International Journal of Biological Macromolecules 88 (2016) 280–287

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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
Received 10 December 2015 gave total carbohydrate and uronic acid contents of 286.2 mg maltose equivalent/g and 374.3 mg Gal/g,
Received in revised form 29 February 2016 respectively. The Mw of PBPP was 270.6 kDa with intrinsic viscosity of 0.262 dm3 /g, which composed
Accepted 1 April 2016
of mannose (2.5%), galacturonic acid (15.0%), rhamnose (4.0%), glucose (9.0%), galactose (62.2%), xylose
Available online 1 April 2016
(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
Keywords:
that PBPP has a similar structure as commercial pectin with an esterification degree of 59.9%, whereas
␣-Amylase inhibitor
Pod
scanning electron microscopy study showed that the crude polysaccharide formed a thin layer of film
Polysaccharide 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.

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 removed and discarded. The efforts to study the effects of extraction
beans, which is highly consumed and commercialised worldwide. processes on polysaccharide as well as their molecular structure,
It was originated in Latin America more than 7000 years ago, and chemical properties and biological activities have not yet been per-
it has been rapidly cultivated throughout the world. According to formed. Therefore, the pod of Pinto bean was chosen as a source of
the Food and Agriculture Organization of the United Nations, the macromolecule carbohydrate and its potentials, as an alternative
top five dry bean producers are India, Brazil, Myanmar, China and macromolecule for commercial uses, need to be evaluated.
the United States of America, which produced approximately 3.3, Due to the structural features (e.g. degree of substitutions, steric
3.0, 2.1, 1.5 and 1.2 million tonnes of dry bean per year, respec- configuration, linkages of monosaccharides and substitutes, and
tively, from the year 1993 to 2014 (http://faostat3.fao.org/browse/ molar mass and its distribution), vegetable polysaccharides play a
Q/QC/E). To our surprise, the leading class of dry common beans critical role in their physical properties, such as thickening, gelling,
produced in the United States is Pinto bean at approximately and stabilizing ability [8]. These properties have extended their
42% of the total production (http://www.ers.usda.gov/topics/crops/ applications in food and biomedical industries. Modern pharma-
vegetables-pulses/dry-beans.aspx). cological research has identified that polysaccharide as one of
Bean consumption has been reported to minimize risk of chronic the major active components in herbs, which is responsible for
diseases, such as diabetes [1], coronary heart disease [2], colon can- various pharmacological activities, such as antioxidant, antiviral,
cer [3], prostate and breast cancer [4,5]. This could be due to the immunostimulatory, antitumour, antifatigue, radioprotection and
presence of polysaccharides. Previous studies also showed that the hepatoprotection activities [9–13]. Clinical studies also suggested
bean has the potential in producing functional protein and bioac- 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,
∗ Corresponding author at: Analytical Biochemistry Research Centre, Universiti hypercholesterolemia, hyperlipidemia, obesity, and colorectal can-
Sains Malaysia, 11800 USM, Penang, Malaysia. cer [14–19]. Sulfated polysaccharides from algae were also reported
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

to possess a higher anticoagulant activity than those of heparin [20] with 75 ␮L of 0.05 kg/dm3 phenol solution on an ice bed and sul-
whereas galactoglucomannans and pectins from woody materials phuric acid (375 ␮L) was then added. The mixtures were incubated
have also been reported to exhibit immunostimulating and free at 80 ◦ C for 30 min and absorbance was measured at 495 nm using a
radicals scavenging activities [21,22]. Several studies have reported spectrophotometer (Spectamax M5, Moleular Devices, USA). Mal-
the possibility of using waste products as polysaccharide sources. tose was used as a standard.
To date, polysaccharides have been extracted from several under Uronic acid content was determined using the method as
utilised biomasses, such as mango waste, sunflower head residues, described by Blumenkrantz and Asboe-Hansen [29]. Crude polysac-
sugar beet, soy hull and sweet potato residues [23–25]. Nonethe- charide (250 ␮L) at concentration of 1 mg/mL was mixed with
less, not all sources are suitable for commercial uses. The sources as 1.5 mL of 0.0125 mol/dm3 sulphuric acid/sodium tetraborate
well as their extraction and purification methodologies may affect solution on an ice bed and then heated at 100 ◦ C for 5 min m-
the structure and composition of the produced pectin. Therefore, a Hydroxydiphenyl (25 ␮L) was then added and the absorbance was
continuous search for suitable pectin is aggressively performed by measured after 5 min at 520 nm using a spectrophotometer (Spec-
researchers. tamax M5, Moleular Devices, USA).
The main objective of this study was to explore the potential
of Pinto bean pod as an alternative source of polysaccharide. The 2.5. Functional groups and degree of esterification (DE)
specific objectives of the study were to extract polysaccharide from determinations
the pods of Pinto bean using various extraction parameters; and to
evaluate the molecular structure, chemical properties as well as the FTIR spectra of crude pectic-polysaccharide (powder form) were
biological activities of the extracted macromolecule carbohydrate. recorded from 650 to 4000 cm−1 using Cary 670 FTIR spectrom-
eter with an attenuated total reflectance (ATR) system (Agilent
2. Materials and methods Technologies, CA, USA). The spectra were analysed using Agilent
Resolutions Pro software. DE was determined using the following
2.1. Materials equation:

Fresh Pinto bean pods were collected from different markets A1730
DE = × 100 (1)
(∼10 kg from each market) in Penang. The pods were cleaned with A1730 + A1600
deionised water and were kept frozen at −20 ◦ C. They were subse-
where A1730 was defined as the area of the band at 1730 cm−1 and
quently freeze-dried. The lyophilised pods were ground into fine
A1600 was defined as the area of the band at 1600 cm−1 [30].
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). 2.6. Determination of molecular weights

2.2. Crude polysaccharide extraction The molecular weight of PBPP was examined using a gel per-
meation chromatography (GPC) equipped with Viscotek Model
Briefly, 6 g of lyophilised pod powders were added in 300 mL of TDA 305 Triple Detector Array incorporated Refractive index, Light
0.1 mol/dm3 citrate-phosphate buffer at different pH values (i.e. pH scattering and viscosity detectors (Malvern, UK). CLM 3021 col-
2, pH 4 and pH 6) using solid to buffer ratio of 1:50. The mixture was umn (A6000 M, 300 × 7.8 mm, 13 ␮m beads size, Malvern, UK) was
then incubated at different extraction durations (i.e. 1 h, 3 h and 5 h) used. A 100 ␮L of sample (0.01 kg/dm3 ) in 0.1 mol/dm3 NaNO3 ) was
in an incubator shaker (IKA KS 4000i Control, Staufen, Germany) injected and the flow rate and temperature were maintained at
which constantly shaking at 250 rpm at different temperature set- 1.0 mL/min and 30 ◦ C, respectively. The elution was carried out with
tings (i.e. 50 ◦ C, 60 ◦ C and 70 ◦ C). The resulting slurries were then 0.1 mol/dm3 NaNO3 containing 0.003 kg/dm3 NaN3 to prevent bac-
filtered through a muslin cloth (2 layers) to remove solid particles. teria growth. Polyethylene Oxide (18670 Da) was used as working
Thereafter, three volumes of ethanol were added to one volume of calibration standard. A second standard, dextran (65333 Da) was
extract and incubated at 4 ◦ C for 5 h to precipitate polysaccharide. applied to verify the calibration accuracy with high level of con-
The precipitates obtained were filtered through a muslin cloth and fidence. The molecular weight of the sample was determined by
then washed with ethanol to remove the small molecular weight comparing with calibration curve. The chromatogram obtained was
molecules in the extract. The crude polysaccharide (PBPP) was then analyzed using the OmniSEC software.
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-
Samples were mounted onto SEM specimen stub with a double- matography (HPLC) system equipped with an UV detector. Sample
sided tape and coated with gold using a Polaron SC 515 Sputter (10 mg) was hydrolyze using 3 mol/dm3 trifluoroacetic acid (1 mL)
Coater (Fisons Instruments, UK). Subsequently, the samples were at 95 ◦ C for 8 h, vacuum dried and re-dissolved in 1 mL of deionized
photographed using Leo Supra 50VP Field Emission Scanning Elec- water. Sample (100 ␮L) was then added with 3-methyl-1-phenyl-
tron Microscope equipped with Oxford INCA 400 energy dispersive 2-pyrazoline-5-one (PMP, 200 ␮L) and 0.3 mol/dm3 NaOH (300 ␮L)
X-ray Microanalysis system (Oxford Instruments Analytical, UK). 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
2.4. Protein content, lipid content, total carbohydrate content 1 mL of chloroform for 3 times. The aqueous layer was collected
and uronic acid content determinations and filtered through a 0.45 ␮m membrane prior to HPLC analysis.
The HPLC system was prepared as the following condition: Zor-
Protein content of PBPP was determined using Bradford method bax SB-C18 reversed-phase column (250 × 4.6 mm, 5 ␮m, Agilent,
[26], lipid content was determined using Soxhlet method [27] and USA). Mobile phase: (A) Acetonitrile; (B) 3.3 mmol/dm3 KH2 PO4 -
total carbohydrate content was determined using the method as 3.9 mmol/dm3 Tris-acetate-EDTA buffer containing 0.10 kg/dm3
described by Dubois et al. [28]. Sample (75 ␮L, 1 mg/mL) was mixed ACN. The gradient: 0–4 min: 94% B; 4–9 min: from 94% to 88%B;
282 F. Kamarudin, C.-Y. Gan / International Journal of Biological Macromolecules 88 (2016) 280–287

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 Fig. 1. Morphology of (A) outer layer and midsection of Pinto bean pod, (B) inner
layer of the paint bean pod and (C) PBPP.
activity was calculated as follows:
 
Apos − (As − Asb ) Emulsifying activity (EA) and emulsion stability (ES) were deter-
%inhibiton =   × 100 (2)
Apos − Aneg 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
where Apos was the absorbance of positive control, Aneg was the 2 min. Corn oil (10 mL) was then added and homogenized for 2 min.
absorbance of negative control, As was the absorbance mixture of ␣- The emulsion was centrifuged (Multifuge X1R, Thermo Scientific,
amylase solution and PBPP solution, and Asb was the sample blank USA) at 1200g for 5 min and the emulsion volume measured. EA
[35]. and ES were expressed in percentage.
The thickening capacity was measured based on the viscosity
2.10. Functional properties determination of the sample (0.005 kg/dm3 ) at different pH conditions using a
rheometer (AR2000ex, TA Instruments, USA) with shear rate rang-
Water- and oil-holding capacity (WHC and OHC) were deter- ing from 0.01 to 1000 s−1 in 5 min, as described previously [38].
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 2.11. Statistical analysis
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 The experiment was first conducted using a factorial design in
extract. Corn oil density is 0.92 g/ml. order to study the suitable condition for the extraction. Results
 F. Kamarudin, C.-Y. Gan / International Journal of Biological Macromolecules 88 (2016) 280–287 283

Table 1
PBPP yields (g/100 g) at different extraction conditions.

Buffer pH (pH) Temperature (◦ C) Extraction duration (h)

1 3 5

2 50 20.8 ± 0.3b 27.1 ± 0.5bc 28.2 ± 0.9bc

60 27.5 ± 0.5bc 33.7 ± 0.3c 35.7 ± 3.0c
70 29.1 ± 0.6bc 38.5 ± 1.2cd 35.8 ± 0.8c
4 50 15.2 ± 2.4a 22.7 ± 3.8b 20.0 ± 1.0b
60 24.0 ± 2.3b 26.9 ± 1.9b 29.1 ± 1.5bc
70 24.4 ± 0.9b 32.1 ± 3.3c 34.0 ± 0.4c
6 50 28.7 ± 0.9bc 30.8 ± 0.8c 21.4 ± 3.7b
60 25.5 ± 0.9b 33.7 ± 5.9c 33.8 ± 4.3c
70 26.6 ± 0.7b 32.1 ± 1.9c 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 Protein content (g/100 g) Lipid content (g/100 g) Carbohydrate content (mg maltose equivalent/g) Uronic acid content (mg Gal equivalent/g)

PBPP 0.2 ± 0.0 0.0 ± 0.0 286.2 ± 11.3 374.3 ± 67.3

were expressed as mean values ± standard deviation of three the pod. Such phenomenon demonstrated an existence of inter-
separated determinations. Comparison of means was performed action between pH, temperature and time, where ANOVA showed
using one-way analysis of variance (ANOVA). When comparing the that there was a significant (p < 0.05) interaction between pH and
functional properties of the PBPP with commercial pectin, t-test temperature as well as between pH and time (p < 0.01) in affecting
was used with significant level of p < 0.05 (SPSS for Windows, Ver- the extraction yield. In this case of study, extraction condition at
sion 19.0, SPSS Institute Inc., Cary NC). pH 2, temperature of 70 ◦ C and extraction time of 3 h, was selected
as the most suitable condition for extracting PBPP because this
3. Results and discussion condition performed better (i.e. gave the highest yield) from the
economic point of view, although minor alteration of the structure
3.1. Microscopic examination of Pinto bean pod 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
Fig. 1A shows the morphology of the outer layer and the mid- citrus peels (16.1 g/100 g) and apple pomace (14 g/100 g) [41,42].
section of Pinto bean pod. The presence of micro-filaments on the Therefore, it was suggested that Pinto bean pod is a good source of
surface of the outer layer was observed, whereas multiple layers macromolecule carbohydrate.
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 3.3. Morphology and chemical compositions of PBPP
thin film, which could be consisted of cellulose, hemicellulose or
pectin. Therefore, Pinto bean pod was suggested to be a good source Fig. 1C shows the morphology of PBPP and it was found that
for the extraction of macromolecule carbohydrate. this extracted polysaccharide was similar to the thin layer found
in the inner layer of the pod (Fig. 1B). It could also be observed
3.2. Effect of pH, temperature and extraction time in extraction that this thin layer of film was made of multiple micro strands of
yield fibre. Table 2 shows the protein, lipid, carbohydrate and uronic con-
tents of PBPP. It was found that PBPP contained very low amounts
Results showed that the PBPP yield was depending on the of protein (0.2 g/100 g) and lipid (0.0 g/100 g) with high amounts
extraction condition (Table 1). According to ANOVA, pH, temper- of carbohydrate (286.2 mg maltose equivalent/g) and uronic acid
ature and extraction time were found significantly (p < 0.0001) (374.3 mg Gal equivalent/g), suggesting that high purity of the
affecting the yield values. In general, the extraction condition at polysaccharide was successfully extracted from Pinto bean pod.
pH 2 resulted in higher extraction yields. It could be due to the The result also suggested that this extracted polysaccharide could
presence of large amounts of hydrogen ions stimulated the hydrol- be a type of pectic-polysaccharide and therefore, FTIR analysis was
ysis of polysaccharide by breaking the glycosidic bonds between conducted and then the IR spectra of PBPP was compared with the
polysaccharide molecules and cell wall constituent at low pH, and structural information of commercial pectin.
thus permitting dissolution of polysaccharide into extracting sol-
vent [39]. In terms of the effect of time, 3 h of extraction period 3.4. Functional groups and esterification degree (DE) of PBPP
sufficiently gave a high yield of ∼38.5% at a temperature of 70 ◦ C.
The result also showed that higher temperature was preferable in The structural information of PBPP was confirmed based on
this case of study. It was suggested that a substantial time (i.e. 3 h) FTIR spectra by comparing the fingerprint region (800–1400 cm−1 )
and a higher temperature were required for extraction because the (Fig. 2). Result showed that the spectrum was similar to the com-
high extraction temperature could enhance the rate of extraction mercial pectin IR spectra, as reported previously, where the peaks
by increasing the solubility of polysaccharide into the extracting at 757, 830, 890, 917, 973, 1019, 1050, 1072, 1094, 1143, 1231,
medium. Increase in temperature would also result in the increase 1330, 1368, 1413 and 1440 cm−1 were found, which correspond-
of the diffusion coefficient, which will improve the rate of dif- ing to C H3 deformation, C O C stretching, O H bending, C H
fusion [40], whereas a substantial extraction duration may allow deformation, O C O bending, O C O bending as well as C O O
the buffer solution to penetrate the dried pods, to dissolve the bending in the pectin chain [43]. In addition, a broad band of the
macromolecules and then to diffuse the macromolecules out of OH stretching (2500–3600 cm−1 ), which is due to the inter/intra-
284 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-
Number average molecular weight (Mn ), weight average molecular weight (Mw ),
ing mole percentages were 2.5%: 15.0%: 4.0%: 9.0%: 62.2%: 2.9%:
polydispersitty index (PDI), and intrinsic viscosity (IV) of PBPP.
4.3%, respectively. It was noted that galactose was the predominant
Property PBPP Commercial pectin monosaccharide in PBPP. Therefore, it was confirmed that PBPP was
Mn (kDa) 148.7 20.7 a highly branched heteropolysaccharide.
Mw (kDa) 270.6 55.9
PDI 1.82 2.70
IV (dm3 /g) 0.262 0.289 3.6. TPC, antioxidant activities and ˛-amylase inhibitory activity
of PBPP

molecular hydrogen bonding of the galacturonic acid backbone and It was interesting to note that the extracted PBPP contained
the presence of methyl ester group (2750–2950 cm−1 ) were also approximately 10× higher TPC compared to apple pomace (8.56 mg
found [44]. The peaks of 1730 and 1620 cm−1 , which represented GAE/100 g) but ∼50% less than citrus peel (169.6 mg GAE/100 g)
the esterified and free carboxyl groups, respectively, were found [48,49]. Results also showed that PBPP exhibited relatively low
and the ratio between these peaks represented the degree of ester- DPPH free radical scavenging activity (7.7%/mg), however, high
ification. By comparing the citrus peel pectin (∼68%) and apple FRAP (2.0 mmol/dm3 FeSO4 /mg) and high ␣-amylase inhibitory
pomace pectin (∼69%) [45,46], PBPP gave a lower DE value (59.9%). activity (97.6%/mg) were observed (Table 4). The IC50 for ␣-amylase
Hence, it could be anticipated that the functional properties would inhibitory activity was ∼0.46 mg. This finding revealed that PBPP
be different from those in the commercial markets. exhibited high ␣-amylase inhibitory activity compared to a typical
anti-hyperglycemic drug (i.e. acarbose). The antioxidative activi-
3.5. Molecular weight and monosaccharide composition of PBPP ties could be closely related to its phenolic components because
they are able to effectively donate electrons to free radicals. Apart
Table 3 shows that PBPP gave values for number average from that, it was recently reported that these components are also
molecular weight (Mn ), weight average molecular weight (Mw ), categorized as one of the largest group of the critical digestive
polydispersity index (PDI), and intrinsic viscosity (IV) of 148.7 kDa, enzyme inhibitors and they were widely used as functional foods
270.6 kDa, 1.819, and 0.262 dm3 /g, respectively. These results indi- [50]. This inhibition activity was due to the phenolic components
cated that PBPP had a higher degree of polymerization with a could strongly interact with proteins and inhibit the ␣-amylase by
narrower molecular weight distribution compared to commercial forming complexes with the enzyme and by changing their confor-
pectin. It could also be found that PBPP had a lower IV, suggesting mations [51]. Further study showed that these small and low polar
that this polysaccharide might have a more spherical conformation phenolic compounds could easily interact with the hydrophobic
comparing to commercial citrus pectin which has a linear rod con- residues of the active sites of ␣-amylase, thus caused the inhibi-
formation [47]. This could be due to the presence of high amount tion of its activity [52]. However, the content of polyphenols in
of neutral sugars that acted as the branches, which attached to the PBPP is relatively low (approximately 0.8 ␮g of TPC was used in
PBPP backbone. The chromatogram in Fig. 3 shows that the PBPP the antioxidative activity determination and 0.08 ␮g of TPC in ␣-
composed of mannose, galacturonic acid, rhamnose, glucose, galac- amylase inhibitory activity), therefore, it was strongly believed that
tose, xylose and arabinose with molar ratio of 1.0: 6.1: 1.6: 3.6: 25.0: the activities were mainly contributed by the polysaccharide. As
 F. Kamarudin, C.-Y. Gan / International Journal of Biological Macromolecules 88 (2016) 280–287 285

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 denatured and lose its activity. Hsu et al. [53] also reported that
suggested that PBPP have the capability in donating electrons or high percentage of galactose polysaccharides were linearly corre-
hydrogen atoms, which was the mechanism of these attributes. lated with anti-hyperglycemic effect. Thus, it could be used as a
It was also suggested that PBPP altered the pH condition of the promising natural antioxidant and as a ␣-amylase inhibitor.
␣-amylase hydrolysis environment, thus the enzyme could be
286 F. Kamarudin, C.-Y. Gan / International Journal of Biological Macromolecules 88 (2016) 280–287

Table 4
TPC, DPPH free radical scavenging activity, FRAP and ␣-amylase inhibitory activity of PBPP.

TPC (mg GAE/100 g) DPPH free radical scavenging activity (%/mg) FRAP (mmol/dm3 FeSO4 /mg) ␣-amylase inhibitory activity (%/mg)

80.6 ± 14.2 7.7 ± 0.6 2.0 ± 0.0 97.6 ± 6.7

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