食品商务网-领先的网上食品贸易市场,食品行业门户网站(http://www.21food.

cn)

45.4.06A (f) Column oven.—Maintaining 40 ± 0.5°C.

AOAC Official Method 997.08 (g) Vacuum oven.—Maintaining 55 ± 3°C.
Fructans in Food Products (h) Reaction tubes.—15 mL, equipped with screw caps.
Ion Exchange Chromatographic Method (i) pH meter.—Temperature compensated, standardized with
First Action 1997 pH 4.0, 7.0, and 9.0 buffer solutions.
Final Action 1999
(j) Glass microfiber filters.—Retaining 0.7 µm particle size.
(k) Filter holder.—25 mm diameter, for low pressure syringe, or
(Applicable to the determination of added fructans in processed
foods.) equivalent.
(l) Syringes.—Luer lock, 10 mL, low pressure.
(m) Desiccator.—With SiO2, or equivalent desiccant. Every
See Table 997.08A for the results of the interlaboratory study sup- 2 weeks dry desiccant overnight at 130°C.
porting the acceptance of the method.
(n) Glass bottles.—100 or 150 mL with screw caps.
A. Principle (o) Magnetic stirrers and stir bars.
Fructans are extracted from the product with boiling water. (p) Water baths.—With shaker, maintaining 85 ± 2°C and
Aliquot of extract is hydrolyzed using lyophilized amyloglucosidase 60 ± 2°C.
to remove starch. Part of that hydrosylate is treated with inulinase (q) Volumetric flasks.—100 mL, 1 L, and 2 L.
followed by determination of released sugars. (r) Mortar with pestle.
The initial test portion and first and second hydrosylates are analyzed (s) Blender.
using high performance anion exchange chromatography with pulsed (t) Knife and scissors.
amperometric detection (HPAEC–PAD). In sugar analysis 1, free fruc- (u) Mixing rods.
tose and sucrose are determined in initial extract. In sugar analysis 2, (v) Vortex mixer.
sum of free glucose and glucose from maltodextrins and starch are de- (w) Pasteur pipets.—3 mL plastic pipets with large tip, or equivalent.
termined in the first hydrosylate. In sugar analysis 3, total amount of
glucose and total amount of fructose from the hydrosylate plus glucose C. Reagents
and fructose from the second hydrosylate are determined. For extraction, and for mobile phase and reagents preparation, use
Fructans are calculated from concentrations of glucose and fructose. H2O, ASTM quality Type 1, or ACS (reagent grade), or equivalent.
(a) Acetate buffer.—pH 4.5. Pipet 28.0 mL 0.2M acetic acid
B. Apparatus
(11.46 mL/L) and 22.0 mL 0.2M Na acetate (28.23 g trihydrate/L)
(a) Anion exchange chromatograph (HPAEC).—Liquid into 100 mL volumetric flask. Dilute to volume with H2O.
chromatograph gradient pump with eluent degas module, mi- (b) HCl solution.—0.05M. Dilute stock solution of known titer,
cro-injection valve, pulsed electrochemical detector working in e.g., 50 mL 1M HCl (86 mL/L) to 1 L with H2O.
pulsed amperometric detection mode (PAD), or equivalent, and au- (c) KOH solution.—0.05M. Dilute stock solution of known titer,
tomated sampler. e.g., 50 mL 1M KOH (56 g/L) to 1 L with H2O.
HPAEC conditions: column temperature, 40 ± 0.5°C; flow rate, (d) Lyophilized amyloglucosidase.—Containing <0.02% glu-
1.0 mL/min; injection volume, 50 µL; detector sensitivity, analog cose, fructose, and sucrose. Store at 4°C when not in use. (Sigma
range 1–3 µC. See Table 997.08B for eluent gradient and Ta- A7420, or equivalent.)
ble 997.08C for detector time program. (Note: Vary parameters to (e) Inulinase solution.—Containing <0.005% glucose, fructose,
optimize chromatography.) and sucrose (available as Fructozyme SP 230® solution from Novo
(b) HPAEC column.—250 × 4 mm id with pellicular an- Nordisk A/S Novo Alle, 2880 Dagsvaerd, Denmark). Store at 4°C.
ion-exchange resin with similar guard column (50 × 4 mm id). (f) NaOH solution.—50%, carbonate-free, density 1.54. NaOH
(c) Data integrator.—Carbopac PA1. solution is stable indefinitely stored under He.
(d) Analytical balance.—Accurately weighing to 0.1 mg. (g) Mobile phase A.—10mM NaOH solution, carbonate-free. Pre-
(e) Membrane filters.—0.2 µm porosity. pare carbonate-free solution as follows: Degas 2 L H2O at least

Table 997.08A Interlaboratory study results for determination of fructans in food and food products by ion exchange chromatography

Mean, g/100g
Samplea initial product sr sR RSDr, % RSDR, % rb Rc
Low-fat spread 8.1 0.23 0.39 2.9 4.8 0.65 1.10
Cheese spread 4.6 0.16 0.51 3.4 11.1 0.44 1.4
Chocolate 9.4 0.54 0.86 5.8 9.2 1.5 2.4
Wine gum 41.6 1.7 2.8 4.0 6.7 4.6 7.8
Powder drink mix 15.5 0.68 0.72 4.4 4.7 1.9 2.0
Biscuits 12.2 0.55 0.94 4.5 7.7 1.5 2.6
a
Blind duplicates.
b
r = 2.8 × sr.
c
R = 2.8 × sR.

© 2000 AOAC INTERNATIONAL

Table 997.08B Eluent grade for determination of fructans in (r) Sugars working standard solutions.—(1) S1.—50 mg of each
foods by ion exchange chromatography sugar/kg (glucoheptose, maltitol, dextrose, galactose, lactose, fruc-
% Mobile phase tose, and sucrose). Prepare by diluting 5.0 g glucoheptose internal
Time, min A B
standard solution, (q), and 5.0 g sugar standard solution, (p), to 100 g
with H2O. (2) S2.—50 mg glucoheptose/kg and 25 mg/kg of each
0 100 0
sugar: dextrose, maltitol, galactose, lactose, fructose and sucrose.
46 100 0 Prepare by diluting 5.0 g glucoheptose internal standards solution,
50 0 100 (q), and 2.5 g sugar standard solution, (p), to 100 g with H2O.
60 0 100 (3) S3.—50 mg glucoheptose/kg and 5 mg/kg of each sugar: dex-
69 100 0 trose, maltose, galactose, lactose, fructose and sucrose. Prepare by
diluting 5.0 g glucoheptose internal standard solution, (q), and 0.5 g
83 100 0
sugar standard solution, (p), to 100 g with H2O.

D. Preparation of Test Products

15 min with N or He in bottle of de-gas module. Without shaking or Homogenize test samples immediately before analysis as follows:
mixing 50% NaOH solution, (f), and while blowing He on liquid sur- Before mixing in blender, freeze sticky or fatty products (e.g.,
face, pipet 1.04 mL from the middle of bottle and add gently to de- chocolate and bars that form a paste-like mass in blender).
gassed H2O. Continue degassing solution 30 min before use. Cut gummy, sticky, and paste-like products that cannot be mixed
(h) Mobile phase B.—1M NaOH, carbonate-free. Degas H2O as in blender into small pieces with a knife or scissors so that particle
in (g). Without shaking or mixing 50% NaOH solution, (f), and size is ≤5 mm diameter.
while blowing He on liquid surface, pipet 109.5 mL from the middle Shatter hard products in mortar (e.g., hard candies), so particle
of bottle and add gently to degassed H2O. Continue degassing solu- size is ≤5 mm diameter.
tion 30 min before use.
(i) Glucose.—D(+)-Dextrose anhydrous, reagent grade. E. Extraction
(j) Fructose.—Levulose, reagent grade. See Figure 997.08 for flow diagram of extraction and hydrolysis.
(k) Sucrose.—Reagent grade. Accurately weigh to the nearest 0.1 mg test portion (M1) from D
(l) Glucoheptose.—D-Glucoheptose (Pfanstiehl Laboratories, Inc., containing ca 1 g fructan (but not exceeding 30 g and/or 5 g starch);
1219 Glen Rock Ave, Waukegan, IL 60085, USA, or equivalent). place in 100 mL beaker containing mixing rod. [Note: For starchy
(m) Lactose.—Monohydrate, reagent grade. products (e.g., biscuits, cakes, other bakery products and cereals)
(n) Galactose.—Reagent grade. weigh 5 g test portion (M1) in 250 mL beaker containing mixing
(o) Maltitol.—ca 98%. Store at 4°C when not in use (Sigma Chemi- rod.] See Table 997.08D for guide values.
cal Co., 3050 Spruce St, St. Louis, MO 63103, USA, or equivalent). Add ca 40 mL boiling H2O (add 100 mL to starchy products) and im-
(p) Sugars standard solution.—Dry glucose, fructose, sucrose, mediately check pH (Mecotrode, Hamilton P/H 238801/03, or equiva-
glucoheptose, and galactose reference sugar standards in vacuum lent) with mild agitation. pH should be between 6.5 and 8.0. If
oven 48 h at 55 ± 3°C. (Note: Do not dry lactose and maltitol.) Weigh necessary, adjust pH immediately with 0.05M KOH or 0.05M HCl.
100 mg maltitol, 100 mg dextrose, 100 mg galactose, 100 mg fruc- (Note: Continue pH measurement and adjustment until test portion is
tose, 100 mg lactose, and 100 mg sucrose and transfer into single completely dissolved.) Rinse electrode with boiling H2O.
100 mL volumetric flask. Dissolve sugars completely in H2O using Transfer solution quantitatively into weighed 100 mL volumetric
magnetic stirrer bar and plate. Add H2O to weight of 100 g. flask (for starchy products transfer solution to 250 mL flask), rinsing
(q) Glucoheptose internal standard solution.—Weigh 100 mg beaker with boiling H2O. Place flask in water bath for 10 min at
glucoheptose (l) and transfer into 100 mL volumetric flask. Dissolve 85 ± 2°C with continuous stirring.
sugar completely in H2O using magnetic stirrer. Add H2O to weight Let solution cool to room temperature. Dilute to 100 mL, weigh,
of 100 g. and homogenize. (Weight of solution is M2). From this step on, en-
sure that solutions are maintained at or above room temperature.
Because of possible presence of insoluble matter and/or fat in
some extracts, it may be difficult to homogenize them well. To en-
sure proper homogenization, proceed as follows: Shake volumetric
flask with extract very vigorously. Transfer extract into beaker and
Table 997.08C Detector time program for determination of fruc- continue very vigorous mixing using magnetic stirrer. While mix-
tans in foods by ion exchange chromatography ing vigorously, withdraw 2 aliquots using Pasteur pipet: 50 g aliquot
Time, s Tension, V (for direct analysis, A0), and 15 g aliquot (for hydrolysis, M3).
Dilute aliquots containing fructan to ca 1% fructan without any
0.00 0.05
heating.
0.20 0.05
0.40 0.05 F. Enzymatic Hydrolysis
0.41 0.65 Weigh 50 g homogenized mixture from E and set aside for direct
0.60 0.65 analysis (assay A0).
0.61 –0.10 (1) First hydrolyzation.—Transfer ca 15 g (weighed to nearest
10 mg) homogenized mixture (M3) into tared glass bottle with
1.00 –0.10
screw-cap and add the same amount of acetate buffer. pH of solution

© 2000 AOAC INTERNATIONAL

 enzyme for 50 mg starch, since 1 Unit liberates 1 mg glucose from
starch). If amount of starch and maltodextrins is unknown, consider
unknown part of test portion as 100% starch. For paste-like products
use 35 mg amyloglucosidase (with activity of 51 Units/mg); for other
products and starchy products use 10 mg.
Incubate mixture 30 min in water bath at 60 ± 2°C with constant,
mild agitation. Start timing 30 min from the moment reaction mix-
ture reaches 60°C. Ensure that during agitation no foam forms and
no air bubbles are brought into suspension. Let cool to room temper-
ature and weigh (net weight is M4). Weigh ca 10 g first hydrolyzate
and set aside for analysis (assay A1).
(2) Second hydrolyzation.—To the remaining part of the first
hydrolyzate (net weight is M5), add sufficient amount of inulinase solution,
taking into account amount of fructan present in test portion and enzyme
concentration (e.g., for Fructozyme SP 230® with activity of 1.8 Units/mg,
use 56 mg enzyme for 100 mg fructan, since 1 Unit hydrolyzes 1 mg
fructan; guide value 150 mg Fructozyme). If amount of fructans is un-
known, consider unknown part of test portion as 100% fructan.
Incubate again 30 min in water bath at 60 ± 2°C with constant,
mild agitation. Start timing 30 min from the moment reaction mix-
ture reaches 60°C. Ensure that during agitation no foam forms and
no air bubbles are brought into suspension. Let cool to room temper-
ature and weigh (net weight is M6; assay A2)
G. Determination of Mono- and Disaccharides
(a) Preparation of extract and hydrolyzates for HPAEC–PAD analy-
sis.—Dilute homogenized mixture from E, and first and second
hydrolyzates from F, so that glucose, fructose, and sucrose contents are
Figure 997.08—Flow diagram of extraction and within concentration range of sugars working standard solutions, and add
hydrolysis for determination of fructans in foods by ion glucoheptose internal standard solution as follows:
exchange chromatography. (1) Dilute 2.0 g glucoheptose internal standard solution, C(q), and
amount of homogenized mixture (M7), within range of glucose stan-
dard solution, to 100 g with H2O (assay A0).
should be 4.5 ± 0.05. If necessary, adjust with 0.05M KOH or 0.05M (2) Dilute 2.0 g glucoheptose internal standard solution, C(q), and
HCl. amount of first hydrolyzate (M8), within range of fructose standard
Add sufficient amount of amyloglucosidase, taking into account solution, to 100 g with H2O (assay A1).
amount of starch and maltodextrins present, and enzyme concentra-
tion (e.g., for amyloglucosidase with activity of 51 Units/mg, use 1 mg

Table 997.08D Guide values for dilutions for HPAEC-PAD fructan analysis for amounts of A0, A1, and A2 to be diluted to 100 g with
H2O

Product g M1 g A0 (M7) g A1 (M8) g A2 (M3)

Low-fat spread (8% inulin) 12.5 5 10 1
Cheese or cheese spread (5% inulin) 20 10 0.5 1 and 5
Chocolate (10% inulin) 10 1 and 10 4 1
Chocolate paste (10% inulin) 10 0.2 and 10 2 0.5
Bakery products (5% oligofructan) 5/250 0.5 and 25 4 2
Dry ice-mixa (15% oligofructan) 7 0.5 and 2 4 1
Vegetables (5% inulin) 20 10 10 1
Dairy products (3% oligofructan) 30 1.5 and 10 3 1
Fruit preparatesb (20% oligofructan) 5 2 1 0.25
Cereals (10% inulin) 5/250 15 1.5 3
Confectionery, sweet (35% oligofructan) 2.5 1 2 2
a
A powder for ice cream preparation.
b
Marmalade and fructan.

© 2000 AOAC INTERNATIONAL

 (3) Dilute 2.0 g glucoheptose internal standard solution, C(q), and Table 997.08E Calculations of sugar contents in test samples
amount of second hydrolyzate (M9), within range of sucrose stan- Assay
dard solution, to 100 g with H2O (assay A2).
Sugar A0 A1 A3
Locate guide values in Table 997.08D. (Note: Prepare 2 different
a
dilutions of the same test solution if large difference between concen- Glucose G1 Gtb
trations of different sugar compounds to be analyzed is expected.) Fructose Ffc Ftd
e
For foods with fat content of more than 5× fructan content, defat Sucrose S
diluted, filtered solutions before injection as follows: Transfer ca 4 g Maltitol Mal1f Mal2g
diluted solution in reaction tube and add 4 mL hexane. Agitate 5 min Galactose Gal1 h
Gal2i
on vortex mixture and centrifuge to separate hexane phase. Discard a
G1 = Sum of free glucose (Gf) and glucose from maltodextrins and starch (Gm).
hexane phase. b
Gt = Total amount of glucose.
Filter aqueous phase through glass microfiber filters, B(j), and c
Ff = Amount of free fructose in g/100 g initial test portion.
then through 0.2 µm membrane filter before injection. d
Ft = Total amount of fructose.
(Note: Use of internal standardization method is optional. Alter- e
S = Amount of sucrose in g/100 g initial test portion.
natively, external standardization method can be applied. Use the f
Mal1 = Amount of maltitol before inulinase hydrolysis.
same procedure as internal standardization method but without us- g
Mal2 = Amount of maltitol after inulinase hydrolysis.
ing internal standard. The response factor is then related to the re- h
Gal1 = Amount of galactose before inulinase hydrolysis.
sponse of standard compound itself.) i
Gal2 = Amount of galactose after inulinase hydrolysis.
(b) Determination.—Ensure that the same type of integration is
used on unknown test and standard working solutions by choosing
peak width, threshhold settings, and other integration parameters.
Carefully control baseline selection. Use peak height or peak area
for quantification. where Cs = concentration of sugar in diluted test solution, mg/kg; R =
Establish system’s linearity by running sugars working standard response factor; C s′ = concentration of glocoheptose (internal stan-
solutions S1, S2, and S3. Run analysis by bracketing duplicate test so- dard) in diluted test solution, mg/kg; Ps = height of sugar peak in di-
lutions with sugars working standard solutions (e.g., S1, test 1A0, test luted test solution; Ps′ = height of glucoheptose peak in diluted test
1A1, S2, test 1A2, test 2A0, S3, test 2A1, test 2A2, S1, test 3A0, test solution.
3A1, S2, etc.). Continue until all solutions have been analyzed. Calculate free fructose (Ff) and sucrose (S) contents; glucose (G1),
Use average response factors from sugars working standard solu- maltitol (Mal1), and galactose (Gal1) contents after hydrolysis with
tions bracketing tests to calculate sugar concentrations for each test amyloglucosidase; and total glucose (Gt) and total fructose (Ft) con-
solution. tents; and maltitol (Mal2) and galactose (Gal2) contents after hydroly-
(c) Possible interferences.—For products containing maltitol sis with inulinase solution in percent of initial test portion as follows:
and/or lactose (e.g., some chocolate, chocolate paste, cheese, and
breakfast drinks), glucose results after hydrolysis may be overesti- C Ff × M 2
Ff =
mated. Therefore, calculate maltitol content in products and correct M1 × M 7 × 100
glucose results for that part of maltitol that has been hydrolyzed by
inulinase solution. Correct glucose results also for that part of
where CFf = mg fructose/kg diluted solution assay A0.
galactose that has been formed by hydrolysis of lactose by inulinase
solution.
CS × M2
S=
H. Calculations M1 × M 7 × 100
Calculate response factors, R, of fructose, glucose, sucrose,
maltitol, and galactose as follows: where CS = mg sucrose/kg diluted solution assay A0.

C G1 × M 2 × M 4
R = (C/C¢) ´ (P¢/P) G1 =
M1 × M 3 × M 8 × 100

where C = concentration of sugar standard in working standard solu- where CG1 = mg glucose/kg diluted solution assay A1.
tion, mg/kg; C¢ = concentration of glucoheptose (internal standard),
mg/kg; P = height of sugar peak; P¢ = height of glucoheptose peak. C Mal1 × M 2 × M 4
Mal1 =
Using Table 997.08E calculate fructose and sucrose content in di- M1 × M 3 × M 8 × 100
luted solutions from assay A1, and fructose and glucose content in
diluted solutions from assay A2.
where CMal1 = mg maltitol/kg diluted solution assay A1.
Calculate maltitol and galactose contents in diluted solutions from
assays A1 and A2 as follows (use corresponding response factors and
C Gal1 × M 2 × M 4
peak height [or peak area] of the components): Gal1 =
M1 × M 3 × M 8 × 100

Cs = R × C s′ × (Ps/Ps′ ) where CGal1 = mg galactose/kg diluted solution assay A1.

© 2000 AOAC INTERNATIONAL

 C Gt × M 2 × M 4 × M 6 Gi = Gt – Gs – G1 – GMal – Glac
Gt =
M1 × M 3 × M 5 × M 9 × 100
Fi = Ft – Fs – Ff

where CGt = mg glucose/kg diluted solution assay A2. where Gs = glucose released from sucrose = S/1.9; GMal = glucose re-
leased from maltitol = [(Mal1 – Mal2)/1.9]; GLac = glucose released from
C Ft × M 2 × M 4 × M 6 lactose = (Gal2 – Gal1); Fs = fructose released from sucrose = S/1.9.
Ft =
M1 × M 3 × M 5 × M 9 × 100 Calculate fructan content (i) in % as follows:

i = k (Gi + Fi)
where CFt = mg fructose/kg diluted solution assay A2.
180 + 162(n − 1)
k=
C Mal2 × M 2 × M 4 × M 6 180n
Mal2 =
M1 × M 3 × M 5 × M 9 × 100
where n = average degree of polymerization = [(Fi/Gi) + 1] for pure
GFn mixtures. For inulin from chicory n = 10 can be used (k = 0.91).
where CMal2 = mg maltitol/kg diluted solution assay A2. For oligofructose, n = 4 can be used (k = 0.925).
Amount of glucose formed by hydrolysis of lactose may be calcu-
C Gal2 × M 2 × M 4 × M 6 lated by dividing difference in lactose content before and after
Gal2 =
M1 × M 3 × M 5 × M 9 × 100 inulinase hydrolysis by 1.9. Amount of glucose formed by hydroly-
sis of maltitol is the same as the amount of sorbitol formed by hydro-
lysis of maltitol by inulinase hydrolysis.
where CGal2 = mg galactose/kg diluted solution assay A2.
Calculate glucose related fructans (Gi) and fructose released from Reference: J.AOAC Int. 80, 1029(1997).
fructans (Fi) as follows: Revised: June 2000

© 2000 AOAC INTERNATIONAL