Pagina 7 van 11
Take 200 µl and add 25 µl Beta-galactosidase (hydrolyse sample)
Take 200 µl phosphate buffer and add 25 µl Beta-galactosidase (blanc
hydrolyse sample)
Take 200 µl and add 25 µl 40mM phosphate buffer (no ribose) (blanc sample
to check evaporation inwaterbaths)
Homogenize the solutions and incubate for 30min at 60
°C
Warm up another waterbath to 100°C
Incubate the hydrolyzed samples and blancs for 10minutes at 100°C
Centrifuge the hydrolyzed samples for 10minutes at 13000 rpm
Add the centrifuged samples in special small volume vials
Analyse the 25 µl of the samples withmethod GOS quantification
Calculation:
Make polynomial order 2 calibration curves for galactose and lactose by setting the
concentration of the component (on the y axis) out against the area ratio of the
component and internal standard (on the x-axis) and force the calibration line
through zero. (the formula looks like y= ax
2
+ bx) Calculate the concentration in the
samples from these calibration curves. Correct the galactose concentration for the
blanc hydrolyse sample and calculate the amount of GOSwith the values of the
hydrolysed and blanc sample. GOS is calculated as followed (gram galactose/ 100
gram IMF)
(mL)
flask
c
volumetri
volume
e
step e
preparativ
before
sample
of
dilution
d
atement
column tre
Carbograph
/
collection
fraction
e
preparativ
by
dilution
c
galactose
as
expressed
lactose
b
mg/mL n to
calculatio
a
IMF)
g.
Gal/100
(g. GOS
) (
g. 100 50
dilution
25
500
342
180
1000
/
100g.)
gal/
(g.
Blanc
) (
g. 100 50
dilution
25
500
1000
blanc)
hydrolysed
-
(
/
100g.)
gal/
(g.
Incubation
blanc -
Incubated
IMF)
g. 100 gal/
. (
e
d
c
e
d
c
x
mg
Sample
ml
µg lac
mg
Sample
sample
hydrolysed
gal
ml
µg
g GOS
b
a
a
GOS-02
FORWORKINGGROUP/ERPUSEONLY
DONOTDISTRIBUTE
