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INTEGRATED
TOTAL DIETARY FIBER
ASSAY PROCEDURE
INCLUDING
RESISTANT STARCH
AND NON-DIGESTIBLE
OLIGOSACCHARIDES

K-INTDF 04/20

AOAC Method 2009.01 & 2011.25

&
AACC Method 32-45.01 & 32-50.01
(100 Assays per Kit)

© Megazyme 2020
GENERAL INTRODUCTION:
It is generally believed1-3 that an increased consumption of dietary
fiber (DF) will lead to a reduction in conditions such as constipation,
diabetes, obesity, coronary heart disease and others. In the 1970’s
Trowell had developed a definition of dietary fiber which evolved in
19763 to:
“Dietary fiber consists of the remnants of edible plant cells, polysaccharides,
lignin, and associated substances resistant to digestion by the alimentary
enzymes of humans.”
This definition defines a macro constituent of foods which includes cellulose,
hemicellulose, lignin, gums, modified celluloses, mucilages, oligosaccharides,
pectins and associated minor substances such as waxes, cutin, and suberin.
On the basis of this definition,3 appropriate methodology for the
measurement of DF was developed by a consortium of researchers
in Europe and USA. This led to AOAC Official Method 985.29 (the
Prosky method),4,5 and to subsequent modifications of this method,
including AOAC Official Method 991.434 in which the buffers
were changed. The aim of these methods was to give an accurate
measurement of the content of total dietary fiber in plant products
and food materials. More specifically, the methodology aimed at
hydrolysing and removing starch and protein. Fats were removed
by the solvents employed to recover the non-hydrolysed material.
From the outset, it was realised that all protein was not hydrolysed,
so each sample was then analysed in duplicate and residues recovered
and weighed. One of these residues is analysed for ash content
and the other for protein. These weights are subtracted from the
average of the residue weights. It was also realised that, in the
analytical procedure, starch also was not completely hydrolysed and
removed. This in turn led to the discovery of so-called “resistant
starch (RS)”. The question then was, “should RS be measured and
added to the total dietary fiber value, or should it be analytically removed
and ignored?” Since RS escapes digestion in the human small intestine,
the general consensus is that it should be accurately measured
and included. Research in the 1990’s showed that AOAC Official
Method 991.43 underestimates RS, so alternative methods for the
measurement of this component were developed and evaluated.
While most of these new methods gave similar results for a range
of RS containing samples, none of the methods survived the rigours
of interlaboratory evaluation except that of McCleary et al.6 (AOAC
Method 2002.02), which also gave results in line with those obtained
from ileostomy patients.
In the mid-1990’s it was generally agreed that dietary fiber should
also include non-digestible oligosaccharides (NDO), as these behaved
physiologically as dietary fiber. Specific methods were developed for
fructan [and fructo-oligosaccharides (FOS)] (AOAC Methods 997.08

1
and 999.03),7,8 galacto-oligosaccharides (GOS) (AOAC Method
2001.03),9 resistant maltodextrins (RMD) (AOAC Method 2001.03)10
and Polydextrose® (AOAC Method 2000.11).11 The development
of these methodologies was very useful for ingredient developers,
food manufacturers and analysts measuring the specific component.
However, for those interested in measuring the total dietary fiber
content of a material, a problem was introduced. For many of
these specific carbohydrates (including RS, inulin and resistant
maltodextrins) a portion of the component is also measured by
AOAC Official method 985.29 and 991.43. Thus if the value for a
component determined using the specific method is added to the
total dietary fiber value, some of the component is double counted.
This is clearly depicted in Figure 1.

Galacto-oligosaccharides
Raffinose/Stachyose
Inulin FOS

Polydextrose
Fibersol 2
Total Dietary Fiber
(AOAC Method 985.29)
(AOAC Method 991.43)
Pectin
Arabinogalactan Cellulose
Beta-Glucan Resistant Starch
Galactomannan
Arabinoxylan

Figure 1. Components measured, and not measured, by AOAC

Official Methods 985.29 and 991.43.
This problem of potential double counting led us to research and
develop a procedure that allows the measurement of total dietary
fiber, which includes RS and NDO. This integrated total dietary
fiber (INTDF) procedure is depicted in Figure 2 and is modelled on
AOAC Official Methods 2002.02 (resistant starch), 991.43 (total
dietary fiber) and 2001.03 (resistant maltodextrins). The theory
of this method is discussed in detail in McCleary (2007)12 and has
been successfully subjected to an interlaboratory evaluation through
AOAC International (AOAC Method 2009.01).13 A modified method
for separately measuring insoluble dietary fiber (IDF), dietary fiber
soluble in water but precipitated in 78% aqueous ethanol (SDFP),
and dietary fiber soluble in water and not precipitated in 78%
aqueous ethanol (SDFS) has also been successfully evaluated (AOAC
Method 2011.25).14 SDFP was previously termed high molecular
weight soluble dietary fiber (HMWSDF) and SDFS was previously
termed low molecular weight soluble dietary fiber (LMWSDF) and
is also referred to as non-digestible oligosaccharides (NDO).
Several minor modifications to ensure complete measurement of FOS
and complete removal of non-resistant maltodextrins are described
2
by McCleary et al. (2013),15 as also are the problems in measurement
of RS4 (starch granules chemically cross-linked with phosphate
groups).
Sample (1.00 g) in sealed
250 mL bottle (in duplicate)

Add Maleate buffer + pancreatic α-amylase (PAA) + AMG

Incubate in shaking water bath at 37°C for 16 h.
Add Tris base solution to adjust pH to ~ 8.2
Incubate at 100°C for 20 min. Cool to ~ 60°C.
Add protease
Incubate at 60°C for 30 min. Cool to room temperature.
Add Acetic acid (to adjust pH to ~ 4.3) + Sorbitol (Internal Standard)

Remove 1 mL for Available

Filter through crucible with celite
CHO Determination

IDF residue SDFP + SDFS in filtrate

Add 4 volumes of ethanol to precipitate SDFP

SDFP (precipitate) SDFS (ltrate)

Figure 2. Principle of the Integrated (Codex compliant) Total

Dietary Fiber assay procedure showing separate measurement of IDF,
SDFP and SDFS.
In November 2008, the Codex Committee on Nutrition and Foods
for Special Dietary Uses (CCNFSDU) established a definition for
dietary fiber. This definition was accepted by the Codex Alimentarius
Commission (CAC) in 2009 (FAO, 2009),16 marking the achievement
of international consensus:
“Dietary fiber consists of carbohydrate polymers with ten or more
monomeric units, which are not hydrolyzed by the endogenous enzymes
in the small intestine of humans and belong to the following categories:
edible carbohydrate polymers naturally occurring in the food as consumed;
carbohydrate polymers which have been obtained from food raw material
by physical, enzymatic or chemical means and which have been shown
to have a physiological effect of benefit to health as demonstrated by
generally accepted scientific evidence to competent authorities, and;
synthetic carbohydrate polymers which have been shown to have a
physiological effect of benefit to health as demonstrated by generally
accepted scientific evidence to competent authorities.
a) When derived from a plant origin, dietary fiber may include fractions
of lignin and/or other compounds when associated with polysaccharides in
the plant cell walls and if these compounds are quantified by the AOAC
gravimetric analytical method for dietary fiber analysis: fractions of lignin
and the other compounds (proteic fractions, phenolic compounds,
3
waxes, saponins, phytates, cutin, phytosterols, etc.) intimately “associated”
with plant polysaccharides in the AOAC 991.43 method.
b) Decision on whether to include carbohydrates of 3 to 9 monomeric
units should be left up to national authorities.”

The Integrated TDF method described in this booklet, as

originally published in 2007,12 has been successfully subjected to
interlaboratory evaluation through AOAC International (Methods
2009.0113 and 2011.2514), and accepted by Codex Alimentarius as a
Type I method (March, 2011).

SCOPE:
Applicable to all samples containing dietary fiber, including RS and
NDO (also referred to as low molecular weight soluble dietary fiber;
LMWSDF; SDFS), e.g. cereal grains, fruit and vegetables, cereal and
fruit products and foods.

ASSAY KIT FOR INTEGRATED TDF:

Kits suitable for performing 100 assays of TDF (Integrated Procedure)
are available from Megazyme. The kits contain the full assay method
plus:
Bottle 1: Concentrated pancreatic α-amylase (E-PANAA);
4 g, 75,000 Ceralpha Units/g.
Stable for > 5 years when stored dry below -10°C.
Bottle 2: Amyloglucosidase (E-AMGDF) (20 mL,
3,300 Units/mL).
Stable for > 3 years at 4°C.
Bottle 3: Puried protease (E-BSPRT) (10 mL, 350 tyrosine
units/mL).
Stable for > 3 years below -10°C.
Bottle 4: LC Retention Time Standard [maltodextrins
plus maltose (4:1 ratio), approx. 5 g].
Stable for > 3 years; store sealed at room
temperature.
Bottle 5: D-Sorbitol (approx. 12 g, dry).
Stable for > 3 years; store sealed at room
temperature.

Celite 545®, acid washed, in 100 g or 500 g packages (cat. no.

G-CELITE), Amberlite® FPA53 (OH-) (cat. no. G-AMBOH) and
Ambersep® 200 (H+) (cat. no. G-AMBH) ion exchange resins are
available separately from Megazyme.

4
 AN INTEGRATED PROCEDURE FOR THE
MEASUREMENT OF TOTAL DIETARY FIBER
(INCLUDING RESISTANT STARCH AND
NON-DIGESTIBLE OLIGOSACCHARIDES)

A. PRINCIPLE:
An integrated procedure (AOAC Method 2009.01 and 2011.25) is
described for the measurement of total dietary fiber, including RS
and SDFS (i.e. NDO) of DP > 3. This method combines the key
attributes of AOAC Official Methods of Analysis 2002.02, 985.29,
991.43 and 2001.03. Duplicate test portions are incubated with
pancreatic α-amylase (PAA) and amyloglucosidase (AMG) for 16 h
at 37°C in sealed 250 mL bottles in a shaking water bath (Figure 3,
page 18) while mixing with sufficient vigour to maintain continuous
suspension. Alternatively, a 2mag Mixdrive 15® submersible magnetic
stirrer (www.2mag.de) can be used (Figure 4, page 18). During
this step, non-resistant starch is solubilised and hydrolysed to
D-glucose, maltose and traces of partially resistant maltodextrins by
the combined action of the two enzymes (These partially resistant
maltodextrins are subsequently hydrolysed before analysis of SDFS by
HPLC).15, 17 The reaction is terminated by adjustment of the pH to
8.2 and temporary heating. Protein in the sample is denatured and
digested with protease. Specific dietary fiber fractions are measured
as follows:
i. Insoluble dietary fiber (IDF), Higher Molecular Weight Soluble
Dietary Fiber (SDFP) and Lower Molecular Weight Soluble
Dietary Fiber (SDFS) determination. (AOAC Method 2011.25).
IDF is recovered by filtration of the aqueous reaction mixture and
the residue is washed, dried and weighed. SDFP in the filtrate is
precipitated with ethanol or industrial methylated spirits (IMS),
recovered, dried and weighed. Both the IDF and SDFP residues are
corrected for protein, ash and blank values for the final calculation of
IDF and SDFP. The aqueous ethanol filtrate from the SDFP fraction
is concentrated, desalted and analysed by HPLC for SDFS.
ii. Total High Molecular Weight Dietary Fiber (HMWDF)
and SDFS determination. (AOAC Method 2009.01). Four
volumes of 95% EtOH are added to the incubation mixture and
stirred. SDFP is precipitated from the incubation mixture and the
suspension is filtered. The HMWDF (comprising IDF and SDFP)
recovered on the crucible is washed, dried and weighed. This residue
weight is corrected for protein, ash and the blank value for the final
calculation. The aqueous ethanol filtrate is concentrated, desalted,
reconcentrated and analysed by HPLC for SDFS. Alternatively,
this fraction can more simply be deionised and analysed as
described in K-RINTDF (AOAC Method 2017.16).
5
The enzymes used in these methods are of very high purity; they are
effectively devoid of contaminating enzymes active on β-glucan, pectin
and arabinoxylan. NDO such as FOS and GOS are not hydrolysed,
and the degree of hydrolysis of Polydextrose® and Fibersol-2® is in
line with the information provided by the suppliers.
To ensure the presence of the appropriate enzyme activity, and
absence of undesirable enzyme activity, the materials listed below
(available in the kit, K-TDFC, from Megazyme) are analysed using
the entire procedure. Each new lot of enzymes should be tested, as
should enzymes that have not been tested for the previous 6 months.
Activity Sample Wt Expected
Test Sample Tested (g) Recovery (%)
Citrus pectin Pectinasec 0.1 87c
β-Glucan (barley) β-Glucanasea 0.1 95-100
Wheat starch α-Amylaseb 1.0 0-1
Casein Proteaseb 0.3 0-2
High amylose starchd α-Amylase 1.0 ~ 48
Galactan (larch) Pectinasec 0.1 ~ 84c
a This activity should not be present in the tests.
b This activity should be fully functional in the tests.
c Low values are mainly due to the moisture content of samples.
Similar values are obtained with AOAC Method 991.43 even
with no enzymes in the incubations.
d This material contains a high level of “enzyme resistant” starch.
This value is higher than TDF (29.3%) with AOAC Method
991.43.
B. APPARATUS:
a. Grinding mill.— Centrifugal, with 12-tooth rotor and 0.5 mm
sieve, or similar device. Alternatively, a cyclone mill can be used
for small test laboratory samples provided the mill has sufficient
air flow or other cooling to avoid overheating of samples.
b. Digestion Bottles.— 250 mL Fisherbrand® soda glass, wide
mouth bottles with with polyvinyl lined cap (cat. no. FB73219,
www.fisher.co.uk).
c. Fritted crucible.— Gooch, fritted disk, Pyrex® 50 mL, pore
size coarse, ASTM 40-60 µm, Corning® No. 32940-50C, or
equivalent.
Prepare four replicates for each sample as follows:
i. Ash overnight at 525°C in muffle furnace, cool furnace to 130°C
before removing crucibles to minimise breakage.
6
ii. Remove any residual Celite® and ash material by using a vacuum.
iii. Soak in 2% cleaning solution, [C(r)], at room temperature for 1 h.
iv. Rinse crucibles with water and deionised water.
v. For final rinse, use 15 mL acetone and air dry.
vi. Add approx. 1.0 g Celite® to dried crucibles and dry at 130°C to
constant weight.
vii. Cool crucible in desiccator for approx. 1 h and record mass of
crucible containing Celite®.
d. Filtering flask.— Heavy-walled, 1 L Büchner flask with side arm
(Figure 11, page 22).
e. Rubber ring adaptors.— For use to join crucibles with filtering
flasks (Figure 11, page 22).
f. Vacuum source.— Vacuum pump or aspirator with regulator
capable of regulating vacuum (e.g. Edwards XDS 10; single-phase
115/230V; product code: A72601906).
g. Water bath(s).— Rotary motion (150 rpm), large-capacity
(20-24 L) with covers; capable of maintaining temperature of 37
± 1°C and 60 ± 1°C; (e.g. Grant® OLS 200 shaking incubation
bath) (Figure 3, page 18). Alternatively, use a 2mag Mixdrive 15®
submersible magnetic stirrer with a 30 x 7 mm stirrer bar, set
at 170 rpm In a Megazyme water bath (cat. no. D-TDFBTH)
(Figure 4, page 18).
h. Balance.— 0.1 mg readability, accuracy and precision.
i. Ovens.— Two, mechanical convection, set at 103 ± 2°C and
130 ± 3°C.
j. Timer.
k. Desiccator.— Airtight, with silica gel or equivalent desiccant.
Desiccant dried biweekly overnight in 130°C oven.
l. pH meter.
m. Positive displacement pipettor.— e.g. Eppendorf Multipette®
- with 5.0 mL Combitip® (to dispense 0.1 and 0.3 mL aliquots
of AMG and 0.1 mL of protease solution).
- with 25 mL Combitip® (to dispense 3 mL aliquots of 0.75 M
Tris buffer solution and 4 mL aliquot of 2 M acetic acid).
n. Dispensers.— To dispense (1) 15 ± 0.5 mL of 78% v/v EtOH (or
IMS), 95% v/v EtOH and acetone, or (2) 40 ± 0.5 mL of buffer.
o. Cylinder.— Graduated, 100 mL and 500 mL.
p. Magnetic stirrers and stirring bars.— (7 x 30 mm; plain
magnetic stirrer bars; VWR International cat. no. 442-0269).
7
q. Rubber policeman spatulas.— VWR International cat. no.
53801-008 (Figure 11, page 22).
r. Muffle furnace.— 525 ± 5°C.
s. Polypropylene columns.— Bio-Rad, Econo-PacTM Disposable
Chromatography Columns (cat. no. 732-1010) with an Alltech
One-Way Stopcock (cat. no. 211524) (Figure 7, page 20).
t. Liquid Chromatograph (LC).— With oven to maintain a
column temperature of 90°C and a 50 µL injection loop.
System must separate maltose from maltotriose.
u. Guard column (or pre-column).— Waters Guard Pak® LC
pre-column inserts (part no. WAT015209) or equivalent.
v. LC column.— Waters Sugar-Pak® 6.5 x 300 mm column
(part no. WAT085188) or equivalent. Mobile phase distilled
water plus ethylene diamine tetraacetic acid; disodium calcium
salt (Na2CaEDTA) (50 mg/L) [C(l)]; flow rate 0.5 mL/min;
column temp. 90°C; run time 30 min to assure column cleaned
out.
w. Detector.— Refractive index (RI); maintained at 50°C.
x. Data integrator or computer.— For peak area measurement.
y. Filters for disposable syringe.— Millipore Millex® Syringe
Driven Filter Unit 0.45 µm (low protein binding Durapore
PVDF), 25 mm or 13 mm or equivalent.
z. Filters for water.— Millipore, 0.45 µm Durapore® Membrane
Filters type HVLP, 47 mm.
aa. Filter apparatus.— To hold 47 mm, 0.45 µm filter, [B(z)]; to
filter larger volumes of water.
bb. Syringes.— 10 mL, disposable, plastic.
cc. Syringes.— Hamilton® 100 µL, 710SNR syringe.
dd. Rotary evaporator.— Heidolph Laborota® 4000 or equivalent.
ee. Thermometer.— Capable of measuring to 110°C.

C. REAGENTS:
a. Ethanol (or IMS) 95% v/v.
b. Ethanol (or IMS) 78% v/v.— Place 821 mL 95% v/v ethanol
(or IMS) into a 1 L volumetric flask. Dilute to volume with
deionised water. Mix well. Check the level and if necessary add
more deionised water to bring it back up to the 1 L mark.
c. Acetone, reagent grade.
8
d. Stock amyloglucosidase (AMG) solution.—
(Bottle 2, page 4) 3,300 Units/mL in 50% v/v glycerol – Solution
is viscous; dispense using a positive displacement dispenser.
(Note: One Unit of enzyme activity is the amount of enzyme
required to release 1 micromole of D-glucose from soluble
starch per minute at 40°C and pH 4.5). AMG solution should
be essentially devoid of β-glucanase, β-xylanase and detectable
levels of free D-glucose. Stable for > 5 years at 4°C or below
-10°C.
e. Pancreatic α-amylase (50 Units/mL)/AMG (3.4 Units/mL).—
Immediately before use, dissolve 0.20 g of purified porcine
pancreatic α-amylase (75,000 Units/g; AOAC Method 2002.01)
(Bottle 1, page 4) in 290 mL of sodium maleate buffer (50 mM,
pH 6.0 plus 2 mM CaCl2 and 0.02% sodium azide) [C(h)] and
stir for 5 min. Add 0.3 mL of AMG [C(d)]. Stable for > 2 years
below -10°C.
f. Protease (50 mg/mL; 350 Tyrosine Units/mL) in 50% v/v
glycerol.— (Bottle 3, page 4). Solution is viscous; dispense
using a positive displacement dispenser. Protease must be
devoid of α-amylase and essentially devoid of β-glucanase and
β-xylanase. Use as supplied. Stable for > 3 years at 4°C.
g. Deionised water.
h. Sodium maleate buffer.— 50 mM, pH 6.0 plus 2 mM CaCl2
and 0.02% sodium azide. Dissolve 11.6 g of maleic acid in
1600 mL of deionised water and adjust the pH to 6.0 with
4 M (160 g/L) NaOH solution. Add 0.6 g of calcium chloride
dihydrate (CaCl2.2H2O) and 0.4 g of sodium azide and adjust
the volume to 2 L. Stable for > 1 year at 4°C.

NOTE: Do not add the sodium azide until the pH has been
adjusted. Acidification of sodium azide releases a poisonous gas.
Handle sodium azide and maleic acid with caution only after
reviewing SDS, using appropriate personal protective gear and
laboratory hood.
i. Tris buffer solution, 0.75 M.— Add 90.8 g of Tris buffer
salt (Megazyme cat. no. B-TRIS500) to approx. 800 mL of
deionised water (pH ~ 10.5) and dissolve. Adjust volume to
1 L. Stable for > 1 year at room temperature.
j. Acetic acid solution, 2 M.— Add 115 mL of glacial acetic acid
(Fluka 45731) to a 1 L volumetric flask. Dilute to 1 L with
deionised water. Stable for > 1 year at room temperature.

9
k. HCl solution, 150 mM.— Add 25 mL of conc. HCl (Sigma cat.
no. 258148 - 2.5 L, 37% v/v, 12 M) to 1.9 L of distilled water
and adjust to 2.0 L. Stable for > 3 years at room temperature.
l. Deionised water containing Na2CaEDTA (50 mg/L).—
Weigh 50 mg of Na2CaEDTA into a 1 L Duran bottle and
dissolve in 1 L distilled water. Prepare fresh weekly; filter
through 0.45 mm filter [B(z)] before use.
m. Sodium azide solution (0.02% w/v).— Add 0.2 g of sodium
azide to 1 L of deionised water and dissolve by stirring.
Stable for > 2 years at room temperature.
NOTE: Do not add sodium azide to solutions of low pH.
Acidification of sodium azide releases a poisonous gas. Handle
sodium azide with caution only after reviewing SDS, using
appropriate personal protective gear and laboratory hood.

n. D-Glucose LC standards (5, 10, 20 mg/mL).— Accurately

weigh 0.5, 1.0, and 2.0 g portions of high purity (> 99.5%)
D-glucose (Sigma cat. no. G5767) and transfer to 3 separate
100 mL volumetric flasks. To each flask pipette 10 mL of
internal standard [C(o)]. Bring to volume with 0.02% sodium
azide solution [C(m)]. Transfer solutions to 100 mL Duran®
bottles. Stable for 1 year at room temperature.
o. D-Sorbitol (Internal standard for SugarPak® column).—
100 mg/mL containing sodium azide (0.02% w/v). Weigh 10 g
of analytical grade (> 99%) D-sorbitol (Bottle 5, page 4) into
a 100 mL volumetric flask. Dissolve in 80 mL of 0.02% (w/v)
sodium azide solution [C(m)] and adjust to volume with 0.02%
sodium azide solution. Mix well. Stable for > 2 years at room
temperature or stable for > 4 years below -10°C .
NOTE: Handle sodium azide with caution only after reviewing
SDS, using appropriate personal protective gear and laboratory
hood.
p. LC retention time standard.— Standard having the distribution
of oligosaccharides (DP > 3) corn syrup solids (DE 25; Matsutani
Chemical Industry Co., Ltd., Itami City, Hyogo, Japan; www.
matsutani.com) analysed by LC plus maltose in a ratio of 4:1
(w/w). Dissolve 2.5 g of oligosaccharide mixture (Bottle 4,
page 4) in 80 mL of 0.02% sodium azide solution [C(m)] and
transfer to 100 mL volumetric flask. Pipette 10 mL of internal
standard [C(o)] into the flask. Bring to volume with 0.02%
sodium azide solution [C(m)]. Transfer solutions to 50 mL
polypropylene storage bottles®. Stable for > 1 year at room
temperature or stable for > 4 years below -10°C.
10
q. pH standards.— Buffer solutions at pH 4.0, 7.0 and 10.0.
r. Cleaning solution.— Micro-90® (International Products Corp.,
USA, www.ipcol.com). Make a 2% solution with deionised
water.
s. Mixed-bed ion exchange resins for each test portion.—
(1) m-1. — approx. 4 g Amberlite® FPA53 (OH-) resin (Rohm
and Haas, France S.A.S.) (see also Megazyme G-AMBOH),
ion exchange capacity 1.6 meq/mL (min) or equivalent (R-OH
exchange capacity data supplied by manufacturer).
(2) m-2.— approx. 4 g Ambersep® 200 (H+) resin or
equivalent, (Rohm and Haas, France S.A.S.) (see also Megazyme
G-AMBH), ion exchange capacity: 1.6 meq/mL (minimum).
Mix the two resins just prior to use and pack in column [B(s),
Bio-Rad disposable chromatography column] for analysis of each
test portion (see Figure 7, page 20). After mixing and packing,
overlay the resin with a small layer (wad) of cotton wool and
then wash the resin with 20 mL of deionised water.
t. Celite®.— acid-washed, pre-washed (Megazyme cat. no.
G-CELITE).

D. PREPARATION OF TEST SAMPLES:

Collect and prepare samples as intended to be eaten, i.e. baking
mixes should be prepared and baked, pasta should be cooked,
etc. Defat per AOAC 985.29 if > 10% fat. For high moisture
samples (> 25%) it may be desirable to freeze dry. Grind
~ 50 g in a grinding mill [B(a)] to pass a 0.5 mm sieve. Transfer
all material to a wide mouthed plastic jar, seal, and mix well by
shaking and inversion. Store in the presence of a desiccant.

E. ENZYME PURITY:
To ensure absence of undesirable enzymatic activities and
effectiveness of desirable enzymatic activities, run standards
(K-TDFC) each time the enzyme lot changes or after the
enzyme has been stored for more than 6 months.

F. ENZYME DIGESTION OF SAMPLES

(AOAC Methods 2009.01 and 2011.25):
(1) Blanks
With each assay, run two blanks along with samples to measure
any contribution from reagents to residue.

11
(2) Samples
(a) Weigh duplicate.— 1.000 ± 0.005 g samples accurately into
250 mL Fisherbrand® soda glass, wide mouth bottles [B(b)].
(b) Addition of Enzymes.— Wet the sample with 1.0 mL of
ethanol and add 40 mL of pancreatic α-amylase/AMG mixture
[C(e)] to each bottle. Cap the bottles. Transfer the bottles to a
Grant OLS 200 shaking incubation bath (or similar) [B(g)] and
secure the bottles in place with the springs in the shaker frame.
Alternatively, use a 2mag Mixdrive 15® submersible magnetic
stirrer [B(g)] with 7 x 30 mm stirrer bars (Figures 3 and 4, page
18).
(c) Incubation with pancreatic α-amylase/AMG.— Incubate
the reaction solutions at 37°C and 150 rpm in orbital motion
in a shaking water bath [B(g)]; or at 170 rpm on a 2mag
Mixdrive 15® submersible magnetic stirrer (to ensure complete
suspension) for exactly 16 h (e.g. 5.00 pm to 9.00 am).
(d) Adjustment of pH to approx. 8.2 (pH 7.9-8.4), Inactivation
of α-amylase and AMG.— After 16 h, remove all sample
bottles from the shaking water bath and immediately add 3.0 mL
of 0.75 M Tris buffer solution [C(i)] to terminate the reaction.
(At the same time, if only one shaker bath is available, increase
the temperature of the shaking incubation bath to 60°C in
readiness for the protease incubation step). Slightly loosen the
caps of the sample bottles and immediately place the bottles in a
water bath (non-shaking) at 95-100°C, and incubate for 20 min
with occasional shaking (by hand). Using a thermometer, ensure
that the final temperature of the bottle contents is > 90°C
(checking of just one bottle is adequate).
(e) Cool.— Remove all sample bottles from the hot water bath
(use appropriate gloves) and cool to approx. 60°C.
(f) Protease treatment.— Add 0.1 mL of protease solution [C(f)]
with a positive displacement dispenser (solution is viscous).
Incubate at 60°C for 30 min.
(g) pH adjustment.— Add 4.0 mL of 2 M acetic acid [C(j)] to each
bottle and mix. This gives a final pH of approx. 4.3.
(h) Internal standard.— Add 1.0 mL of D-sorbitol internal
standard solution (100 mg/mL) [C(o)] to each bottle and mix
well.
(i) Proceed to step [G(a)] for determination of HMWDF/
SDFS (AOAC Method 2009.01) or to step [H(a)] for
determination of IDF/SDFP/SDFS (AOAC Method
2011.25).
12
G. DETERMINATION OF HMWDF (IDF plus SDFP):
(AOAC Method 2009.01):

(a) Precipitation SDFP.— Pre-heat the sample to 60°C and add

192 mL (measured at room temperature) of 95% (v/v) EtOH
(or IMS) pre-heated to 60°C. Mix thoroughly and allow the
precipitate to form at room temperature for 60 min.
(b) Filtration setup.— Tare crucible containing Celite® {from [B(c)],
page 6} to the nearest 0.1 mg. Wet and redistribute the bed of
Celite® in the crucible, using 15 mL of 78% (v/v) EtOH or IMS
from wash bottle. Apply suction to crucible to draw Celite®
onto fritted glass as an even mat (see Figure 11, page 22).
(c) Filtration.— Using vacuum, filter precipitated enzyme digest
[G(a)] through crucible. Using a wash bottle with 78% (v/v)
EtOH (or IMS) [C(b)], quantitatively transfer all remaining
particles to crucible. Retain filtrate and washings and proceed to
step [I(a)] on page 15 for determination of SDFS.
(d) Wash.— Using a vacuum, wash residue sequentially with two
15 mL portions of the following: 78% (v/v) EtOH (or IMS), 95%
(v/v) EtOH (or IMS) and acetone.
(e) Dry crucibles containing residue overnight in 105°C oven. If
a forced air oven is used, loosely cover the crucibles with
aluminium foil to prevent loss of dried sample.
(f) Cool crucible in desiccator for approx. 1 h. Weigh crucible
containing dietary fiber residue and Celite® to nearest 0.1 mg.
To obtain residue mass, subtract tare weight, i.e. weight of dried
crucible and Celite®.
(g) Protein and ash determination.— The residue from one
crucible is analysed for protein and the second residue of the
duplicate is analysed for ash. Perform protein analysis on residue
using Kjeldahl or combustion methods (Caution should be
exercised when using a combustion analyser for protein in the
residue. Celite® volatilised from the sample can clog the transfer
lines of the unit). Use 6.25 factor for all cases to calculate mg
of protein. For ash analysis, incinerate the second residue for
5 h at 525°C. Cool in desiccator and weigh to nearest 0.1 mg.
Subtract crucible and Celite® weight to determine ash.
(h) Determination of HMWDF.— Subtract ash and protein from
average residue weight and proceed to step [ J ] for calculation
of HMWDF.

13
H. DETERMINATION OF IDF and SDFP SEPARATELY
(AOAC Method 2011.25):

IDF
(a) Filtration setup.— Tare crucible containing Celite® {from
[B(c)], page 6} to nearest 0.1 mg. Wet and redistribute the bed
of Celite® in the crucible, using 15 mL of 78% (v/v) EtOH (or
IMS) [C(b)] from wash bottle. Apply suction to crucible to draw
Celite® onto the fritted glass as an even mat (see Figure 11,
page 22).
(b) Filtration.— Using vacuum, filter the enzyme digest from step
[F(h)] through the crucible. Using a wash bottle with 60°C
deionised water, rinse the incubation bottle with a minimum
volume of water (approx. 10 mL) and use a rubber policeman
(spatula) to dislodge all particles from the walls of the container.
Transfer this suspension to the crucible. Wash the bottle with
a further 10 mL of water at 60°C and again transfer to the
crucible. Collect the combined filtrate and washings and adjust
the volume to 70 mL and retain this for determination of SDFP
[H(f)] and SDFS [I(a)].
(c) Wash.— Using a vacuum, wash the residue successively with
two 15 mL portions of the following: 78% (v/v) EtOH (or IMS),
95% (v/v) EtOH (or IMS) and Acetone. Discard the washings.
(d) Dry crucibles containing residue overnight in 105°C oven.
(e) Cool crucibles in desiccators for approx. 1 h. Weigh crucible
containing IDF residue and Celite® to nearest 0.1 mg. To
obtain residue mass, subtract tare weight, i.e. weight of dried
crucible and Celite®. Calculate IDF; step [ J ], as shown on
page 17.
SDFP
(f) Precipitation of SDFP.— Pre-heat the filtrate of each sample
(approx. 70 mL) to 60°C and add 280 mL (measured at room
temperature) of 95% (v/v) EtOH (or IMS) [C(a)] pre-heated to
60°C and mix thoroughly. Allow the precipitate to form at
room temperature for 60 min.
(g) Recovery of SDFP and SDFS.— Proceed according to step
[G(b)] to [G(h)] on page 13.
(h) For determination of SDFS.— Proceed according to step
[I(a)] to [I(f)] on pages 15-16.

14
I. DETERMINATION OF SDFS:
Note 1: Since the development and publication of the
INTDF method, an improved procedure for measurement
of TDF has been developed, namely, the Rapid Integrated
TDF method (AOAC Method 2017.16; Megazyme
cat no. K-RINTDF). We strongly recommend the use
of this method to simplify analyses and obtain more
physiologically relevant analyses.

Note 2: Proper deionisation is an essential part of

obtaining quality chromatographic data on SDFS.— To
obtain familiarity regarding the appearance of salt peaks in
the SDFS chromatograms, dissolve 10 mg of sodium chloride
in 9 mL of deionised water and add 1 mL of 100 mg/mL LC
internal standard [C(o)] and proceed to step [I(c)] at “Transfer
the solution to a 10 mL disposable……”. To ensure the resins
being used are of adequate deionising capacity, dissolve 10 mg
of sodium chloride in 1 mL of deionised water. Add 1 mL of
100 mg/mL LC internal standard [C(o)], and proceed to step
[I(b)] at “Transfer 2 mL of this solution to the top of.....”. The LC
chromatogram of this solution should show no peaks in the
time range corresponding to carbohydrates of DP3 or greater.
(a) Filtrate recovery and concentration.— {Set aside the filtrate
from one of the sample duplicates [G(c)] to use in case of spills
or if duplicate SDFS data is desired}. Transfer one half of filtrate
[G(c)] of the other sample duplicate to a 500 mL evaporator
flask and evaporate to dryness under vacuum at 60°C.
(b) Deionisation of sample.— Add 5 mL of deionised water to the
evaporator flask and swirl the flask for approx. 2 min to dissolve
the sample. Transfer the solution to a sealable polypropylene
20 mL container. Transfer 2 mL of this solution to the top
of the Bio-Rad disposable column [B(c)] containing 4 g each
of freshly prepared and thoroughly mixed, Amberlite FPA 53
(OH-) [C(s)] and Ambersep 200 (H+) [C(s)] (see Figure 7, page
22). Elute the column at a rate of 1.0 mL/min into a 100 mL
Duran® bottle. When the sample has entered the resin, add 2
mL of distilled water to the resin and allow this to percolate in.
Then add approx. 20 mL of deionised water to the top of the
column and continue to elute at a rate of 1.0 mL/min. Transfer
the eluate to a 250 mL round bottom rotary evaporator flask
and evaporate to dryness under vacuum at 60°C. Add 2 mL
of deionised water to the flask and redissolve the sugars by
swirling the flask for approx. 2 min. Using a Pasteur pipette,
transfer the solution to a polypropylene storage container.

15
(c) Preparation of samples for LC analyses.— Transfer the
solution to a 10 mL disposable syringe [B(bb)] and filter through
a 0.45 µm filter [B(y)]. Use a 100 µL LC glass syringe [B(cc)]
to fill the 50 µL injection loop on the LC [B(t)]. Perform this
analysis in duplicate. Column: Waters Sugar-Pak® (6.5 x 300
mm). Solvent: distilled water containing Na2Ca-EDTA
(50 mg/L). Flow rate: 0.5 mL/min. Temperature: 90°C.
(d) Determine the response factor for D-glucose.— Since
D-glucose provides an LC refractive index (RI) response
equivalent to the response factor for the non-digestible
oligosaccharides that make up SDFS the LC is calibrated using
D-glucose, and the response factor is used for determining the
mass of SDFS. Use a 100 µL LC syringe to fill a 50 µL injection
loop for each standard D-sorbitol/D-glucose solution. Inject in
triplicate. Obtain the values for the peak areas of D-glucose and
internal standard from the 3 chromatograms. The reciprocal of
the slope obtained by comparing the ratio of peak area of
D-glucose/peak area of D-sorbitol internal standard (y-axis) to
the ratio of the mass of D-glucose/mass of D-sorbitol (x-axis) is
the “response factor”. Determine the average response factor
(typically 0.97 for D-sorbitol).
Response factor (Rf) = (PA-IS) / (PA-Glu) x (Wt-Glu / Wt-IS)
where:
PA-IS = peak area internal standard (D-sorbitol).
PA-Glu = peak area D-glucose.
Wt-Glu = mass of D-glucose in standard.
Wt-IS = mass of D-sorbitol in standard.
(e) Calibrate the area of chromatogram to be measured
for LMWSDF.— Use a 100 µL LC syringe [B(cc)], to fill the
50 µL injection loop with retention time standard [C(p)]. Inject
in duplicate. Determine demarcation point between DP 2 and
DP 3 oligosaccharides (disaccharides maltose versus higher
oligosaccharides) (see Figure 10, page 22).
(f) Determine peak area of SDFS (PA-SDFS) and internal
standard (PA-IS) in chromatograms of sample
extracts.— Inject sample extracts [I(c)] on LC. Record area of
all peaks of DP greater than the DP2/DP3 demarcation point as
PA-SDFS. Record the peak area of internal standard as PA-IS.

NOTE: An in-line desalting procedure has been published.17 This

procedure is more convenient, but the disposable desalting
cartridges are costly and relatively few samples can be desalted with
one cartridge.

16
J. CALCULATIONS FOR HMWDF, IDF and SDFP:
Blank (B) determination (mg):

= BR1 + BR2 - P - P
B A
2

where:
BR1 and BR2 = residue mass (mg) for duplicate blank
determinations respectively.
PB and PA = mass (mg) of protein and ash respectively,
determined on first and second blank residues.

HMWDF, IDF or SDFP (mg/100 g):

R1 + R2 - PB - PA - B
2
= X 100
M1 + M2
2
where:
R1 = residue mass 1 from M1 in mg.
R2 = residue mass 2 from M2 in mg.
M1 = test portion mass 1 in g; M2 = test portion mass 2 in g.
PA = ash mass from R1 in mg; PB = protein mass from R2 in mg.

HMWDF (%) = HMWDF (mg/100 g)/1000

IDF (%) = IDF (mg/100 g)/1000
SDFP (%) = SDFP (mg/100 g)/1000

K. CALCULATIONS FOR SDFS:

SDFS (mg/100 g)
= Rf x (Wt-IS, mg) x (PA-SDFS)/(PA-IS) x 100/M
where:
Rf is the response factor (see page 16).
Wt-IS is weight in mg of internal standard contained in 1 mL of
internal standard solution pipetted into sample mixture (100 mg).
PA-SDFS is the peak area of the SDFS.
PA-IS is the peak area of the internal standard (D-sorbitol).
M is the test portion mass (M1 or M2) in grams of the sample whose
filtrate was concentrated and analysed by LC.

17
L. CALCULATION OF INTEGRATED TDF:
Integrated TDF (%) = HMWDF (%) + SDFS (%)

NOTE: These calculations can be simplified by using the Megazyme

Mega-CalcTM, downloadable from where the product appears on
the Megazyme web site (www.megazyme.com).

Figure 3. Picture showing mixing of samples (in rotary motion)

in a Grant® OLS 200 shaking incubation bath at 37°C.

Figure 4. Picture showing a 2mag Mixdrive 15® submersible

magnetic stirrer in a custom made water bath (Megazyme
water bath (cat. no. D-TDFBTH). This allows stirring of
15 samples at controlled speed (170 rpm) and 37°C.

18
 

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Figure 5. Rate of hydrolysis (and apparent resistant starch level) of

regular maize starch (RMS), high amylose maize starch (HAMS) and
native potato starch (PS) on incubation at 37°C with α-amylase/AMG
in Duran® bottles either shaken in rotary motion at 150 rpm in a
Grant® OLS 200 shaking incubation bath (Figure 3) or stirred at 170
rpm with α-amylase/AMG in Fisherbrand® bottles in a water bath at
37°C on a 2mag Mixdrive 15® submersible magnetic stirrer (Figure 4).
Note: The values for HAMS and RMS are the same with both mixing
and stirring. This was found for all samples studied except native
potato starch, which has a fragile granule structure.

Figure 6. HPLC traces for Raftilose P-95® dissolved in water and

analysed directly (A), compared with Raftilose P-95® recovered
as SDFS after running through the Integrated TDF procedure (B).
Column: Waters Sugar-Pak® (6.5 x 300 mm); solvent: distilled water
containing Na2Ca-EDTA (50 mg/L); flow rate: 0.5 mL/min; temp.
90°C. Note: No breakdown of the fructo-oligosaccharides. Also
note that F3 (fructo-triose from hydrolysed inulin) elutes after
sucrose (at the same point as lactose). For analysis of samples
containing F3, a separate pre-treatment before HPLC is required (see
Figure 10, page 22; also refer to the data sheet for E-SUCRBG).

19
Figure 7. Deionisation of samples with mixed bed resin [~ 4 g
Amberlite® FPA53 (OH-) and ~ 4 g Ambersep® 200
(H+)] in Bio-Rad, Econo-Pac® Disposable Chromatography
Columns connected to a Gilson Minipuls® Evolution pump.


 

 

 




 

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! 



 

!
 


 
 
! 






   

!
!



!

Figure 8. Elution of D-sorbitol and fructo-oligosaccharides from the

mixed bed resin column. Note that the rate of elution of
the D-sorbitol and fructo-oligosaccharides is the same and
that essentially all carbohydrate is eluted with 20 mL of
water.

20
 Sample (1.000 g) in duplicate

+
40 mL of maleate buffer (pH 6.0)
containing pancreatic α-amylase plus AMG

Incubate samples in a shaking water bath or on 2mag stirrer

at 37ºC for 16 h

Add 3.0 mL of 0.75 M tris buffer solution

(final pH ~ 8.2)
Heat at 95-100ºC for 20 min

Cool to 60ºC
Add 100 µL protease solution
Incubate at 60ºC for 30 min

Remove from water bath

Add 4.0 mL of 2 M acetic acid and 1 mL of internal standard, mix
(final pH approx. 4.3)

Precipitate by adding 192 mL of 95% (v/v) EtOH

pre-heated to 60ºC

Filter
Recover filtrate for
Dry Residue SDFS determination

2 residues

Protein Ash

High Molecular Weight

Dietary Fiber
(HMWDF)

Figure 9. Analytical scheme for the determination of HMWDF

(IDF + SDFP) and SDFS. If IDF and SDFP are to be
separately determined, filter the reaction mixture before
the addition of ethanol or IMS. The residue collected
on the filter is IDF. Four volumes of ethanol or IMS
are added to the filtrate to precipitate SDFP, which is
recovered on a Celite® filter. The filtrate contains SDFS.

21
Figure 10. Chromatography of a mixture of maltodextrins, maltose,
D-sorbitol and glycerol on a Waters Sugar-Pak® (6.5 x 300 mm);
solvent: distilled water containing Na2Ca-EDTA (50 mg/L); flow
rate: 0.5 mL/min; temperature 90°C. The arrows show demarcation
between DP 2 (maltose) and DP 3 (higher maltodextrins). NOTE:
F3, the fructosyl-trisaccharide produced on hydrolysis of inulin by
endo-inulinase or acid, elutes from the Sugar Pak® column after
maltose (see arrow, F3, in above figure and see Figure 6). Samples
containing this oligosaccharide, which is readily identified from the
HPLC pattern, require pre-treatment with sucrase/maltase/
β-galactosidase (see Frequently Asked Questions; FAQ) or see
recommended method in McCleary et al. (2013).15

Crucible

Rubber sleeve

Vacuum manifold
arrangement

Rubber policeman
on glass rod

Figure 11. Büchner flask, crucible, rubber sleeve and vacuum

manifold arrangement for filtration of dietary fiber samples. Picture
also shows sample bottle and rubber policeman on a glass rod.

22
M. REFERENCES:
1. Hippsley, H. (1953). Br. Med. J., 2, 420-422.
2. Burkitt, D. P., Walker, A. R. P. & Painter, N. S. (1972). Lancet ii, 1408-1412.
3. Trowell, H. C., Southgate, D. A .T., Wolever, T. M. S., Leeds, A. R., Gassull, M. A.
& Jenkins, D. J. A. (1976). Lancet a, 967.
4. Official Methods of Analysis of AOAC International (W. Horwitz, Ed.), 18th Ed
2005. AOAC International, Gaithersberg, USA.
5. Prosky, L., Asp, N-G., Furda, I., DeVries. J. W., Schweizer, T. F. & Harland, B. F.
(1985). J. AOAC Int., 68, 677-679.
6. McCleary, B. V., McNally, M. & Rossiter, P. (2002). J. AOAC Int., 85, 1103-1111.
7. Hoebregs, H. (1997). J. AOAC Int., 80, 1029-1037.
8. McCleary, B. V., Murphy, A. & Mugford, D. C. (2000). J. AOAC Int., 83, 356-364.
9. De Slegte, J. (2002). J. AOAC Int., 85, 417-423.
10. Gordon, D. T. & Okuma, K. (2002). J. AOAC Int., 85, 435-444.
11. Craig, S. A. S., Holden, J. F. & Khaled, M. J. (2000). J. AOAC Int., 83, 1006-1012.
12. McCleary, B. V. (2007). An integrated procedure for the measurement of total
dietary fibre (including resistant starch), non-digestible oligosaccharides and
available carbohydrates. Anal. Bioanal. Chem., 389, 291-308.
13. McCleary, B. V., DeVries, J. W., Rader, J. I., Cohen, G., Prosky L., Mugford, D. C.,
Champ, M. & Okuma, K. (2010). Determination of Total Dietary Fiber (CODEX
Definition) by Enzymatic-Gravimetric Method and Liquid Chromatography:
Collaborative Study. J. AOAC Int., 93, 221-233.
14. McCleary, B. V., Sloane, N., Draga, A. & Lazewska, I. (2013). Measurement of
total dietary fiber using AOAC method 2009.01 (AACC method 32-45.01):
evaluation and updates, Cereal Chemistry, 90, 394-414.
15. FAO 2009. Food and Agriculture Organization of the United Nations. Guidelines
for the use of nutrition claims: Table of conditions for nutrient contents (part b)
dietary fibre. ALINORM 09/32/26. Appendix II, page 46.
16. Brunt, K. & Sanders, P. (2013). Improvement of AOAC 2009.01 total dietary
fibre method for bread and other high starch containing matrices. Fd. Chem., 140,
547-580.
17. Post, B. E., Marshak, M. R. & DeVries, J. W. (2010). Simultaneous ion removal and
quantitation of low-molecular-weight dietary fiber from high-molecular-weight
dietary fiber filtrates using liquid chromatography. J. AOAC Int., 93, 234-242.

Bray Business Park, Bray,

Co. Wicklow,
A98 YV29,
IRELAND.
Telephone: (353 1) 286 1220
Facsimile: (353 1) 286 1264)
Internet: www.megazyme.com
E-Mail: [email protected]
WITHOUT GUARANTEE
The information contained in this booklet is, to the best of our knowledge, true and accurate, but since the
conditions of use are beyond our control, no warranty is given or is implied in respect of any recommendation or
suggestions which may be made or that any use will not infringe any patents.

23