LWT - Food Science and Technology 58 (2014) 396e403

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LWT - Food Science and Technology

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Emulsifying and structural properties of pectin enzymatically

extracted from pumpkin
Steve W. Cui a, Yoon Hyuk Chang b, *
a
Food Research Program, Agriculture and Agri-Food Canada, 93 Stone Road West, Guelph, Ontario N1G 5C9, Canada
b
Department of Food and Nutrition, Kyung Hee University, Seoul, 130-701, Republic of Korea

a r t i c l e i n f o a b s t r a c t

Article history: The present study investigated the emulsifying and structural properties of pectin enzymatically
Received 13 January 2014 extracted from pumpkin. Pumpkin pectin fraction A was obtained from raw pumpkin with an enzymatic
Received in revised form preparation of cellulase and a-amylase. Pumpkin pectin fraction B was achieved by treating the fraction A
31 March 2014
solution with pronase to reduce protein content. According to the findings (on protein content, gal-
Accepted 6 April 2014
Available online 13 April 2014
acturonic acid content, neutral sugar composition, and molecular weight distribution), the pronase
treatment could remove protein from the fraction A without considerably influencing any other chemical
and molecular properties. Moreover, the fraction A exhibited emulsifying properties in water and oil
Keywords:
Pumpkin pectin
mixture, whereas the removal of protein in the fraction B resulted in the loss of emulsifying properties.
Emulsifying properties The FT-IR and 1D NMR analysis revealed that the backbone of pumpkin pectin is mainly composed of a-
Structural characterization 1,4-D-galacturonic acid in which a considerable portion of galacturonic acid residues is present as methyl
esters, and some L-rhamnose are involved in the linear region of the backbone through a-1,2-linkages.
Crown Copyright Ó 2014 Published by Elsevier Ltd. All rights reserved.

1. Introduction
Pectin, the most abundant polysaccharides in the middle
lamella and cell wall of many higher plants, has been widely used in
the food industry as a gelling agent, thickener, and stabilizer of jam,
jelly, and acid milk products (Singthong, Cui, Ningsanond, & Goff,
2004; Tamaki, Konishi, Fukuta, & Tako, 2008). The identification
of the molecular structure for pectin is of fundamental importance
because the physicochemical and functional properties of pectin
are highly dependent on its structure. However, pectin is very
heterogeneous in its composition, which renders the structural
study of the polysaccharides extremely challenging.
Pectin is considered as an alternation of homogalacturonan re-
gions, generally called “smooth regions”, and rhamnogalacturonan
regions I, also called “hairy regions” (Waldron, Parker, & Smith,
2003). Homogalacturonan is a repetition of (1 / 4)-linked a-D-
galactopyranosyluronic acid (GalpA) residues. Rhamnogalactur-
onan region I is constituted by an alternating sequence of (1 / 4)-
linked a-D-GalpA and (1 / 2)-linked a-L-rhamnopyranose (Rhap)
residues. Various side chains (arabinan, galactan, and arabinoga-
lactan) can be linked to the rhamnose residues (Vincken et al.,
2003; Voragen, Pilnik, Thibault, Axelos, & Renard, 1995).
* Corresponding author. Tel.: þ82 2 960 0264; fax: þ82 2 961 0261.
E-mail address: yhchang@khu.ac.kr (Y.H. Chang).
Citrus peel and apple pomace are the major raw materials used
for the production of commercially acceptable pectin. Other raw
materials, including pumpkin (Matora et al., 1995), sugar beet
(Levigne, Ralet, & Thibault, 2002), sunflower (Iglesias & Lozano,
2004), honey pomelo (Guo et al., 2014), and papaya peels
(Koubala, Christiaens, Kansci, Loey, & Hendrickx, 2014) have been
considered as possible pectin sources and their potential has been
discussed.
Several studies on the extraction of pumpkin pectin have been
performed using different extraction methods, such as an acidic
extraction method and enzymatic extraction method (Fissore,
Ponce, Stortz, Rojas, & Gerschenson, 2007; Matora et al., 1995;
Ptichkina, Markina, & Rumyantseva, 2008; Shkodina, Zeltser,
Selivanov, & Ignatov, 1998). Matora et al. (1995) observed that the
enzyme extraction method (multi-enzyme culture supernatants
from various strains of Bacillus polymyxa) gave much bigger yields
of pumpkin pectin than did the acid extraction method (0.1 M HCl).
Moreover, the current acid pectin extraction methods not only do
not allow pectin to be extracted fully with no damage to its struc-
ture, but also lead to the environmental concerns due to acid usage
(Ptichkina et al., 2008).
Different enzymes, like cellulase, hemicellulase, protease,
amylase, and so on, have been employed for the enzymatic
extraction of pumpkin pectin (Fissore et al., 2007; Shkodina et al.,
1998). Shkodina et al. (1998) employed cellulase (Trichoderma

http://dx.doi.org/10.1016/j.lwt.2014.04.012
0023-6438/Crown Copyright Ó 2014 Published by Elsevier Ltd. All rights reserved.
 S.W. Cui, Y.H. Chang / LWT - Food Science and Technology 58 (2014) 396e403
viride) or hemicellulase (Aspergillus niger)] for the extraction of
pectin from pumpkin pulp (following juice extraction), and re-
ported that the cellullase treatment led to an increase in pectin
yields and in galacturonic acid contents, as compared to that of the
hemicellulose treatment. The same result was also observed in the
literature performed by Fissore et al. (2007), who found that the
amounts of galacturonic acid in pumpkin pectin obtained from
cellulase were greater than those obtained from hemicellulase.
The emulsifying properties of pectin have been demonstrated in
a few literatures (Dickinson, 2003; Funami et al., 2011; Leroux,
Langendorff, Schick, Vaishnav, & Mazoyer, 2003; Siew, Williams,
Cui, & Wang, 2008). A small percentage of hydrophobic protein
(about 2%) in sugar beet pectin plays major role in stabilizing oil-in-
water emulsions (Siew et al., 2008). Leroux et al. (2003) also noted
that beet pectin and citrus pectin may efficiently decrease the
interfacial tension between oil and water phase in the emulsions;
however, the emulsion stabilizing features of pectin change due to
the contents difference of calcium ions, acetyl groups, and proteins.
As far as the authors are aware, no studies have apparently re-
ported the effect of pronase treatment on the emulsifying proper-
ties of pumpkin pectin, nor have elucidated the structural features
of pectin enzymatically extracted from pumpkin. In the present
study, pectin fraction A was obtained from raw pumpkin using
cellulase and a-amylase, and then pumpkin pectin fraction B was
produced after pronase treatment with the fraction A. The main
objectives of the present study are to (1) evaluate the potential of
pumpkin for the enzymatic extraction of pectin and (2) investigate
the emulsifying and structural features of pectin enzymatically
extracted from pumpkin.
2. Materials and methods
2.1. Materials
Raw pumpkin (Cucurbita mixta), harvested in ON, Canada, was
used in the present study. Cellulase from Trichoderma viride (5 U/
mg) and a-amylase from Bacillus licheniformis (3500 U/mL) were
obtained from Sigma (Oakville, ON, Canada). Pronase from Strep-
tomyces griseus (7.0 U/mg) was provided by Roche Molecular Bio-
chemicals (Indianapolis, USA). Standards for rhamnose, arabinose,
galactose, glucose, xylose, mannose, and galacturonic acid were
purchased from Sigma (Oakville, ON, Canada). All chemicals were of
reagent grade unless otherwise specified.
2.2. Enzymatic extraction for the production of pumpkin pectin
fractions A and B
Pumpkin pectin fractions A and B were extracted from raw
pumpkin using the procedure described by Fissore et al. (2007)
with some modifications. Raw pumpkin was first cut, and the
seeds and skin were removed. The remainder was reduced to
fragments (about 4 cm  0.5 cm), freeze-dried, and ground to make
dried pumpkin powder. Fifty g of pumpkin powder were suspended
in 1.5 L of 50 mmol/L sodium citrate buffer (pH 5.2), and then
cellulase (600 mg) and a-amylase (500 mg) were added. Our pre-
liminary study indicated that starch contents of dried pumpkin
powder were 7.93 g/100 g dry solids; therefore, a-amylase was
employed to remove starch existed in pumpkin powder. The
mixture was stirred at 30  C for 20 h and centrifuged at 15,000 g
for 30 min. The supernatant was collected. The remaining pellet
was resuspended in 100 mL of distilled water and subjected to a
further centrifugation. The supernatants were combined and
treated with two volumes of absolute ethanol (Sigma, Oakville, ON,
Canada). The resulting precipitate was collected, solubilised in
397
distilled water, freeze-dried, and ground. This procedure produced
pumpkin pectin fraction A.
To produce pumpkin pectin fraction B, 0.5 g/100 mL solution of
pumpkin pectin fraction A was stirred with pronase for 60 min at
50  C to reduce protein content. The solution was heated at 85  C
for 30 min, cooled, and centrifuged at 15,000 g for 20 min. The
supernatant was collected and then treated with two volumes of
absolute ethanol (Sigma, Oakville, ON, Canada). The resulting pre-
cipitate was collected, solubilised in distilled water, freeze-dried,
and ground. The resulting protein-reduced pectin fraction was
termed as pumpkin pectin fraction B. The details of enzymatic
extraction for pumpkin pectin fractions A and B are summarised in
Fig. 1.
2.3. Moisture content, protein content, galacturonic acid content,
degree of esterification, and degree of acetylation
Pumpkin pectin fraction A and fraction B were analysed for their
moisture contents using AACCI Approved Method 44-15A (AACCI,
2000). The protein contents of pumpkin fraction A and fraction B
were analysed by NA2100 Nitrogen and Protein Analyser (Strada
Rivoltana, Milan, Italy) using the factor of 5.7 to convert measured
nitrogen to protein. The galacturonic acid contents of pumpkin
pectin fraction A and fraction B were determined by the m-
hydroxybiphenyl method (Blumenkrantz & Asboe-Hansen, 1973).
Standard galacturonic acid solutions (10e100 mg/mL) were used to
construct the standard curve for the determination. The degree of
esterification (DE) and degree of acetylation for the fractions A and
B were determined by the titrimetric method of Food Chemical
Codex (FCC, 1981, pp. 283e286) and the method of Matora et al.
(1995), respectively.
2.4. Neutral sugar composition
The neutral sugar composition of pumpkin pectin fraction A and
fraction B was determined by modifying the procedure of Wood,
Weisz, and Blackwell (1994). Each sample was hydrolysed in
1 mol/L H2SO4 at 100  C for 4 h and diluted 20 times. The diluted
samples were passed through a 0.45 mm filter and injected to a
high-performance anion exchange chromatography with pulsed
amperometric detection (HPAEC-PAD) (Dionex-5500, Dionex Cor-
poration, Canada). Separation of each neutral sugar was performed
on a CarboPac PA1 column (250  4 mm I.D., Dionex Corporation,
Canada) and a guard column (3  25 mm, Dionex Corporation,
Canada). The solvents were A: 100 mmol/L NaOH, B: 300 mmol/L
NaOH, and C: water (distilled water filtered through Nanopure In-
finity, Model: D8971, Dubuque, IA, USA). Elution was with 8% A and
92% C for 7 min, then with a gradient to 100% eluent C for 28 min.
The column system was cleaned after each analysis with 100%
eluent B for 15 min. After cleaning, the initial conditions were
maintained for 10 min between each injection of sample. The sol-
vent flow rate was 1.0 mL/min and the injection volume was 50 mL.
A post-column delivery system of 600 mmol/L NaOH with a flow-
rate of 0.5 mL/min was added to the HPAEC-PAD system. Stan-
dard solutions of the individual neutral sugar (rhamnose, arabi-
nose, galactose, glucose, xylose, and mannose) were used at varying
concentrations (10e50 mg/mL) for identification and quantification.
The instrument was controlled and data were processed using
Dionex AI 450 software (Dionex Corporation, Canada).
2.5. Molecular weight distribution
The molecular weight distribution of pumpkin pectin fraction A
and fraction B was evaluated by high performance size exclusion
chromatography (HPSEC) equipped with a refractive index (RI)
398 S.W. Cui, Y.H. Chang / LWT - Food Science and Technology 58 (2014) 396e403

Raw pumpkin

Cutting
freeze-drying
grinding

Pumpkin powder

Sodium citrate buffer (pH 5.2)

cellulase
α-amylase
centrifuge

Residue Supernatant

Ethanol precipitation
solubilization
freeze-drying

Fraction A

Solubilization
pronase
centrifuge

Residue Supernatant

Ethanol precipitation
solubilization
freeze-drying

Fraction B
Fig. 1. Scheme of enzymatic extraction for pumpkin pectin fraction A and fraction B from raw pumpkin.

detector (Model Dual 250, Viscotek, Houston, TX, USA). The chro-
matographic system includes a Shimadzu SCL-10Avp pump, auto-
matic injector (Shimadzu Scientific Instruments Inc., Columbia, MD,
USA), and two columns in series: a Shodex OHpak KB-806M (Showa
Denko K.K., Tokyo, Japan) and an Ultrahydrogel linear (Waters,
Milford, CT, USA). The columns and RI detector were maintained at
40  C. The mobile phase, injection volume, and flow rate were
0.1 mol/L NaNO3, 100 mL, and 0.6 mL/min, respectively. Each sample
was solubilized in distilled water at 90  C for 1 h, cooled, and
filtered through a 0.45 mm filter prior to injection onto the column.
The molecular weight distribution was estimated by the pullulan
standards (molecular weights: 10, 20, 50, and 400 kDa) (Sigma,
Oakville, ON, Canada).
2.6. Surface tension
2.7. Emulsion capacity and stability
The emulsion capacity and emulsion stability of pumpkin pectin
fraction A, fraction B, and pectin standard (citrus pectin, DE: 56%,
Sigma, Oakville, ON, Canada) were investigated according to
Yasumatsu et al. (1972) with some modifications. The sample (0.01 g)
was suspended in 10 mL of distilled water before mixing with 10 mL of
vegetable oil. The water/oil mixture was emulsified using a homoge-
nizer at 10,000 rpm for 1 min, and then the mixture was centrifuged at
1300 g for 5 min. Emulsion capacity was calculated as follows:
ðHeight of emulsion layer=total height of fluidÞ  100% (1)
Emulsion stability was determined by heating the emulsion at
80  C for 30 min, cooling with tap water for 15 min, and centrifuging
at 1300  g for 5 min. Emulsion stability was calculated as follows:

The surface tension of pumpkin pectin fraction A, fraction B, and ðHeight of remaining emulsion layer=total heightof fluidÞ
pectin standard (citrus pectin, DE: 56%, Sigma, Oakville, ON, Can-
 100% (2)
ada) was determined using a semi-automatic surface tensionmeter
model Surface Tensionmat 21 (Fisher Scientific, Toronto, Canada) at
21e22  C. This tensionmeter employed the Du Nouy ring method to 2.8. Structural analysis
measure static surface tension. Distilled water was used to calibrate
the tensionmeter for surface tension measurements. Sample con- Only pumpkin pectin fraction B was used to analyse the struc-
centrations of 0.05, 0.1, 0.2, 0.3, 0.4, 0.6, 0.8, and 1.0 g/100 mL were tural characteristics because pectin with little or no proteins is
used for surface tension measurements. better for the structural analysis.
 S.W. Cui, Y.H. Chang / LWT - Food Science and Technology 58 (2014) 396e403 399

2.8.1. Fourier transform-infrared spectroscopy Table 1

The pectin standard (citrus pectin, DE: 56%, Sigma, Oakville, ON, Chemical composition of pumpkin pectin fraction A and fraction B.a

Canada) and pumpkin pectin fraction B were dried in a vacuum Pectin sample
oven for 5 h and stored in a desiccator prior to Fourier transform- Fraction A Fraction B
infrared (FT-IR) analysis. FT-IR spectra of each sample were ob- a
Moisture (g/100 g) 9.61  0.27 9.65  0.12a
tained according to the procedure of Singthong et al. (2004).
Protein (g/100 g) 4.58  0.32 NDþ
Degree of esterification (%) 47.30  0.12a 47.10  0.15a
2.8.2. 1H and 13C NMR spectroscopy Degree of acetylation (%) 6.87  0.22a 6.79  0.20a
Pumpkin pectin fraction B was dissolved in deuterium oxide Galacturonic acid (g/100 g) 75.02  0.93b 78.22  0.45a
(D2O, 80  C, 1 h) and freeze-dried for three times to replace the Neutral sugar (g/100 g)
Rhamnose 1.73  0.09a 1.72  0.04a
exchangeable protons with deuterons before being finally redis- Arabinose 1.77  0.09a 1.78  0.04a
solved in D2O (3 g/100 mL) for NMR analysis. High-resolution 1H Galactose 3.15  0.03a 3.15  0.05a
and 13C NMR spectra were recorded in D2O at 500.13 and Glucose 2.26  0.01a 2.24  0.03a
125.78 MHz, respectively, on a Bruker AM500 NMR spectrometer Xylose 0.41  0.02a 0.41  0.04a
Mannose 0.31  0.00a 0.30  0.05a
operating at 25  C. A 5 mm inverse geometry 1H/13C/15N probe was
þ
used. The chemical shifts were reported relative to external stan- ND: not detectable.
a
Results are the means of two determinations  standard deviation. Values with
dards, trimenthysily propionate (TSP in D2O, 4.76 ppm, for 1H) and
different letters within the same row differ significantly (P < 0.05).
1,4-dioxane (in D2O, 66.5 ppm, for 13C).

2.9. Statistical analysis
All statistical analyses were performed using SAS version 9.1
(SAS Institute Inc., Cary, NC, USA). Analysis of variance (ANOVA) was
performed using the general linear models (GLM) procedure to
determine significant differences among the samples. Means were
compared by using Fisher’s least significant difference (LSD) pro-
cedure. Significance was defined at the 5% level.
galactomannans. In the present study, the observations (on protein
content, galacturonic acid content, neutral sugar composition, and
molecular weight distribution) suggest that the treatment of pro-
nase could selectively remove the residual proteinaceous compo-
nents from pumpkin pectin without considerably influencing any
other chemical and molecular properties.
3.3. Surface tension

3. Results and discussion Changes in surface tension of water by pumpkin pectin fraction
A and fraction B with different concentrations (0.05e1.00 g/100 g)
3.1. Chemical composition at 21e22  C are shown in Fig. 3. The surface tension of water was
0.0723 N/m. Adding pumpkin pectin fraction A, pumpkin pectin
The yields of pumpkin pectin fractions A and B were calculated fraction B, or pectin standard at all the concentrations studied into
as a percentage of the weight of dried pumpkin powder, which water led to a decrease in the surface tension of water, indicating
were 10.03 and 8.08 g/100 g, respectively. The protein content that the pectin fractions A and B as well as pectin standard have the
(derived from nitrogen) of pumpkin pectin fraction A was 4.55 g/ surface activity. Moreover, the surface tension of water decreased
100 g, while no nitrogen was detected in pumpkin pectin fraction B with increasing the concentration of pumpkin pectin fractions
(Table 1). The DE values (about 47%) of pumpkin pectin fractions A (fraction A from 0 to 0.2 g/100 g; fraction B from 0 to 0.1 g/100 g)
and B were not significantly different. The galacturonic acid con- and finally reached a plateau. Similar trend was also found in other
tents of pumpkin fractions A and B represent 75.02 and 78.22 g/ polysaccharide samples, such as arabinoxylan and arabinogalactan
100 g, respectively. This finding indicated that both of the fractions (Izydorczyk, Biliaderis, & Bushuk, 1991), Portulaca oleracea gum
are mainly composed of homogalacturonan (Vincken et al., 2003; (Garti, Slavin, & Aserin, 1999), and fenugreek gum (Brummer et al.,
Waldron et al., 2003). Small amounts (about 10 g/100 g) of six 2003).
different neutral sugars were found in both of the pectin fractions,
including rhamnose, arabinose, galactose, glucose, xylose, and
mannose (Table 1). The presence of rhamnose, galactose, and
arabinose in the pumpkin pectin fractions could indicate the exis-
tence of rhamnogalacturonan I with side chains, such as arabinan,
galactan, and arabinogalactan (Tamaki et al., 2008; Wu, Ai, Wu, &
Cui, 2013). The presence of glucose and xylose in the pumpkin
pectin fractions could explain the presence of xyloglucan as one of
the side chains and the presence of xyloglacturonan (Fissore et al.,
2007; Kost’álová, Hromádková, & Ebringerová, 2013; Vincken et al.,
2003). More specific structural analysis for pumpkin pectin will be
discussed in the next section.

3.2. Molecular weight distribution

The molecular weight distribution of pumpkin pectin fraction A

and fraction B was very similar (Fig. 2). The finding was consistent
with Brummer, Cui, and Wang (2003), who applied pronase for the
purification of fenugreek gum. They found that treating fenugreek
gum solution with pronase considerably decreased the protein
content of fenugreek gum from 2.36 to 0.57 g/100 g without Fig. 2. Molecular weight distribution of pumpkin pectin fraction A (–) and fraction
affecting the molecular weight and galactose/mannose ratio of the B (d).
400 S.W. Cui, Y.H. Chang / LWT - Food Science and Technology 58 (2014) 396e403

Table 2
Emulsion capacity and stability of pumpkin pectin fraction A, fraction B, and pectin
standard.a

Pectin sample Emulsifying property

Emulsion capacity (%) Emulsion stability (%)

a
Fraction A 63.7  2.0 58.3  0.2a
Fraction B NDþ ND
Pectin standard 62.5  0.4a 58.0  0.3a
þ
ND: not detectable.
a
Results are the means of two determinations  standard deviation. Values with
different letters within the same column differ significantly (P < 0.05).

polysaccharides can adsorb at oilewater interfaces to form stabi-

lizing layers around oil droplets (Siew et al., 2008). Leroux et al.
(2003) noted that the emulsifying ability of beet pectin and citrus
pectin is due to a small amount of protein which is covalently
Fig. 3. Changes in surface tension of water by pumpkin pectin fraction A (,), fraction bound to a highly branched polysaccharide structure. Therefore, in
B (>), and pectin standard (6) with different concentrations at 21e22  C. the present study, it is evident that the emulsifying properties of
pumpkin pectin fraction A can be considerably related to the
In the present study, the surface tension values of water for presence of the residual hydrophobic proteinaceous components in
pumpkin pectin fraction A at all the concentrations were lower the pectin structure.
than those for pectin standard, indicating that the effect of fraction
A on the surface activity of water was superior to that of pectin 3.5. Structural characterization
standard. In particular, the surface tension values of water for
pumpkin pectin fraction A at all the concentrations were remark- 3.5.1. Fourier transform-infrared spectroscopy
ably lower than those for pumpkin pectin fraction B. This finding The FT-IR spectrum of pumpkin pectin fraction B was similar to
suggests that pumpkin pectin fraction A had better capability to that of pectin standard (citrus pectin), as illustrated in Fig. 4. In
decrease the surface tension of water than did pumpkin pectin particular, both of pumpkin pectin fraction and pectin standard
fraction B.
Until now, the effects of residual protein existing in poly-
saccharides on the surface activity of water are still controversial.
For instance, Garti, Madar, Aserin, and Sternheim (1997) studied the
surface activity of non-purified fenugreek gum and purified fenu-
greek gum which was obtained by physical separation of contam-
inating protein from the non-purified fenugreek gum. They
reported that reducing the protein content did not influence the
surface tension of water. On the other hand, Brummer et al. (2003)
used a purification step to remove the contaminating protein from
fenugreek gum with pronase, and they compared the surface ac-
tivity of the non-purified fenugreek gum and purified fenugreek
gum. According to them (Brummer et al., 2003), the surface tension
of water for non-purified fenugreek gum was much lower than that
for purified fenugreek gum, explaining that the proteins closely
associated with the galactomannans can enhance the surface ac-
tivity of fenugreek gum. Dickinson, Murray, Stainsby, and Anderson
(1988) also showed positive correlation between the surface ac-
tivity and protein content of Acacia gum (nitrogen contents in the
range from 0.1 to 7.5 g/100 g). Thus, it is inferred in the present
study that the existence of the residual proteinaceous matters in
pumpkin pectin can play a major functional role in improving its
surface activity.

3.4. Emulsion capacity and stability

The emulsion capacity and stability of pumpkin pectin fraction A

were 63.7 and 58.3%, respectively, and were not significantly
different from those of pectin standard while both of them were not
detected in pumpkin pectin fraction B (Table 2). In general, it has
been widely known that many commercial polysaccharide samples
include a small amount of protein, either as a contaminant or as an
intrinsic part of the polysaccharide structure. The protein compo-
nents can provide the polysaccharides with good emulsifying
ability, since those are usually strongly hydrophobic (Dickinson, Fig. 4. Fourier transform-infrared spectra of pumpkin pectin fraction B and pectin
2003). That is, the hydrophobic protein components in the standard.
 S.W. Cui, Y.H. Chang / LWT - Food Science and Technology 58 (2014) 396e403
Fig. 5. 1H NMR spectrum of pumpkin pectin fraction B.
exhibited strong absorbances between 950 and 1200 cm1 wave
number regions. The regions between 950 and 1200 cm1, gener-
ally called as the “finger print” region for each polysaccharide,
provide information regarding the identification of major chemical
groups in polysaccharides since the position and intensity of the
individual band in the regions are unique for each polysaccharide
(Kalapathy & Proctor, 2001). According to Kost’álová et al. (2013),
the fingerprint region of pumpkin pectin represented the charac-
teristic bands at 1145, 1103, 1077, 1050, and 1017 cm1. Thus, it is
confirmed in the present study that pectin was successfully
extracted from pumpkin using our enzymatic extraction method
(amylase, cellulase, and pronase).
Chatjigakis et al. (1998) reported that the regions between 1500
and 1800 cm1 wave number are the most important regions for the
examination of structural features of pectin because carboxylic acid
and carboxylic ester groups of pectin are found in the regions. More
specifically, according to the work conducted by Manrique and Lajolo
(2002), the band at 1630 cm1 wave number regions indicated the
ionized carboxylate groups (COO), whereas the band at 1740 cm1
wave number regions was assigned to carbonyl groups (C]O) from
COOCH3 groups. Furthermore, the bands at 1730 and 1600 cm1
correspond to esterified carboxylic acid groups and nonesterified
carboxylic acid groups, respectively (Singthong et al., 2004; Winning,
Viereck, Salomonsen, Larsen, & Engelsen, 2009). Therefore, in the
present study, the occurrence of two distinguished bands at 1730 and
1600 cm1 apparently indicated the nonesterifed and esterified
carboxyl groups of galacturonic acid in pumpkin pectin.
3.5.2. 1H and 13C NMR analysis
The 1H NMR spectrum of pumpkin pectin fraction B is shown in
Fig. 5. From the data, it was possible to distinguish and assign the
resonances originating from a- and b-configured residues. In the
anomeric region, the strong signals at 5.08 and 5.11 ppm were
assigned to H-1 of 4-linked a-D-GalpA and H-1 of 2elinked a-L-
Rhap, respectively (Tamaki et al., 2008; Wu et al., 2013). The signals
at 4.45 and 5.17 ppm were attributed to H-1 of b-galactose and a-
arabinose, respectively (Duan, Wang, Dong, Fang, & Li, 2003;
Golovchenko, Ovodova, Shashkov, & Ovodov, 2002).
The very large signal at 3.81 ppm was derived from methyl
groups binding to carboxyl groups of 4-linked a-D-GalpA (Cozzolino
401
et al., 2006; Kost’álová et al., 2013; Tamaki et al., 2008). Two signals
at around 2.15 ppm were attributed to acetyl groups binding at O-2
and O-3 of GalpA. The identification of the acetyl groups was ach-
ieved by comparison of the observed spectrum against literature
values (Bédouet, Courtois, & Courtois, 2003; Cozzolino et al., 2006;
Kost’álová et al., 2013; Perrone et al., 2002). The methyl rhamnose
signals at 1.13 and 1.25 ppm were associated with the 1,2-linked
and 1,2,4-linked Rhap (Perrone et al., 2002; Wu et al., 2013).
The 13C NMR spectrum of pumpkin pectin fraction B explained
the resonances from C-1 of 4-linked a-D-GalpA at 100.2 and
101.0 ppm which were the most intensive in the anomeric region
(Fig. 6). Both of the signals can be related to C-1 of nonesterifed and
esterified carboxyl groups of 4-linked a-D-GalpA, respectively, by
comparison with literature values of the anomeric chemical shifts
(Cozzolino et al., 2006; Westerlund, Åman, Andersson, &
Andersson, 1991). The respective corresponding typical reso-
nances of C-6 of the carboxyl groups of 4-linked a-D-GalpA were
observed at 171.7 and 171.5 ppm, and the existence of two carboxyl
signals confirmed the presence of nonesterifed and esterified
carboxyl groups of 4-linked a-D-GalpA (Cozzolino et al., 2006; Wu
et al., 2013). The carbon signals at 21.08 ppm were assigned to
CH3 of the O-acetyl groups (Westerlund et al., 1991).
Based on the findings obtained from the galacturonic acid
content, neutral sugar composition, FT-IR spectra, and positions of
signals in the 1H and 13C NMR spectra of pumpkin pectin, it is
suggested in the present study that the backbone of pumpkin
pectin proved to mainly represent linear a-1,4-D-galacturonic acid
in which a considerable portion of galacturonic acid residues is
present as methyl esters, and some L-rhamnose are involved in the
linear region of the backbone through a-1,2-linkages. Furthermore,
it is suggested that the side chains representing galactans and
arabinans are attached to the 4-position of the rhamnose residues
of the backbone.
4. Conclusion
The present study focused on the potential of pumpkin as a
source for the extraction of pectin using enzymatic methods. In
particular, the effect of pronase treatment on emulsifying proper-
ties of pumpkin pectin and the structural properties of pumpkin
402 S.W. Cui, Y.H. Chang / LWT - Food Science and Technology 58 (2014) 396e403
13
Fig. 6. C NMR spectrum of pumpkin pectin fraction B.
pectin were investigated. The emulsifying properties of pumpkin
pectin resulted from the presence of hydrophobic proteinaceous
components in the pectin structure. Pumpkin pectin could be used
as an emulsifier in oil-in-water emulsions for the beverage industry
unless residual hydrophobic proteinaceous components are
removed. The backbone of pumpkin pectin was considered as an
alternation of homogalacturonan regions, repetition of (1 / 4)-
linked a-D-GalpA, and rhamnogalacturonan regions. The GalpA
residues of the backbone were partially methyl esterified and
partially acetylated on C-2 and/or C-3.
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