Commission Regulation (EC) No 273/2008 (repealed)Show full title

QUANTITATIVE DETERMINATION OF SKIMMED-MILK POWDER IN COMPOUND FEEDINGSTUFFS BY ENZYMATIC COAGULATION OF PARA-CASEIN

1. PURPOSE

Quantitative determination of skimmed-milk powder in compound feedingstuffs by enzymatic coagulation of para-casein.

2. SCOPE

This method applies to compound feedingstuffs containing at least 10 % skimmed-milk powder; large quantities of buttermilk and/or of certain non-milk proteins may lead to interferences.

3. PRINCIPLE OF THE METHOD

3.1. Dissolving of casein contained in the compound feedingstuff by extraction with sodium citrate solution

3.2. Adjustment of the calcium ion concentration to the required level to precipitate para-casein by addition of rennet

3.3. The nitrogen content of the para-casein precipitate is determined by the Kjeldahl method as described by standard ISO 8968-2:2001/IDF 20-2:2001; the quantity of skimmed-milk powder is calculated on the basis of a minimum casein content of 27,5 % (see 8.1).

4. REAGENTS

The reagents used must be of analytical grade. The water used must be distilled water or water of equivalent purity. With the exception of the rennet (4.5), all the reagents and solutions must be free of nitrogenous substances.

4.1. Trisodium citrate, dihydrate (1 % w/v solution)

4.2. Calcium chloride (about 5 M solution)

Dissolve 75g of CaCl₂ · 2 H₂O in 100 ml of distilled water by shaking (draw attention to exothermic reaction). Leave overnight and then filter the solution. Store the solution in a refrigerator.

4.3. 0,1 N sodium hydroxide

4.4. 0,1 N hydrochloric acid

4.5. Liquid calf rennet (strength about 100 IMCU/ml according to standard ISO 11815/IDF 157). Store in a refrigerator at 4 to 6 °C

4.6. Reagents for the quantitative determination of nitrogen according to the Kjeldahl method as described by ISO 8968-2:2001/IDF 20-2:2001.

5. APPARATUS

Common laboratory apparatus, including:

5.1. Mortar or homogeniser

5.2. Analytical balance capable of weighing to the nearest 1 mg, with a readability of 0,1 mg.

5.3. Bench-top centrifuge (500 g or 2 000 to 3 000 rpm) with 50 ml tubes and 2 000 g

5.4. Magnetic stirrer with (10 to 15 mm) followers

5.5. 150 to 200 ml beakers

5.6. 250 and 500 ml flasks

5.7. Glass funnels of 60 to 80 mm diameter

5.8. Fast-filtering ashless filters of diameter 150 mm (Whatman No 41 or equivalent)

5.9. Pipettes of various nominal volume

5.10. Thermostatically controlled water bath at 37 °C ± 1 °C

5.11. pH meter, accurate to 0,1 units

5.12. Thermometers, accurate to 1 °C.

6. PROCEDURE

6.1. Preparation of the sample

Grind in the mortar or homogenise in the mill 10 to 20 g of the sample to obtain a homogeneous mixture.

6.2. Dissolving of milk powder and separation of the insoluble residue

6.2.1. Weigh 1,000 ± 0,002 g of well-homogenised compound feedingstuff (6.1) directly into a 50 ml centrifuge tube. Add 30 ml of trisodium citrate solution (4.1) previously heated to 45 °C ± 2 °C. Mix with the aid of the magnetic stirrer for at least five minutes or by vigorous manual shaking.

6.2.2. Centrifuge at 500 g (2 000 to 3 000 rpm) for 10 minutes and decant the clear aqueous supernatant into a 150 to 200 ml beaker, taking care that no loose material on the bottom goes over.

6.2.3. Carry out two further extractions on the residue, according to the same procedure, adding the extracts to the first one.

6.2.4. If a layer of oil forms at the surface, cool in the refrigerator until the fat solidifies and remove the solid layer with a spatula.

6.3. Coagulation of casein with the enzymes of rennet

6.3.1. While stirring continuously, add dropwise 2 ml of calcium chloride (4.2) to the total aqueous extract (about 100 ml). Adjust the pH to 6,4-6,5 with solutions of NaOH (4.3) or HCl (4.4). Place in the thermostatically-controlled water bath at 37 °C ± 1 °C for 15 to 20 minutes to obtain saline balance. It becomes more evident by the formation of a light turbidity.

6.3.2. Transfer the liquid into one centrifuge tubes and centrifuge at 2 000 g for 10 minutes in order to remove the precipitated material. Transfer the supernatant, without washing the sediment, into one another centrifuge tubes.

6.3.3. Bring the temperature of the supernatant back to 37 °C ± 1 °C. While stirring the extract, add, dropwise, 0,5 ml of the liquid rennet (4.5). Coagulation occurs in two minutes.

6.3.4. Return the sample to the water bath and leave at a temperature of 37 °C ± 1 °C for 15 minutes. Remove the sample from the bath and break the coagulum by stirring. Centrifuge at 2 000 g for 10 minutes. Filter the supernatant through a suitable filter paper (5.8) and retain the filter paper. Wash the precipitate in the centrifuge tube with 50 ml of water at approximately 35 °C by stirring the precipitate.

Centrifuge again at 2 000 g for 10 minutes. Filter the supernatant through the filter paper retained previously.

6.4. Determination of casein nitrogen

6.4.1. After washing, transfer quantitatively the precipitate to the filter paper retained from 6.3.4 using distilled water. Transfer the dried filter paper to the Kjeldahl flask. Determine the nitrogen by the Kjeldahl method as described by standard ISO 8968-2:2001/IDF 20-2:2001.

7. BLANK TEST

7.1. A blank test shall be made regularly by submitting to mineralisation by the Kjeldahl method as described at standard ISO 8968-2:2001/IDF 20-2:2001. An ashless filter paper (5.8) moistened with a mixture of 90 ml (4.1) sodium citrate solution, 2 ml solution of calcium chloride (4.2), 0,5 ml of liquid rennet (4.5), and washed with 3 × 15 ml of distilled water

7.2. The volume of acid used for the blank test must be subtracted from the volume of acid (4.4) used for titration of the sample.

8. EXPRESSION OF RESULTS

8.1. The percentage of skimmed-milk powder in the compound feedingstuff is calculated by the following formula:

where:

N is the percentage of para-casein nitrogen;

27,5 is the factor for converting determined casein into the percentage of skimmed-milk powder;

2,81 and 0,908 are correction factors obtained from regression analysis.

9. ACCURACY OF THE METHOD

9.1. Repeatability

In at least 95 % of the cases studied, duplicate analysis of the same sample by the same operator in the same laboratory must give differences in the results equivalent to no greater than 2,3 g of skimmed-milk powder in 100 g of compound feedingstuff.

9.2. Reproducibility

In at least 95 % of the cases studied, the same sample analysed by two laboratories, must give differences in the results no greater than 6,5 g of skimmed-milk powder in 100 g of compound feedingstuff.

10. OBSERVATIONS

10.1. The addition of large percentages of certain non-milk proteins and especially of soya proteins, when heated together with skimmed-milk powder, may lead to too high results due to co-precipitation with the para-casein of milk.

10.2. Addition of buttermilk may lead to somewhat low figures, due to the fact that only the non-fat portion is determined. Addition of certain acid buttermilk may give considerably low figures, due to incomplete dissolving in the citrate solution.

10.3. Lecithin additions of 0,5 % or more may also lead to low results.

10.4. Incorporation of high-heat skimmed-milk powder may lead to too high figures due to the co-precipitation of certain whey proteins with the para-casein of milk.