Commission Regulation (EC) No 152/2009Show full title

K. DETERMINATION OF LACTOSE

1. Purpose and scope

This method makes it possible to determine the level of lactose in feed containing more than 0,5 % of lactose.

2. Principle

The sugars are dissolved in water. The solution is subjected to fermentation by the yeast Saccharomyces cerevisiae which leaves the lactose intact. After clarification and filtration the lactose content of the filtrate is determined by the Luff-Schoorl method.

3. Reagents

3.1. Suspension of Saccharomyces cerevisiae: suspend 25 g of fresh yeast in 100 ml of water. The suspension will keep for a maximum period of one week in a refrigerator.

3.2. Carrez solution I: dissolve in water 21,9 g of zinc acetate, Zn (CH₃ COO)₂ 2H₂O and 3 g of glacial acetic acid. Make up to 100 ml with water.

3.3. Carrez solution II: dissolve in water 10,6 g of potassium ferrocyanide K₄Fe (CN)₆ 3H₂O. Make up to 100 ml with water.

3.4. Luff-Schoorl reagent:

Stirring carefully, pour the citric acid solution (3.4.2) into the sodium carbonate solution (3.4.3). Add the copper sulphate solution (3.4.1) and make up to 1 litre with water. Leave to settle overnight and filter. Check the concentration of the reagent thus obtained (Cu 0,05 mol/litre; Na₂ CO₃ 1 mol/litre). The solution's pH shall be approximately 9,4.

3.4.1. Copper sulphate solution: dissolve 25 g of copper sulphate Cu SO₄ 5H₂O, free from iron, in 100 ml of water.

3.4.2. Citric acid solution: dissolve 50 g of citric acid C₆H₈O₇·H₂O in 50 ml of water.

3.4.3. Sodium carbonate solution: dissolve 143,8 g of anhydrous sodium carbonate in approximately 300 ml of warm water. Leave to cool.

3.5. Granulated pumice stone boiled in hydrochloric acid, washed in water and dried.

3.6. Potassium iodide, solution 30 % (w/v).

3.7. Sulphuric acid 3 mol/litre.

3.8. Solution of sodium thiosulphate 0,1 mol/litre.

3.9. Starch solution: add a mixture of 5 g of soluble starch in 30 ml of water to 1 litre of boiling water. Boil for three minutes, leave to cool, and if necessary add 10 mg of mercuric iodide as a preservative.

4. Apparatus

Water bath with thermostat set at 38-40 ^oC.

5. Procedure

Weigh 1 g of the sample to the nearest mg and place this portion of the sample in a 100 ml volumetric flask. Add 25 to 30 ml of water. Place the flask in a boiling water bath for 30 minutes and then cool to approximately 35 ^oC. Add 5 ml of yeast suspension (3.1) and homogenise. Leave the flask to stand for two hours in a water bath, at a temperature of 38-40^o C. Cool to approximately 20 ^oC.

Add 2,5 ml of Carrez solution I (3.2) and stir for 30 seconds, then add 2,5 ml of Carrez solution II (3.3) and again stir for 30 seconds. Make up to 100 ml with water, mix and filter. Using a pipette, remove an amount of filtrate which does not exceed 25 ml and which preferably contains from 40 to 80 mg of lactose and transfer it to a 300 ml Erlenmeyer flask. If necessary, make up to 25 ml with water.

Carry out a blank test in the same way with 5 ml of yeast suspension (3.1). Determine the lactose content according to Luff-Schoorl, as follows: add exactly 25 ml of Luff-Schoorl reagent (3.4) and two granules of pumice stone (3.5). Stir by hand-while heating over a free flame of medium height and bring the liquid to the boil in approximately two minutes. Place the Erlenmeyer immediately on an asbestos-coated wire gauze with a hole approximately 6 cm in diameter under which a flame has been lit. The flame shall be regulated in such a way that only the base of the Erlenmeyer is heated. Fit a reflux condenser to the Erlenmeyer flask. Boil for exactly 10 minutes. Cool immediately in cold water and after approximately five minutes titrate as follows:

Add 10 ml of potassium iodide solution (3.6) and immediately afterwards (carefully, because of the risk of abundant foaming) add 25 ml of sulphuric acid (3.7). Titrate with sodium thiosulphate solution (3.8) until a dull yellow colour appears, add the starch indicator (3.9) and complete titration.

Carry out the same titration on an accurately measured mixture of 25 ml of Luff-Schoorl reagent (3.4) and 25 ml of water, after adding 10 ml of potassium iodide solution (3.6) and 25 ml of sulphuric acid (3.7) without boiling.

6. Calculation of results

Using the attached table, establish the amount of lactose in mg which corresponds to the difference between the results of the two titrations, expressed in ml of sodium thiosulphate 0,1 mol/litre.

Express the result of anhydrous lactose as a percentage of the sample.

7. Observation

For products containing more than 40 % of fermentable sugar, use more than 5 ml of yeast suspension (3.1).

Table of values for 25 ml of Luff-Schoorl reagent

ml of Na₂ S₂ O₃0,1 mol/litre, two minutes' heating, 10 minutes' boiling

Na2 S2 O30,1 mol/litre	Glucose, fructose invert sugarsC6 H12 O6		LactoseC12 H22 O11		MaltoseC12 H22 O11		Na2 S2 O30,1 mol/litre
ml	mg	difference	mg	difference	mg	difference	ml
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23	2,4 4,8 7,2 9,7 12,2 14,7 17,2 19,8 22,4 25,0 27,6 30,3 33,0 35,7 38,5 41,3 44,2 47,1 50,0 53,0 56,0 59,1 62,2	2,4 2,4 2,5 2,5 2,5 2,5 2,6 2,6 2,6 2,6 2,7 2,7 2,7 2,8 2,8 2,9 2,9 2,9 3,0 3,0 3,1 3,1	3,6 7,3 11,0 14,7 18,4 22,1 25,8 29,5 33,2 37,0 40,8 44,6 48,4 52,2 56,0 59,9 63,8 67,7 71,7 75,7 79,8 83,9 88,0	3,7 3,7 3,7 3,7 3,7 3,7 3,7 3,7 3,8 3,8 3,8 3,8 3,8 3,8 3,9 3,9 3,9 4,0 4,0 4,1 4,1 4,1	3,9 7,8 11,7 15,6 19,6 23,5 27,5 31,5 35,5 39,5 43,5 47,5 51,6 55,7 59,8 63,9 68,0 72,2 76,5 80,9 85,4 90,0 94,6	3,9 3,9 3,9 4,0 3,9 4,0 4,0 4,0 4,0 4,0 4,0 4,1 4,1 4,1 4,1 4,1 4,2 4,3 4,4 4,5 4,6 4,6	1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23