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First Commission Directive of 25 October 1985 on methods of analysis for edible caseins and caseinates (85/503/EEC)

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This method determines the protein content of:

  • acid caseins,

  • rennet caseins,

  • caseinates,

except those containing ammonium caseinate or other ammonium or nitrogenous non-protein compounds.


The protein content: the nitrogen content as determined by the method specified and then multiplied by 6,38 and expressed as a percentage by mass.


A test portion is digested with a mixture of potassium sulphate and sulphuric acid, in the presence of copper (II) sulphate as catalyst, to convert organic nitrogen to ammoniacal nitrogen. The ammonia is distilled and absorbed into boric acid solution and then titrated with standard hydrochloric acid solution. The nitrogen content is converted to protein content by multiplying by 6,38.

4.1. Sulphuric acid, concentrated, S2O 1,84 g/ml.U.K.
4.2. Potassium sulphate, anhydrous (K2SO4).U.K.
4.3. Copper (II) sulphate pentahydrate (CuSO45H2O).U.K.
4.4. Sucrose (C12H22O11).U.K.
4.5. Boric acid, 40-g/l solution.U.K.
4.6. Sodium hydroxide, concentrated aqueous solution 30 % (m/m), carbonate free.U.K.
4.7. Hydrochloric acid, 0,1 mol/1.U.K.
4.8. Mixed indicator. Mix equal volumes of a 2 g/1 solution of methyl red in at least 95 % (V/V) ethanol and a 1 g/1 solution of methylene blue in at least 95 % (V/V) ethanol.U.K.
5.1. Analytical balance U.K.
5.2. Kjeldahl flask, 500 ml capacity.U.K.
5.3. Digestion apparatus to hold the Kjeldahl flask (5.2) in an inclined position and with a heating device which will not heat the part of the flask above the surface of the liquid contents.U.K.
5.4. Condenser with straight inner tube.U.K.
5.5. Outlet tube with safety bulb connected to the lower end of the condenser (5.4) by a ground glass joint or a rubber tube. If rubber tubing is used, the glass ends must be near one another.U.K.
5.6. Splash-head connected to the Kjeldahl flask (5.2) and to the condenser (5.4) by soft, close-fitting rubber or other appropriate stoppers.U.K.
5.7. Conical flask, 500 ml capacity.U.K.
5.8. Graduated cylinders, 50 ml and 100 ml capacity.U.K.
5.9. Burette, 50 ml capacity, graduated in 0,1 ml.U.K.
5.10. Boiling aids: U.K.
5.10.1.For the digestion: small pieces of hard porcelain, or glass beads.U.K.
5.10.2.For the distillation; freshly calcined pieces of pumice.U.K.
6.1. Preparation of the test sample U.K.

As described in Section 1.2 of the General Provisions.

6.2. Test for presence of ammoniacal nitrogen U.K.

If the presence of ammonium caseinate or other ammonium compounds is suspected, carry out the following test. Add to 1 gram of sample in a small conical flask 10 ml of water and 100 mg of magnesium oxide. Rinse down any magnesium oxide adhering to the walls and close the flask with a cork stopper, inserting a piece of moistened red litmus paper between the stopper and the neck of the flask. Mix the contents of the flask carefully and heat the flask in a water bath at 60 to 65 o C. If the litmus paper colours blue within 15 minutes ammonia is present, and the method is not applicable (see Section 1).

6.3. Blank test U.K.

At the same time as the determination of the nitrogen content of the sample perform a blank determination using 0,5 grams of the sucrose (4.4) instead of the test portion, using the same apparatus, the same quantities of all reagents and the same procedure as described in 6.5. If the titration in the blank determination exceeds 0,5 ml of 0,1 mol/1 acid, the reagents shall be checked and the impure reagent or reagents purified or replaced.

6.4. Test portion U.K.

Transfer to the Kjeldahl flask (5.2) 0,3 to 0,4 grams of the test sample (6.1), weighed to the nearest 0,1 mg.

6.5. Determination U.K.
6.5.1.Transfer to the flask a few pieces of porcelain or a few glass beads (5.10.1) and about 10 grams of the anhydrous potassium sulphate (4.2).U.K.

Add 0,2 g of the copper (II) sulphate (4.3) and wash down the neck of the flask with a little water. Add 20 ml of the concentrated sulphuric acid (4.1). Mix the contents of the flask.

Heat gently on the digestion apparatus (5.3) until any frothing has ceased, boil gently until the solution is clear and a pale green-blue colour persists. During heating, swirl the flask occasionally.

Continue the boiling, regulating the heating so as to condense the vapours in the middle of the flask neck. Continue the heating for 90 minutes avoiding local overheating.

Allow to cool to room temperature. Carefully add about 200 ml of water and a few pieces of pumice (5.10.2). Mix and cool again.

6.5.2.Transfer into the conical flask (5.7) 50 ml of the boric acid solution (4.5) and four drops of the indicator (4.8). Mix. Place the conical flask under the condenser (5.4) so that the tip of the outlet tube (5.5) is immersed in the boric acid solution. Using a graduated cylinder (5.8), add to the Kjeldahl flask 80 ml of the sodium hydroxide solution (4.6). During this operation, hold the flask in an inclined position so that the sodium hydroxide solution runs down the side of the flask to form a bottom layer.U.K.

Immediately connect the Kjeldahl flask to the condenser by means of the splash-head (5.6).

Gently rotate the Kjeldahl flask to mix its contents. Boil gently at first, avoiding any frothing. Continue the distillation so that 150 ml of distillate are collected in approximately 30 minutes. The distillate should have a temperature below 25 o C. About two minutes before the end of the distillation, lower the conical flask so that the tip of the outlet tube is no longer immersed in the acid solution, and rinse the tip with a little water. Stop heating, remove the outlet tube and rinse its outer and inner walls with a little water, collecting the washings in the conical flask.

6.5.3.Titrate the distillate in the conical flask, using the standard volumetric hydrochloric acid solution (4.7).U.K.
7.1. Formula and method of calculation U.K.

The protein content of the sample, expressed as a percentage by mass, is given by:



is the volume, in millilitres, of the standard volumetric hydrochloric acid solution (4.7) used in the determination (6.5);


is the volume, in millilitres, of the standard volumetric hydrochloric acid solution (4.7) used in the blank test (6.3);


is the strength of the standard volumetric hydrochloric acid solution (4.7) in mol/1;


is the mass, in grams, of the test portion.

Calculate the protein content to the nearest 0,1 %.

7.2. Repeatability U.K.

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 0,5 grams of protein per 100 grams of product.

This repeatability interval should be achieved in 95 % of the times that the method is correctly carried out.

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