Food Research International xxx (xxxx) xxx–xxx

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Food Research International

journal homepage: www.elsevier.com/locate/foodres

Cotyledon pectin molecular interconversions explain pectin solubilization

during cooking of common beans (Phaseolus vulgaris)
⁎
Claire M. Chigwedere , Cornelius M. Nkonkola, Shrijana Rai, Clare Kyomugasho,
Zahra Jamsazzadeh Kermani, Andrea Pallares Pallares, Ann M. Van Loey, Tara Grauwet,
⁎
Marc E. Hendrickx
Leuven Food Science and Nutrition Research Center (LFoRCe), Department of Microbial and Molecular Systems, Laboratory of Food Technology, KU Leuven, Kasteelpark
Arenberg 22, Box 2457, Heverlee, 3001, Belgium

A R T I C LE I N FO A B S T R A C T

Keywords: Dynamics of pectin extractability in cotyledons and seed coats were explored for mechanistic insight into pectin
Bean cotyledons changes due to aging and cooking of beans. In addition, changes in mineral distribution during cooking were
Bean seed coats determined in order to investigate their retention in the matrix. Pre-soaked fresh and aged beans were cooked in
Aging demineralized water for different times and the cotyledons, seed coats and cooking water were lyophilized. From
Cooking
cotyledon and seed coat powders, alcohol insoluble residue (AIR) was extracted and sequentially fractionated
Pectin solubilization
Mineral distribution
into water-, chelator- and sodium carbonate-extractable pectin (WEP, CEP and NEP, respectively).
Characterization of pectin in AIR and pectin fractions revealed inherent structural differences between cotyledon
and seed coat pectin with the latter exhibiting a lower degree of methylesterification (DM) and being more
linear. Due to aging, WEP decreased whilst NEP substantially increased and the CEP fraction and DM of pectin in
AIR did not change significantly, suggesting a more crucial role of increased covalent bonding than cation-
mediated crosslinking in aging-induced hardening of beans. During cooking, some NEP was converted into WEP
and no pectin depolymerization was observed from molar mass distribution profiles. Pectin changes due to aging
and cooking of beans were more pronounced in the cotyledon compared to the seed coat. Whilst Ca2+, Fe2+ and
Zn2+ were largely retained in the bean matrix during cooking, Mg2+ was largely leached from cotyledons into
the cooking water. In conclusion, aging-induced hardening of beans and softening during cooking were found to
be premised on interconversion of pectin fractions in cotyledons.

1. Introduction dietary fiber polymers, particularly pectin, a structure-determining

Seeds of the legume family are consumed worldwide and constitute
a significant part of the diet of populations in developing nations, due to
their nutritionally-rich composition. They are a good source of proteins,
which complement those from cereals, complex carbohydrates as well
as vitamins and minerals such as calcium, magnesium, zinc and iron
(Siddiq & Uebersax, 2013). Complex carbohydrates make up a large
fraction of legumes and include dietary fiber which constitutes of
pectin, hemicellulose, lignin and/or cellulose (Padayachee, Day,
Howell, & Gidley, 2017). On the one hand, dietary fiber promotes
various physiological effects that are beneficial to human health such as
high digesta viscosity which leads to a blood glucose-lowering effect.
On the other hand, it may hinder bioavailability of vitamins, minerals
and proteins by complexation (Kumar, Sinha, Makkar, de Boeck, &
Becker, 2012; Tosh & Yada, 2010). Moreover, changes in some of the
polysaccharide in the cell wall and middle lamella, occurring in situ
during maturation, storage and cooking of plant-derived foods are be-
lieved to alter texture, an important property for acceptability of food
(Prasanna, Prabha, & Tharanathan, 2007; Sila et al., 2008).
Structural changes in pectin, as a consequence of inappropriate
storage of legumes in general and common beans (Phaseolus vulgaris) in
particular, are hypothesised to contribute to development of a textural
defect, hard-to-cook (HTC). During HTC development, cell-cell adhe-
sion is enhanced, necessitating prolonged cooking for attainment of
tenderness of affected beans. As such, desired texture is not readily
achieved, rendering the beans unacceptable to consumers (Paredes-
López, Carabez-Trejo, Palma-Tirado, & Reyes-Moreno, 1991). Pectin
structural changes are considered as the classical mechanism of HTC
development among other postulations involving phenolic compounds,
proteins and starch (El-Tabbey Shehata, 1992). Pectin is a complex

⁎
Corresponding authors.
E-mail addresses: clrchigwedere1@gmail.com (C.M. Chigwedere), marceg.hendrickx@kuleuven.be (M.E. Hendrickx).

https://doi.org/10.1016/j.foodres.2018.08.062
Received 29 March 2018; Received in revised form 4 August 2018; Accepted 18 August 2018
0963-9969/ © 2018 Elsevier Ltd. All rights reserved.

Please cite this article as: Chigwedere, C.M., Food Research International (2018), https://doi.org/10.1016/j.foodres.2018.08.062
C.M. Chigwedere et al. Food Research International xxx (xxxx) xxx–xxx

heteropolysaccharide comprised of three structurally different regions Aging

Fresh beans Aged beans
including homogalacturonan (HG), a smooth region due to absence of
side chains (neutral sugars) and two hairy regions namely, rhamnoga-
Determination of cooking profiles for selection of cooking times
lacturonan (RG) I and II. The HG and RG II have a backbone of α-1,4-
linked galacturonic acid (GalA) moieties which can be methylesterified,
giving rise to degree and pattern of methylesterification, important Soaking and cooking for selected times in demineralized water

properties largely determining pectin functionality (Voragen, Coenen, Peeling Peeling

Lyophilization of
Verhoef, & Schols, 2009). It has been suggested that during aging of Lyophilization of cotyledons and seed coats cooking water
common beans, demethylesterification of pectin by pectin methyles-
terase results in low DM pectin which engages in cation-mediated
crosslinking thereby promoting cell-cell adhesion and consequently, Extraction and fractionation of AIR Quantification of
minerals
enhanced texture. The involved cations are reportedly released from
hydrolysis of phytate by phytase before migration to the middle lamella Pectin characterization
and cell wall to interact ionically with the demethylesterified pectin (El-
Degree of methylesterification (DM)
Tabbey Shehata, 1992; Paredes-López et al., 1991). This is termed the Composition of sugars
pectin-cation-phytate hypothesis of HTC development.
Understanding the texture evolution of beans during cooking ne- Fig. 1. Schematic illustration of the experimental approach of this study.
cessitates a deeper insight into the role of structure-determining com-
ponents, particularly pectin. Pectin-related studies on the cooking be-
(95 °C) in demineralized water followed by hardness measurement on a
havior of P. vulgaris are focused mainly on analysis of whole bean
single cotyledon per seed for each of 20 sampled seeds. Average values
material. Although Yi et al. (2016) examined cotyledons and seed coats
of hardness for each sampling time were plotted against cooking time to
separately, they did not consider changes in the time domain (they
obtain the cooking profiles. These profiles are not considered as results
analyzed non-cooked and fully-cooked states only). Therefore, a de-
in this work since their purpose was for selection of samples for AIR
tailed characterization of pectin from cotyledons and seed coats with
extraction as shown in Fig. 2. The reader is referred to Chigwedere et al.
evolution of texture (samples cooked for different times) was explored
(2018) for a discussion on these profiles. Soaked, non-cooked beans
in the current study. Our previous study (Chigwedere et al., 2018)
(cooked for 0′) represented control samples (Fig. 2A). Fresh and aged
provided evidence that the rate-limiting step in softening of beans
beans each cooked for 60′ and 270′ (Fig. 2B and C) were selected to
during cooking is related to changes in mainly cotyledon cell walls and
determine the influence of differences in hardness (due to HTC) on
middle lamellae which influence pectin solubilization. However, seed
pectin solubilization at short and long cooking times, respectively.
coats were analyzed in this work in order to obtain deeper insights into
Cooking times of 60′ and 210′ as well as 120′ and 330′ for fresh and
localized changes thereby providing a more complete picture of pectin
aged beans, respectively in each case, were chosen to represent beans
changes in beans during cooking. It was also intended to gain insight
having similar hardness (Fig. 2D and E). This was aimed at determining
into how HTC would influence pectin changes hence beans without
the influence of processing (cooking) time on pectin solubilization at
(fresh) and with (aged) HTC were analyzed. Hereto, AIR (rich in cell
relatively short and long cooking times. Samples of 40 g of fresh and
wall polymers) was extracted from cotyledons and seed coats obtained
aged beans were soaked then cooked in demineralized water for each of
from fresh and aged beans cooked in demineralized water for different
the selected cooking times. Then, the beans were peeled to generate a
times, followed by characterization of inherent pectin in terms of DM.
sample of each of cotyledons and seed coats per cooking time. These
The AIR was sequentially fractionated into pectin fractions based on
materials were then lyophilized together with the cooking water, using
extractability in different media and pectin solubilization was de-
a Christ Alpha Plus 2–4 lyophiliser (Osterode, Germany). The lyophi-
termined as changes in relative amounts of pectin in the fractions with
lized samples were ground using pestle and mortar and then sieved
increase in cooking time. Given the suggested relation between pectin
using a Retsch sieve (mesh size 500 μm) to obtain fine powders.
and cations during aging of beans (Paredes-López et al., 1991), changes
in mineral distribution during cooking were determined along with
2.3. Extraction and fractionation of alcohol insoluble residue
changes in pectin solubilization.

In duplicate, AIR was extracted from cotyledon and seed coat

2. Materials and methods
25000
2.1. Raw materials A B C

Common beans grown, harvested and sun-dried at Thika Station 20000

(Kenya) were sorted and cleaned prior to being denoted as ‘fresh’ beans,
a part of which was incubated above saturated potassium chloride so-
lution at 35 °C to achieve and maintain a relative humidity of 83% for 15000
Hardness (g)

three months. These beans are herein referred to as ‘aged’ beans. Since
Fresh beans
both the fresh and aged beans were from the same batch, no biological Aged beans
10000
variability from the raw material was expected. The fresh and aged F60' vs 210' D
beans were then stored in a freezer at −40 °C until use. Fig. 1 is a
schematic illustration of the experimental work carried out in this 5000 F120' vs A330' E
study. All chemicals used were of analytical grade.

2.2. Preparation of samples for extraction of alcohol insoluble residue 0

0 30 60 90 120 150 180 210 240 270 300 330 360
Cooking time (min)
Based on cooking profiles of fresh and aged beans generated as
described by Chigwedere et al. (2018), a selection of cooking times of Fig. 2. Profiles of fresh and aged beans cooked in demineralized water high-
beans prior to AIR extraction was made. In short, to obtain the cooking lighting control samples (A), samples cooked either for same times (B and C) or
profiles, fresh and aged beans were soaked (25 °C, 16 h) then cooked to similar hardness (D and E).

2
C.M. Chigwedere et al.
powders based on the method of Njoroge et al. (2014), with mod-
ifications. Unlike seed coats, cotyledons are rich in starch and proteins
hence these biopolymers were hydrolyzed in order to increase the cell
wall material content in the extracted AIR. Thereafter, the AIR was
fractionated into different pectin-rich fractions.
2.3.1. Extraction of alcohol insoluble residue
Approximately 4 g was suspended in 50 mL of 0.1 M sodium acetate
buffer (pH 4.3). The low pH was to diminish possibility of pectin de-
gradation/solubilization by β-elimination during a subsequent heat
treatment at 95 °C for 15 min in order to gelatinize starch. The impact of
this additional treatment as a cooking effect was not assessed but the
short time rendered it less harsh compared to the cooking process. The
suspensions were subsequently cooled and the pH was adjusted to 5.2
using 2 M NaOH. For starch degradation, 5.333 mL of amyloglucosidase
from Aspergillus niger (300 U/mL, Sigma Chemical Co., St. Louis,
Missouri) and 3.904 mL of Type II-A α-amylase from Bacillus species
(410 U/mL buffer, Sigma Chemical Co., St. Louis, Missouri) were added
followed by incubation at 62.5 °C for 4 h in a water bath, with shaking.
The thermal load due to this treatment was not expected to influence
pectin solubilization in the samples. However, the samples were cooled
in an ice bath and technical ethanol (96%) was added to 70% saturation
followed by incubation at 4 °C for 16 h in order to precipitate any pectin
(polymer of interest) that could have been solubilized during heat
treatment, especially at 95 °C. Thereafter, a JS-HS centrifuge
(Beckmann, American Instrument Exchange, Inc.) was utilized at
10000 g for 15 min to obtain pellets. The pellets were suspended in
25 mL of 0.1 M sodium phosphate buffer (pH 7.5) and incubated with
2 mL of protease from Bacillus licherniformis (50 U/mL buffer, Chemical
Co., St. Louis, Missouri) at 60 °C for 1 h in a water bath, with shaking.
Prior to and after a second protein hydrolysis, ethanol precipitation for
2 h and centrifugation were performed as described above.
Subsequently, the pellets were homogenized for 3 × 6 s first in
128 mL, then in 64 mL of technical ethanol using a Büchi mixer (B-400,
Flawil, Switzerland). After each homogenization step, samples were
vacuum filtered through MN615 filter papers (Ø 90 mm, Machery-
Nagel). The residues were then homogenized in 64 mL of acetone at 4 °C
for 10 min followed by vacuum filtration and the final residues (AIR)
were dried at 40 °C for 16 h. The dry AIR was suspended in deminer-
alized water (1:100, w/v) and dialyzed against demineralized water for
96 h using dialysis membranes with a molecular weight cut off of
3.5 kDa (Spectra/Por®, Spectrum Laboratories, Inc., Rancho
Dominguez). Finally, the AIR was lyophilized and stored in a desiccator
until further use. For seed coat powders, AIR extraction was performed
as described above, without starch and protein hydrolysis.
2.3.2. Fractionation of alcohol insoluble residue
In duplicate, AIR was sequentially fractionated into WEP, CEP and
NEP as described by Njoroge et al. (2014). First, WEP was extracted by
suspension of 0.5 g of AIR in 90 mL of boiling demineralized water (at
pH 4) followed by boiling of the suspension for 5 min. The samples were
cooled in an ice bath and vacuum filtered as described in Section 2.3.1.
The filtrate, WEP, was then adjusted to pH 5 and 100 mL. Using 90 mL
of 0.1 M potassium acetate solution (pH 5) containing 0.05 M cyclo-
hexane-trans-1, 2-diaminetetraacetic acid, CEP was extracted from the
residue at 28 °C for 6 h. Lastly, NEP was extracted using 90 mL of
0.05 M sodium carbonate solution containing 0.02 M sodium borohy-
dride first at 4 °C for 16 h then at 28 °C for 6 h. For CEP and NEP, fil-
tration and, pH and volume adjustments were conducted as described
for WEP. The WEP and NEP fractions were dialyzed against deminer-
alized water at 4 °C for 72 h as described in Section 2.3.1 whilst CEP
was dialyzed first against 0.1 M sodium chloride for 36 h and then
against demineralized water for the same time. Following dialysis, the
pectin fractions were lyophilized and stored in a desiccator until ana-
lysis.
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2.4. Determination of the degree of methylesterification of pectin in alcohol
insoluble residue
To determine the DM, which is the molar ratio of methanol to ga-
lacturonic acid (GalA), methanol and galacturonic acid were quantified
using chromatographic and colorimetric assays, respectively.
2.4.1. Determination of galacturonic acid content
Galacturonic acid content was determined through hydrolysis of the
sample following the procedure of Ahmed and Labavitch (1978) and
then quantification according to a procedure described by
Blumenkrantz and Asboe-Hansen (1973). For hydrolysis, in duplicate,
8 mL of 98% sulphuric acid was added to 10 mg of AIR in a beaker (in
an ice bath) followed by dropwise addition of 2 mL of demineralized
water whilst stirring. After 5 min, another 2 mL of demineralized water
was added dropwise and the mixture was stirred for 1 h. Using demi-
neralized water, the hydrolysate was diluted to 20 mL from which
0.6 mL was transferred into each of four Pyrex tubes in an ice bath.
Exactly 3.6 mL of 98% sulphuric acid containing 0.0125 M sodium
tetraborate was added to the tubes and the mixtures were vortexed
followed by incubation in an oil bath at 100 °C for 5 min. After cooling,
to the blank, 60 μL of 0.5% NaOH was added with subsequent vortexing
for 1 min and reading of absorbance at 520 nm after another min using
a UV–Vis spectrophotometer (Ultrospec 1100 pro, Amersham Bios-
ciences Uppsala, Sweden). The rest of the tubes were treated in a similar
way except that 60 μL of m-hydroxydiphenyl solution was added in-
stead of 0.5% NaOH. For quantification of GalA in the samples, a ca-
libration curve was built using mono-GalA (0 to 100.66 μg GalA/mL
demineralized water).
2.4.2. Determination of methanol content
Methanol (MeOH) content was determined through saponification
of the methanol-GalA ester bonds in pectin as described by Ng and
Waldron (1997) followed by quantification of the released MeOH using
a chromatographic method as described by Willemsen et al. (2017).
Hereto, in duplicate, approximately 50 mg of AIR was suspended in
1 mL of ultra-pure water (organic free, 18 MΩ cm resistance) in a 10 mL
amber glass vial (VWR International, Radnor, USA) and 10 μmol of
deuterated (d3) methanol was added as internal standard. Saponifica-
tion was done using 0.4 mL of 2 M NaOH at 20 °C for 1 h followed by
neutralization using 0.4 mL of 2 M HCl, addition of 3.1 mL of ultra-pure
water and equilibration at 20 °C for 15 min. For each AIR sample, a
correction for free methanol was facilitated by including a blank for
which NaOH and HCL were replaced with ultra-pure water.
For MeOH quantification, a gas chromatography (GC) system cou-
pled to a mass spectrometer (MS) (5977 N, Keysight Technologies,
Diegem, Belgium) was utilized. The samples were transferred to a
cooling tray (4 °C) of the CTC Combi PAL autosampler (CTC Analytics,
Zwingen, Switzerland). Each sample was incubated at 40 °C for 5 min
with agitation at 500 rpm. Through headspace-solid phase micro-
extraction, a pre-conditioned (according to manufacturer's instructions)
fiber (Supelco, Bellefonte, Pennsylvania) with an 85 μm carboxen/
polydimethylsiloxane sorptive coating extracted the volatile fraction for
5 min. The extract was injected into the injection port of the GC at
300 °C for 2 min then onto a 30 m × 320 μm × 20 μm HP-Plot Q
column (Keysight Technologies, Santa Rosa, California) in split mode
(1/10). For separation and elution of volatile compounds, the GC oven
temperature was maintained at 40 °C for 5 min, temperature was
ramped up to 250 °C at 70 °C/min and maintained for 5 min before
cooling back to 40 °C. Helium was used as the carrier gas at a constant
flow rate of 1.5 mL/min. For the quadrupole MS, an electron ionization
mode at 70 eV was used. The temperatures of the quadrupole and MS
ion source were 150 °C and 230 °C, respectively. A combination of se-
lective ion monitoring and scan modes was employed with a scanning
range of mass-to-charge ratio (m/z) of 10–200 at 7.1 scans/s. The se-
lected ions included one for methanol (m/z 31) and another for d3
C.M. Chigwedere et al.
methanol (m/z 35). Dilutions of a stock solution of MeOH (0–20 μmol/
mL ultra-pure water) wherein spiking with 10 μmol of d3 methanol was
done were analyzed. By plotting the ratio of peak areas of the selected
ions (methanol/d3 methanol) against concentration of MeOH, a cali-
bration curve was obtained.
2.5. Determination of pectin content in pectin fractions
In order to investigate pectin solubilization during cooking, pectin
in the pectin fractions was quantified through determination of GalA
and pectin-related neutral sugar content as described by Santiago et al.
(2017) with slight modifications. Hereto, in duplicate, a 40 μL aliquot of
a 2000 ppm solution of a pectin fraction sample in ultra-pure water was
dried at 45 °C under nitrogen (N2) gas prior to addition of 2 mL of an-
hydrous 2 M HCl (methanolic) for methanolysis at 80 °C for 16 h in an
oil bath. After cooling, the methanolic HCl was evaporated at 30 °C
under N2, after which 2 mL of trifluoroacetic acid (2 M) was added for
hydrolysis at 121 °C for 1 h. After cooling and drying at 45 °C under N2,
the hydrolysate was dissolved in 200 μL of ultra-pure water and was
filtered using a 0.45 μm filter (Chromaphil® A-45/25, Macherey-Nagel,
Germany). For quantification, a mixture of commercial sugar standards
(1–50 ppm) including L-fucose, L-rhamnose, L-arabinose, D-glucose, D-
galactose, D-xylose, D-mannose and D-GalA was used. Furthermore, to
correct for degradation during hydrolysis, mixtures of the sugars were
hydrolyzed in the same way as the samples. Recovery values were
calculated as a ratio of hydrolyzed to non-hydrolyzed sugar standards.
For profiling of the monosaccharides, a high performance anion
exchange chromatography system equipped with an autosampler of a
Dionex Bio-LC system (DX600), a GS50 gradient pump, a 150 × 3 mm
CarboPac™ PA20 column and its corresponding 30 × 3 mm guard
column (Dionex, Sunnyvale, CA, USA) was utilized. The column, at
30 °C, was first equilibrated using 100 mM, then 0.5 mM NaOH for
5 min each, at a constant flow rate of 0.5 mL/min. Following this, 10 μL
of the hydrolysate was injected onto the column and isocratically eluted
with 0.5 mM NaOH for 20 min followed by column regeneration using
500 mM NaOH for 10 min. Each sample was also isocratically eluted
with 25 mM NaOH in order to ensure good peak resolution of rhamnose
and arabinose. The sugars were detected using an ED50 electrochemical
detector equipped with a silver/silver chloride reference pH electrode
and a gold electrode, in the pulsed amperometric detection mode. To
identify and quantify sugars in the samples, sugar standards were
analyzed in the same way. Pectin extractability was calculated as the
ratio of the sum of GalA and pectin-related neutral sugars (fucose,
rhamnose, arabinose, galactose and xylose) in a specific pectin fraction
to the sum of these in all pectin fractions. To gain more insight into
cotyledon and seed coat pectin structure, the extent of linearity of
pectin in each fraction was determined as the ratio of GalA to pectin-
related neutral sugars.
2.6. Determination of molar mass distribution of pectin fractions
Molar mass distribution of pectin in WEP and NEP extracted from
cotyledons and seed coats of fresh and aged beans cooked for different
times was determined as described by Njoroge et al. (2016). To this
end, the eluent, 0.1 M acetic acid buffer (pH 4.4) containing 0.1 M so-
dium nitrate, was filtered through a 0.1 μm filter and used to prepare
0.2% (w/v) sample solutions in duplicate. After filtration through a
0.45 μm filter (Millex-HV, Merck Millipore Ltd., Cork, Ireland), an ali-
quot of 100 μL of sample solution was injected onto an Agilent 1200
high performance liquid chromatography system (Agilent technologies,
Diegem, Belgium) equipped with three Ultrahydrogel size exclusion
columns (2000, 1000 and 250) having exclusion limits of 1 × 107,
4 × 106 and 8 × 104 g/mol, respectively (Waters, Milford, MA). Elution
was performed at 0.5 mL/min and 35 °C followed by detection using
multi-angle laser light scattering (PN3621, Postnova Analytics, Ger-
many), refractive index (Shodex RI-101, Showa Denko K. K., Kawazaki,
4
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Japan) and a diode array detector (G1316A, Agilent Technologies 1200
Series, Diegem, Belgium). Data obtained was analyzed (calculation of
concentration and molar mass) with Nova Mals software (Version
1.0.0.18, Postnova Analytics) using a 2nd order regression Debye fitting
method. A refractive index increment of 0.146 mL/g was used.
2.7. Determination of mineral content in cotyledon, seed coat and cooking
water powders
Calcium (44Ca), magnesium (24Mg), iron (56Fe) and zinc (66Zn) were
quantified in cotyledons, seed coats and cooking water. In duplicate,
approximately 20 mg of a sample was incinerated in a muffle furnace at
550 °C for 20 h, then the ash was dissolved in 10 mL of ultra-pure water
before acidification with 0.1 mL of 65% nitric acid. Thereafter, the
solutions were filtered using a 0.45 μm filter (Chromaphil® A-45/25,
Macherey-Nagel, Germany) before analysis using inductively coupled
plasma-optical emission spectrometry (ICAP 7000 series, ThermoFisher
Scientific). Measurements were performed at wavelengths of 318, 285,
238 and 206 nm for 44Ca, 24Mg, 56Fe and 66Zn, respectively. Multi-
element standards were analyzed to obtain a calibration curve.
2.8. Data analysis
Data from DM and pectin solubilization analyses was statistically
analyzed. One way analysis of variance followed by a student's t-test for
pairwise comparison of means at 5% significance level was performed
using JMP Pro 13.1.0 software (SAS Institute, Inc., 2016). A regression
analysis was conducted to determine the influence of cooking on DM
using SAS version 9.4 TS1M4 (SAS Institute, Inc., Cary, NC, USA).
3. Results and discussion
3.1. Degree of methylesterification of pectin in alcohol insoluble residue
The DM of pectin in AIR extracted from cotyledons and seed coats of
fresh and aged beans cooked for different times is shown in Fig. 3A and
B. For all samples, the DM of cotyledon pectin was much higher than
that of seed coat pectin, suggesting inherent differences in the DMs.
According to Thakur, Singh, Handa, and Rao (1997), pectin is classified
as highly or lowly methylesterified if DM is higher or lower than 50,
respectively. Therefore, beans are characterized by highly methyles-
terified cotyledon pectin and lowly methylesterified seed coat pectin. Yi
et al. (2016) reported similar DM results for Rose coco common beans.
The low DM of seed coat pectin renders it much more sensitive to
crosslinking compared to cotyledon pectin, especially given the much
higher quantity of Ca2+ in seed coats compared to cotyledons (Section
3.4). However, our previous results showed that the contribution of
seed coats to HTC development is insignificant unlike that of cotyledons
(Chigwedere et al., 2018). Of interest is that for both cotyledon and
seed coat pectin, there were no significant changes in DM as a con-
sequence of aging of the beans (F0′ vs A0′ in Fig. 3, statistical results not
shown), suggesting that changes in DM do not play a crucial role in HTC
development. Njoroge et al. (2015) found similar results for DM of
pectin in AIR extracted from whole bean flour. In addition, similar re-
sults were reported for other legumes which are prone to HTC devel-
opment, including cowpeas (Liu, Phillips, Hung, Shewfelt, &
McWatters, 1992) and lentils (Bhatty, 1990).
During cooking of fresh beans in the current study, there was no
influence of cooking on DM of cotyledon pectin whilst for seed coat
pectin, DM decreased (Fig. 3A, Table 1). In aged beans, a minor influ-
ence of cooking on DM of cotyledon pectin was observed and for seed
coat pectin, it was the same as for fresh beans (Fig. 3B, Table 1). Due to
the decrease in DM of seed coat pectin during cooking, which implies
increased cation-binding capacity, it is highly unlikely that the minerals
(cations) under study could migrate to the cotyledons where DM was
rather constant. Values of DM of pectin in AIR from fresh and aged
C.M. Chigwedere et al.
Fig. 3. Degree of methylesterification (DM) of pectin in alcohol insoluble re-
sidues extracted from cotyledons and seed coats of fresh beans (A) and, coty-
ledons and seed coats of aged beans (B) cooked for different times in demi-
neralized water: F0′ and A0′ represent fresh beans cooked for 0 min and aged
beans cooked for 0 min, respectively. Error bars represent standard deviations.
Table 1
Values of slopes of the estimated regression lines for changes in degree of
methylesterification (DM) of cotyledon and seed coat pectin from fresh and
aged beans with cooking time.
Slope of regression line for change in DM
Materials from fresh beans
Cotyledons −0.0024 ± 0.0200
Seed coats −0.0570 ± 0.0070
Materials from aged beans
Cotyledons 0.0280 ± 0.0170
Seed coats −0.0320 ± 0.0060
samples cooked for the same time (F60′ vs A60′ and F270′ vs A270′)
were not significantly different (results not shown) indicating that de-
velopment of HTC did not significantly influence the DM of pectin
during cooking of the beans. However, for AIR from fresh and aged
beans of similar texture, results were inconsistent. A significant differ-
ence in DM was found between F60′ and A210′ but not between F120′
and A330′. In general, the changes in DM are possibly due to solubili-
zation and subsequent leaching of pectin of different DM from the co-
tyledons and seed coats into the cooking medium.
3.2. Pectin solubilization during cooking of beans
The relative amounts of the different pectin fractions extracted from
cotyledon and seed coat AIR of beans cooked for different times are
shown in Fig. 4A-D. Details on statistics are included in Supplementary
Table 1. Comparing pectin fractions obtained from non-cooked samples
(0′) of fresh bean cotyledon and seed coat materials (Fig. 4A and B), the
amounts of WEP in cotyledons and seed coats were not significantly
different, CEP in cotyledons was only 58% of that in seed coats and NEP
of seed coats was 78% of that in cotyledons. Yi et al. (2016) also found
higher amounts of CEP in seed coats than in cotyledons of non-cooked
Rose coco common beans. The abundance of CEP in seed coat pectin
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Food Research International xxx (xxxx) xxx–xxx
indicates that a greater part of pectin was ionically crosslinked and this
corroborates the very low DM of pectin in seed coat AIR (Section 3.1).
The linearity of all pectin fractions from cotyledons and WEP from seed
coats was rather comparable while CEP and NEP from seed coats was
much more linear as shown in Supplementary Fig. 1A-D. In conclusion,
cotyledon and seed coat pectin was structurally different.
Due to aging, a drastic decrease (by 55%) in WEP coupled with an
increase of comparable magnitude (58%) in NEP and a non-significant
increase (10%) in CEP were observed in cotyledons (Fig. 4A and C, and
Supplementary Table 1). This observation can be attributed to the in-
terconversion of loosely bound pectin (WEP) into covalently (ester)
bound pectin (NEP). Shiga, Lajolo, and Filisetti (2004) found a decrease
in water extractable cell wall polysaccharides from cotyledons of aged
Carioca common beans. In the current study, a similar interconversion
of pectin fractions as for cotyledon pectin but to a much lower extent
was observed in seed coat pectin, with WEP decreasing by 13% and
NEP increasing by 17% whilst CEP had a non-significant change
(Fig. 4B and D, and Supplementary Table 1).
The non-significant changes in both the DM (Section 3.1) and CEP
fraction, and the interconversions of WEP and NEP fractions revealed in
the current study undermine the classical pectin-cation-phytate hy-
pothesis of HTC development which was described in the introduction.
This hypothesis requires the DM of pectin to decrease with concomitant
increase in cation-mediated pectin crosslinking. In this case, the CEP
fraction would be expected to increase. Since CEP did not increase, a
more plausible explanation for the pectin-related mechanism of bean
hardening would be: the inherent DM of cotyledon pectin (~60) facil-
itates pectin crosslinking in the presence of divalent cations released
from phytate hydrolysis by phytase in the cotyledon. In this way, mi-
gration of cations from the seed coat to the cotyledons where they are
thought to contribute to pectin crosslinking is futile. This is more so as
seed coat pectin has a higher cation-binding capacity than cotyledon
pectin due to its low DM (Section 3.1).
Results from this study show that loosely-bound pectin became
strongly covalently crosslinked or was crosslinked to other cell wall
components due to aging. Such interactions may include (i) esterifica-
tion of ferulic acid dehydrodimers to arabinan and galactan side chains
of RG I (ii) uronyl ester formation between a carboxyl group of a ga-
lacturonosyl residue of one HG chain and a hydroxyl group of the same
residue of another HG chain and (iii) dimerization of RG II through
borate diester bonds leading to HG crosslinks since RG II is covalently
linked to HG, as reviewed by Voragen et al. (2009). An interesting case
of carrots which can be likened to aging in beans (according to the
pectin-cation-phytate hypothesis) was reported by Sila, Smout, Elliot,
Van Loey, and Hendrickx (2006) whereby pretreatment conditions that
induced pectin demethylesterification were applied in the presence of
Ca2+ resulting in reduced texture loss. A great increase in NEP was
observed instead of the expected increase in CEP. This raises a possi-
bility that CEP could have been entrapped in other cell wall polymers,
making it difficult to extract with a chelator-containing solution. Ra-
ther, it could have become extractable together with NEP. In the current
study, this entrapment proposition is unlikely since the DM did not
change significantly unlike in the study by Sila, Smout, et al. (2006).
However, to test this proposition, there would be need to explore vi-
sualization and/or identification of the location of CEP and NEP in situ.
During cooking, both cotyledons and seed coats of fresh beans ex-
hibited an increase in WEP coupled with a decrease in NEP (Fig. 4A and
B). Similar thermal processing-induced conversion between NEP and
WEP was observed in other plant-based systems as reviewed by Sila,
Van Buggenhout, Duvetter, Van Loey, and Hendrickx (2009). There
were no significant changes in the amount of CEP from seed coats
whereas in cotyledons, it decreased slightly but significantly. These
interconversions of covalently bound and ionically bound pectin into
loosely-bound pectin are evidence of pectin thermosolubilization in
beans during cooking. For aged beans (Fig. 4C and D), similar results as
for fresh beans were observed with respect to differences in relative
C.M. Chigwedere et al. Food Research International xxx (xxxx) xxx–xxx

Fig. 4. Profiling of water-extractable pectin (WEP), chelator-extractable pectin (CEP) and sodium carbonate-extractable pectin (NEP) fractions extracted from
cotyledons (A) and seed coats (B) of fresh beans as well as cotyledons (C) and seed coats (D) of aged beans, after the beans were cooked for different times in
demineralized water: F0′ and A0′ represent fresh beans cooked for 0 min and aged beans cooked for 0 min, respectively.

amounts of pectin fractions and pectin thermosolubilization. For all
samples, the tendency for WEP to decrease at long cooking times can be
attributed to leaching into the cooking solution. The amount of GalA
that was leached into the cooking water increased with increasing
cooking time as shown in Supplementary Fig. 2 for aged beans. It in-
dicates the trend of leaching of water-extractable pectin but cannot be
attributed to leaching from either the seed coat or cotyledons since
whole beans were cooked (with seed coats on). Neutral sugars asso-
ciated with pectin were not quantified along with GalA because some
oligosaccharides which are leached out during soaking and cooking
have some of the neutral sugars as for pectin so their presence would
influence results.
Considering samples from cotyledons of fresh and aged beans
cooked for the same time (F60′ vs A60′ and F270′ vs A270′) thus dif-
ferent hardness, the fresh samples exhibited significantly higher WEP
but lower CEP and NEP compared to their aged counterparts (Fig. 4A
and C, and Supplementary Table 1). It is clear from these results that
HTC (aged) samples were characterized by more strongly-bound pectin
compared to fresh samples. On the other hand, for cotyledons of fresh
and aged beans cooked to similar hardness, results were inconsistent.
There were no significant differences between pectin fractions from
F60′ and A210′. However, for F120′ and A330′, WEP from F120′ was
significantly higher while NEP was significantly lower than that from
A330′ and CEP remained relatively unchanged. The lower amount of
WEP in A330′ can be attributed to its leaching into the cooking water
due to the long cooking time (Supplementary Fig. 2). In the case of seed
coats (Fig. 4B and D, and Supplementary Table 1), significant differ-
ences in WEP were noted between F60′ and A60′, F60′ and A210′, and
F120′ and A330′, with WEP generally being more abundant in fresh
than aged samples. NEP of A60′ was significantly higher than that of
F60′ and there were no changes in CEP in all cases.
From changes in cotyledon and seed coat pectin, it is clear that the
most prominent changes occurred in the cotyledons. This is in agree-
ment with our previous findings that the cotyledon plays a more sig-
nificant role in HTC development compared to seed coats (Chigwedere
et al., 2018). Moreover, bean softening which was attributed to pectin
solubilization as a rate-limiting step, could be premised on inter-
conversion of covalently bound pectin into loosely bound pectin.
3.3. Molar mass distribution of pectin fractions
Considering that the molar mass distribution profiles of a specific

6
C.M. Chigwedere et al. Food Research International xxx (xxxx) xxx–xxx

1.E+07 50 1.E+07 10
A C
45 9
1.E+06 1.E+06

Concentration (μg/mL) (solid lines)

40 8

Concentration (μg/mL) (solid lines)

Molar mass (Da) (broken lines)

1.E+05

Molar mass (Da) (broken lines)

1.E+05 35 7

30 6
1.E+04 1.E+04
25 5
1.E+03 1.E+03
20 4

1.E+02 15 1.E+02 3

10 2
1.E+01 1.E+01
5 1

1.E+00 0 1.E+00 0
30 35 40 45 50 55 60 30 35 40 45 50 55 60

1.E+07 50 1.E+07 10

Concentration (μg/mL) (solid lines)

B D

Concentration (μg/mL) (solid lines)

45 9
1.E+06 1.E+06

Molar mass (Da) (broken lines)

40 8
Molar mass (Da) (broken lines)

1.E+05 35 1.E+05 7

30 6
1.E+04 1.E+04
25 5
1.E+03 1.E+03
20 4

1.E+02 15 1.E+02 3

10 2
1.E+01 1.E+01
5 1

1.E+00 0 1.E+00 0
30 35 40 45 50 55 60 30 35 40 45 50 55 60
Elution time (min) Elution time (min)

F0ʹ F120ʹ F270ʹ F0ʹ F120ʹ F270ʹ

Fig. 5. Molar mass (broken lines) and concentration (solid lines) profiles for water-extractable pectin from cotyledons (A) and seed coats (B), and sodium bi-
carbonate-extractable pectin from cotyledons (C) and seed coats (D) of fresh beans cooked in demineralized water for different times: F0′ denotes fresh beans cooked
for 0 min.

fraction (WEP/NEP) from cotyledons and seed coats of fresh beans were Looking at profiles of NEP from cotyledons, one pectin polymer
similar to those from aged beans, only the profiles from fresh beans are population (concentration curves) corresponding to the high molar
shown. For WEP from cotyledons, two pectin populations were evident, mass pectin polymers was present (Fig. 5C) and all the extracts were of
a low concentration of high molar mass polymers (first peak) and high similar molar mass. NEP from seed coats (Fig. 5D) exhibited similar
concentration of low molar mass polymers (second peak) (Fig. 5A). profiles to those for WEP from seed coats (Fig. 5B). It can be concluded
Only a slight shift in elution time maxima of the high molar mass that there were differences in profiles between seed coat and cotyledon
polymers to a slightly longer elution time was observed with cooking pectin fractions but in each case no influence of aging on the profiles
time. In fact, the molar mass profiles (broken lines) were nearly su- during cooking was observed. The similarity in molar mass profiles of
perimposable, highlighting a high degree of similarity in pectin molar WEP and NEP from either cotyledons or seed coats suggests that NEP
mass despite differences in cooking time. These observations suggest that was solubilized into WEP (Section 3.2) was of similar molar mass
limited (if any) cooking-induced depolymerization of the pectin poly- as the WEP.
mers as further confirmed by absence of β-elimination products which
absorb ultraviolet light at 235 nm (results not shown). According to
3.4. Changes in distribution of minerals during cooking of beans
Sila, Smout, et al. (2006), during thermal processing, pectin solubili-
zation along with depolymerization due to β-elimination which is ex-
Some of the fresh beans exhibited a hard shell problem (partial or no
tensive at pH > 4.5 influence tissue texture. In the current study,
soaking) hence fresh beans were not considered in the investigation of
softening of beans during cooking can be attributed to mainly pectin
changes in distribution of minerals during cooking. Fig. 6 shows the
solubilization (Section 3.2) without depolymerization. Unlike the con-
relative quantities of minerals in cotyledons, seed coats and cooking
centration profiles from cotyledons, peaks from the seed coats did not
water during cooking of aged beans whilst Supplementary Table 2
separate clearly and no shifts in elution time maxima were observed
shows the absolute quantities and the associated standard deviations.
(Fig. 5B). In addition, they showed a difference between non-cooked
Looking at the non-cooked sample (A0′), it is clear that Mg2+, Zn2+ and
(F0′) and cooked (F120′ and F270′) samples which can be attributed to
Fe2+ were abundant in the cotyledon, with limited leaching into the
low recoveries of pectin in F0′. This could have contributed to the ob-
soaking water. Calcium was more abundant in the seed coat than the
served difference in molar mass profiles. According to Sila, Doungla,
cotyledon. Using elemental mapping from proton-induced X-ray emis-
Smout, Van Loey, and Hendrickx (2006), the likelihood of cooking-in-
sion spectrometry, Kruger, Minnis-Ndimba, Mtshali, and Minnaar
duced depolymerization by β-elimination is highly diminished for low
(2015) also found the concentration of Mg2+ to be much higher than
DM pectin. Thus, in the current study, seed coat pectin was not ex-
that of Ca2+ in cotyledons. In the current study, the high quantity of
pected to undergo β-elimination due to its low DM.
Ca2+ coupled with presence of highly linear and low DM pectin explain

7
C.M. Chigwedere et al.
Fig. 6. Mineral ion distribution during cooking of aged beans in demineralized water expressed as relative amounts of minerals in cotyledons, seed coats and cooking
water: A0′ denotes aged beans cooked for 0 min.
the presence of more CEP in the seed coat than cotyledons (Section 3.3)
as these properties promote cation-mediated pectin crosslinking via
ionic bonds. Although Kyomugasho et al. (2017) showed the strength of
a pectin-divalent cation interaction to decrease in the order
Fe2+ > Zn2+ > Ca2+ > Mg2+, the abundance of Ca2+ in seed coats
in the current study implies that it is responsible for most of the ionic
crosslinks between the seed coat pectin polymers. During cooking of the
beans, except for Mg2+, the rest of the minerals were largely retained in
either cotyledons or seed coats. Approximately 40% of Mg2+ ions lea-
ched from cotyledons into the cooking water, mainly during the first 60′
of cooking while the Mg2+ content of seed coats remained rather
constant. Leaching of Mg2+ can be attributed to this ion being the least
interactive with low DM pectin given its low electronegativity and
higher effective hydrated diameter (Kyomugasho et al., 2017) in com-
parison with other minerals under study.
4. Conclusion
This study revealed that storage-induced hardening of beans and
their subsequent softening during cooking is related to interconversions
between pectin fractions. Moreover, a comparison of cotyledon and
seed coat pectin from non-cooked fresh and aged beans showed in-
herent molecular structure differences. Seed coat pectin was mainly
CEP, characterized by a low DM and high linearity but also an appre-
ciable amount of covalently bound pectin (NEP) which was more linear
than cotyledon pectin. Upon aging, a decrease in loosely-bound pectin
(WEP) coupled with an increase in NEP was observed and attributed to
interactions of loosely-bound pectin with other cell wall components
through covalent linkages. This interconversion was more pronounced
in cotyledons than in seed coats, indicating a greater role of the former
in bean hardening. For both cotyledon and seed coat, CEP remained
rather constant. Even though DM did not change significantly, there is
need to confirm that no CEP was formed and entrapped in more
covalently bound polymers of the cell wall such that it became difficult
to extract with a chelator-containing solution. To this end, visualization
and/or identification of the location of CEP and NEP in situ can be
explored.
Our detailed investigation of evolution of pectin changes in coty-
ledons and seed coats separately yielded a far more complete picture of
8
Food Research International xxx (xxxx) xxx–xxx
the pectin solubilization mechanism during cooking of beans as so far
available in open literature. During cooking, interconversion of NEP
into loosely bound pectin (WEP) was observed and this was more
pronounced in cotyledons compared to seed coats, leading to bean
softening. In general, CEP remained relatively unchanged with a few
exceptions. Molar mass distribution results revealed that softening of
beans due to solubilization of covalently-bound pectin was not ac-
companied by its depolymerization. With respect to mineral content,
except for Ca2+ which was more abundant in seed coats, other minerals
(Mg2+, Zn2+ and Fe2+) were abundant in cotyledons. During cooking,
apart from Mg2+ which leached greatly from cotyledons into the
cooking medium, the other minerals (Ca2+, Zn2+ and Fe2+) were lar-
gely retained in the bean matrix. Thus, common beans still remain a
good source of most minerals regardless of the extent of cooking.
However, these findings could be of particular interest in exploring the
bioaccessibility of these minerals from cotyledons and/or seed coats.
This detailed study on mechanistic insight into pectin solubilization and
mineral distribution during cooking may also be relevant for other le-
gume matrices such as cowpeas (Vigna unguiculata) and Bambara
groundnuts (Vigna subterranea) that are known to be susceptible to HTC
development.
Acknowledgements
Chigwedere C.M. is a Ph.D. fellow funded by the Interfaculty
Council for Development Cooperation (IRO, Grant number
000000003746). Kyomugasho C. is a postdoctoral researcher funded by
the Onderzoeksfonds KU Leuven post-doctoral fellowship (PDM).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.foodres.2018.08.062.
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