2
4
Reagents andmaterials
4.1Reagents
Note: unless otherwise stated, the analytical reagents and 18.2MΩ deionizedwaterwere
used in thismethod.
4.1.1Sodium hydroxide
4.1.2Maleic acid (purity
＞
99%, Sigma)
4.1.3FructanAssayKit
（
Megazyme, K-FRUC
）
4.1.3.1Sucrase (100U)
/
β-amylase (500U)/pullulanase (100U)/maltase (1000U)mixed enzyme
4.1.3.2Fructanase.Highlypurified
exo
-inulinase at ca 10000U and
endo
-inulinase at ca 100U as
a freeze-dried powder.
4.1.4Sodium borohydride
4.1.5Acetic acid
4.1.6Sodium acetate trihydrate
4.1.7Sodium hydroxide solution,50%. Fluka
4.1.8Sodium acetate, anhydrous. Sigma.
4.1.9 Hexane
4.1.10 Nitrogen gas: purity≥99.99%
4.1.11Methanol
4.2Reagent preparation
4.2.1 Sodium hydroxide solution (1mol/L): dissolve 40g (with an accuracyof 0.01g) sodium
hydroxide (4.1.1) and dilute the solution to 1000mLusingwater.
4.2.2 Sodiummaleate buffer solution (100mmol/L, pH6.5): 1.16gmaleic acid is placed in a
150mLbeaker and dissolved in nearly70mLwater, the pH value is adjusted to 6.5 by1mol/L
sodium hydroxide solution, then the solution is diluted to 100mL. The solution can be stored for
3months at 4
℃
.
4.2.3 Sucrase (4.5U/mL)/β-amylase (23U/mL)/pullulanase (4.5U/mL)/maltase
(45U/mL):Sucrase (100U)/β-amylase (500U)/pullulanase (100U)/maltase(1000U) are dissolved
in 22mL sodiummaleate buffer solution and divided into 2mLpolypropylene tubes, which can
be stored for sixmonths at -20
℃
.
4.2.4 Sodium hydroxide solution (50mmol/L): dissolve 2 g (with an accuracyof 0.01g)
sodium hydroxide (4.1.1) and dilute the solution to1000mLusingwater.
4.2.5 Sodium borohydride solution(10mg/mL): 50mg (with an accuracyof 0.1mg) sodium
borohydride is placed in polypropylene tube(with screw cap), dissolved using50mmol/L sodium
hydroxide solution to the final concentration of 10mg/mL, which ismade upwhen used.
4.2.6 Acetic acid solution (200mmol/L):0.6mL acetic acid is diluted to 50mLusingwater.
4.2.7 Sodium acetate solution (200mmol/L): dissolve 1.36 g (with an accuracyof 0.01g)
sodium acetate trihydrate and dilute the solution to50mLusingwater.
4.2.8 Sodium acetate buffer solution (pH4.5
）：
mix 14mL acetic acid solutionwith 11mL
sodium acetate solution, and then dilute the solution to 50mLusingwater.
4.2.9Fructanase solution (
exo
-inulinase at ca 455U/mL,
endo
-inulinase at ca 4.55U/mL):
fructanase is dissolved in 22mL sodium acetate buffer solution and divided into 2mL
polypropylene tubes, which can be stored for sixmonths at -20
℃
.
FOS-02
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