2.1.1.

Wherever mention is made to water for solution, dilution or rinsing purposes, distilled water, deionized water or demineralized water of at least equivalent purity, shall be used unless otherwise specified.

2.1.2.

Wherever reference is made to ‘solution’ or ‘dilution’ without further indication, ‘solution in water’ or ‘dilution with water’ is meant.

5.1.1.

Repeatability (r) is the value below which the absolute difference between two single test results obtained with the same procedure on identical test material, under the same conditions (same operator, same apparatus, same laboratory, and a short interval of time) lies.

5.1.2.

Reproducibility (R) is the value below which the absolute difference between two single test results obtained with the same procedure on identical test material, under different conditions (different operators, different apparatus, different laboratories and/or different time) lies.

5.1.3.

Unless otherwise specified for each individual method the values for the repeatability- and reproducibility-criteria of each procedure represent the 95% confidence level intervals as defined by ISO 5725: 2'ed. 1986.

5.1.4.

The necessary collaborative trials and studies should be planned and conducted in accordance with international guidelines.

4.2.2.1.

Mix the milk by an appropriate procedure, before sampling.

To mix the contents of large vessels or of storage, rail or road tanks, the use of mechanical agitation is advised (4.2.2.2.).

The extent of mixing shall be appropriate to the period of time over which the milk has been at rest. The efficiency of the procedure of mixing applied in any particular circumstances shall be demonstrated as being adequate for the purposes of the analysis envisaged; the criterion of mixing efficiency particularly influences the similarity between analytical results from samples taken either from different parts of the consignments, or from the outlet of the tank at intervals during discharge. A procedure of mixing milk (untreated milk or whole milk) shall be considered efficient if the difference in fat content between two samples, taken under these conditions, is less than 0,1 %.

In a large vessel with a bottom discharge outlet there may be, at the discharge point, a small quantity of milk which is not representative of the whole contents even after mixing. Therefore samples should preferably be taken through a manhole. If samples are taken from the discharge outlet, run off sufficient milk to ensure that the samples are representative of the whole.

4.2.2.2.

Mixing of the contents of large vessels or of storage, rail or road tanks can be carried out:

—

During transport and storage, precautions shall be taken to prevent exposure to contaminating odours and to direct sunlight. If the container used for samples is transparent, it shall be stored in a dark place.

4.1.

Analytical balance

4.2.

Desiccator, provided with an efficient desiccant (for example freshly dried silica gel with a water content indicator).

4.3.

Drying oven, ventilated, thermostatically controlled at 102 ± 2 oC throughout the total working space.

4.4.

Flat-bottom dishes, of height 20 to 25 mm, diameter 50 to 75 mm, and of appropriate material provided with well-fitting, readily removable lids.

4.5.

Boiling water bath

4.6.

Homogenizer

6.3.1.

Pre-dry the dish for 30 minutes by heating it on the boiling water bath (4.5).

6.3.2.

Heat the dish, with its lid alongside, in the oven (4.3), controlled at 102 ± 2 o C, for two hours. Place the lid on the dish and remove from the oven.

6.3.3.

Allow to cool in the desiccator (4.2.) to room temperature (i.e. for at least 30 minutes) and weigh to the nearest 0,1 mg.

6.3.4.

Heat the dish again, with its lid alongside, in the oven for one hour. Place the lid on the dish and remove from the oven. Allow to cool for approximately 30 minutes in the desiccator and weigh to the nearest 0,1 mg.

6.3.5.

Repeat the operations described in 6.3.4. until the difference in mass between two consecutive weighings does not exceed 0,5 mg. Record the lowest mass.

WT

=

the total solids content in g per 100 g,

m0

=

the mass, in grams, of the dish and lid (see 6.1.),

m1

=

the mass, in grams, of the dish, lid and test portion (see 6.2.),

m2

=

the mass, in grams, of the dish, lid and dried test portion (see 6.3.5.).

7.2.1.

Repeatability (r): 0,10 g of total solids per 100 g of product.

7.2.2.

Reproducibility (R): 0,20 g of total solids per 100 g of product.

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.).

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.

4.3.

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.

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.).

4.5.

Light petroleum, having any boiling range between 30 and 60 oC.

4.6

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

5.1.

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.

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

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.

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.

5.5.

Water bath, capable of being maintained at a temperature of 35 -40 oC.

5.6.

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.

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.).

5.8.

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

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.

5.10.

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

5.11.

Measuring cylinders, of capacities 5 and 25 ml.

5.12.

Pipettes, graduated, of capacity 10 ml.

5.13.

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

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.

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.).

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

7.2.1.

7.2.2.

4.1.

4.2.

Copper sulphate solution. Dissolve 5,0 g of copper (II) sulphate pentahydrate (CuSO4, 5H2O) in water and dilute to 100 ml (at 20 oC) in a volumetric flask.

4.3.

Sulphuric acid, at least 98,0% (m/m) H2SO4.

4.4.

Note: A less concentrated sodium hydroxide solution may be used for example: 40% (m/m) 572 g/1, 20 oC; or 30% (m/m) 399 g/1, 20 oC.

4.5.

Boric acid solution. Dissolve 40 g of boric acid (H3BO3) in one litre of hot water, allow to cool, and store in a borosilicate glass bottle.

4.6.

Indicator solution. Dissolve 0,01 g methyl red, 0,02 g bromothymol blue and 0,06 g bromocresol green in 100 ml of ethanol. Store the solution in a brown closed bottle, in a cool, dark place.

4.7.

c (1/2 H2SO4) or c (HC1) = 0,1 mol/1 standardized to the nearest 0,0001 mol/1.

4.8.

4.9.

Ammonium salt, pure, such as ammonium oxalate (NH4)2C2O4,H2O or ammonium sulfate (NH4)2SO4.

4.10.

Tryptophan (C11H12N2O2), phenacetin (C10H7CH2CONH2) or lysine mono- or di-hydrochloride (C6H14N2O2 HCl or C6H14N2O2 · 2HCl).

Note: The purity of reagents in 4.9. and 4.10. should be of higher quality than ‘analytical grade’. If available, certified ammonium salt solution (4.9.) should be used.

5.1.

Kjeldahl flasks of capacity 500 ml.

5.2.

Suitable boiling aids, for example, glass beads of diameter approximately 5 mm, Hengar granules, pumice.

5.3

Burette or automatic pipette, to deliver 1,0 ml.

5.4.

Graduated measuring cylinders, glass, of capacities 50, 100 and 250 ml.

5.5.

Digestion apparatus in an inclined position (approximately 45o), with electric heaters or gas burners that do not heat the flasks above the level of their contents, with a fume extraction system.

5.6.

Distillation apparatus, made of borosilicate glass, to which a Kjeldahl flask (5.1.) can be fitted, consisting of an efficient splash-head connected to an efficient condenser with straight inner tube and an outlet tube attached to its lower end; the connecting tubing and stopper (s) shall be close-fitting and preferably of neoprene rubber.

5.7.

Pipette or automatic pipette, to deliver 0,10 ml.

5.8.

Conical flasks, of capacity 500 ml, graduated at 200 ml.

5.9.

Burette of capacity 50 ml, gratuated in 0,1 ml, maximum error ± 0,05 ml.

5.10.

Magnifying lens, for reading the burette (5.9.).

5.11.

pH meter

5.12.

Automatic burette.

6.1.

To the Kjeldahl flask (5.1.) add boiling aid (5.2.) (eg. three glass beads), 15 g of potassium sulphate (4.1.), 1,0 ml of copper sulphate solution (4.2.), approximately 5 g of milk sample (weighed to the nearest 0,001 g) and 25 ml of sulphuric acid (4.3.). Use the acid to wash down any copper sulphate solution, potassium sulphate or milk on the neck of the flask, and gently mix the contents of the flask.

Note: Because organic matter consumes sulphuric acid during boiling, use 30 ml of H2SO4 (4.3.), instead of 25 ml for digestion, if the milk contains more than 5,0% (m/m) of fat. This should also be done in the blank test.

6.2.

Heat each Kjeldahl flask on the digestion apparatus (5.5.), very gently at first so that all the black froth stays within the bulb. When the initial frothing has ceased and copious white vapour appears, boil vigorously (acid vapour will condense half-way up the neck of the flask) until no black particles remain and until the digest becomes clear pale blue-green in colour. Then boil gently for at least 1,5 hours. Note the following requirements:

6.3.

When the Kjeldahl flasks are cool, add 300 ml of water (see note) to each so as to wash carefully down the neck of the flask, and mix the contents thoroughly ensuring that the crystals which have separated out are dissolved. Add some boiling aid (5.2.) to ensure uniform boiling. Then to each flask, add 70 ml of sodium hydroxide solution (4.4.) (see note) by gently pouring the solution down the inclined neck of the flask to form a bottom layer in the bulb; do not wet the top of the neck with the sodium hydroxide solution.

Note: It is necessary that the combined volume of water and sodium hydroxide solution total 370 ml to enable approximately 150 ml of distillate to be collected just before irregular boiling (‘bumping’) ensues (6.4.). Thus, if a larger equivalent volume of a sodium hydroxide solution which is less concentrated than 47 % (m/m) is added, the volume of water added shall be reduced accordingly. For example, if 85 ml of 40 % (m/m) or 125 ml of 30% (m/m) sodium hydroxide solution are to be added, the volume of water added shall be 285 ml or 245 ml respectively.

6.4.

Immediately connect each Kjeldahl flask to a distillation apparatus (5.6.). Ensure that the tip of the condenser outlet-tube is immersed in 50 ml of boric acid solution (4.5.) together with 0,20 ml (5 - 6 drops) of indicator solution (4.6.) all contained in a conical flask (5.8.). Swirl the contents of each Kjeldahl flask to mix thoroughly and boil, but gently at first to prevent excessive frothing. When 100 to 125 ml of distillate have been collected, lower each conical flask until the tip of the condenser outlet-tube is approximately 40 mm above the 200 ml mark. Continue each distillation until irregular boiling (‘bumping’) starts and then immediately stop the heating. Disconnect each Kjeldahl flask and rinse the tip of each condenser outlet-tube with a little water, collecting the rinsings in the conical flask. Note the following requirements:

6.5

Titrate each distillate with standard volumetric solution (4.7.) until the pH is 4,6 ± 0,1, using a pH meter and if desired an automatic burette. Addition of an indicator helps to check whether the titration is proceeding correctly. Take each burette reading to the nearest 0.01 ml with the aid of a magnifying lens (5.10.) avoiding errors of parallax.

The titrating may be carried out with the indicator only. Titrate until the colour of the distillate corresponds to that of a solution recently prepared from 150 ml of water to which has been added 50 ml of the boric acid solution and 0,20 ml of the indicator contained in a conical flask (5.8).

6.6.

Carry out a blank test according to 6.1. to 6.5. inclusive, taking 5 ml of distilled water together with about 0,1 g of sucrose (4.8.) through the procedure instead of the milk sample.

Note: The titration of the blank distillate will require only a very small volume of the standard volumetric solution (4.7.).

6.7.

Regularly check the accuracy of the procedure by using two recovery trials following the procedure according to 6.1. to 6.5. inclusive.

6.7.1.

Check that no loss of nitrogen occurs as a result of excessive heat or mechanical leaks during distillation, by using a test portion of 0,15 g of ammonium oxalate or sulphate (4.9.) weighed to the nearest 0,001 g together with 0,1 g of sucrose (4.8.).

The percentage of nitrogen recovered shall be between 99,0 and 100,0 %.

Lower or higher results will indicate failures in the procedure and/or inaccurate concentration of the standard solution (4.7.).

6.7.2.

Check that the digestion procedure is sufficient to release all the protein nitrogen by using a test portion of 0,20 g of pure tryptophan, 0,35 g of phenacetin or 0,20 g of lysine hydrochloride (4.10.). All weighings should be to the nearest 0,001 g. At least 98—99% of the nitrogen should be recovered.

WN

=

the nitrogen content.

V

=

the volume in millilitres of the standard volumetric solution of acid used in the determination.

Vo

=

the volume in millilitres of the standard volumetric solution of acid used in the blank test,

c

=

the concentration, expressed as moles per litre of the acid standard volumetric solution (4.7.).

m

=

the mass in grams of the test portion.

8.2.1.

Repeatability (r): 0,007 g per 100 g.

8.2.2.

Reproducibility (R): 0,015 g per 100 g.

9.1.

Use a block digestion apparatus fitted with cylindrical flasks, instead of the digestion apparatus and the Kjeldahl flasks described in 5.5. and 5.1. In this case to identify potential trouble, each spot has to be checked individually (6.7.).

9.2.

Use of steam distillation instead of direct heating of the flasks (6.4.). When the apparatus does not allow the use of distilled water, care should be taken that the water does not contain acid or alkaline volatiles.

9.3.

A test portion of 1 g of milk (semi-macro Kjeldahl) can be used instead of 5 g (6.1.) provided:

Note: Using one or more of these options is acceptable only if the repeatability value (8.2.1.) and the two accuracy tests results (6.7.) are in accordance with the requirements given in this method.

4.1.

The specific gravity hydrometer is an instrument consisting of a glass float, which, at its lower end, is wide and heavy. There is a cylindrically shaped glass rod attached to the upper and of the float and coaxially orientated to it; the upper end of the rod is closed.

The glass float contains the load (lead, mercury, etc.) intended to adjust the hydrometer mass. The rod includes a graduated scale from 1,025 to 1,035 g/ml.

The hydrometer should he checked by the pycnometric method, using a pycnometer of a capacity of approximately 100 ml equipped with a precision thermometer.

4.2.

Cylinders (glass or stainless steel).

Minimum dimensions should be:

4.3.

Water bath regulated at 20 ± 0,1 oC.

4.4.

Water bath regulated at 40 ± 2 oC.

4.5.

Thermometer, graduated to 0,5 oC.

5.1.

Mix the sample by inversion to disperse the fat and place in the water bath (4.4.). Allow the sample to reach a temperature of 40 oC and maintain at that temperature for five minutes. Mix thoroughly by careful inversion to ensure that the fat is homogeneously distributed. Cool to 20 oC in the second water bath (4.3).

5.2.

Mix the sample thoroughly by careful inversion to avoid inclusion of air. Pour the milk into the cylinder (4.2.), held inclined so as to avoid the formation of foam or of bubbles. Use sufficient milk sample to ensure that some will overflow from the cylinder when the hydrometer (4.1.) is placed in it. Carefully lower the hydrometer into the milk and allow to float freely when it reaches its balance position. The cylinder is to be placed vertical. The hydrometer must be positioned in the middle of the liquid column and should not touch the sides of the cylinder.

5.3.

When the hydrometer reaches equilibrium read the graduation at the top of the meniscus.

5.4.

Immediately after taking the hydrometer reading introduce the thermometer (4.5.) into the sample and read the temperature with an accuracy of 0,5 oC. The temperature must not differ more than ± 2 oC from ± 20 oC.

6.1.

If the temperature of the milk sample is not exactly 20 oC when the measurement of its specific mass is made, then the result obtained must be corrected adding to the determined specific mass 0,0002 for each degree Celcius above 20 oC, or subtracting 0,0002 for every degree Celsius below 20 oC. This correction is only valid if the temperature of the milk sample differs by not more than 5 oC from 20 oC.

mv

=

the specific mass of the sample read on the hydrometer (5.4) in g/l

MG

=

the fat content of the sample in g/1

0,92

=

the density of fat.

8.1.

Repeatability (r): 0,0003 g/ml.

8.2.

Reproducibility (R): 0,0015 g/ml.