the weighing room (for glass vessels allow at least one hour, for metal dishes allow at least 30 minutes) and weigh to the nearest 0,1 mg. U.K.

Do not wipe the vessel immediately before weighing. Place the vessel on the balance using tongs and avoid, in particular, temperature variations.

6.5.14. Repeat the operations described in 6.5.13. until the mass of the fat-collecting vessel decreases by 0,5 mg or less, or increases, between two successive weighings. Record the minimum mass observed as the mass of the fat-collecting vessel and extracted matter. U.K.

6.5.15. Add 25 ml of light petroleum to the fat-collecting vessel in order to verify whether or not the extracted matter is wholly soluble. Warm gently and swirl the solvent until all the fat is dissolved. U.K.

If the extracted matter is wholly soluble in the light petroleum, take the mass of fat as the difference between the final mass of the vessel containing the extracted matter (6.5.14.) and its initial mass (6.4.).

6.5.16. If the extracted matter is not wholly soluble in the light petroleum, or in case of doubt, extract the fat completely from the vessel by repeatedly washing with warm light petroleum. U.K.

Allow any trace of insoluble material to settle and carefully decant the light petroleum without removing any insoluble material. Repeat this operation three more times, using the light petroleum to rinse the inside of the neck of the vessel.

Finally, rinse the outside of the top of the vessel with mixed solvent so that the solvent does not spread over the outside of the vessel. Remove light petroleum vapour from the vessel by heating the vessel for one hour in the oven, allow to cool and weigh, as described in 6.5.13. and 6.5.14.

Take the mass of fat as the difference between the mass determined in 6.5.14. and this final mass.

7. EXPRESSION OF RESULTS U.K.

7.1. Calculation and formula U.K.

Calculate the fat content as a percentage by mass by:

where

F

=

the fat content,

m₀

=

the mass, in grams, of the test portion (6.2.),

m₁

=

the mass, in grams, of the fat-collecting vessel and extracted matter determined in 6.5.14.,

m₂

=

the mass, in grams, of the prepared fat-collecting vessel or, in the case of undissolved material, of the fat-collecting vessel and insoluble residue determined in 6.5.16.,

m₃

=

the mass, in grams, of the fat-collecting vessel used in the blank test (6.3.) and any extracted matter determined in 6.5.14.,

m₄

=

the mass, in grams, of the prepared fat-collecting vessel (see 6.4.) used in the blank test (6.3.), or in the case of undissolved material, of the fat-collecting vessel and insoluble residue determined in 6.5.16.

Report the result to the nearest 0,01 %.

7.2. Precision U.K.

7.2.1. Repeatability (r): U.K.

• for whole milk and partly skimmed milk: 0,02 g of fat per 100 g of product,

• for skimmed milk: 0,01 g of fat per 100 g of product.

7.2.2. Reproducibility (R): U.K.

• for whole milk: 0,04 g of fat per 100. g of product,

• for partly skimmed milk: 0,03 g of fat per 100 g of product,

• for skimmed milk: 0,025 g of fat per 100 g of product.