J Sci Food Agric 1998, 76, 107È110

A Rapid Determination of the Total Sugar

Content and the Average Inulin Chain Length
in Roots of Chicory (Cichorium intybus L)
Chris Van Waes,* Joost Baert, Lucien Carlier and Erik Van Bockstaele
Plant Breeding Institute, Burg Van Gansberghelaan 109, 9820 Merelbeke, Belgium
(Received 29 July 1996 ; revised version received 1 April 1997 ; accepted 16 May 1997)

Abstract : A method is proposed to the chicory breeder for the fast screening of
total sugar content and average inulin chain length of lots of chicory roots. The
determination of the refractive index of the chicory root juice by refractometer is
the Ðrst step. This rapid analysis gives a good estimation of the total sugar
content (r \ 0É85 ; RSD \ 3É8%). In the next step extracts of chicory roots with a
high total sugar content are hydrolysed and analysed by chromatography. The
calculated fructose/glucose ratio predicts the average inulin chain length well
(r \ 0É91 ; RSD \ 5É3%). The molecular weight distribution of the inulin was
analysed by anion exchange HPLC with pulsed amperometric detection. ( 1998
SCI

J Sci Food Agric 76, 107È110 (1998)

Key words : chicory ; sugar content ; inulin ; chain length ; refractometer ; anion
exchange HPLC with pulsed amperometric detection

INTRODUCTION means of an anion exchange HPLC with pulsed

In the past the root of chicory has only been used as a
co†ee surrogate. Now, chicory is increasingly cultivated
for the extraction of inulin and its hydrolysis product
fructose. The chicory root contains small amounts of
free glucose, free fructose, sucrose and a lot of inulin, a
polymer consisting of 2È60 fructose units and a terminal
glucose. Inulin chains of most of all these various
lengths are present in the chicory root. A high total
sugar content and long inulin chains are preferred by
the sugar industry. Selecting genotypes that combine
these two features is a great part of the breeding work
(Baert and Van Bockstaele 1993). A method is proposed
for the fast screening of many chicory root samples.
We estimate the total sugar content as the brix value
by a refractometer. Samples with a high brix value are
hydrolysed. Their fructose and glucose content is mea-
sured by ion chromatography. The fructose/glucose
(F/G) ratio predicts the average inulin chain length in
the root. The composition of inulin was analysed by
* To whom correspondence should be addressed.
107
( 1998 SCI. J Sci Food Agric 0022-5142/98/$17.50. Printed in Great Britain
amperometric detection. The method is described in
detail, based on our own experience. We preferred this
method because of its ease and power of separation,
detection and quantiÐcation in comparison to other
chromatographic and enzymatic methods (Pontis 1990 ;
Chatterton et al 1993 ; Timmermans et al 1994).
This rapid determination technique is reliable if
I there is a good correlation between the brix-value
and the total sugar content.
I there is a good relationship between the F/G ratio
and the average inulin chain length.
The present work investigates these two conditions.
MATERIALS AND METHODS
Plant material and sample preparation
Samples were taken from plots of Ðeld trials with
chicory varieties and diploid and tetraploid halfsib fam-
ilies. Longitudinal quarters of 30 washed roots of each
108
plot were sliced, shredded by means of a cheese grater
to get small pieces of maximum 2 mm thickness, and
mixed. The juice of a pressed subsample was imme-
diately analysed by the refractometer. Two other sub-
samples were frozen and stored at [20¡C for the later
sugar analyses. Data of 718 plots of 11 trials, harvested
at di†erent dates, were used for the comparison of the
brix value with the total sugar content. We determined
the average inulin chain length of samples from 63
selected plots.
Refractometer
The refractometer analyses were performed on a
“Schmidt ] Haensch [ DURÏ digital refractometer tem-
perature controlled at 20¡C. The amount of soluble
components in the juice of chicory root was expressed
as the brix-value : this is the total sugar concentration in
% (w/v) of a solution with the same refractive index.
One measurement takes only a few seconds.
Ion chromatography
Carbohydrates are separated as anions using sodium
hydroxide, sometimes in combination with sodium
acetate, as eluent (Lee 1990). They are detected by oxi-
dation at a gold electrode using a repeating sequence of
three potentials (pulsed amperometric detection) to
avoid electrode fouling (Rocklin and Pohl 1983). Chro-
matography was performed on a “Dionex DX-300 ICÏ
equipped with a pulsed amperometric detector (PAD)
using “Dionex AI-450Ï software.
T otal sugar
Total sugar is deÐned as the amount of total glucose
and fructose after hydrolysis of the chicory root
samples. Shredded chicory root is treated with hydro-
chloric acid (0É2 M) at 85¡C for 1 h. Afterwards sodium
hydroxide is added to obtain neutral pH. After centrifu-
gation for 10 min at 1400 g the supernatant is frozen
and stored at [20¡C. Thawed samples are diluted so
that the total amount of sugar is about 40 mg litre~1.
Before injection the samples are Ðltered through a
45 km membrane Ðlter. The chromatographic condi-
tions are :
Analytical column CarboPac PA1 (4 ] 250 mm)
Guard Column : CarboPac PA1 (4 ] 50 mm)
Eluent : 100 mM NaOH ] 10 mM NaAc
Flow rate : 1 ml min~1
Injection volume : 25 kl
Detection : PAD at a gold electrode. The applied
potentials are 0É1 V (400 ms), 0É7 V (200 ms) and
[0É1 V (400 ms).
Under these conditions, separation of glucose and fruc-
tose is performed within 6 min. Standard samples con-
C V an W aes et al
tained 4 mg litre~1 glucose, 36 mg litre~1 fructose and
20 mg litre~1 fucose.
It has been observed that the detector response to
carbohydrates increases with time and that the rate of
increase is not identical for all detected sugars. To reach
the highest degree of precision each sample injection is
followed by a standard injection. The sample concentra-
tion is calculated based on the average response value
of the before and the after injected standard (Thompson
1990).
Inulin
The inulin present in the chicory roots is extracted by
use of hot deionised water (Berghofer et al 1993). There-
fore approximately 85 g deionised water at 85¡C is
added to 11É5 g defrosted shredded chicory root. The
mixture is put into a water bath at 85¡C for 1 h. During
this time it is shaken six times by hand. After cooling to
room temperature, deionised water is added to obtain
exactly 100É0 g. Then the samples are centrifuged for
10 min at 1400 g. The supernatant is frozen and
stored at [20¡C. The thawed samples are injected in a
concentration of about 23 g FW litre~1. The chromato-
graphic conditions are the same as described for the
analysis of total glucose and fructose except for the
eluent. Three eluents are used (E1 : 80 mM NaOH ; E2 :
80 mM NaOH ] 400 mM NaAc ; E3 : 1 M NaOH). Gra-
dients are used to obtain separation of the inulin
chains :
T ime (min) % E1 % E2 % E3
0È5 0 0 100
5È22 100 0 0
22È40 100È70 0È30 0
40È60 70È30 30È70 0
60È75 30È10 70È90 0
75È85 10È0 90È100 0
All gradient steps are linear. During the Ðrst step strong
alkali is used to clean the column of a previous injec-
tion. Afterwards, stabilisation with weak sodium
hydroxide is required to allow the separation of free
glucose and fructose. The injection takes place at
minute 11. A gradient is established by mixing E1 with
E2 in various ratios to separate the inulin chains of
increasing degree of polymerisation (DP). The concen-
tration of sodium hydroxide is kept constant. This
ensures an optimal pH for the detection (Chatterton et
al 1989 ; Churms 1990). Using this gradient method,
separation of fructans up to DP 60 is possible in about
90 min. In the same run, the amount of free glucose,
fructose and sucrose are determined.
Methods for quantitative analysis of inulin oligomers
are rare because of the lack of commercial available
standards. The average inulin chain length is expressed
as the number average degree of polymerisation (DP )
n
Determination of total sugar and average inulin chain length 109

Fig 1. Relationship between total sugar content (y) and brix-

value (x) (n \ 718). y \ 8É9 x [ 13É9. r \ 0É85***,
RSD \ 7É1 g kg~1. *** : signiÐcant at the 0É001 probability
level.

using the relative detector response values calculated by

Timmermans et al (1994). They have determined the
relative detector responses for DP 2 to 5 and for DP 10
to 17 followed by interpolation for DP 6 to 9 and
extrapolation for DP larger than 17.

RESULTS AND DISCUSSION

Brix-value and total sugar content

The mean of the total sugar content of the 718 plots

was 188 g kg~1 FW ranging from 134 g kg~1 to
233 g kg~1 with a standard deviation of 14 g kg~1 and
the average brix-value was 22É6 ranging from 17É9 to
27É4 with a standard deviation of 1É3.
The data of the 11 trials were pooled because regres- Fig 3. Chromatograms of the distribution of inulin chains in
sion coefficients were homogeneous (Gomez and Gomez chicory root. (A) chicory root with low DP (7É0). (B) chicory
n
root with high DP (11É0). (a) glucose ; (b) fructose ; (c) sucrose ;
n (d) DP2 ; (e) DP30.

1984). Figure 1 shows the relationship between the total

sugar content and the brix-value. The correlation coeffi-
cient was 0É85*** and the residual standard deviation
7É1 g kg~1 or 3É8%. As the F/G ratio may a†ect the
refractive index we also calculated the multiple regres-
sion relating brix-value and F/G ratio to the total sugar
content. Although the additional regression was signiÐ-
cant, the correlation coefficient only increased from 0É85
to 0É87 and the residual standard deviation only
decreased by 0É2%. The contribution of the F/G ratio
to the regression was six times smaller than the contri-
bution of the brix-value.

Fructose/glucose ratio and average inulin chain length

Fig 2. Relationship between the number average degree of
polymerisation (y) and the F/G ratio (x) for chicory roots
(n \ 63). y \ 0É97 x ] 1É83. r \ 0É91***, RSD \ 0É50. *** : The mean F/G ratio of the 63 analysed samples was 7É9,
signiÐcant at the 0É001 probability level. varying from 5É2 to 9É8 with a standard deviation of
110
1É1 ; and the average inulin chain length with a mean of
9É5 ranged from 6É9 to 11É5 with a standard deviation of
1É2. These results are comparable to those obtained by
Timmermans et al (1994) for chicory inulin. Figure 2
shows the relationship between DP and F/G. The
n Containing Crops, ed Fuchs A. Elsevier Science Publishers
correlation coefficient was 0É91***, the residual stan-
dard deviation was 0É50 units or 5É3%. Excluding the
free fructose, free glucose and sucrose from the F/G
ratio, the correlation coefficient increased to 0É92,
whereas the residual standard deviation decreased with
0É3%. Figure 3(A) and 3(B) show chromatograms of
inulin with low (7É0) and high (11É0) DP , respectively.
n mers by anion exchange chromatography. In : Studies in
They originate from samples of two chicory varieties
harvested at two extreme dates. Both variety and
harvest date a†ect the inulin chain length. Also the con-
tents of free fructose and sucrose increase with harvest
time whereas the contents of free glucose and inulin
decrease. (Baert 1997). The di†erence in average DP
corresponds to a shift in the chain length distribution
pattern.
CONCLUSIONS
The brix-value of shredded chicory roots gives a good
estimation of the total sugar content. Sample prep-
aration and analysis by refractometer is very simple and
fast. The F/G ratio measured after hydrolysis predicts
the average inulin chain length, determined by ion chro-
matography, very well.
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