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Food Chemistry 104 (2007) 948–954
www.elsevier.com/locate/foodchem
Received 7 July 2006; received in revised form 26 December 2006; accepted 28 December 2006
Abstract
The proximate composition and dietary fibre (DF) content of a fibre-rich product obtained from cocoa were studied. This product
contained 60.54% (dry matter basis) of DF, made of mainly insoluble fibre although with appreciable amounts of soluble dietary fibre
(10.09% d.m.). The presence of associated polyphenolic compounds (1.32% and 4.46% of soluble polyphenols and condensed tannins,
respectively) provides this fibre material with intrinsic antioxidant capacity as determined by the FRAP and TEAC methods. Hydration
properties (swelling and water holding capacity) and the glucose retardation index of cocoa fibre were similar to other natural commer-
cial insoluble fibres. The antioxidant capacity of this fibre-rich cocoa powder and its physico-chemical properties render it a suitable
product to be used in the preparation of low-calorie, high-fibre foods.
Ó 2007 Elsevier Ltd. All rights reserved.
Keywords: Cocoa; Dietary fibre; Antioxidant capacity; Nutritional properties; Functional food
0308-8146/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2006.12.054
E. Lecumberri et al. / Food Chemistry 104 (2007) 948–954 949
peroxidation, platelet activation or cyclo-oxygenase and cellulose, citrus pectins and apple pectins were all from
lipoxygenase activities, and enhancing levels of the endothe- Sigma Chemical Co. (Madrid, Spain), as were all enzymes
lial-derived relaxing factor, nitric oxide (Karim, McCor- and standards, except for the carob pod condensed tannins
mick, & Kappagoda, 2000; Rein et al., 2000; Schewe, Kühn, standard that was supplied by Nestlé S.A. Trolox (6-
& Sies, 2002; Steinberg, Bearden, & Keen, 2003; Wan hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), a
et al., 2001; Wiswedel et al., 2004). Also, cocoa polyphenols water soluble analogue of vitamin E, was from Aldrich
have shown to have antimutagenic activity (Yamagishi Chemicals Co. (Guillingam, Dorset, UK). ABTS (2,20 -azin-
et al., 2000), and decreased levels of 8-hydroxy-20 -deoxygua- obis-3-ethylbenzothiazoline-6-sulphonic acid, diammonium
nosine, a biomarker of oxidative damage to DNA, have salt) and TPTZ (2,4,6-tripyridyl-s-triazine) were from Fluka
been reported in rats after consumption of cocoa suggesting Chemicals (Madrid, Spain). All other reagents were of chro-
a potential role in cancer (Orozco, Wang, & Keen, 2003). matographic or analytical quality.
Besides flavonoids, cocoa is rich in other component
of remarkable nutritional interest such as dietary fibre 2.2. Analytical procedures
(DF). According to the updated definition of DF put for-
ward by the American Association of Cereal Chemists Dietary fibre was analysed in defatted fibre-rich cocoa
Expert Committee on dietary fibre (De Vries, 2003), this samples by the AOAC method modified in our laboratory
consists of plant cell wall polysaccharides, lignin and (Mañas, Bravo, & Saura-Calixto, 1994). Briefly, samples
associated substances resistant to hydrolysis by the diges- were treated with heat-stable a-amylase (Sigma, A-3306),
tive enzymes of humans. DF has well-documented bene- protease (P-3910) and amyloglucosidase (A-9913), fol-
ficial effects on human health and body function; thus, lowed by centrifugation (15 min, 3000g) instead of filtra-
a high consumption of DF is associated with a reduced tion to separate the soluble and insoluble fractions
incidence of disorders and diseases common in developed obtained after enzymatic hydrolysis of digestible com-
societies such as chronic bowel disorders, obesity, diabe- pounds. Supernatants were quantitatively collected and
tes, cardiovascular disease and cancer (Bessesen, 2001; pellets were washed twice with 10 mL of distilled water,
Johnson, 2004; Kris-Etherton et al., 2002; Sungsoo Cho centrifuged and all supernatants combined. These were
& Dreher, 2001). transferred into dialysis tubes (12000–14000 MWCO, Dial-
It has been suggested that cocoa hull may be a good ysis Tubing Visking, Medicell International Ltd., London,
source of dietary fibre, with reported values ranging from UK), and dialysed against water for 48 h at 25 °C (water
38% to 44% of total dietary fibre as non-starch polysaccha- flow 7 L/h in a 43 L reservoir). Dialysates (soluble dietary
rides plus Klason lignin (Martı́n-Cabrejas, Valiente, Este- fibre, SDF) were hydrolysed with 1 M sulphuric acid at
ban, Mollá, & Waldron, 1994; Serra-Bonvehı́ & Aragay- 100 °C, 90 min and non-starch polysaccharides (NSP)
Benerı́a, 1998). Considering the health benefits associated determined in the hydrolysate. The residues obtained after
to the consumption of dietary fibre and polyphenols in enzymatic hydrolysis of samples (insoluble dietary fibre,
the diet, the presence of both bioactive components in IDF) were dried overnight at room temperature, and
cocoa bean husks could highlight the interest of such a hydrolysed with sulphuric acid (12 M H2SO4, 1 h, 30 °C,
product as a potential ingredient for the functional food then diluted to 1 M H2SO4 100 °C, 90 min with shaking).
industry. After acid hydrolysis, samples were centrifuged (15 min,
In the present work we studied the composition of a 3000g), pellets washed twice with distilled water, and com-
fibre-rich product obtained from cocoa bean husks, as well bined supernatants collected for NSP determination. Resi-
as some properties related to its nutritional quality, such as dues were dried at 105 °C overnight and gravimetrically
its polyphenolic content, antioxidant capacity and physico- quantified as Klason lignin (KL).
chemical properties. Uronic acids (UA) in hydrolysates from both SDF and
IDF were quantified spectrophotometrically by the Scott
2. Experimental (1979) method using galacturonic acid as standard. Neutral
sugars (NS) were analysed chromatographically by GLC as
2.1. Samples and reagents alditol acetates (Englyst, Quigley, Hudson, & Cummings,
1992), using inositol as internal standard. A Shimadzu
The dietary fibre rich cocoa product was supplied as a GC-14A gas chromatograph (Shimadzu Co., Kyoto,
fine powder by Nutrexpa S.A. (Barcelona, Spain). This Japan) fitted with a flame ionization detector was used.
product was obtained as follows: clean cocoa beans were Separation of sugars was achieved in a SP-2330 capillary
cracked to loose the shells from the nibs. After this breaking column (30 m 0.32 mm i.d., Cat No. 2-4073, Supelco,
step, the fractions obtained go to the winnowing cabinets PA, USA). Column temperature was set at 240 °C (isother-
were the husks are air-separated from the kernels. Then, mal), and injector and detector temperature was 270 °C.
husks go through a sterilization process and they are finally Nitrogen was used as carrier gas. Data were collected
ground to a particle size of 75 lm. Carob pod fibre (Cerato- and processed with an Agilent ChemStation software sys-
nia siliqua L.) was obtained from Compañı́a General del tem (Agilent Technologies, Waldrom, Germany). Total
Algarrobo de España S.A. (Valencia, Spain). Guar gum, dietary fibre (TDF) was calculated as IDF + SDF; IDF
950 E. Lecumberri et al. / Food Chemistry 104 (2007) 948–954
was calculated as NSP + KL, and SDF as NSP (NSP = (12000–14000 MWCO, Dialysis Tubing Visking, Medicell
UA + NS). International Ltd., London, UK) were loaded with
Soluble polyphenols were extracted by sequentially wash- 7.5 mL of distilled water (control) or 500 mg of sample plus
ing 1 g of sample with 40 mL of 16 mM hydrochloric acid 7.5 mL of distilled water, and hydrated in a chamber at
in 50% aqueous methanol (50:50, v/v, 1 h at room temper- 100% relative humidity for 60 min. After this time, a glu-
ature, constant shaking) and 40 mL of acetone–water cose solution was added into the bags to a final concentra-
(70:30, v/v, 1 h room temperature, constant shaking) in tion of 2 mg glucose mL1, thoroughly mixed with the
50 mL centrifuge tubes (Bravo & Saura-Calixto, 1998). solid samples, and the bags transferred into reservoirs con-
After centrifugation (15 min, 3000g), supernatants from taining 400 mL distilled water and held in a thermostatic
each extraction step were combined and made up to water bath at 37 °C for 1 h with constant shaking. At
100 mL. Polyphenols in the extracts were analysed spectro- 15 min intervals, 0.5 mL of dialysate was collected and glu-
photometrically as total polyphenols by using the Folin- cose concentration determined spectrophotometrically
Ciocalteau’s reagent and gallic acid as standard; also, the using a glucose oxidase–peroxidase method (Peridochrom
soluble tannins fraction in these extracts was quantified Glucose, GOD-POP, Böehringer Mannheim, Germany).
by treating the samples for 3 h at 100 °C with 5% HCl– The retardation of glucose diffusion from the dialysis bag
butanol (v/v) (Reed, McDowell, Van Soest, & Horvath, into the dialysate was calculated as follows:
1982). This same method was used to determine insoluble
Total glucose diffused from fibre sample
condensed tannins in the residues obtained after extraction GRI ¼ 100 100
Total glucose diffused from control sample
of soluble polyphenols. The proanthocyanidin solutions
thus obtained were quantified spectrophotometrically at Swelling: sample was accurately weighed (200 mg) and
553 nm, using carob pod condensed tannins as standard. transferred into a calibrated cylinder (1.5 cm diameter),
Antioxidant capacity of the fibre-rich cocoa powder was and 10 mL of distilled water containing 0.02% sodium
evaluated in the soluble polyphenols extract by two differ- azide as a bacteriostat were added. After thoroughly mix-
ent methods: the FRAP assay as an evaluation of the ing, cylinders were let to stand undisturbed for 18 h at
reducing power of the sample, and the TEAC method to room temperature. Then, the bed volume was recorded
assess its free radical scavenging capacity. and swelling was calculated as mL per gram of dry sample
FRAP assay: the ferric reducing/antioxidant power (Robertson et al., 2000).
(FRAP) as described in Pulido, Bravo, and Saura-Calixto Water holding capacity (WHC): sample (250 mg) was
(2000) was used. Briefly, FRAP reagent (containing accurately weighed in 50 mL centrifuge tubes, and 25 mL
10 mM TPTZ in 40 mM HCl, 20 mM FeCl3 6H2O and of distilled water containing 0.02% sodium azide were
0.3 M acetate buffer, pH 3.6), prepared daily and kept at added, thoroughly mixed and let to stand at room temper-
37 °C, was mixed with test samples, water or Trolox. ature for 60 min. After this time, samples were centrifuged
Absorbance readings at 595 nm were taken after 30 min (15 min, 3000g) and the supernatant carefully removed.
reaction using a Beckman DU640 spectrophotometer Sample fresh weight was recorded and WHC expressed as
(Beckman Instruments Inc., Fullerton, CA) equipped with the amount of water retained per gram of dry sample
a thermostatized auto-cell holder set at 37 °C. Increases in (g g1 dry matter) (Robertson et al., 2000).
absorbance are due to Fe3+ reduction by antioxidants and
the subsequent formation of a coloured TPTZ–Fe2+ com- 2.3. Other analyses
plex with an absorbance maximum at 595 nm. A Trolox
reference curve (0.1–0.8 mM) was used as a standard and Protein was quantified using an automated nitrogen
the results were expressed as lmol of Trolox per gram of analyser (LECO FP-2000, LECO Corp., Michigan, USA).
dry matter. Protein was calculated as N 5.75. Fat was quantified
TEAC assay: the capacity of samples to scavenge the after extraction with light petroleum in a Soxtec System
free radical cation ABTS+ was assessed using the method HT (Soxtec Extraction Unit 1043 and Service Unit 1046,
of Re et al. (1999). ABTS+ was preformed by the reaction Tecator, Höganäs, Sweden). Ash content was determined
of 7 mM ABTS with 2.54 mM potassium persulphate dur- gravimetrically after calcination in a muffle furnace at
ing 12–16 h at room temperature in the dark. Decrease of 550 °C for 16 h. Soluble sugars were measured spectropho-
ABTS+ absorbance at 658 nm (initially adjusted by dilu- tometrically in the extracts obtained after washing samples
tion in ethanol to an absorbance of 0.7 ± 0.02) in the pres- with 85% ethanol using anthrone/thiourea as reagent and
ence of the polyphenol extract was monitored during glucose as standard following the conditions described else-
6 min. Trolox (0–0.8 mM) was used for calibration and where (Mañas et al., 1994).
results expressed as lmol of Trolox per gram of dry matter.
Glucose retardation index (GRI) was determined by the 2.4. Statistics
procedure of Jenkins, Jenkins, Wolever, Taylor, and Gha-
fari (1986) slightly modified (Bravo, 1999). Prior to the Samples were analysed in triplicate unless stated other-
assay, samples were extracted with 85% ethanol to ensure wise. Results are expressed as mean values ± standard
removal of soluble sugars. For the GRI assay, dialysis bags deviations. To assess for differences in the physico-chemical
E. Lecumberri et al. / Food Chemistry 104 (2007) 948–954 951
properties between the cocoa-fibre product and other solu- close to 60%. Total polyphenols (as the sum of soluble
ble or insoluble dietary fibre sources, multiple sample com- polyphenols plus insoluble condensed tannins) in this
parison was performed using the Statgraphics Plus cocoa fibre amounted to close to 6% of the dry matter.
program version 2.1 (Statistical Graphics Corp., Rockville, Total dietary fibre (TDF), as well as the content of the
MD). Analysis of variance (ANOVA) followed by Dun- major constituents of the soluble and insoluble DF frac-
can’s multiple comparison test were used to contrast tions (neutral sugars, uronic acids and Klason lignin), are
groups. Cochran’s test was performed to test for homoge- shown in Table 2. TDF content of this cocoa product
neity of variances, and to discriminate among means the was extremely high, over 60% of the dry matter. This
Fisher’s least significant difference procedure was applied. implies that this product might be of interest for the food
No transformation of raw data was required for swelling industry, considering its potential application as a func-
and GRI values, although logarithmic transformation of tional ingredient in confectionery, bakery or in the prepa-
raw data was required in the case of WHC values to ensure ration of low-fat, high-fibre dietetic products. As for the
variance homogeneity. The level of significance was constituent fractions, SDF accounted for less than 17%
P < 0.05. of the TDF content of the fibre-rich cocoa product, made
of mainly pectic substances as shown by the high uronic
3. Results and discussion acids content (7.13% of the dry matter). Quantitatively,
IDF was the main component of this cocoa product,
Dietary guidelines recommend a minimum daily intake accounting for over 80% of the TDF and 50% of the total
of DF of 25 g (equivalent to 12.5 g DF per 1000 calories dry weight. Close to one third of this IDF fraction corre-
consumed) (Marlett et al., 2002; USDA, 2000), which is sponded to non-starch polysaccharides (18.1% of the dry
considerably higher than the estimated intakes in Western matter), the remaining being Klason lignin (KL). Serra-
countries (Sungsoo Cho & Dreher, 2001). Therefore, there Bonvehı́ and Aragay-Benerı́a (1998) reported a TDF con-
is a need to increase fibre intake, which has prompted the tent of 43.9% in cocoa husks, with values for SDF higher
consumption of dietary supplements or fibre-enriched food than those analysed in the present work. These authors
products. DF components like pectins, gums, cellulose and did not mention the KL residue, yet Chung, Iiyama, and
others have been used as functional ingredients by the food Han (2003) reported values of 32% in cocoa hulls, which
industry, with an extensive market of food by-products as are similar to the values in this fibre-rich cocoa product.
DF sources. In the present work we report the composi-
tion, as well as the antioxidant and some physico-chemical
properties of a fibre-rich product obtained from cocoa
Table 2
bean husks, which might be used as an ingredient in the
Content and composition of the soluble and insoluble dietary fibre
preparation of different food products and/or dietetic, fractions of the cocoa product (% dry matter)
low-calorie high-fibre foods.
Fibre-rich cocoa product
Table 1 shows the proximate composition of the dietary
(% Dry matter) % Relative to TDF
fibre-rich cocoa powder. This product had a relatively low
fat content in comparison with cocoa beans where fat con- Soluble dietary fibre 10.09 ± 0.38 16.67
Rhamnose 0.29 ± 0.04 –
stitutes over 50% of the dry weight (Valiente, Esteban,
Fucose n.d. –
Mollá, & López-Andréu, 1994). Martı́n-Cabrejas et al. Arabinose 0.29 ± 0.01 –
(1994) reported a fat content in cocoa hulls of 18.5% (dry Xylose 0.09 ± 0.01 –
matter basis), higher than the values found in the fibre-rich Mannose 0.51 ± 0.01 –
cocoa powder (less than 7%). Also, the content of soluble Galactose 1.36 ± 0.02 –
Glucose 0.41 ± 0.03 –
sugars was negligible (lower than 0.5%), which together
Neutral sugarsa 2.96 ± 0.10 4.89
with the low fat content represent an advantage of this Uronic acids 7.13 ± 0.29 11.78
product, with a reduced caloric value. Protein and ash were
Insoluble dietary fibre 50.42 ± 0.70 83.32
higher than those reported by Martı́n-Cabrejas et al. Rhamnose 0.15 ± 0.01 –
(1994), as well as total dietary fibre content, which was Fucose 0.06 ± 0.02 –
Arabinose 0.94 ± 0.01 –
Xylose 0.97 ± 0.01 –
Table 1 Mannose 0.96 ± 0.01 –
Proximal composition of the fibre-rich cocoa product (% dry matter) Galactose 0.91 ± 0.01 –
Fibre-rich cocoa product Glucose 10.53 ± 0.15 –
Neutral sugarsa 14.53 ± 0.16 24.01
Protein 16.71 ± 0.18
Uronic acids 3.48 ± 0.13 5.75
Fat 6.62 ± 0.38
Klason lignin 32.41 ± 0.40 53.56
Ash 11.42 ± 0.04
Soluble sugars 0.35 ± 0.04 Total dietary fibre (TDF) 60.51 ± 0.32 100
Dietary fibre 60.54 ± 0.32 n.d.: Not detected; TDF: total dietary fibre.
Polyphenols 5.78 ± 0.29 Mean values ± STD (n = 3).
a
Mean values ± STD (n = 3). Sum of constituents sugars (by GLC).
952 E. Lecumberri et al. / Food Chemistry 104 (2007) 948–954
As for the constituent sugars, the IDF fraction was rich by digestive enzymes, increasing fecal bulk, and protein
in glucose (Table 2), suggesting that cellulose was the major and fat excretion (Bravo, 1998; Bravo, Abia et al., 1994;
non-starch polysaccharide (NSP) of cocoa fibre (about 10% Saura-Calixto et al., 2000). But polyphenols are also well-
of the dry matter), which agrees with reports from other known bioactive compounds that have shown to be protec-
authors (Chung et al., 2003; Redgwell et al., 2003; Serra- tive against diseases like coronary heart disease, cancer,
Bonvehı́ & Aragay-Benerı́a, 1998). Minor amounts of neurodegenerative disorders, etc., mostly through their
monosaccharides such as arabinose, xylose, mannose or antioxidant and free radical scavenging capacities (Bravo,
galactose, together with a significant content of uronic 1998; Wan et al., 2001). The content of PP naturally pres-
acids were indicative of the presence of hemicelluloses ent in the cocoa fibre product is shown in Table 3, as well
(xyloglucans, arabinoxylans, glucuronoxylans) and pectic as the reducing power and the scavenging capacity against
substances associated to the cell wall matrix in cocoa bean the ABTS+ radical cation of polyphenolic extracts
shells. Together neutral sugars plus uronic acids in the obtained from this fibre. The content of both CT and sol-
fibre-rich cocoa powder amounted to 18.01% of the dry uble PP was lower than expected. Especially the concentra-
matter in the IDF fraction (Table 2). As to SDF (10.09% tion of soluble PP, with only 1.32% of the dry matter (with
of the dry matter), it showed appreciable amounts of galac- nearly 80% of these soluble phenols corresponding to pro-
tose and mannose, which may be suggestive of the presence anthocyanidins), was very low with a value nearly five
of minor amounts of galactomannans or else these mono- times smaller than that found in cocoa powder (Vinson
saccharides may be part of pectins, which are the major et al., 1999). Still, the total polyphenolic content of this
component of SDF. Pectic substances, both soluble and cocoa product was higher than that reported in other foods
associated to insoluble cell wall polysaccharides in the like cereals, legumes, vegetables, nuts or fruits (Saura-Cal-
IDF fraction and determined as uronic acids, amounted ixto & Goñi, 2006). As a consequence of the low soluble
to nearly 10% of the dry cocoa fibre product. This is in polyphenolic content of the fibre-rich cocoa product, the
accordance with results reported by Redgwell et al. antioxidant activity of the extracts was also relatively low
(2003), who found that cellulose and pectins were the major (Table 3). However, the reducing power of these extracts
cell wall polysaccharides in cocoa shells. (FRAP values) was higher than that found in the men-
The NSP fraction of the cocoa fibre powder showed a tioned groups of plant foods, while the free radical scav-
rather balanced content of both soluble (close to 40% of enging capacity (determined by the TEAC assay) was
the total NSP) and insoluble NSP, which can be considered comparable to that found in fruits, vegetables and legumes,
as another appreciable attribute of this fibre product. and higher than in cereal foods (Saura-Calixto & Goñi,
Many commercial DF sources, usually obtained as by- 2006). It is worth noting that the antioxidant capacity of
products of the food industry (e.g. cereal brans, wine by- the phenolic compounds in foods is usually attributed to
products), mostly provide IDF and little SDF, a fraction the soluble fraction. This soluble, low-molecular weight
of nutritional and technological interest due to its func- polyphenolic fraction, in spite of its limited bioavailability
tional properties. Taken together, NSP in soluble and (reviewed in Williamson & Manach, 2005), is partly
insoluble DF made up nearly half the TDF content of absorbed in the gastrointestinal tract and thus it would
the cocoa fibre (28.1% dry matter), the other half being contribute to the antioxidant status in vivo, whilst the
Klason lignin (Table 2). This fraction is not considered as high-molecular weight condensed tannins would remain
a DF constituent by some authors or in countries where in the intestinal tract, therefore limiting their potential bio-
the definition of DF is that of indigestible non-starch poly- logical effects to this location.
saccharides of plant origin (Englyst & Cummings, 1990), Although the content of polyphenolic compounds and
although it is considered as an integral component of DF the antioxidant capacity of the fibre-rich cocoa product
as officially defined by the AOAC (Prosky et al., 1988) were relatively low, it is important to emphasize that these
and also as a component of the indigestible fraction of are remarkable attributes of this product, especially in
foods (De Vries, 2003; Saura-Calixto, Garcı́a-Alonso,
Goñi, & Bravo, 2000). KL encompasses not only lignin, Table 3
but also other polyphenols – including condensed tannins Phenolic content and antioxidant activity of the fibre-rich cocoa product
–, resistant protein, Maillard reaction products, etc. (Bravo Fibre-rich cocoa product
& Saura-Calixto, 1998), being a non-digestible constituent
Soluble polyphenols (% d.m.)
of plant foods that might account for some of the physio- As total phenolsa 1.32 ± 0.02
logical properties of DF (Bravo, Abia, & Saura-Calixto, As soluble tanninsb 1.04 ± 0.06
1994; Saura-Calixto et al., 2000).
Condensed tannins (% d.m.) 4.46 ± 0.50
Polyphenols (PP), especially highly polymerized com- FRAP (lmol TE/g d.m.) 72.32 ± 0.67
pounds like condensed tannins (CT) and phenols bound TEAC (lmol TE/g d.m.) 7.73 ± 0.47
to protein and polysaccharides from cell walls, are some TE: Trolox equivalents.
of the substances associated to DF and partly quantified Mean values ± STD (n = 4).
a
in the Klason lignin residue. Some polyphenolic com- Analysed by the Folin-Ciocalteau’s method.
b
pounds behave as DF constituents, resisting hydrolysis Analysed by the method of Reed et al. (1982).
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