Molecular structure, chemical properties and biological activities of Pinto bean pod polysaccharide

Article abstract of DOI:10.1016/j.ijbiomac.2016.04.003

Pinto bean pod polysaccharide (PBPP) was successfully extracted with yield of 38.5 g/100 g and the PBPP gave total carbohydrate and uronic acid contents of 286.2 mg maltose equivalent/g and 374.3 mg Gal/g, respectively. The M_w of PBPP was 270.6 kDa with intrinsic viscosity of 0.262 dm³/g, which composed of mannose (2.5%), galacturonic acid (15.0%), rhamnose (4.0%), glucose (9.0%), galactose (62.2%), xylose (2.9%) and arabinose (4.3%) with trace amount of ribose and fucose. The result suggested that PBPP has a spherical conformation with a highly branched structure. Fourier Transform Infrared analysis showed that PBPP has a similar structure as commercial pectin with an esterification degree of 59.9%, whereas scanning electron microscopy study showed that the crude polysaccharide formed a thin layer of film that was made of multiple micro strands of fibre. PBPP exhibited substantial free radical scavenging activity (7.7%), metal reducing capability (2.04 mmol/dm³) and α-amylase inhibitory activity (97.6%) at a total amount of 1 mg. PBPP also exhibited high water- and oil-holding capacities (3.6 g/g and 2.8 g/g, respectively). At a low concentration, PBPP exhibited emulsifying activity of 39.6% with stability of 38.6%. Apart from that, PBPP was able to show thickening capability at low concentration (0.005 kg/dm³).

Full text of DOI:10.1016/j.ijbiomac.2016.04.003

International Journal of Biological Macromolecules 88 (2016) 280–287
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules
journal homepage: www.elsevier.com/locate/ijbiomac
Molecular structure, chemical properties and biological activities of
Pinto bean pod polysaccharide
Fazlina Kamarudin, Chee-Yuen Gan∗
Analytical Biochemistry Research Centre, 11800 USM, Penang, Malaysia
a r t i c l e i n f o
a b s t r a c t
Article history:
Pinto bean pod polysaccharide (PBPP) was successfully extracted with yield of 38.5 g/100 g and the PBPP
gave total carbohydrate and uronic acid contents of 286.2 mg maltose equivalent/g and 374.3 mg Gal/g,
respectively. The Mw of PBPP was 270.6 kDa with intrinsic viscosity of 0.262 dm3/g, which composed
of mannose (2.5%), galacturonic acid (15.0%), rhamnose (4.0%), glucose (9.0%), galactose (62.2%), xylose
(2.9%) and arabinose (4.3%) with trace amount of ribose and fucose. The result suggested that PBPP has
a spherical conformation with a highly branched structure. Fourier Transform Infrared analysis showed
that PBPP has a similar structure as commercial pectin with an esterification degree of 59.9%, whereas
scanning electron microscopy study showed that the crude polysaccharide formed a thin layer of film
that was made of multiple micro strands of fibre. PBPP exhibited substantial free radical scavenging
activity (7.7%), metal reducing capability (2.04 mmol/dm3) and ␣-amylase inhibitory activity (97.6%) at
a total amount of 1 mg. PBPP also exhibited high water- and oil-holding capacities (3.6 g/g and 2.8 g/g,
respectively). At a low concentration, PBPP exhibited emulsifying activity of 39.6% with stability of 38.6%.
Apart from that, PBPP was able to show thickening capability at low concentration (0.005 kg/dm3).
© 2016 Elsevier B.V. All rights reserved.
Received 10 December 2015
Received in revised form 29 February 2016
Accepted 1 April 2016
Available online 1 April 2016
Keywords:
␣-Amylase inhibitor
Pod
Polysaccharide
1. Introduction
tive peptides [6,7]. However, Pinto bean is purchased as canned
pre-cooked or plastic packaged dry beans. The pods are usually
Pinto bean (Phaseolus vulgaris cv. Pinto) is a variety of common
beans, which is highly consumed and commercialised worldwide.
It was originated in Latin America more than 7000 years ago, and
it has been rapidly cultivated throughout the world. According to
the Food and Agriculture Organization of the United Nations, the
top five dry bean producers are India, Brazil, Myanmar, China and
the United States of America, which produced approximately 3.3,
3.0, 2.1, 1.5 and 1.2 million tonnes of dry bean per year, respec-
tively, from the year 1993 to 2014 (http://faostat3.fao.org/browse/
Q/QC/E). To our surprise, the leading class of dry common beans
produced in the United States is Pinto bean at approximately
42% of the total production (http://www.ers.usda.gov/topics/crops/
vegetables-pulses/dry-beans.aspx).
removed and discarded. The efforts to study the effects of extraction
processes on polysaccharide as well as their molecular structure,
chemical properties and biological activities have not yet been per-
formed. Therefore, the pod of Pinto bean was chosen as a source of
macromolecule carbohydrate and its potentials, as an alternative
macromolecule for commercial uses, need to be evaluated.
Due to the structural features (e.g. degree of substitutions, steric
configuration, linkages of monosaccharides and substitutes, and
molar mass and its distribution), vegetable polysaccharides play a
critical role in their physical properties, such as thickening, gelling,
and stabilizing ability [8]. These properties have extended their
applications in food and biomedical industries. Modern pharma-
cological research has identified that polysaccharide as one of
the major active components in herbs, which is responsible for
various pharmacological activities, such as antioxidant, antiviral,
immunostimulatory, antitumour, antifatigue, radioprotection and
hepatoprotection activities [9–13]. Clinical studies also suggested
that moderate or high intake of dietary fibre, such as cellulose,
hemicellulose and lignin, can effectively reduce the risks for devel-
oping diseases like diabetes, cardiovascular disease, hypertension,
hypercholesterolemia, hyperlipidemia, obesity, and colorectal can-
cer [14–19]. Sulfated polysaccharides from algae were also reported
Bean consumption has been reported to minimize risk of chronic
diseases, such as diabetes [1], coronary heart disease [2], colon can-
cer [3], prostate and breast cancer [4,5]. This could be due to the
presence of polysaccharides. Previous studies also showed that the
bean has the potential in producing functional protein and bioac-
∗
Corresponding author at: Analytical Biochemistry Research Centre, Universiti
Sains Malaysia, 11800 USM, Penang, Malaysia.
E-mail address: mattgan81@gmail.com (C.-Y. Gan).
http://dx.doi.org/10.1016/j.ijbiomac.2016.04.003
0141-8130/© 2016 Elsevier B.V. All rights reserved.

F. Kamarudin, C.-Y. Gan / International Journal of Biological Macromolecules 88 (2016) 280–287
281
with 75 ␮L of 0.05 kg/dm3 phenol solution on an ice bed and sul-
phuric acid (375 ␮L) was then added. The mixtures were incubated
at 80 ◦C for 30 min and absorbance was measured at 495 nm using a
spectrophotometer (Spectamax M5, Moleular Devices, USA). Mal-
tose was used as a standard.
Uronic acid content was determined using the method as
described by Blumenkrantz and Asboe-Hansen [29]. Crude polysac-
charide (250 ␮L) at concentration of 1 mg/mL was mixed with
1.5 mL of 0.0125 mol/dm3 sulphuric acid/sodium tetraborate
solution on an ice bed and then heated at 100 ◦C for 5 min m-
Hydroxydiphenyl (25 ␮L) was then added and the absorbance was
measured after 5 min at 520 nm using a spectrophotometer (Spec-
tamax M5, Moleular Devices, USA).
to possess a higher anticoagulant activity than those of heparin [20]
whereas galactoglucomannans and pectins from woody materials
have also been reported to exhibit immunostimulating and free
radicals scavenging activities [21,22]. Several studies have reported
the possibility of using waste products as polysaccharide sources.
To date, polysaccharides have been extracted from several under
utilised biomasses, such as mango waste, sunflower head residues,
sugar beet, soy hull and sweet potato residues [23–25]. Nonethe-
less, not all sources are suitable for commercial uses. The sources as
well as their extraction and purification methodologies may affect
the structure and composition of the produced pectin. Therefore, a
continuous search for suitable pectin is aggressively performed by
researchers.
The main objective of this study was to explore the potential
of Pinto bean pod as an alternative source of polysaccharide. The
specific objectives of the study were to extract polysaccharide from
the pods of Pinto bean using various extraction parameters; and to
evaluate the molecular structure, chemical properties as well as the
biological activities of the extracted macromolecule carbohydrate.
2.5. Functional groups and degree of esterification (DE)
determinations
FTIR spectra of crude pectic-polysaccharide (powder form) were
recorded from 650 to 4000 cm−1 using Cary 670 FTIR spectrom-
eter with an attenuated total reflectance (ATR) system (Agilent
Technologies, CA, USA). The spectra were analysed using Agilent
Resolutions Pro software. DE was determined using the following
equation:
2. Materials and methods
2.1. Materials
A1730
Fresh Pinto bean pods were collected from different markets
(∼10 kg from each market) in Penang. The pods were cleaned with
deionised water and were kept frozen at −20 ◦C. They were subse-
quently freeze-dried. The lyophilised pods were ground into fine
powder using a blender, sieved (30 Mesh) and stored at 4 ◦C prior
to extraction. All chemicals used in this study were of analytical
grade (Sigma-Aldrich, USA).
DE =
× 100
(1)
A1730 + A1600
where A1730 was defined as the area of the band at 1730 cm−1 and
A1600 was defined as the area of the band at 1600 cm−1 [30].
2.6. Determination of molecular weights
The molecular weight of PBPP was examined using a gel per-
meation chromatography (GPC) equipped with Viscotek Model
TDA 305 Triple Detector Array incorporated Refractive index, Light
scattering and viscosity detectors (Malvern, UK). CLM 3021 col-
umn (A6000 M, 300 × 7.8 mm, 13 ␮m beads size, Malvern, UK) was
used. A 100 ␮L of sample (0.01 kg/dm3) in 0.1 mol/dm3 NaNO3) was
injected and the flow rate and temperature were maintained at
1.0 mL/min and 30 ◦C, respectively. The elution was carried out with
0.1 mol/dm3 NaNO3 containing 0.003 kg/dm3 NaN3 to prevent bac-
teria growth. Polyethylene Oxide (18670 Da) was used as working
calibration standard. A second standard, dextran (65333 Da) was
applied to verify the calibration accuracy with high level of con-
fidence. The molecular weight of the sample was determined by
comparing with calibration curve. The chromatogram obtained was
analyzed using the OmniSEC software.
2.2. Crude polysaccharide extraction
Briefly, 6 g of lyophilised pod powders were added in 300 mL of
0.1 mol/dm3 citrate-phosphate buffer at different pH values (i.e. pH
2, pH 4 and pH 6) using solid to buffer ratio of 1:50. The mixture was
then incubated at different extraction durations (i.e. 1 h, 3 h and 5 h)
in an incubator shaker (IKA KS 4000i Control, Staufen, Germany)
which constantly shaking at 250 rpm at different temperature set-
tings (i.e. 50 ◦C, 60 ◦C and 70 ◦C). The resulting slurries were then
filtered through a muslin cloth (2 layers) to remove solid particles.
Thereafter, three volumes of ethanol were added to one volume of
extract and incubated at 4 ◦C for 5 h to precipitate polysaccharide.
The precipitates obtained were filtered through a muslin cloth and
then washed with ethanol to remove the small molecular weight
molecules in the extract. The crude polysaccharide (PBPP) was then
lyophilised and stored in a desiccator prior to analysis. The PBPP
yield was expressed in g/100 g (w/w, dry basis).
2.7. Determination of monosaccharide composition
2.3. Scanning electron microscopic (SEM) analysis
Monosaccharides composition was determined using the
method of Lv et al. [31] using a high performance liquid chro-
matography (HPLC) system equipped with an UV detector. Sample
(10 mg) was hydrolyze using 3 mol/dm3 trifluoroacetic acid (1 mL)
at 95 ◦C for 8 h, vacuum dried and re-dissolved in 1 mL of deionized
water. Sample (100 ␮L) was then added with 3-methyl-1-phenyl-
2-pyrazoline-5-one (PMP, 200 ␮L) and 0.3 mol/dm3 NaOH (300 ␮L)
followed by incubation at 70 ◦C for 1 h. HCl (300 ␮L, 0.3 mol/dm3)
was then added and the resulting solution was extracted with
1 mL of chloroform for 3 times. The aqueous layer was collected
and filtered through a 0.45 ␮m membrane prior to HPLC analysis.
The HPLC system was prepared as the following condition: Zor-
bax SB-C18 reversed-phase column (250 × 4.6 mm, 5 ␮m, Agilent,
USA). Mobile phase: (A) Acetonitrile; (B) 3.3 mmol/dm3 KH2PO4-
3.9 mmol/dm3 Tris-acetate-EDTA buffer containing 0.10 kg/dm3
ACN. The gradient: 0–4 min: 94% B; 4–9 min: from 94% to 88%B;
Samples were mounted onto SEM specimen stub with a double-
sided tape and coated with gold using a Polaron SC 515 Sputter
Coater (Fisons Instruments, UK). Subsequently, the samples were
photographed using Leo Supra 50VP Field Emission Scanning Elec-
tron Microscope equipped with Oxford INCA 400 energy dispersive
X-ray Microanalysis system (Oxford Instruments Analytical, UK).
2.4. Protein content, lipid content, total carbohydrate content
and uronic acid content determinations
Protein content of PBPP was determined using Bradford method
[26], lipid content was determined using Soxhlet method [27] and
total carbohydrate content was determined using the method as
described by Dubois et al. [28]. Sample (75 ␮L, 1 mg/mL) was mixed

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9–20 min: 88% B. The flow rate was set at 1.0 mL/min and the injec-
tion volume of 20 ␮L. The UV detector was set at 250 nm. A mixed
standard, which composed of glucoronic acid, mannose, galactur-
onic acid, ribose, rhamnose, glucose, galactose, xylose, arabinose
and fucose were prepared. The result was expressed as a molar
percentage.
2.8. Total phenolic content and antioxidant activity
determinations
In the total phenolic content (TPC) analysis, sample (1 mg)
was mixed with 1.8 mL of Folin-Ciocalteau reagent and incu-
bated at room temperature for 5 min. Sodium bicarbonate (1.2 mL,
0.075 kg/dm3) was then added and incubated for 60 min at room
temperature. The absorbance was measured at 765 nm using a
spectrophotometer (Spectamax M5, Moleular Devices, USA) and
the result was expressed as mg Gallic acid equivalents (GAE)/100 g
extracts [32].
In the 2,2-Diphenyl-1-picrylhydrazyl (DPPH) scavenging activ-
ity determination, sample (1 mg) was added to 500 ␮L of ethanolic
DPPH solutions (0.1 mmol/dm3) and incubated in the dark for
30 min at 30 ◦C. The discoloration was measured at 517 nm using
a spectrophotometer (Spectamax M5, Moleular Devices, USA) and
the result was expressed as %/mg extract [33].
In the ferric reducing/antioxidant power (FRAP) analysis, the
FRAP reagent (400 ␮L) was pre-warmed at 37 ◦C for 30 min and then
added to 1 mg of sample. The reaction mixtures were incubated for
1 h at 37 ◦C and the absorbance at 593 nm was measured using a
spectrophotometer (Spectamax M5, Moleular Devices, USA). The
result was expressed as mole of FeSO4/g of extracts [34].
2.9. ˛-Amylase inhibitory activity
␣-Amylase solution (100 ␮L, 1 mg/mL) and PBPP solution
(1 mg/mL) were mixed and incubated at 25 ◦C for 10 min. Starch
solution (100 ␮L) in 0.02 mol/dm3 sodium phosphate containing
6.0 mmol/dm3 sodium chloride at pH 6.9 was then added into the
mixture, followed by incubation at 25 ◦C for 10 min. Dinitrosali-
cylic acid (200 ␮L) was subsequently added into the mixture in
order to terminate the reaction and heated in boiling water for
5 min. The mixture was cooled to room temperature and diluted
with 3 mL of deionized water. The absorbance was measured at
540 nm using UV–vis spectrophotometer (Spectramax M5, Molec-
ular Devices, USA). Sample blank (mixture of PBPP solution and
buffer solution), negative control (only buffer solution), positive
control (mixture of ␣-amylase and buffer solution) were prepared
and analyzed using the same procedure. The ␣-amylase inhibitory
activity was calculated as follows:
Fig. 1. Morphology of (A) outer layer and midsection of Pinto bean pod, (B) inner
layer of the paint bean pod and (C) PBPP.
ꢀꢁ
ꢂꢃ
Apos − A − A
Apos − Aneg
(
)
s
sb
Emulsifying activity (EA) and emulsion stability (ES) were deter-
mined according to Betancur-Ancona et al. [37]. Sample (10 mL,
0.01 kg/dm3) was homogenized (TH-02, Omni) at 35,000 rpm for
2 min. Corn oil (10 mL) was then added and homogenized for 2 min.
The emulsion was centrifuged (Multifuge X1R, Thermo Scientific,
USA) at 1200g for 5 min and the emulsion volume measured. EA
and ES were expressed in percentage.
ꢁ
ꢂ
%inhibiton =
× 100
(2)
where Apos was the absorbance of positive control, Aneg was the
absorbance of negative control, As was the absorbance mixture of ␣-
amylase solution and PBPP solution, and Asb was the sample blank
[35].
The thickening capacity was measured based on the viscosity
of the sample (0.005 kg/dm3) at different pH conditions using a
rheometer (AR2000ex, TA Instruments, USA) with shear rate rang-
ing from 0.01 to 1000 s−1 in 5 min, as described previously [38].
2.10. Functional properties determination
Water- and oil-holding capacity (WHC and OHC) were deter-
mined using the method described by Chau et al. [36]. Sample (0.1 g)
and 10 mL of distilled water or corn oil were added and incubated
for 5 h at room temperature. Subsequently, the mixture was cen-
trifuged at 5000g for 10 min. WHC and OHC were expressed as g/g
extract. Corn oil density is 0.92 g/ml.
2.11. Statistical analysis
The experiment was first conducted using a factorial design in
order to study the suitable condition for the extraction. Results

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283
Table 1
PBPP yields (g/100 g) at different extraction conditions.
Buffer pH (pH)
Temperature (◦C)
Extraction duration (h)
1
3
5
2
4
6
50
60
70
50
60
70
50
60
70
20.8 0.3b
27.5 0.5bc
29.1 0.6bc
15.2 2.4a
24.0 2.3b
24.4 0.9b
28.7 0.9bc
25.5 0.9b
26.6 0.7b
27.1 0.5bc
28.2 0.9bc
33.7 0.3c
38.5 1.2cd
22.7 3.8b
26.9 1.9b
32.1 3.3c
30.8 0.8c
33.7 5.9c
32.1 1.9c
35.7 3.0c
35.8 0.8c
20.0 1.0b
29.1 1.5bc
34.0 0.4c
21.4 3.7b
33.8 4.3c
33.8 2.4c
Note: Comparison within the column and row in Table with the data written as mean. (n = 3). Means within the same column not followed by the same letter are significantly
different at p < 0.01 level of significance, according to Duncan’s Multiple-Range Test.
Table 2
Protein, lipid, carbohydrate and uronic acid contents of PBPP.
Sample
PBPP
Protein content (g/100 g)
0.2 0.0
Lipid content (g/100 g)
0.0 0.0
Carbohydrate content (mg maltose equivalent/g)
286.2 11.3
Uronic acid content (mg Gal equivalent/g)
374.3 67.3
were expressed as mean values standard deviation of three
separated determinations. Comparison of means was performed
using one-way analysis of variance (ANOVA). When comparing the
functional properties of the PBPP with commercial pectin, t-test
was used with significant level of p < 0.05 (SPSS for Windows, Ver-
sion 19.0, SPSS Institute Inc., Cary NC).
the pod. Such phenomenon demonstrated an existence of inter-
action between pH, temperature and time, where ANOVA showed
that there was a significant (p < 0.05) interaction between pH and
temperature as well as between pH and time (p < 0.01) in affecting
the extraction yield. In this case of study, extraction condition at
pH 2, temperature of 70 ◦C and extraction time of 3 h, was selected
as the most suitable condition for extracting PBPP because this
condition performed better (i.e. gave the highest yield) from the
economic point of view, although minor alteration of the structure
of the PBPP might occur. It should also be noted that the extraction
yields obtained were found to be higher than that of pectin from
citrus peels (16.1 g/100 g) and apple pomace (14 g/100 g) [41,42].
Therefore, it was suggested that Pinto bean pod is a good source of
macromolecule carbohydrate.
3. Results and discussion
3.1. Microscopic examination of Pinto bean pod
Fig. 1A shows the morphology of the outer layer and the mid-
section of Pinto bean pod. The presence of micro-filaments on the
surface of the outer layer was observed, whereas multiple layers
of fibre were observed in the midsection. It was interesting to note
that the surface of the inner layer (Fig. 1B) shows the presence of
thin film, which could be consisted of cellulose, hemicellulose or
pectin. Therefore, Pinto bean pod was suggested to be a good source
for the extraction of macromolecule carbohydrate.
3.3. Morphology and chemical compositions of PBPP
Fig. 1C shows the morphology of PBPP and it was found that
this extracted polysaccharide was similar to the thin layer found
in the inner layer of the pod (Fig. 1B). It could also be observed
that this thin layer of film was made of multiple micro strands of
fibre. Table 2 shows the protein, lipid, carbohydrate and uronic con-
tents of PBPP. It was found that PBPP contained very low amounts
of protein (0.2 g/100 g) and lipid (0.0 g/100 g) with high amounts
of carbohydrate (286.2 mg maltose equivalent/g) and uronic acid
(374.3 mg Gal equivalent/g), suggesting that high purity of the
polysaccharide was successfully extracted from Pinto bean pod.
The result also suggested that this extracted polysaccharide could
be a type of pectic-polysaccharide and therefore, FTIR analysis was
conducted and then the IR spectra of PBPP was compared with the
structural information of commercial pectin.
3.2. Effect of pH, temperature and extraction time in extraction
yield
Results showed that the PBPP yield was depending on the
extraction condition (Table 1). According to ANOVA, pH, temper-
ature and extraction time were found significantly (p < 0.0001)
affecting the yield values. In general, the extraction condition at
pH 2 resulted in higher extraction yields. It could be due to the
presence of large amounts of hydrogen ions stimulated the hydrol-
ysis of polysaccharide by breaking the glycosidic bonds between
polysaccharide molecules and cell wall constituent at low pH, and
thus permitting dissolution of polysaccharide into extracting sol-
vent [39]. In terms of the effect of time, 3 h of extraction period
sufficiently gave a high yield of ∼38.5% at a temperature of 70 ◦C.
The result also showed that higher temperature was preferable in
this case of study. It was suggested that a substantial time (i.e. 3 h)
and a higher temperature were required for extraction because the
high extraction temperature could enhance the rate of extraction
by increasing the solubility of polysaccharide into the extracting
medium. Increase in temperature would also result in the increase
of the diffusion coefficient, which will improve the rate of dif-
fusion [40], whereas a substantial extraction duration may allow
the buffer solution to penetrate the dried pods, to dissolve the
macromolecules and then to diffuse the macromolecules out of
3.4. Functional groups and esterification degree (DE) of PBPP
The structural information of PBPP was confirmed based on
FTIR spectra by comparing the fingerprint region (800–1400 cm−1
)
(Fig. 2). Result showed that the spectrum was similar to the com-
mercial pectin IR spectra, as reported previously, where the peaks
at 757, 830, 890, 917, 973, 1019, 1050, 1072, 1094, 1143, 1231,
1330, 1368, 1413 and 1440 cm−1 were found, which correspond-
ing to C H3 deformation, C
O
C stretching, O H bending, C
O bending as well as C O
H
O
deformation, O O bending, O
C
C
bending in the pectin chain [43]. In addition, a broad band of the
OH stretching (2500–3600 cm−1), which is due to the inter/intra-

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F. Kamarudin, C.-Y. Gan / International Journal of Biological Macromolecules 88 (2016) 280–287
Fig. 2. FTIR spectra of PBPP.
Table 3
1.2: 1.7 with trace amount of ribose and fucose. Their correspond-
ing mole percentages were 2.5%: 15.0%: 4.0%: 9.0%: 62.2%: 2.9%:
4.3%, respectively. It was noted that galactose was the predominant
monosaccharide in PBPP. Therefore, it was confirmed that PBPP was
a highly branched heteropolysaccharide.
Number average molecular weight (Mn), weight average molecular weight (Mw),
polydispersitty index (PDI), and intrinsic viscosity (IV) of PBPP.
Property
PBPP
Commercial pectin
Mn (kDa)
Mw (kDa)
PDI
148.7
270.6
1.82
20.7
55.9
2.70
0.289
IV (dm3/g)
0.262
3.6. TPC, antioxidant activities and ˛-amylase inhibitory activity
of PBPP
molecular hydrogen bonding of the galacturonic acid backbone and
the presence of methyl ester group (2750–2950 cm−1) were also
found [44]. The peaks of 1730 and 1620 cm−1, which represented
the esterified and free carboxyl groups, respectively, were found
and the ratio between these peaks represented the degree of ester-
ification. By comparing the citrus peel pectin (∼68%) and apple
pomace pectin (∼69%) [45,46], PBPP gave a lower DE value (59.9%).
Hence, it could be anticipated that the functional properties would
be different from those in the commercial markets.
It was interesting to note that the extracted PBPP contained
approximately 10× higher TPC compared to apple pomace (8.56 mg
GAE/100 g) but ∼50% less than citrus peel (169.6 mg GAE/100 g)
[48,49]. Results also showed that PBPP exhibited relatively low
DPPH free radical scavenging activity (7.7%/mg), however, high
FRAP (2.0 mmol/dm3 FeSO4/mg) and high ␣-amylase inhibitory
activity (97.6%/mg) were observed (Table 4). The IC50 for ␣-amylase
inhibitory activity was ∼0.46 mg. This finding revealed that PBPP
exhibited high ␣-amylase inhibitory activity compared to a typical
anti-hyperglycemic drug (i.e. acarbose). The antioxidative activi-
ties could be closely related to its phenolic components because
they are able to effectively donate electrons to free radicals. Apart
from that, it was recently reported that these components are also
categorized as one of the largest group of the critical digestive
enzyme inhibitors and they were widely used as functional foods
[50]. This inhibition activity was due to the phenolic components
could strongly interact with proteins and inhibit the ␣-amylase by
forming complexes with the enzyme and by changing their confor-
mations [51]. Further study showed that these small and low polar
phenolic compounds could easily interact with the hydrophobic
residues of the active sites of ␣-amylase, thus caused the inhibi-
tion of its activity [52]. However, the content of polyphenols in
PBPP is relatively low (approximately 0.8 ␮g of TPC was used in
the antioxidative activity determination and 0.08 ␮g of TPC in ␣-
amylase inhibitory activity), therefore, it was strongly believed that
the activities were mainly contributed by the polysaccharide. As
3.5. Molecular weight and monosaccharide composition of PBPP
Table 3 shows that PBPP gave values for number average
molecular weight (Mn), weight average molecular weight (Mw),
polydispersity index (PDI), and intrinsic viscosity (IV) of 148.7 kDa,
270.6 kDa, 1.819, and 0.262 dm3/g, respectively. These results indi-
cated that PBPP had a higher degree of polymerization with a
narrower molecular weight distribution compared to commercial
pectin. It could also be found that PBPP had a lower IV, suggesting
that this polysaccharide might have a more spherical conformation
comparing to commercial citrus pectin which has a linear rod con-
formation [47]. This could be due to the presence of high amount
of neutral sugars that acted as the branches, which attached to the
PBPP backbone. The chromatogram in Fig. 3 shows that the PBPP
composed of mannose, galacturonic acid, rhamnose, glucose, galac-
tose, xylose and arabinose with molar ratio of 1.0: 6.1: 1.6: 3.6: 25.0:

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285
Fig. 3. Chromatogram of: (A) mixed standard composed of (1) glucoronic acid, (2) mannose, (3) galacturonic acid, (4) ribose, (5) rhamnose, (6) glucose, (7) galactose, (8)
xylose, (9) arabinose and (10) fucose, and (B) PBPP. *PMP = 3-methyl-1-phenyl-2-pyrazoline-5-one.
aforementioned that PBPP is an acidic heteropolysaccharide, it was
suggested that PBPP have the capability in donating electrons or
hydrogen atoms, which was the mechanism of these attributes.
It was also suggested that PBPP altered the pH condition of the
␣-amylase hydrolysis environment, thus the enzyme could be
denatured and lose its activity. Hsu et al. [53] also reported that
high percentage of galactose polysaccharides were linearly corre-
lated with anti-hyperglycemic effect. Thus, it could be used as a
promising natural antioxidant and as a ␣-amylase inhibitor.

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Table 4
TPC, DPPH free radical scavenging activity, FRAP and ␣-amylase inhibitory activity of PBPP.
TPC (mg GAE/100 g)
80.6 14.2
DPPH free radical scavenging activity (%/mg)
7.7 0.6
FRAP (mmol/dm3 FeSO4/mg)
2.0 0.0
␣-amylase inhibitory activity (%/mg)
97.6 6.7
Table 5
shear rate dependent and the existence of weak structures in pectin
molecules can be easily destroyed by the low shear rate.
Functional characteristics of PBPP.
Apart from that, the viscosity of these samples were depend-
ing on the pH condition given. At pH 2, PBPP gave a low viscosity
solution (0.72 Pa s) whereas commercial pectin gave a highly vis-
cous solution (15.25 Pa s). However, when pH increased to pH 4.5,
both samples showed similar viscosity (∼2.5-2.7 Pa s). As the pH
increased further (pH 6.5), commercial pectin solution gave a very
low viscosity (0.32 Pa s) compared to PBPP (1.54 Pa s).
Functional properties
PBPP
3.6 0.9a
Commercial pectin
WHC (g of water/g of extracts)
OHC (g of oil/g of extract)
Emulsifying activity (%)
Emulsion stability (%)
7.0 0.6b
2.3 0.5a
2.8 0.8a
39.6 0.9b
38.6 0.3a
0.72 0.31a
2.72 0.06a
1.54 0.06b
5.4 1.7a
64.1 3.3b
15.25 1.12b
2.51 0.26a
0.32 0.17a
Viscosity (Pa.s)
pH 2
pH 4.5
pH 6.5
In general, the viscosity of PBPP solution was lower than com-
mercial pectin. According to Thakur et al. [56], the gel formation of
HM pectic-polysaccharide involves a combination of the hydropho-
bic interactions between the pectin molecules as well as hydrogen
bonds between the free carboxyl groups on the pectin molecules.
Therefore, it could be explained that highly branched confor-
mation of PBPP induced a steric hindrance, which interrupted the
hydrophobic interactions between pectic chains, whereas the rod
shape structure of commercial pectin encourage the hydropho-
bic bonds. PBPP was therefore depending on the hydrogen bonds
formed between the molecules, which was pH dependent. At a mild
acidic pH (i.e. pH 4.5), PBPP would tend to ionize and a substan-
tial amount hydrogen bond (or an adequate charge density) was
formed, which encouraged the gelation of PBPP. As the pH increased
(pH 6.5), the repulsive effect of the negative charge between the
molecules was apparent, thus reduced the viscosity of PBPP solu-
tion. From the results, it could be observed that gelling behaviour of
PBPP primarily involved hydrogen bond compared to commercial
pectin where hydrophobic interaction was more profound.
Note: The data were written as mean
umn not followed by the same letter are significantly different at p < 0.01 level of
significance, according to t-test.
SD. (n = 3). Means within the same col-
3.7. Functional properties
Table 5 shows that PBPP obtained a lower WHC (3.6 g/g) com-
pared to commercial pectin (7.0 g/g). Galacturonic acid content in
PBPP could be the cause of the low WHC [38]. It was reported
that carboxyl groups in galacturonic acid were responsible to form
hydrogen bonds with water molecules in order to bind them tightly.
At such low amounts of galacturonic acid in PBPP (15%, mole per-
centage) may result in a lower WHC. This property is important
in the food industry due to its ability to increase bulk volume, to
inhibit food syneresis, as well as to modify food texture.
The result showed that OHC for both PBPP and commercial
pectin were similar (2.3–2.8 g/g). Therefore, PBPP can be used to
formulate food products, particularly high-fat products, which con-
fer foods with mouth feel and greasy sensation. Although the
methyl ester content, which was responsible for the hydrophobicity
and surface porosity of pectin, was higher (∼68%) in commer-
cial pectin compared to PBPP (59.9%), it was suggested that the
highly branched structure of PBPP contributed to the hydropho-
bicity property and the surface exposure to the oil molecules.
Therefore, a similar OHC was observed.
In terms of emulsifying activity, PBPP apparently showed a
higher activity (39.6%) compared to commercial pectin (5.4%).
This could be due to the balance between the hydrophobicity
and hydrophilicity properties of the polysaccharide. The branched
structure (hydrophobic component) of PBPP could be adsorbed
onto the oil surface, whereas the carboxylic group (hydrophilic
component) could be solubilized in the aqueous phase. On the other
hand, commercial pectin, which contains high amount of galatur-
onic acids, was more favourable in the aqueous phase. Therefore,
PBPP could be considered as an active agent that has the capacity to
stabilize the emulsion by reducing the interfacial tension between
oil and water. However, the result showed that the stability of the
PBPP emulsion was relatively low (38.6%), which indicated that the
interactions between PBPP and oil or water were rather weak and
they could be easily disrupted by heat. Once heat treatment was
induced, the protective barrier at the interface was ruptured.
In terms of gelling capacity, both PBPP and commercial pectin
showed pseudoplastic shear-thinning behaviour. According to
Wang and Cui [54], the viscosity of a solution decreases at high
shear rate, given that the number of chain entanglements reduces
as the chain adjust themselves with the flow direction. Forma-
tion of new entanglements cannot counterbalance for those being
disentangled, hence shear thinning flow behaviour was observed.
Sato et al. [55] also reported that viscosity of polysaccharide was
4. Conclusion
PBPP was successfully extracted from the Pinto bean pod. It
is a highly branched heteropolysaccharide with a high degree
of polymerization. It is composed of multiple monosaccharides,
predominantly galactose, and FTIR analysis confirmed that the
structure is similar to commercial pectin with a DE of 59.9%.
PBPP exhibited substantial antioxidative and ␣-amylase inhibitory
potentials with desired functional properties. It was therefore
believed that PBPP could be used as a food additive commercially.
To our knowledge, these data were first reported and would provide
not only the rationale of utilization of the Pinto bean pod in general,
but also a base study in the food and pharmaceutical industries.
Acknowledgement
This project was supported by RUI Grant (1001/CAATS/814257).
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