4
increased 50μLenzyme), shake the tube and place it in a 40
℃
water bath shaker, shake it by
150r/min for 60minutes; then add 300μL sodium borohydride solution, shake the tube andplace
it in a 40
℃
water bath shaker, shake it by150 r/min for another 30minutes; take it out and cool
down to room temperature, add 750μLacetic acid solution, shake and stand for 10minutes, then
add 100μL fructanase solution (
exo
-inulinase 455U/mL,
endo
-inulinase 4.55U/mL)
(appropriately increase the amount of enzyme solution if the fructan content level is relatively
high in sample solution. If the 200μLbackup sample solution containing140μg fructan, 100μL
enzyme solution is enough. But if fructan content ismore than 140μg, for each additional 15μg
increased 10μLenzyme), shake the tube and place it in a 40
℃
water bath shaker, shake it
again by150 r/min for 30minutes, take it out and cool down to room temperature; transfer the
solution into a 10mLvolumetric flask, rinse the tube three timeswithwater, and dilute the
solution to themark and set-aside. The sample is passed sequentially through a 0.2μmmillipore
membrane and a purification column, discard the first three column volumes of solution and
collect the following solution, set-aside for test. The purification column should be activated
before use bypassing10mLofmethanol and then rinsingwith 15mLwater, set aside for 30
minutes.
The sample should be defatted according to the following stepswhen the fat content of the
sample is higher than 30%: 1g~5g (with an accuracyof 0.1mg, contains at least 25mg fructan)
homogenized sample is placed in a 100mLcentrifuge tubewith plug, add 50mLhexane, shake
the tube vigorously for 2minutes, centrifuge it by3000 r/min for 10minutes, discard the hexane,
repeat the above steps three times and evaporate the residual hexane, smash the sample using
glass rod and transfer it into a 150mLbeaker, rinse the centrifuge tube three times using80
℃
hotwater, thenmix thewashing liquors in the beaker. The following steps are referred to the
extraction process.
5.2 Instrument reference conditions
5.2.1 EluentA:water; Eluent B: sodium hydroxide (200mmol/L); Eluent C: sodium hydroxide
(150mmol/L), sodium acetate (500mmol/L). Gradient elution conditions are shown inTable 1:
Table1 Gradient elution conditions
Time,min
Eluent,%
curve
A
B
C
0~20
80
20
0
linear
20.1~30
0
0
100
linear
30.1~40
0
100
0
linear
40.1~50
80
20
0
linear
5.2.2 Detector: pulsed amperometric detector, Auworking electrode, Ag /AgCl reference
electrode, detection cell temperature: 30
℃
, the sugar detectionwaveform is shown inTable 2:
Table2 Sugar detectionwaveform
FOS-02
FORWORKINGGROUP/ERPUSEONLY
DONOTDISTRIBUTE
