ISO 26462:2010 pdf free download.Milk — Determination of lactose content — Enzymatic method using difference in pH.
ISO 26462:2010 specifies an enzymatic method for the determination of the lactose content of milk and reconstituted milk by measurement of the difference in pH (differential pH measurement).
3 Principle
B-Galactosidase is added to cleave lactose into glucose and galactose. At pH 7, 8, glucose is phosphorylated by glucokinase, thereby releasing protons that induce a change in pH. The pH change varies as a function of the lactose content of the sample and is measured by using a differential pH analyser.
4 Reagents
During the analysis, unless otherwise stated, use only reagents of recognized analytical grade and distilled or demineralized water or water of equivalent purity.
4.2 Enzyme solutions
4.2.1 Glucokinase enzyme solution
Dissolve 2, 57 mg of lyophilized glucokinase-1(GK1: 1 mg=350 U: EC 2.7.1.2)in 3 ml of glycerol with a volume fraction of 50. The activity of the glucokinase solution obtained shall be 290 U/ml +30 U/ml(see2.2).
The glucokinase enzyme solution can be kept for 6 months if stored at C
4.2.2 B-Galactosidase enzyme solution.
Dilute a concentrated -galactosidase (EC 3.2.1. 23) extract purified from contaminating enzymes with glycerol with a volume fraction of 50 % The activity of the -galactosidase solution obtained shall be 1500/ml±200u/ml.
4.3 Lactose standard solution(150 mmol/l)
Before use, determine the water content of lactose monohydrate powder by a Karl Fischer titration method, in order to correct for the quantity of lactose monohydrate used for the lactose standard solution. The correction should be based on the percentage of the determined water content in order to prepare a lactose standard solution containing 5, 404 g lactose monohydrate per 100 ml.