Food Chemistry 81 (2003) 103–112

www.elsevier.com/locate/foodchem

Dietary fibre in cocoa shell: characterisation of component

polysaccharides
R. Redgwella,*, V. Trovatoa, S. Merinata, D. Curtia, S. Hedigera,b, A. Maneza,c
a
Nestlé Research Center, Nestec Ltd., Vers-chez-les-Blanc, PO Box 44, CH-1000 Lausanne 26, Switzerland
b
Laboratoire de Stéréochimie et des Interactions Moléculaires, Ecole Normale Superieure de Lyon, 46 Allee d’Italie, 69364 Lyon cedex 07, France
c
Nestlé R&D Centre, York, N.Yorkshire, YO 91 1XY, UK

Received 3 May 2002; received in revised form 29 July 2002; accepted 29 July 2002

Abstract
Polysaccharides were isolated from cocoa shells and characterised by compositional and linkage analysis. The polysaccharide
types were diverse and included pectic polysaccharides (  45%) which were made up of a heterogeneous mixture of rhamnoga-
lacturonans with variable degrees of branching. Hemicelluloses (  20%) consisted of a mixture of a fucosylated xyloglucan, galac-
toglucomannans, and glucuronoarabinoxylan. Cellulose accounted for  35% of the cell wall polysaccharides. The total dietary
fibre content was approximately 40%, not as high as previous reports. This was attributed to the fact that previous studies have
included a ‘‘Klason Lignin’’ fraction in estimates of fibre. A solid state NMR study of the cocoa shell ‘‘Klason Lignin’’ fraction
provided evidence that it contained little if any lignin and presumably consisted principally of protein–Maillard–tannin complexes.
# 2002 Elsevier Science Ltd. All rights reserved.
Keywords: Cocoa shells; Polysaccharides; Pectic polysaccharides; Hemicelluloses; Roasting; Lignin; Dietary fibre

1. Introduction 50% for total dietary fibre estimated by the method of

Currently, tonnes of cocoa (Theobroma cacao L.)
shells are disposed of as waste every year, a compelling
reason for researchers to come up with a more useful
and profitable outlet for this by-product of the cocoa
bean processing industry. The interest in functional
foods and the focus on potential health benefits of ele-
vated levels of dietary fibre in the human diet, invites the
speculation that cocoa bean shell could provide a ready
source of inexpensive dietary fibre in chocolate products.
The idea of using cocoa shell as an additive to human
foods is not new and several patents already exist for the
use of cocoa shell in this regard (Eggen, 1979; Hess,
1969; Kleinert, 1982; Manez & Barfuss, 1998). The
claims that cocoa shell contains high amounts of dietary
fibre are based on publications where the fibre in cocoa
shell has been estimated by standard procedures for
dietary fibre determination. Martin-Cabrejas, Valiente,
Estaban, Molla, and Waldron (1994), gave a value of
* Corresponding author. Tel.: +41-21-785-8681; fax: +41-21-785-
8554.
E-mail address: robert.redgwell@rdls.nestle.com (R. Redgwell).
0308-8146/03/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0308-8146(02)00385-0
Posky, Asp, Schweizer, De Vries, and Furda (1988)
while in a more recent publication, Bonvehi and Beneria
(1998), using the method of Englyst and Hudson (1987),
determined that total dietary fibre was 57%. Each of
these papers also published additional data on the neu-
tral sugar and uronic acid composition of the poly-
saccharide content of the shells. From these data, the
main constituents of insoluble fibre are glucose and
uronic acid with lesser amounts of galactose, arabinose,
xylose and mannose. These sugars indicate that the cell
wall polysaccharides in cocoa shell are predominantly
cellulose with lesser amounts of pectin and hemi-
cellulose also present. No attempt had been made to
characterise the structural features of these poly-
saccharides further.
The standard protocols for dietary fibre determina-
tion, as set out in the AOAC official methods of analysis
(1984), provide convenient, dependable procedures
which allow comparisons of dietary fibre content in dif-
ferent plant tissues to be made, for the most part, with a
measure of accuracy and reliability. However, the rela-
tively crude nature of the measurements, based as they
are on gravimetric determinations of insoluble fibre
104 R. Redgwell et al. / Food Chemistry 81 (2003) 103–112
fractions, can be subject to gross errors where plant tis-
sues contain tannin/protein complexes and /or products
of Maillard reactions (Bartolomé, Jiménez-Ramsey, &
Butler, 1995). Many of these components are insoluble
in 72% sulphuric acid and so are recovered as ‘‘Klason
Lignin’’, giving an artificially elevated figure for inso-
luble fibre in tissues which contain them. To date, no
effort has been made to validate the lignin values pub-
lished for cocoa shells.
While it is customary to ascribe the health benefits of
dietary fibre to the total dietary fibre content of a plant
tissue, there is mounting evidence that specific compo-
nents are more effective than others for benefiting
human health. Failure to distinguish different types of
cell walls, or polysaccharides with a particular structural
feature, may provide a major limitation to current
assessments of the role of dietary fibre in human nutri-
tion and disease.
The aim of the present study was therefore two-fold:
to compare the dietary fibre value of cocoa shells
obtained by a standard gravimetric procedure with that
determined by chemical and spectroscopic means
designed specifically for the estimation of poly-
saccharide and detection of lignin, and to determine the
relative proportions of the various chemical types of
polysaccharide in cocoa shell and characterise their
major structural features.
2. Materials and methods
2.1. Plant material
Cocoa (Theobroma cacao L.) shells were taken from
the winnowing of debacteriarised roasted West African
beans (Forastero). They were washed, boiled, dried, and
stored at 4 C pending analysis.
For a comparison of polysaccharide composition in
shell and nibs, whole fermented, non-roasted beans
(nibs plus shells, Forestero) were used. The shells were
separated from the nibs and each examined separately
for polysaccharide composition.
2.2. General
CWM and polysaccharide fractions were hydrolysed
with 2 M TFA for 1 h at 110 C. Sugar mixtures were
converted into alditol acetates for GLC analysis. The
column, SP-2330 fused silica (30 m  0.32 mm), was
maintained at 120  C for 2 min and then raised to 220
C at 25 C/min. The celluose content was determined by
the procedure of Updegraff (1969). The TFA-resistant
material was dissolved in 72% H2SO4 and the glucose
content determined after Saeman hydrolysis (Selven-
dran, March, & Ring, 1979) by GLC of the alditol
acetates. Uronic acid was determined by the method of
Blumenkrantz and Asboe-Hansen (1973). Protein con-
tent was determined by estimating the N content by
GLC and multiplying by a factor of 6.25.
Methylation of hemicellulosic polysaccharides was
performed using a modification of the method of Ciu-
canu and Kerek (1984). Carboxyl moieties of uronosyl
residues in polysaccharides were reduced to the corre-
sponding 6,6-dideuterioglycosyl residues prior to
methylation using the procedure of Kim and Carpita
(1992). GLC-MS of the partially methylated alditol
acetates was accomplished using an SP-2330 column
maintained at 70 C for 4 min, raised to 150 C at 25
C/min and then to 220  C at 4 C /min. Identifications
were based on peak retention times and comparison of
their electron impact mass spectra with published spec-
tra. The molar response factors reported by Sweet,
Shapiro, and Albersheim (1975) were used.
2.3. Dietary fibre measurement
Dietary fibre content was determined by a gravimetric
method (AOAC Collaborative Study 1984). Triplicate
samples of de-fatted cocoa shells were gelatinised with
Termamyl (heat stable a-amylase), then enzymatically
digested with protease and amyloglucosidase to remove
the protein and starch present. Total dietary fibre
(TDF) was calculated as the sum of soluble dietary fibre
(SDF) and insoluble dietary fibre (IDF) after correcting
for ash and undigested protein.
Dietary fibre was also measured by determining the
non-starchy polysaccharides by chemical means. The
polysaccharide content of all polymer fractions was
determined by acid hydrolysis of the fractions in either 2
M TFA or by Saeman hydrolysis (72% sulphuric acid).
Following Saeman hydrolysis of cell wall material the
insoluble material remaining was washed thoroughly,
freeze-dried and weighed to give ‘‘Klason lignin’’.
2.4. Isolation of cell wall material from shells
The dried shells were cryo-milled in liquid nitrogen
and the powder (10 g) refluxed in 100 ml 80% ethanol
for 15 min. The suspension was centrifuged, filtered and
the residue re-extracted in 100 ml of 80% ethanol for 2
h at room temperature. The residue was extracted in 100
ml chloroform/methanol (1:1) for 1.5 h, the step repe-
ated and followed by a 15 min extraction in 100 ml
methanol. The residue was extracted in 100 ml of phe-
nol/acetic acid/water (2:1:1 w/v/v) (PAW) overnight and
then with a further 100 ml of PAW for 2 h. The residue
after PAW extraction, henceforth referred to as cell wall
material (CWM), was dialysed, and during dialysis fur-
ther solubilisation of polysaccharides occurred. These
were added to those already extracted by PAW to give
the PAW-soluble fraction. The CWM and PAW-soluble
fraction were recovered after freeze-drying.
 R. Redgwell et al. / Food Chemistry 81 (2003) 103–112
2.5. Fractionation of CWM
Cell wall material was extracted sequentially with 0.05
M CDTA (6 h), 0.05 M Na2CO3 (18 h, 4 C, 2 h,
ambient) and 4 M KOH (2 h). All alkaline solvents
contained 20 mM NaBH4. The 4 M KOH-insoluble
material was dialysed against water and freeze-dried.
The solubilised polymers were recovered following neu-
tralisation, dialysis and freeze drying.
2.6. Purification of pectic and hemicellulosic
polysaccharides in 4 M KOH-soluble fraction
2.6.1. Cetyl trimethyl hexadecyltrimethylammoniumbromide
(CTAB) precipitation
In order to characterise the hemicellulosic poly-
saccharides contained in the 4 M KOH-soluble fraction,
it was subjected to CTAB treatment to remove the pec-
tic polysaccharides.
Approximately 1 g of the 4 M KOH-soluble was dis-
solved in 80 ml of water and stirred for 3 h. An equiva-
lent volume of 1% CTAB was added and, after stirring
briefly, the solution was allowed to stand overnight at
ambient temperature to allow the acidic fraction to
precipitate. Following centrifuging (10,000 rpm, 15
min) the supernatant, which contained the neutral
polysaccharides, was decanted and filtered through
glass fibre paper (GFA/3). The supernatant was
washed 3 with chloroform (30–40 ml) in a separat-
ing funnel, dialysed in 50% ethanol (5000 ml) over-
night and then in water for 3 days and freeze-dried
(282 mg).
The precipitate was dispersed in 4 M NaCl (4 ml) and
suspended in 10–15 ml ethanol saturated with sodium
acetate. This solution was changed several times over
two days. The supernatants were discarded and the final
pellet suspended in water, dialysed and freeze-dried (490
mg).
2.6.2. Ba(OH)2 precipitation and gel-permeation
chromatography of hemicellulosic polysaccharides
The hemicelluloses from the 4 M KOH extract were
dissolved in 11 ml 5% NaOH and then 11 ml of 5%
Ba(OH)2 were added. After mixing, the solutions were
left to stand for 3 h at 4  C. The precipitate (mannan-
enriched) was recovered after centrifuging, washed in
5% Ba(OH)2 and then dissolved in 5% NaOH. It was
neutralised with acetic acid and dialysed. The super-
natant (xyloglucan-enriched) was neutralised and dia-
lysed.
Each hemicellulose fraction was then subjected to gel-
permeation chromatography on a column (2.5  100
cm) of Sephacryl S-300 in 0.05M acetate buffer, pH 6.0.
The elution profile was monitored with phenol-sul-
phuric and fractions were recovered after dialysis and
freeze-drying.
105
2.6.3. Ion exchange chromatography of acidic
polysaccharide fraction
The acidic polysaccharide fraction from the 4 M
KOH extract was subjected to ion-exchange chromato-
graphy on a column (2.5  33 cm) of DEAE Sepharose
CL-6B, using a linear gradient consisting of 250 ml of
0.05 M and 250 ml of 1M imidazole buffer, pH 6.5.
Fractions (8 ml) were monitored for their carbohydrate
content by the phenol-sulphuric assay and appropriate
fractions recovered after dialysis and freeze-drying.
2.7. Enzyme treatment of 8 M KOH-insoluble residue
b-Mannanase (EC 3.2.1.78) from A. niger, Cellulase II
(endo-1,4-b-glucanase, EC 3.2.1.4) and cellobiohy-
drolase II (EC 3.2.1.91) from Trichoderma long-
ibrachiatum were purchased from Megazyme
International (Ireland).
The 8 M KOH-insoluble residue (100 mg) was sus-
pended in 5 ml pH 4.0 0.05 M acetate buffer containing
0.02% sodium azide. Mannanase (30 U), cellulase II (15
U) and cellobiohydrolase (15 U) were added and the
mixture stirred for 48 h at 37  C. The supernatant was
recovered, the pH adjusted to 5.5 and the solution
heated at 100 C for 10 min to inactivate the enzymes.
The solution, containing solubilised pectic poly-
saccharides was dialysed (MWCO 6–8 kDa) and freeze-
dried.
13
2.8. Solid state C-NMR measurements of lignin
NMR experiments were performed with a 4 mm CP/
MAS Bruker probehead on a DSX400 Bruker spectro-
meter operating at a proton frequency of 400MHz. The
magic angle sample spinning (MAS) frequency was set
to 15 kHz. To ensure a good sensitivity of carbonyl and
methyl group, which was essential to discriminate lignin
and tannin, adiabatic cross polarization (Hediger,
Meier, & Ernst, 1995; Metz, Wu, & Smith, 1994) with a
12 kHz ramp on the proton channel was used. Hart-
mann–Hahn matching was set on a cross-polarisation
(CP) sideband with a proton and carbon rf-field
strength set approximately on 53 and 68 kHz, respec-
tively. The contact time was 3 ms. TPPM decoupling
(Bennett, Rienstra, Auger, Lakshmi, & Griffin, 1995) at
80 kHz was applied.
3. Results and discussion
3.1. Preparation of CWM
CWM from cocoa shell was isolated by a modification
of the method (Redgwell & Hansen, 2000) used for the
preparation of CWM from cocoa beans (Fig. 1). The
low levels of starch (1%) in the shells meant that the
106 R. Redgwell et al. / Food Chemistry 81 (2003) 103–112

Fig. 1. Scheme for isolation of CWM from cocoa shell.

extraction step with 90% DMSO, which was used for
the beans, could be omitted. The procedure was
designed to isolate the cell wall polysaccharides (CWM)
in as pure as form as possible by using reagents which
selectively removed non-cell wall components, such as
low molecular weight metabolites and intracellular pro-
teins. In practice, some loss of a proportion of the cell
wall polysaccharides to some of the extractants is inevi-
table. However, these were recovered separately and
their polysaccharide contents determined.
In the present procedure, 80% ethanol was used to
solubilise the low molecular weight compounds (sugars,
amino acids and organic acids) and this fraction
accounted for approximately 10% of the shells (Table 1).
PAW is a reagent specifically designed to inactivate
endogenous enzyme activity (preventing inadvertent
degradation of the polysaccharides during their isola-
tion). In the case of the cocoa shells, this was unneces-
Table 1
Yield of chloroform/methanol-soluble, PAW-soluble and CWM frac-
tions isolated during CWM preparation of cocoa bean shells
(N=3S.E.)
Fraction Yield (g/10 g)
80% Ethanol 0.90
Methanol/chloroform 0.40
PAW-soluble 0.82
CWM 7.0
sary since the roasting had effectively inactivated all
endogenous enzyme activity. However, PAW is also a
very effective solvent for proteins and removes many of
the intracellular proteins which could contaminate the
final CWM by binding to the acidic cell wall poly-
saccharides. On a dry weight basis, the PAW-soluble
fraction accounted for 8.2% and the CWM 70% of the
starting material (Table 1).
3.2. Composition of CWM and other fractions
The major components of the CWM were glucose and
uronic acid indicating that pectin and cellulose were the
predominant polysaccharides (Table 2). Amounts of
pectic polysaccharide were also solubilised in the PAW-
soluble fraction and were characterised by a relatively
high rhamnose content. In the CWM, polysaccharides
accounted for only 45% of the dry weight. The remain-
ing material in the CWM could be accounted for as
protein (18.6%) ash (4.0%) and ‘‘Klason Lignin’’
(40%). The combined percentages total 107%, but some
of the 18.6% protein of the CWM is also recovered in
the ‘‘Klason lignin’’ fraction. Protein accounted for
approximately 27% of the ‘‘Klason lignin’’.
0.02
0.02 3.3. Fractionation and composition of CWM
0.03 polysaccharides
0.03

Total 9.1 0.05 Triplicate samples of the CWM were fractionated by

sequential extraction in 0.05 M CDTA, 0.05 M Na2CO3
 R. Redgwell et al. / Food Chemistry 81 (2003) 103–112 107

Table 2
Yield and monosaccharide composition of cocoa shell PAW-soluble fraction, CWM and fractions solubilised from CWM by extraction in 0.05 M
CDTA, 0.05 M Na2CO3 and 4 M KOH (n=3)

Fraction Yield Monosaccharide composition (mol%)

g/10 g CWM
Rha Fuc Ara Xyl Man Gal Glc UA Total mg/mg

PAW-sol – 7.0 0.2 2.8 0.8 3.9 9.4 4.1 71.0 697
CWMa – 3.3 0.4 4.0 4.5 5.0 6.4 42.0 34.5 450
CWM fractions
CDTA-sol 0.7 2.6 0.23 2.1 1.0 1.4 3.3 2.2 87.2 726
Na2CO3 -sol 1.2 13.5 0.1 1.5 0.5 1.1 14.9 1.8 66.0 663
4M KOH 1.7 2.0 1.8 4.7 23.5 9.9 9.6 21.8 26.2 363
Residue 5.1 2.0 0.2 6.2 2.3 4.2 3.0 66.9 15.5 420

and 4 M KOH. The polysaccharides solubilised by these
reagents and the fraction solubilised during dialysis of
the 4 M KOH-insoluble residue, were recovered and
their compositions determined.
Moderate amounts of pectic polysaccharide were
extracted in CDTA and 0.05 M Na2CO3. The CDTA-
soluble pectic polysaccharide fraction was very high in
uronic acid. This is consistent with a homo-
galacturonan-type structure, often associated with mid-
dle lamella pectin in the cell walls of higher plants. In
contrast, the Na2CO3-soluble fraction contained a
rhamnogalacturonan, as evidenced by a higher rham-
nose content, which is typically incorporated into the
galacturonic acid backbone.
Levels of uronic acid, xylose, glucose and mannose in
the 4 M KOH soluble fraction indicated the presence of
a mixture of hemicelluloses and pectic polysaccharides
in these fractions. To investigate the nature of the poly-
mers further, the acidic polysaccharides were separated
from the neutral polysaccharides by precipitating the for-
mer with CTAB. The monosaccharide compositions of
the acidic polymer fractions (CTAB ppt) and the neutral
polymer fractions (CTAB-soluble) are shown in Table 3.
The monosaccharide composition of the poly-
saccharides in the neutral 4 M KOH fraction was con-
sistent with the presence of a mixture of fucosylated
xyloglucan (XG) and galactoglucomannan (GGM).
Enriched fractions, containing various proportions of
XG and GGM, were obtained following Ba(OH)2 pre-
cipitation and gel-permeation chromatography on
Sephacryl S-300 (Fig. 2, Table 4). In previous studies,
this procedure has resulted in a good separation of XG
and GGM. However, in cocoa shell there was consider-
able co-precipitation of the XG with GGM during pre-
cipitation with Ba(OH)2 and, as the gel-permeation
profile shows, each polysaccharide was very poly-
dispersed with regard to molecular weight. Both the XG
and GGM polymers ranged in molecular size from 500
kDa (void volume of the column) to 10 kDa. Never-
theless, at least one gel-permeation fraction for the XG
(F2) and GGM (F3) was relatively enriched in XG and
GGM, respectively. Their monosaccharide composi-
tions are typical for a fucosylated xyloglucan and GGM
previously reported in the cell walls of dicotyledenous
plants (Redgwell & Fischer, 2002).
The acidic 4 M KOH fraction contained large pro-
portions of xylose (Table 3), which suggested the pre-
sence of either a xylogalacturonan (polysaccharide with
a rhamnose-galacturonic acid backbone with the rham-
nose substituted with single residues of xylose and/or, a
glucuronoxylan (polysaccharide with xylan backbone,
substituted with arabinose and glucuronic acid residues.
The fraction was subjected to ion-exchange chromato-
graphy on DEAE Sepharose CL 6B, using a buffer gra-
dient and several fractions were obtained (Fig. 3). The
first fraction to be eluted, once the gradient had com-
menced, was rich in xylose (F1) with lesser amounts of
uronic acid and this suggested the presence of an acidic
xylan rather than a xylogalacturonan. Subsequent frac-
tions contained less xylose but were rich in uronic acid
and rhamnose, indicating the presence of a rhamnoga-
lacturonan type molecule.
The residue remaining after 4 M KOH extraction also
showed evidence of the presence of pectic poly-

Table 3
Composition of acidic and neutral polysaccharide fractions from 4 M KOH-extract

Fraction Composition (mol%)

Rha Fuc Ara Xyl Man Gal Glc UA Total mg/mg

4 M KOH neutral 2.0 3.9 6.4 25.2 25.2 17.1 41.3 4.0 667
4 M KOH acidic 4.3 0.4 5.0 32.7 4.0 7.4 9.5 36.9 400
108 R. Redgwell et al. / Food Chemistry 81 (2003) 103–112

saccharides (Table 2). Some of these were released by

treating the 4 M KOH-insoluble residue with a mixture
of cellulase and mannanase and then subjecting the
solubilised fraction to ion-exchange chromatography to
remove neutral sugars. The recovered pectic fraction
showed a similar monosaccharide composition to that
of the F3 and F4 fractions (Table 5).

3.4. Structural features of polysaccharides

3.4.1. Pectic polysaccharides

Selected pectic fractions were subjected to carboxyl-
reduction by carbodiimide and their linkage types were
determined by methylation analysis (Table 6).
The Na2CO3-soluble fraction contained pre-
dominantly 4-galacturonosyl residues consistent with
the normal pectin backbone of linear galacturonosyl
residues. The backbone contained unusually high levels
of rhamnosyl residues, two thirds of which were 2,4
linked. These were the likely points of attachment of the
terminal galactose residues. All of the 4-linked galacto-
syl residues were deuterated, indicating that they were
Fig. 2. Gel-permeation profiles of XG- and GGM-enriched fractions derived from reduced galacturonosyl residues. The
on Sephacryl S-300. absence of any native 4-galactosyl residues in the poly-
saccharide suggested that the pectic sidechains consisted
of single terminal galactosyl residues attached to the O-
Table 4 4 position of the backbone rhamnosyl residues.
Monosaccharide composition of GGM, XG fractions, following gel- The 4 M KOH F3 fraction contained a mixture of
permeation chromatography on Sephacryl S-300 pectic polysaccharides and glucuronoarabinoxylan
Monosaccharide composition (mol%) (GAX). Linkage analysis indicated a rhamnogalactur-
onan.The presence of 5-arabinosyl residues was evi-
Rha Fuc Ara Xyl Man Gal Glc Total
mg/mg
dence that the molecule had some extended sidechains
of 5-linked arabinosyl residues. The pectin fraction,
XG solubilised from the 4 M KOH-insoluble residue resem-
F1 5.8 4.0 2.8 16.4 22.2 10.9 37.8 635
F2 0.8 5.8 3.0 28.6 11.5 11.9 38.4 843
F3 1.2 2.5 10.0 25.6 13.3 17.6 30.0 605
Table 5
GGM Monosaccharide composition of DEAE-Sepharose CL-6B fractions of
F1 0.8 5.6 0.8 28.6 12.8 10.5 40.9 750 acidic polysaccharides isolated from 4M KOH-extract and solubilised
F2 0.2 5.8 0.7 28.3 13.7 9.2 42.1 944 from the 4 M KOH-residue by enzyme treatment
F3 0.1 1.0 2.7 4.0 39.7 13.8 38.7 920 Fractions Wt.% Monosaccharide composition (mol%)

Rha Fuc Ara Xyl Man Gal Glc UA Total

mg/mg

4M KOH-sol
0.05 M 33.9 2.9 0.5 4.2 31.1 4.1 9.0 15.7 32.5 527
buffer

Gradient
F1 13.1 0.5 0.1 4.8 71.8 3.3 1.6 1.8 16.0 840
F2 10.0 4.7 0.3 8.1 43.7 8.8 9.6 3.6 21.2 750
F3 10.9 9.1 0.7 7.0 22.2 2.7 12.9 2.0 43.5 649
F4 21.3 3.7 0.3 2.2 5.2 1.5 4.6 1.4 81.3 630
1M 13.7 7.0 0.4 5.3 16.3 4.5 12.4 5.9 48.3 369
buffer
Res-sola – 6.9 0.5 6.0 5.6 2.2 7.2 8.9 62.9 534
a
Fig. 3. Ion-exchange chromatography of 4 M KOH-soluble acidic 4M KOH-residue treated with mannase and cellulase to solubilise
fraction on DEAE-Sepharose CL-6B. a pectic fraction.
 R. Redgwell et al. / Food Chemistry 81 (2003) 103–112 109

Table 6 of branching. Thus, it was impossible to separate all the

Glycosyl-linkage analysis of XG-, GGM- and GAX- enriched and GGM from the XG by the procedures used in this
selected pectic polysaccharide fractions
study. However at least one of the GGM fractions (F3
Composition (mol%) Table 4) was considerably enriched in GGM. Its back-
bone of b-1-4 glucosyl and mannosyl residues was much
Hemicellulosic polysaccharides Pectic polysaccharides
less branched than the GGM in the bean (4-man/4,6
XG GGM 4M Na2CO3- 4 M Res- man ratio 1.3:1 and 3.1:1 in the bean and shell respec-
F2 F3 KOH F1 sol KOH F3 sol tively). The degree of branching was not consistent for
t-Araf 1.0 2.7 0.7 tc 7.7 all the GGM of the shell. Thus, the 4-man/4,6 man
t-Arap – 4.4 ratios in GGM F1 and F2 of 4.8: 1and 4.3:1, respec-
2-Ara 1.4 tively, (data not shown) showed the presence of GGM
5-Ara 1.0 3.8 5.5
polymers with even less branching than F3.
t-Xyl 13.2 3.3 The third hemicellulose characterised in the shell was
2-Xyl 8.0 1.7 a GAX, characterised by a backbone of b-1–4 xylosyl
4-Xyl 60.9 16.0 7.3 residues. Approximately 1in 7 of the xylosyl residues
2,4- Xyl 10.5 3.6 was also substituted at O-4 with single glucuronosyl,
arabinosyl (pyranosyl and furanosyl) and xylosyl resi-
t-Fuc 4.1 0.6
dues. Small amounts of 2-galacturonosyl residues were
t-Rha also detected in this fraction. Whether these are struc-
2-Rha 5.4 4.6 5.4 tural components of the GAX, or belong to small
2,4-Rha 10.4 8.4 4.2 amounts of pectic polymers which co-purified with the
GAX, is not known.
t-Gal 7.9 15.9 14.1 9.0 7.5
2-Gal 6.2 4.8
4-Gal – 2.4 3.5. Lignin content of shells
2,4-Gal –
3,4-Gal – 1.7 The term ‘‘Klason lignin’’ has been, and still is used,
3-Gal 1.3
to describe a fraction of plant material which remains
t-Glc 3.1 3.0 3.0 3.1 insoluble after treatment with 12 M H2SO4 for 3 h at
4-Glc 13.2 27.5 2.0 2.7 room temperature and for 2 h in 1 M H2SO4 at 100  C
4,6-Glc 23.2 4.3 (Saeman hydrolysis). In some plants, the insoluble
3-Glc 1.2 – material resulting from this treatment represents lignin.
2-Glc 4.4
However, tannin/protein complexes and Maillard pro-
4-Man 10.6 30.5 1.5 2.2 3.3
4,6-Man 3.1 9.0 ducts from roasting are also, largely, insoluble after this
treatment. Therefore, in material which contains sig-
t-GalAa – nificant amounts of these complexes the term ‘‘Klason
4-GalAa – 2.1 59.1 32.0 60.6 lignin’’ is meaningless as a measurement of actual lignin.
2-GalAa 2.1
This has great significance for the estimation of dietary
3,4-GalAa – 3.8 2.8
fibre in such materials, because lignin is included in the
t-GlcAa – 10.1 3.0 7.0 1.0 definition of dietary fibre, whereas tannin/protein com-
plexes and Maillard products are not.
a
Analysed as 6,6-dideuterioglycosyl residues. When CWM from cocoa shells was subjected to
hydrolysis in 12 M sulphuric acid the recovered ‘‘Kla-
bled F3, except that it contained a higher proportion of son lignin’’ accounted for 36% of the original CWM.
2- rhamnosyl than of 2,4 linked rhamnosyl residues. This material was examined directly by solid state 13C-
NMR for the presence of lignin.
3.4.2. Hemicellulosic polysaccharides Lignin consists of 3 monomeric subunits—p-cou-
The glycosidic linkages in Table 6 are consistent with maryl, guiacyl, and sinapyl-propane. The last two con-
the same three types of hemicellulosic polysaccharide as tain methoxyl groups which give a strong 13C-NMR
previously reported for the cocoa bean (Redgwell & signal at a chemical shift of 55.5 ppm which is well clear
Hansen, 2000), i.e. a fucosylated XG, GGM and a of most of the spectrum for lignin and phenolic com-
GAX. pounds. The third lignin subunit (p-coumaryl) does not
While the XG showed similar structural features to contain a methoxyl group. However, the coumaryl sub-
that of the XG in bean, the GGM of the shell, which unit has not been reported as occurring in the lignin of
was present at higher levels than in the bean, showed dicotyledenous plants, which for the most part consist
some differences. The GGM was more polydispersed in of equal amounts of guiacyl and sinapyl-propane sub-
the shell with regard to its molecular weight and degree units. Therefore, the presence or absence of the 13C-
110 R. Redgwell et al. / Food Chemistry 81 (2003) 103–112

NMR signal for the methoxyl group would be diagnostic
for lignin, providing other sources of methoxyl groups
(esterified pectin) have been removed. Fortunately, hydro-
lysis in 12 M sulphuric acid completely hydrolyses and de-
esterifies all pectin but does not break the ether bond which
links the methoxyl group to the aromatic ring of lignin.
We compared the 13C-CP/MAS spectrum of a sample
of commercial lignins after hydrolysis in 12 M sulphuric
acid, with that of the ‘‘Klason Lignin’’ from cocoa shells
(Fig. 4). The signal for the methoxyl group in the com-
mercial lignin is evident but there was no trace of an
equivalent signal in the cocoa shell. The sensitivity of
NMR is limited, so that components which account for
less than 10% of the material may not be detected.
Nevertheless, the result would suggest that, if lignin
exists at all in cocoa shell, it is present at low levels and
certainly does not account for a significant proportion
of the ‘‘Klason Lignin’’ which is a major component of
dietary fibre reported for cocoa shells.
3.6. Dietary fibre content
Dietary fibre in 12 varieties of unroasted cocoa shells
has previously been determined according to Englyst’s
enzymatic—chemical procedure (Bonvehi & Beneria,
1998). Reported values for total non-starch poly-
saccharide ranged from 39.9 to 49.4%, with soluble
dietary fibre ranging from 13.1 to 18.6% and insoluble
fibre ranging from 24.2 to 30.8%. ‘‘Klason lignin’’
values were between 11.5 and 17.0%. In an earlier study
Martin-Cabrejas et al. (1994), using an AOAC method
(1984), measured insoluble, soluble and total dietary
fibre in roasted cocoa shells and reported values of 35.5,
14.9 and 50.4% respectively.Their ‘‘Klason lignin’’
value was 16.1%. It should be noted that the values in
the latter study were obtained with shells which con-
tained 18.5% fat. In our study, we obtained fat values
of approximately 4% and Bonvehi and Beneria (1998)
reported fat contents between 1.8 and 3.0%. It is possi-
ble that, in the Martin-Cabrejas et al. (1994) study, the
shells were contaminated with beans. The latter have
much higher fat levels than the shells.
In the present study dietary fibre fractions were mea-
sured by the gravimetric AOAC method and also by
determining the total polysaccharide content by GLC
analysis of the monosaccharides, following Saeman
hydrolysis of the polysaccharide fractions isolated dur-
ing CWM preparation (Table 7).
The total polysaccharide level, as determined by acid
hydrolysis of the polysaccharide content of the shells, is
in good agreement with values published by Bonvehi
and Beneria (1998). The dietary fibre levels, as measured
by the gravimetric AOAC method, are higher than
those published by Martin-Cabrejas et al. (1994). How-
ever, one reason for this is that the total fibre included
the ‘‘Klason lignin’’. Since we used roasted shells (the
Martin-Cabrejas study used unroasted shells), the for-
mation of Maillard products could augment this frac-
tion. It may therefore be expected that the ‘‘Klason
Lignin’’ value in our study would be higher.
3.7. Differences in properties of polysaccharides in bean
and shell
A comparison of the properties of polysaccharides
derived from shells and beans showed differences which
related, not only to structural features, but also to the
ease of extractability of the pectic polysaccharides. To
Table 7
Determination of total- (TDF), soluble- (SDF) and insoluble dietary
fibre (IDF) of cocoa shell, by AOAC method, and by summing the
monosaccharide components of all polysaccharide fractions (Values
n=3 S.E.)
Component % Dry weighta
AOAC method
TDF 63.60.4
SDF 11.70.1
IDF 51.90.4

Saeman hydrolysis
Monosaccharide
Composition
Rha 1.3 0.06
Fuc 0.1 0.005
Ara 1.2 0.03
Xyl 1.2 0.03
Man 1.8 0.04
Gal 2.6 0.03
Glc 13.6 0.41
Uronic 16.4 0.62

Total polysaccharide 38.2 1.1

‘‘Klason lignin’’ 27.9 2.1

Fig. 4. CP/MAS spectrum of cocoa bean ‘‘Klason lignin’’ compared
a
with spectrum of commercial lignin. Non-defatted shells.
 R. Redgwell et al. / Food Chemistry 81 (2003) 103–112
test that these differences between shell and beans were
real and did not just reflect two disparate tissue samples
taken from different cocoa beans, CWM was prepared
from the shell and nibs of the same cocoa beans.
Whereas in the polysaccharides of the bean, arabinose
and galactose were the predominant non-cellulosic neu-
tral sugars, in the shell arabinose was a minor component
and mannose a more predominant component (Table 8).
Another important difference was the elevated levels
of rhamnose in the shell, which reflected an increase in
the degree of rhamnosylation of the pectin backbone.
However, this did not lead to an increase in the pro-
portion of neutral sugar residues accommodated in the
side-chains of the pectic polysaccharides. It appears
that, while the shell polymers were more rhamnosylated
than those of the bean, the pectic polysaccharides in the
latter had more extended side-chains of galactosyl and
arabinosyl residues, as shown by the increased presence
of inter-chain residues in their linkage analysis.
The different neutral sugar contents in the pectic
polysaccharides may also be a factor in their differing
degrees of solubility in the bean and shell. In the latter,
much more pectin was solubilised both during isolation
of the CWM and during fractionation of the CWM with
CDTA and Na2CO3. These two reagents solubilised
36% of the pectic polysaccharide content of the shell
CWM but only 9% of the bean.
The increased mannose content of the shell was
reflected in at least three times the level of GGM in the
shell than was found in the bean.
4. Conclusions
4.1. Dietary fibre
This study has shown that roasted non-defatted cocoa
shells contain approximately 40% of total dietary fibre.
Higher values, reported previously, have included
‘‘Klason lignin’’ which can account for another 30% of
the shell. This material does not conform to the present
Table 8
Comparison of monosaccharide composition of polysaccharides in
bean and shell of unroasted cocoa beans
Monosaccharide Mol%
Shell
Rha 4.0
Fuc 0.2
Ara 1.3
Xyl 4.5
Man 6.5
Gal 5.9
Glc 36.6
Uronic acid 41.4
111
definition of what constitutes dietary fibre because it
does not appear to contain lignin. In roasted cocoa
shells the material is essentially tannin–protein–Mail-
lard complexes which are equally resistant to solubilisa-
tion in 72% sulphuric acid. Unfortunately, the
gravimetric procedures currently in use are unable to dis-
tinguish these complexes from legitimate lignin and their
use for determining dietary fibre will invariably lead to
over-estimation of dietary fibre in cocoa-derived material.
The concept of dietary fibre continues to be one which
attracts attention, in part because acceptable definitions
of what is dietary fibre seem to include an increasing
number of dietary components. Resistant starch and
fructan oligosaccharides are now included under the
umbrella of dietary fibre. The current definition is based
largely on resistance to digestion in the small intestine.
It is not clear to what extent protein–Maillard–tannin
complexes are modified during their passage through
the human digestive tract. If further research reveals a
specific health benefit of such complexes, it may be
possible to apply to regulatory authorities for accep-
tance of such materials as dietary fibre components for
food labelling purposes.
4.2. Cell wall polysaccharides
Compositional and linkage analysis demonstrated
that the non-cellulosic polysaccharides in cocoa shell
consisted of a heterogeneous mixture of several types of
pectic and hemicellulosic polymers in the following
approximate amounts:
Pectin 40–45%
GGM 7–10%
XG 5–7%
GAX 4%
Cellulose 34%
The range of cell wall polysaccharides in cocoa shell is
therefore diverse. Currently, our knowledge of the role
of individual polysaccharides in dietary fibre, in pro-
moting a specific health benefit, is limited. However, as
further research sheds more light on the health-related
effects of different types of polysaccharide, it may well
be that the range of polymers, as well as the total
amount in cocoa shell, will be advantageous in promot-
ing it as a source of dietary fibre.
Bean
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