Inulin and FOS
is a mixture of primarily oligofructose (Fm)
analysis: which
method to use in
food and feed
products?
Introduction
Inulin and FOS (fructo-oligosaccharides) are
increasingly used as a health enhancing
ingredient in all kinds of food and also feed
and pet food products. Both inulin and FOS
pass the stomach and small intestine
unchanged and are fermented in the large
intestine where they stimulate the growth
and/or activity of bacteria like lactobacilli and
bifidobacteria which are beneficial to the
health of the body.
Inulin
Inulin molecules consist of a linear series of
-(21) fructose units, and typically have a
terminal non-reducing glucose (GFn). In
native inulins the total number of fructose
units can go up to over 60.
Fructo-oligosaccharides/FOS
FOS is low molecular weight material with
degrees of polymerization of up to 10. There
are two types of FOS material, being
material prepared by hydrolysis of inulin (this
and some small inulin (GFn) material) and
material prepared by enzymatic elongation
of sucrose (primarily GFn, with n ranging
from 2 to 5).
Inulin (GFn) consists of a broad range of
molecules with a number of fructose units
that can range from 2 to over 50. GFn are
non-reducing carbohydrates, Fm has a
reducing fructose end group.
Currently two different AOAC methods are
described for the analysis of FOS and inulin
in foods, being the AOAC 997.08 and the
AOAC 999.03.
Methods of analysis
Both analytical methods for inulin/FOS have
typical different fields of application in the
analysis of (food) samples. The different
underlying principles of analysis are the
reason that there is not one golden
standard method. It is necessary to choose
the best method for different matrices. The
principle and the pros and contras of both
methods are described here. At the end of
this brochure, advice is given for choosing
the best method.
Method 1 AOAC 997.08
In this method first the free fructose and free
sucrose content are quantified. In the next
step after enzymatic conversion of starch
and maltodextrins, total glucose is measured.
The last step includes total conversion of
inulin/FOS and sucrose into glucose and
fructose and analysis of these components.
After three high performance anion
exchange chromatographic (HPAEC-PAD)
measurements the inulin/FOS content is
calculated by subtracting the glucose,
sucrose and fructose contents as measured
in step 1 and 2 from the total fructose and
glucose content as measured in step 3.
This implies that large corrections have to
be made for samples containing large
quantities of fructose, glucose, sucrose,
maltodextrin and/or starch. Subtraction of F3 and F4, so total recovery will be about
two large values in order to calculate much 80% or even lower for these preparations.
lower inulin/FOS values generally results in
less precise data with a large standard The advantages/disadvantages of this
deviation. The advantages/disadvantages of method are summarized in the table.
this method are summarized in the tables
below.

AOAC 997.08 official method inulin/FOS AOAC 999.03 official method inulin/FOS
analysis analysis
Pros Contras Pros Contras
Well suited for pure Large standard Well suited for all Incomplete recovery
ingredients deviation for kind of foods , of oligofructoses
samples high in especially when (Fm) leading to an
glucose, fructose, chromatography is underestimation of
sucrose, applied 10 20 % for certain
maltodextrins and/or FOS ingredients
starch Just one
Three chromatogram is
chromatograms needed for each
needed for each sample to analyse
sample to analyze

Method 2 AOAC 999.03 Chromatographic fingerprinting

The principle of this method differs from Inulin and FOS can be characterized by
AOAC 997.08 in that here all qualitative High Performance Anion
monosaccharides present after the Exchange Chromatography with Pulsed
combined amylases and sucrase treatment amperometric Detection (HPAEC-PAD).
are removed by converting them into alditols This chromatographic fingerprinting can give
(borohydride reduction). So, also samples insight in the GFn and Fm composition of
with high contents of monosaccharides, the inulin/FOS in the sample. This reveals
sucrose, maltodextrin and/or starch can be information whether application of AOAC
accurately measured in this procedure 999.03 will result in underestimation of the
because subtractions are not needed. measured inulin/FOS content.
The standard methodology of end point
measurement is colorimetry (total of
reducing sugars present after inulinase
treatment). Eurofins applies, like in AOAC
997.03, HPAEC-PAD, since this is specific
for glucose and fructose and not hindered by
turbidity, which is characteristic for e.g. dairy
samples.
However, there is a major drawback in this
method since also the reducing end groups
of Fm molecules are reduced into alditol and
so these end groups escape analysis. This
implies that recovery of difructose (F2) is
only 50%, for F3 67 %, for F4 75% etcetera. Chromatographic fingerprinting of a pure
GFn molecules do not have a reducing end short chain inulin (GFn) and a FOS (Fm)
group, so these are recovered completely. ingredient
FOS material prepared by depolymerization
of inulin contains in general high amounts of
Chromatographic fingerprinting of a pure
long chain inulin (GFn) and a FOS (Fm) The carbohydrate composition of inulin/FOS
ingredient enriched products can be very complex as
illustrated in the real life sample milk based
2902322 inuline
INULIN SAMPLE
100
nC powder drink. This product contains at least
Az

sucrose
GFn = inuline three different types of oligosaccharides,
Fn = FOS
75
being starch derived malto-oligosaccharides,
Glucose

GF2 = kestose
fructose

GF3 = nystose (Gn),inulins (GFn) and FOS (Fn).

50 GF10 GF20
GF4
GF5
GF7
GF30 Total dietary fibre analyses for inulin/FOS
25

F3
containing foods and feed
F4 F5 F6
The classical dietary fibre methods, AOAC
0
985.29 and AOAC 991.43 do not detect the
-20 min low molar mass soluble inulins and FOS
0.0 10.0 20.0 30.0 40.0 50.0 60.0
constituents. Applying these methods will
lead to the erroneous conclusion : no
detectable amounts of dietary fibre is
present. In case total dietary fibre including
inulin/FOS has to be determined, it is
advised to apply the AOAC 2009.01 for the
high molar weight dietary fibre (HMWDF)
and low molar weight dietary fibre (LMWDF)
or the AOAC 2011.25 method for soluble
and insoluble high molar weight dietary fibre
Typical
(IHMWDF and SHMWDF) and LMWDF..
Examples of food products enriched with
inulin and/or FOS ingredients
150
nC
Inulin containing
2952710fruit juice
The EUROFINS advice
Az

Glucose + fructose

120 Sucrose 1. Apply AOAC 999.03 for all samples

100
GFn = inulin
Fn = FOS
where GFn material (so normal
80
inulin or enzymatically elongated
sucrose) is the principal FOS/inulin
60
constituent.
GF4

GF5

GF6

GF10
GF7

GF15
GF8

GF20

40
2. Apply the AOAC 997.08 for purity
20 measurements in inulin/FOS
0
F3 F4
ingredients and raw materials
-20 min containing low contents of free
0.0 10.0 20.0 30.0 40.0 50.0 60.0
sugars and maltodextrins/starch.
3. If no knowledge is available about
FOS containing milk
3 powder product
the source of FOS/inulin in your
150
sucrose
glucose/galactose

nC
food sample, determine it by
Az

F3

F4 making a FOS/inulin HPAEC-PAD

Maltose

GFn = inuline
100 Fn = FOS
Gn = malto-oligosaccharide
oligosaccharide fingerprint
fructose

F5 (qualitative composition of all Fm

50 G3
G4
G5 F6 and GFn components).
GF7 4. For food samples containing
G8

0
depolymerized inulin (so with Fm)
G7
GF3
GF5
G6 and high amounts of glucose,
GF4
GF6
fructose, sucrose, maltose or
min
-50
0.0 10.0 20.0 30.0 40.0 50.0 60.0
maltotriose apply AOAC 999.03.
Also analyze your pure FOS
ingredient with this method in order
 to determine the recovery of the
total ingredient. With additional
knowledge on the non FOS
components (in general these are
glucose, fructose, sucrose and
water) in your ingredient a value for
FOS recovery can be calculated.

The inulin /FOS advice matrix

Ingredient type Product type Concentration G, F, S, M + Advised method
maltodextrin + starch
Inulin (GFn) Pure ingredient AOAC 999.03
Food product High AOAC 999.03
low AOAC 999.03

FOS (Fm) Pure ingredient AOAC 997.08

Food product High AOAC 999.03 + recovery of pure FOS ingredient
low AOAC 997.08

Mixture inulin Pure ingredient AOAC 997.08

and FOS Food product High AOAC 999.03 + recovery of pure inulin-FOS mixture
low AOAC 997.08

unknown Pure ingredient AOAC 997.08 in combination with chromatographic

(HPAEC-PAD) fingerprinting
Food product First determine ingredient type by chromatographic
(HPAEC-PAD) fingerprinting
G = glucose, F = fructose, S = sucrose, M = maltose

Description Method
Fingerprinting inulin/FOS Own method
Inulin AOAC 999.02
Inulin/FOS AOAC 997.08
Dietary fibre incl. inulin/FOS (HMWDF + LMWDF) AOAC 2009.01
Dietary fibre incl. inulin/FOS (IHMWDF + SHMWDF + LMWDF) AOAC 2011.25

Contact
Eurofins CarbohydratesTesting
Leeuwarderstraatweg 129
8441 PK Heerenveen
The Netherlands
Tel. +31 88 8310000
Fax +31 88 8310010
Email: carbohydrates@eurofins.com
Web: www.eurofins.nl
Web: www.carbohydratestesting.com