Technical Summary: Lactose Analysis (Bulletin ref. TB010414)
 

A simple two-step electrochemical enzymatic assay for lactose in aqueous solutions or extracts.
 

Name:
Code:
 

Lactose
GMRD-095S (50-100 tests)
 

Instruments:
 

GM10 Series analysers
 

Principle:

i) Lactose (milk sugar), a disaccharide, is stoichiometrically hydrolysed by galactosidase to a-D-glucose and galactose in a simple pre-reaction. Under the special buffer conditions used, mutarotation to ß-D-glucose rapidly occurs:
 

ii) In the presence of molecular oxygen, ß-D-glucose is oxidised by the enzyme glucose oxidase to gluconic acid and hydrogen peroxide:

Under the conditions of the assay, oxygen consumption is directly proportional to glucose concentration, which relates directly to the original lactose concentration. Any glucose originally present is measured directly after aqueous dilution as in the galactosidase pre-reaction.
 

Samples:

Aqueous solutions and extracts

Injected volume:
Pre-Reaction
Time:
Analysis time:
Calibrant:
 

25µl

Approx 15 seconds
20 seconds
5 %W/V lactose
 

Linearity:
 

ca 15 %W/V lactose as extract
 

Reagent Stability:

Shelf-life 9 months unopened stored at 0 - 5°C. Reconstituted ß-galactosidase
7 - 9 days (4 vials) if stored at 0 - 5°C. Aliquots may be frozen for extended life.

Note:

i) Sample opacity or turbidity presents no problem since the sensor system is electrochemical rather than spectrophotometric.
ii) Endogenous glucose, if present, should be determined as a sample 'blank',
i.e. extract diluted pro-rata in water instead of ß-Galactosidase.
iii) Incomplete hydolysis may take place for lactose concentrations greater than 10 %W/V. For greater accuracy at these levels repeat hydrolysis using a 12.5µl sample and scale results proportionally.