Food Hydrocolloids xxx (2013) 1e9

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Food Hydrocolloids
journal homepage: www.elsevier.com/locate/foodhyd

Emulsion stabilizing properties of pectins extracted by high

hydrostatic pressure, high-speed shearing homogenization and
traditional thermal methods: A comparative study
Xingfeng Guo a, b, c, d, Wenting Zhao a, c, d, Xueli Pang a, c, d, Xiaojun Liao a, c, d, Xiaosong Hu a, c, d,
Jihong Wu a, c, d, *
a
College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China
b
Food Science Department, College of Agriculture, Liaocheng University, Liaocheng 252059, China
c
Key Laboratory of Fruits and Vegetables Processing, Ministry of Agriculture, Beijing 100083, China
d
National Engineering Research Center for Fruits and Vegetables Processing, Beijing 100083, China

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

Article history: In the previous studies, two novel methods for pectin extraction, using high hydrostatic pressure (HHP)
Received 26 July 2012 and high-speed shearing homogenization (HSHE), were developed with higher efficiency, less time and
Accepted 22 May 2013 energy consumptions than traditional thermal extraction (THE); however, many functional properties of
these pectins are still unknown. To explore the influence of extraction methods (HHP, HSHE and THE)
Keywords: and pH on the emulsion stabilizing properties of these pectins, many parameters, including zeta po-
High hydrostatic pressure
tential, apparent viscosity, light microscopy, and particle mean diameters of pectins or their emulsions
High-speed shearing homogenization
(1% pectin with 42.8% refined soybean oil), were firstly determined compared with those of two com-
Pectin
Particle-size distribution
mercial pectins (AP and SP). The results revealed that extraction methods have important influence on
Emulsifying stability viscosities of pectins and their emulsions. The emulsions prepared by HHP extracted pectin have the
Atomic force microscopy smallest particle mean diameters (8.96e11.02 mm) and best emulsifying stability (100%) after centrifu-
gation assay or 3 weeks of storage at 4
C with the pH in the range of 3e5. The emulsions prepared by
HSHE and THE extracted pectins have a similar particle mean diameters and emulsifying stabilities better
than those made with AP and SP. The molecular weight distributions and morphological features,
determined by HPSEC-RID and AFM, then indicated their difference at molecular level and their in-
teractions. From the results, it could be concluded that emulsion stabilizing properties of pectin were
greatly influenced by their physicochemical properties, including viscosity, molecular size and their
interactions.
Crown Copyright Ó 2013 Published by Elsevier Ltd. All rights reserved.

1. Introduction
Many food products, including milk, cream, beverages, dress-
ings, dips, sauces, batters and desserts, etc., are oil-in-water (O/W)
or water-in-oil (W/O) emulsions that consist of small lipid droplets
dispersed in a continuous aqueous/oil medium, which can
make and maintain the necessary textures and tastes of foods
(Akhtar, Dickinson, Mazoyer, & Langendorff, 2002; Guzey, Kim, &
McClements, 2004). Emulsions, however, are thermodynamically
unstable systems that are prone to destabilization through a variety
of different physiochemical processes, including gravitational sep-
aration, flocculation, coalescence, or Ostwald ripening (Guzey et al.,
* Corresponding author. No. 17 Qinghua East Road, Haidian District, Beijing
10083, China. Tel./fax: þ86 10 62737434 22.
E-mail address: wjhcau@yahoo.com.cn (J. Wu).
2004). One of the most important and widely used methods
for improving the stability of O/W or W/O emulsions is to
utilize emulsifiers (Dalgleish, 2006; Dickinson, 2009; Neirynck,
Van der Meeren, Bayarri Gorbe, Dierckx, & Dewettinck, 2004), of
which food hydrocolloids from natural resources are the most
commonly used materials, including protein, pectin, gums etc.,
for facilitating the formation, improving the stability, and preparing
desirable quality of the emulsions (Dickinson, 2003; McClements,
2004; Nakauma et al., 2008; Thanasukarn, Pongsawatmanit, &
McClements, 2004).
Pectin, a family of complex heteropolysaccharides consisting
predominantly of partially methoxylated galacturonic acid resi-
dues, is extensively distributed in almost all of the fruits and veg-
etables as the structural unit of fresh cells and the junction between
the cells (Christiaens et al., 2011; Jolie et al., 2012; Ridley, O’Neill, &
Mohnen, 2001; Thakur, Singh, & Handa, 1997). Its structure is based

0268-005X/$ e see front matter Crown Copyright Ó 2013 Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodhyd.2013.05.010

Please cite this article in press as: Guo, X., et al., Emulsion stabilizing properties of pectins extracted by high and traditional thermal methods: A
comparative study, Food Hydrocolloids (2013), http://dx.doi.org/10.1016/j.foodhyd.2013.05.010
2 X. Guo et al. / Food Hydrocolloids xxx (2013) 1e9
on 1, 4-linked a-D-galacturonic acid, interrupted by L-rhamnose
residues with side-chains of neutral sugars (mainly D-galactose and
L-arabinose) (Mohnen, 2008; Ridley et al., 2001). Pectin, as a well-
known food additive, is widely used as thickener, emulsifier and
stabilizer in a variety of food, pharmaceutical and cosmetic prod-
ucts (Thakur et al., 1997). Pectin has been extracted from various
agricultural byproducts, including orange peel, apple pomace,
lemon and sugar beet pulp, specifically, the orange peel and apple
pomace are abundant and contain high levels of pectic poly-
saccharides (Fishman, Chau, Cooke, & Hotchkiss, 2008; Wang et al.,
2007; Yeoh, Shi, & Langrish, 2008). The most commonly used
methods for pectin extraction currently were direct boiling by
water acidified with mineral acid (the so-called traditional thermal/
acid extraction) under the pH, temperature, and duration condi-
tions generally in the range of 1.3e3, 60e100
C, and 20e360 min,
respectively (Liu, Shi, & Langrish, 2006; Yeoh et al., 2008). Due to a
long period of heating under strong acid condition, the extracted
pectin undergoes thermal/acid degradation, which could result in
undesired changes in physicochemical and functional properties of
pectin (Koubala et al., 2008). Currently, new technologies, including
ultrasonication, high hydrostatic pressure, microwave and super-
high frequency electromagnetic field, are employed in pectin
extraction process to give an increased extraction yield, higher
quality, less time or energy consumptions than traditional thermal
extraction (Fishman et al., 2008; Guo et al., 2012; Kratchanova,
Panchev, Pavlova, & Shtereva, 1994; Wang et al., 2007; Yeoh et al.,
2008). Fishman, Chau, Hoagland, and Ayyad (2000) demonstrated
that microwave heating within 3 min at moderate pressure
(50  2 psi) can effectively decrease the thermal/acid degradation
and significantly increase the weight-average molar mass and
intrinsic viscosity of pectin. Our previous study also confirmed that
pectin extracted by high hydrostatic pressure has a significant
higher intrinsic viscosity and viscosity-average molecular weight
than those extracted by traditional thermal methods (Guo et al.,
2012).
Many studies currently have investigated the emulsifying or
emulsion stabilizing properties of pectin in O/W emulsions (Akhtar
et al., 2002; Al-Hakkak & Al-Hakkak, 2010; Dickinson, 2009;
Drusch, 2007; Leroux, Langendorff, Schick, Vaishnav, & Mazoyer,
2003; Neirynck et al., 2004). Leroux et al. (2003) demonstrated
that citrus pectin and beet pectin can efficiently reduce the inter-
facial tension between oil and water phase in the emulsions,
however, their emulsion stabilizing properties of pectin varies due
to the contents difference of acetyl groups, proteins and calcium
ion. Akhtar et al. (2002) also confirmed that depolymerized pectins
with different molecular weights have different emulsion stabilities
in the rapeseed oil/gum arabic emulsion. In our previous study, we
developed two novel extraction technologies for the extraction of
pectin from the peels of pomelo with higher yield, less time and
energy consumptions than the traditional thermal extraction (Guo
et al., 2012). However, the applications of these pectins in food as a
thickener, emulsifier or stabilizer have neither been investigated
nor compared with traditional thermal extracted pectin or com-
mercial pectins.
The purpose of the present study was to investigate the thick-
ening, emulsion stabilizing properties of pectins extracted by high
hydrostatic pressure (HHP) and high-speed shearing homogeniza-
tion (HSHE) compared with pectin extracted by traditional thermal
extraction (THE) in O/W emulsion. Many physicochemical proper-
ties and morphological features, such as apparent viscosities,
droplet-size distributions, micrographs and stabilities, of the pec-
tins or their emulsions with different pH before or after the storage
were first determined to explore the differences of emulsion sta-
bilizing properties among different pectins. The influences of mo-
lecular structure, morphological feature and extraction method on
Please cite this article in press as: Guo, X., et al., Emulsion stabilizing properties of pectins extracted by high and traditional thermal methods: A
comparative study, Food Hydrocolloids (2013), http://dx.doi.org/10.1016/j.foodhyd.2013.05.010
emulsion stabilizing properties of pectins were then investigated to
interpret their interactions.
2. Materials and methods
2.1. Materials and chemical reagents
Honey pomelo (Citrus grandis Osbeck) cultivated in Guanxi,
Fujian province, was purchased from a local market of Beijing in
November 2011. The collected peels of pomelo (ca. 1.5 kg) were first
cut into pieces and steamed at 100
C for 2 min to inactivate the
enzymes using a Vertical Heating Pressure Steam Sterilizer (LDZX-
50KBS, Shen’an Medical Instrument Factory, Shanghai, China) un-
der the ambient pressure, the drying process was then carried out
by a vacuum freeze dryer (LGJ-25C, Four-ring Science Instrument
Co., Ltd, Beijing, China) with the shelf temperature at 18
C for
about 24 h until the water content was reduced to ca. 8%. The dried
sample was then milled by an electric grinder (HY-04A, Beijing
Huanya Tianyuan Machine Technology Co., Ltd, Beijing, China) and
filtrated using a filter sieve (ca. 60 meshes). Finally, the sample was
vacuum-packed and stored at 4
C for the subsequent extraction.
Two types of commercial citrus pectins, including SP9135 (Sigmae
Aldrich, St. Louis, USA) and AP102 (Andre Pectin Co. Ltd, Yantai,
China), extracted by traditional thermal methods with industrial
scale, were purchased from Beijing Biodee Biotechnology Co., Ltd
(Beijing, China) and Andre Pectin Ltd (Yantai, China), respectively.
All chemical reagents, including ethanol, hydrochloric acid, cit-
ric acid, sodium citrate, sodium azide, etc, used in the study were
analytical grade and purchased from Lanyi reagent company (Bei-
jing, China).
2.2. Apparatus
HHP extraction was carried out using a pressure-assisted ther-
mal high hydrostatic pressurization unit (CAU-HHP-700-6, Baotou
Kefa High Pressure Food Processing Inc., Inner Mongolia, China)
with a cylindrical pressure chamber capacity of 7 L. Distilled water
was used as the pressure-transmitting medium. The rate of pres-
sure increase was about 130 MPa/min and the pressure release time
was about 2 s.
HSHE extraction was carried out by an analytical system (JHBE-
50, Jinnai Sci-tech Development Ltd, Zhengzhou, China) contains
rotating cutter, high-speed motor, lifting system, control system
and sample container. The rotation speed of the high-speed motor
could be regulated in the range of 0e10,000 rpm by controlling the
extraction voltage from 0 to 220 V.
2.3. Preparation of pectins
The pectin extracted by HHP, HSHE and THE were based on the
methods of the previous investigations with slight modification
(Guo et al., 2012; Kratchanova, Pavlova, & Panchev, 2004). Extrac-
tion solvent (pH ¼ 1.24) was prepared by distilled water and
0.5 mol/L HCl, the solid to liquid ratio was 1:50. HHP extraction was
carried out at the pressure of 500 MPa, temperature of 55
C and
the pressure-holding time of 10 min. THE extraction was carried out
at 82
C for 80 min using a reciprocating shaker bath. While, HSHE
extraction was carried out with the extraction voltage of 156 V and
extraction time of 240 s following a preheating process at 85
C for
5 min. After extraction, the crude filtrate was precipitated using
double volumes of 95% (v/v) ethanol and kept overnight without
stirring at 4
C. The precipitated pectin was then filtered by a filter
cloth (400 meshes), and washed three times (5 min each time)
using absolute alcohol to remove the monosaccharides, di-
saccharides and other impurities (Masmoudi et al., 2008). The
 X. Guo et al. / Food Hydrocolloids xxx (2013) 1e9
pectin was then obtained after dried by a vacuum freeze dryer (LGJ-
25C, Four-ring Science Instrument Co., Ltd, Beijing, China) with the
shelf temperature at 18
C for 24 h.
2.4. Preparation of emulsion
The aqueous buffer (ionic strength 0.1 M) was prepared using
citric acid, sodium citrate and distilled water with the pH of 3, 4, 5
or 6, respectively. To prevent the spoilage of all the solutions during
storage 0.05% sodium azide were added as a preservative. 0.6 g of
each pectins, including two commercial pectins and pectins
extracted by HHP, HSHE and THE, were dispersed into the buffer
solution, respectively, at the concentration of 1% w/w. After the
samples were dissolved completely under magnetic stirring at
room temperature, the emulsions were then prepared by slowly
mixing 15 g of refined soybean oil (Arawana, Yihai Kerry group,
Shanghai, China) into 20 g of pectin solutions, and emulsifying
the mixtures with a high-speed homogenizer (Ningbo Xinzhi
Biotechnology Co., China) at a speed of 10,000 rpm for 3 min. The
pH of each emulsion was the same as their pectin solution since the
addition of oil had little effect on pH (Huang, Kakuda, & Cui, 2001).
2.5. Determination of physicochemical properties of pectins
The galacturonic acid (GA) content of pectin was determined by
the m-hydroxybiphenyl method (Blumenkrantz et al., 1973). Stan-
dard galacturonic acid solutions (10e100 mg/mL) were used to
construct the standard curve for the determination. The degree of
esterification (DE) of pectin was determined by the titrimetric
method of Food Chemical Codex (FCC, 1981) with slight modifica-
tion. The protein content in pectin was determined by the coo-
massie brilliant blue G250 method (Sedmak & Grossberg, 1977) and
the protein concentration was expressed as the equivalent con-
centration of bovine serum albumin (BSA). The molecular weight
distribution of pectin extracted by different methods was deter-
mined by high performance size exclusion chromatography with a
refractive index detector (HPSEC-RID) (Fishman et al., 2008) with
slight modification. P-82 Pullulan standards (Showa Denko KK Inc.,
Kawasaki, Japan) with 8 kinds of molecular weight, including 5.9,
11.8, 22.8, 47.3, 112, 212, 404, 788 KDa, were selected as the cali-
bration standards.
2.6. AFM determination and image analysis
The nanostructure of pectin was characterized by AFM (MFP-
3D-SA, Asylum Research Inc., Santa Barbara, CA, USA) in tapping
mode (Yang, Lai, An, & Li, 2006; Zhang et al., 2012). The PPP-NCHR
Silicon SPM Sensor (NanosensorsÔ, Neuchatel, Switzerland) was
used with scan rate of 0.5 Hz. The resonance frequency and force
constant of the probe were 330 kHz and 42 N/m, respectively.
Pectin was dissolved in deionized water with the concentration of
10 mg/mL. Samples (20 mL) were drop deposited onto a freshly
cleaved mica sheets (Tozai IS-313B muscovite mica, Toukyou,
Japan). Then the solution was air dried in a dust-free enclosure at
ambient temperature before imaging. The AFM images were
analyzed by AFM offline software (Igor Pro 6.22A, WaveMetrics Inc.,
Portland, OR, USA).
2.7. Viscosity measurement of pectin and emulsion
To evaluate the viscosity of pectin and its relevance to the
emulsion stabilizing properties, 1% pectin solutions were pre-
pared by using 0.1 mol/L citric acid e sodium citrate buffer as
dissolvent with the pH of 3, 4, 5 and 6, respectively. The viscosity
of each samples, including solutions and emulsions, were
Please cite this article in press as: Guo, X., et al., Emulsion stabilizing properties of pectins extracted by high and traditional thermal methods: A
comparative study, Food Hydrocolloids (2013), http://dx.doi.org/10.1016/j.foodhyd.2013.05.010
3
determined using a Controlled Stress Rheometer (AR 550, TA In-
struments, New Castle, DE, USA) with a conical concentric cylin-
der and plate geometry (SST ST 60 mm diameter, cone angle 2
,
52 mm gap) at the angular velocity of 10 s1 at 25
C immediately
after preparation. All viscosity measurements were carried out in
triplicate.
2.8. Zeta potential of pectin
The zeta potential of pectin solution was measured with a
Zetasizer Nano ZS90 (Malvern Instruments Ltd., Worcestershire,
UK). The measurements were carried out at 25
C in triplicate using
0.05% pectin solutions prepared by the aqueous buffer (ionic
strength 0.1 M) using citric acid, sodium citrate and distilled water
with the pH in the range of 3e6 and the data were analyzed by
Zetasizer software 6.20 (Malvern Instruments Ltd., Worcestershire,
UK).
2.9. Light microscopy
The 20 times-diluted emulsions (pH ¼ 3) for light microscopy
were examined and photographed immediately after preparation
under bright field illumination with a 40 objective lens on a
inverted microscope BIO 200-PH (Jingbaizhouxian technology Co.
Ltd., Beijing, China). Microphotographs were taken using a Nikon
D5000 digital camera (Nikon Corporation, Sendai, Japan) that
equipped on the microscope.
2.10. Determination of emulsion droplet-size distribution
Droplet-size distribution of the emulsions before or during 3
weeks of storage at 4
C was measured by computer-controlled
laser diffraction apparatus (LS 230, Beckman Coulter, Inc., Full-
erton, USA) once a week with the measurement range from 0.4 to
2000 mm and analyzed by the installed software (LS32 Version 3.29,
Beckman Coulter, Inc., Fullerton, USA). The average droplet-size was
characterized by the equivalent volume-weighted mean diameter
(d43), defined by:
X
d43 ¼ ni d4i =Xn d3
i i
i
i
where ni is the number of droplets of diameter di. The d43 value was
used to monitor changes in the droplet-size distribution of freshly
made emulsions and emulsions on storage (Akhtar et al., 2002).
2.11. Determination of emulsion stability
The emulsions stability was evaluated using a combination of
centrifugation assay and storage assessment (Huang et al., 2001;
Surh, Decker, & McClements, 2006). The centrifugation assay of
the emulsion was determined by the method of Huang et al.
(2001) with slight modification. Approximately 35 g of each
emulsion was placed into 50 ml plastic centrifugal tube and
centrifuged at 3000 g for 5 min and 20 min, respectively, by a
compact centrifuges (CF 16RXII, Hitachi, Ltd., Tokyo, Japan) at the
temperature of 20
C. Centrifugation produced an aqueous phase
(water/pectin dispersion) on the bottom, an emulsion phase in
the middle or an oil phase on the top. The height of the emulsion
phase after centrifuged for 5 min and 20 min were recorded as H1
and H2, respectively. The initial height of the emulsion before the
centrifugation was noted as H0. The emulsion stability (ES5 or
ES20) was then calculated as a percentage using the following
equation:
4 X. Guo et al. / Food Hydrocolloids xxx (2013) 1e9

ES5 ð%Þ ¼ H1 =H0  100 10

6

30
ES20 ð%Þ ¼ H2 =H0  100
Another set of emulsions were used to investigate the stability 5
20 10
of emulsions during the storage (Surh et al., 2006). Each emulsion

IRU (mV)
(35 g) was placed into 50 ml plastic centrifugal tube, tightly sealed

Mw(Da)
with the plastic cap, and then stored at 4
C for 3 weeks. During the
storage, emulsions were separated into an optically opaque cream 10
SP 4
layer at the top and a transparent (or turbid) layer at the bottom. 10
P
The initial height of the emulsions was recorded as H0. The height
of the emulsions during the storage were measured and recorded 0 HHP

as H3 once a week. The emulsion stability (%) was also calculated HSHE

using the following equation: THE 10

3

10 12 14 16 18 20
ESð%Þ ¼ H3 =H0  100
Retention time (min)

Fig. 1. HPSEC-IRD chromatograms of different pectins and the standard molecular

weight curve. Experimental conditions: column, Shodex OHpak SB-804 HQ
2.12. Statistical analysis (300  8 mm, i.d. 10 mm); column temperature, 35
C; mobile phase, 0.05 mol/L
NaNO3; flow-rate, 0.5 mL/min; injection volume, 20 mL.

Results of three to five replicates were analyzed using analysis of

variance (ANOVA) by OriginPro 7.5 (OriginLab Corporation, North-
constituents in AP and SP were much higher than others. According
ampton, USA), and expressed as mean value  standard deviation.
to the retention time, the peak molecular weights (Mp) of pectins
Statistical analysis was performed by Tukey’s test and the confi-
extracted by HHP, HSHE and THE were 520 KDa, 482 KDa and
dence level for statistical significance was set at a probability value
437 KDa, respectively. However, two individual peaks were pre-
of 0.05.
sented in the commercial pectins and Mp of them were 455 and 184
for AP, 467 and 127 for SP, respectively. The results revealed that
3. Results and discussion extraction method has a significant effect on the average molecular
weight of pectins.
3.1. Physicochemical properties of pectin

3.2. AFM analysis

The contents of GA, DE and protein of the pectins extracted by
different methods are summarized in Table 1. It is clearly revealed
Morphological features of the pectins extracted by different
that the contents of GA, DE and protein of the pectins extracted by
methods can be compared by AFM images as shown in Fig. 2(A)e
HHP, HSHE and THE have no significant difference (p > 0.05).
(E). The color bar legends at the right of the images signify the full
However, GA contents of pectin extracted by HHP (81.32  1.76%),
height of the samples scanned. The effect of extraction methods on
HSHE (80.55  2.02%) and THE (82.56  1.35%) were significantly
the morphological features of pectin could exhibited by comparing
higher than AP (72.99  0.20%) and SP (75.59  0.32%). And DE of
the images within these figures. The AFM image of pectins
pectins extracted by HHP (75.79  0.74), HSHE (74.36  0.85%) and
extracted by HHP, as shown in Fig. 2(A) has much more long chains
THE (76.64  0.92%) was significantly lower than SP (84.91  1.88%)
and large agglomerate compared with others, specifically there are
but higher than AP (72.08  0.16%). HPSEC-RID chromatograms of
mostly short chains and aggregate in the AP and SP. These results
pectins extracted by HHP, HSHE, THE, AP, and SP were presented in
were also fit well with the molecular weight distribution in the
Fig. 1. In addition, the standard molecular weight curve was also
above as well as viscosity-average molecular weight in our previous
plotted against retention time in Fig. 1. From the overlaid chro-
study (Guo et al., 2012). From the above results on molecular
matograms shown in Fig. 1, it can be clearly observed that the
weight distribution and morphological features of pectins, it can be
profiles of the pectins have a significant difference in the molecular
observed that extraction methods have significant effect on the
weight distribution. According to the standard molecular weight
molecular size and molecular interactions of pectins.
curve, it can be found that all pectins have apparent wide molecular
weight distribution from approximately 10 Kda to 800 KDa. Pectin
extracted by HHP has higher percentage of high molecular weight 3.3. Viscosity of pectins and its emulsions
constituents than pectins extracted by HSHE, THE, AP and SP,
however, pectin extracted by HSHE has a wider molecular weight Viscosity, as one of the most important parameters in colloidal
distribution than those extracted by HHP, but still narrower than solution and emulsion, has a significant effect on the stability of
those extracted by THE, AP and SP. The low molecular weight colloidal solution and emulsion. Fig. 3(a) and (b) represent the

Table 1
Contents of galacturonic acid, degree of esterification, and protein in different pectins.

Characteristics Pectin samples

HHP HSHE THE AP SP

GA (%) 81.32  1.76a 80.55  2.02a 82.56  1.35a 72.99  0.20b 75.59  0.32b
DE (%) 75.79  0.74b 74.36  0.85bc 76.64  0.92b 72.08  0.16c 84.91  1.88a
Protein (%) 7.04  0.15a 6.87  0.33a 6.72  0.38a 2.38  0.17b 3.44  0.07c

Each value is expressed as mean  standard deviation of triplicate tests; Means within the same row with different letters are significantly difference at p < 0.05.

Please cite this article in press as: Guo, X., et al., Emulsion stabilizing properties of pectins extracted by high and traditional thermal methods: A
comparative study, Food Hydrocolloids (2013), http://dx.doi.org/10.1016/j.foodhyd.2013.05.010
 X. Guo et al. / Food Hydrocolloids xxx (2013) 1e9 5

Fig. 2. AFM images of the five different pectins. Scan area ¼ 2.0 mm  2.0 mm (A) pectin extracted by HHP; (B) pectin extracted by HSHE; (C) pectin extracted by THE; (D) commercial
pectin SP; (E) commercial pectin AP. Note: lc-long chains; ls-linear single fractions; sc-short chains; ag-aggregate.

(a) 2.5
HHP (b) 7.0
HHP
2.4 HSHE 6.0 HSHE
THE THE
3.0
Apparent Viscosity (Pas)

SP SP
Apparent Viscosity (Pas)

AP AP
1.2

2.0
0.8

1.0
0.4

0.0 0.0
3 4 5 6 3 4 5 6
pH pH

Fig. 3. Effects of pH on the apparent viscosity of 1% pectin solutions (a) and their emulsions (b). Analysis conditions: pH of pectins and emulsions are 3, 4, 5 and 6, respectively, plate
geometry: diameter of 60 mm, cone angle of 2
, gap of 52 mm, shear rate 10 s1, temperature of 25
C (n ¼ 3).

Please cite this article in press as: Guo, X., et al., Emulsion stabilizing properties of pectins extracted by high and traditional thermal methods: A
comparative study, Food Hydrocolloids (2013), http://dx.doi.org/10.1016/j.foodhyd.2013.05.010
6 X. Guo et al. / Food Hydrocolloids xxx (2013) 1e9

Table 2
Zeta potential of pectin under different pH conditions.

Pectins pH of pectin solutions

3 4 5 6
a, B bc, A b, A
HHP 7.05  0.36 9.73  0.39 8.81  0.29 10.60  0.36c, B
HSHE 7.32  0.02a, B
9.63  0.27b, A 9.50  0.32b, AB 9.40  0.48b, AB
THE 6.76  0.20a, A
10.30  0.17c, A 10.05  0.30c, B 8.93  0.30b, A
AP 8.88  0.20a, C
13.63  0.95b, B 13.13  0.46b, C 12.63  0.72b, C
SP 9.41  0.19a, C
13.20  0.98b, B 13.67  0.42b, C 12.90  0.35b, C

Note: Data are expressed as mean  standard deviations (n ¼ 3). Means within the same rows with different lowercase letters are significantly difference at p < 0.05. Means
within the same columns with different capital letters are significantly difference at p < 0.05.

apparent viscosity of 1% pectin solutions (a) and their emulsions (b)
with pH in the range of 3e6. From the results of Fig. 3(a), it is clearly
revealed that the extraction method has a great influence on the
viscosity of solution. The viscosity of pectins extracted by HHP and
HSHE are significantly higher than the viscosity of commercial
pectins and THE extracted pectin at the pH from 3 to 6. In addition,
Fig. 3(a) also showed that the viscosity of all pectin solutions
decreased in a certain extent with the pH increased from 3 to 6.
From the comparison of Fig. 3(a) and (b), it was obviously found
that all emulsions have a higher viscosities than their pectin solu-
tions at the same pH; especially the emulsion made with the pectin
extracted by HHP has the highest viscosity, 6.56  0.10, 3.07  0.04,
2.33  0.046 Pa s, with the pH of 3e5, respectively. However, at the
pH of 6, there was exception in emulsion prepared by the HHP
extracted pectin, because its viscosity (0.75  9.08E-4 Pa s) was less
than emulsions prepared by the HSHE (1.36  4.06E-3 Pa s) and
THE (1.08  4.01E-3 Pa s) extracted pectin. All the pectin solutions
and their emulsions above have significant higher viscosity than AP,
SP and their emulsions. The higher viscosity of solutions and
emulsions can decrease the particle movement and coalescence
effect; therefore, it can effectively increase the stability of emulsion
(Dickinson, 2009; Surh et al., 2006). In the following results con-
cerning the stability assessment, we also confirmed the above
phenomena.
3.4. Zeta potential of pectin
Zeta potential, as a measure of the electrokinetic potential, gives
an indication of the potential stability within the colloidal or
emulsion system according to its magnitude (Nakamura, Fujii, Tobe,

Fig. 4. Photomicrographs of emulsions prepared by pectin extracted by HHP (A), HSHE (B), THE (C) and two commercial pectin, including AP (D) and SP (E) under bright field
illumination (40).

Please cite this article in press as: Guo, X., et al., Emulsion stabilizing properties of pectins extracted by high and traditional thermal methods: A
comparative study, Food Hydrocolloids (2013), http://dx.doi.org/10.1016/j.foodhyd.2013.05.010
 X. Guo et al. / Food Hydrocolloids xxx (2013) 1e9 7

Adachi, & Hirotsuka, 2012). The zeta potentials of all pectin solu-
tions with pH in the range of 3e6 are shown in Table 2. As the
results shown in Table 2, the zeta potentials (negative) of all pectins
decreased with increasing the pH of the solutions from 3 to 6. For
the same pectin, the zeta potentials at the pH ¼ 3 were different
from those with pH of 4e6 significantly (noted with lowercase
letters in Table 2). However, the zeta potentials of the pectins at the
pH of 4e5 have no significant difference. Additionally, the zeta
potentials of pectin solutions at the same pH also have a significant
difference varying with their extraction methods (noted with
capital letters in Table 2). Zeta potentials of pectins extracted by
HHP, HSHE and THE were significantly higher than the two com-
mercial pectins, while the pectins extracted by HHP, HSHE and THE
had a smaller difference with or without significance (p < 0.05).
Nakamura et al. (2012) also investigated the zeta potentials of the
soybean soluble polysaccharide (SSPS) and the soybean soluble
polysaccharide cross-linked via phosphate (SSPS-HC) and found
that the zeta potential also decreased with increasing the pH from
2.5 to 12.5. However, from the results of our investigation and the
previous published researches, it can be concluded that acidic
polysaccharides, such as pectin, SSPS, etc., with low zeta potentials
(þ30 > zeta potential > 30 mV) tend to coagulate or flocculate
and maybe hard to maintain a stable colloidal or emulsion system
only by the electrokinetic potential (Nakamura et al., 2012).
3.5. Light microscopy
To elucidate the microstructure and micro-morphology of
emulsions prepared by different pectins, the photomicrographs of
all emulsions at pH of 3 were examined and photographed
immediately after preparation under bright field illumination with
a 40  objective lens. The typical micrographs for emulsions pre-
pared by HHP, HSHE, THE extracted pectins and the commercial
ones (AP and SP) at the concentration of 1% are presented in
Fig. 4(A)e(E), respectively. Fig. 4 revealed that all the particles of
the pectin emulsions are dispersed throughout the whole emulsion

(A) 150 (B) 40

0week 0week
120 1week 35 1week
2weeks 2weeks
90 3weeks 3weeks
30
14
µ m)

µ m)

25
4,3 (

4,3 (

12
d

20

10
15

8
3.0 3.5 4.0 4.5 5.0 5.5 6.0 10
3.0 3.5 4.0 4.5 5.0 5.5 6.0
pH
pH
(C) 80
0week
(D) 160

1week 0week
2weeks 1week
60 3weeks 2weeks
120
3weeks
µ m)

µ m)

25
80
4,3 (

4,3 (

20
d

40
15

10 0
3.0 3.5 4.0 4.5 5.0 5.5 6.0 3.0 3.5 4.0 4.5 5.0 5.5 6.0
pH
pH

(E) 100
0week
1week
2weeks
80 3weeks
µ m)

60
4,3 (
d

40

20
3.0 3.5 4.0 4.5 5.0 5.5 6.0

pH

Fig. 5. Particle mean diameter (d4,3 in mm) of emulsions prepared by pectin extracted by HHP (A), HSHE (B), THE (C), AP(D), SP (E) with pH in the range of 3e6. Measurements were
made after 0, 1, 2 and 3 weeks of storage at temperature of 4
C.

Please cite this article in press as: Guo, X., et al., Emulsion stabilizing properties of pectins extracted by high and traditional thermal methods: A
comparative study, Food Hydrocolloids (2013), http://dx.doi.org/10.1016/j.foodhyd.2013.05.010
8 X. Guo et al. / Food Hydrocolloids xxx (2013) 1e9

system regularly; however, the emulsions prepared by different Table 3

pectins varied significantly in particle diameters and volumes in the Storage stability of emulsions prepared by 1% of pectin and 42.8% of refined soybean
oil.
micrographs. Emulsion particles prepared by HHP extracted pectin
have a much smaller and uniform droplet diameter than others, Pectins pH Storage stability of emulsions (%)
and the emulsions prepared by HSHE and THE have a similar par- Centrifugation Storage time (W)
ticles constitutes in diameters but slight larger than emulsion assay
particles prepared by HHP extracted pectin. From the Fig. 4(D) and ES5 ES20 1 2 3
(E), it could be clearly observed that the emulsions prepared by AP
HHP 3 100 100 100 100 100
and SP have a relative larger particle diameters and wide size dis- 4 100 100 100 100 100
tributions than all other emulsion particles. During the determi- 5 100 100 100 100 100
nation, the constant flowing motion of the emulsion particles 6 25 25 45 40 37
prepared by AP and SP in view made it different to focus and take HSHE 3 55 40 100 100 100
4 60 45 100 90 87
photographs, however, the emulsions made by HHP, HSHE and THE 5 85 80 100 90 90
extracted pectins just present a tightly packed particles under a 6 80 60 100 92 100
relative static condition, which may resulted from their differences THE 3 40 30 100 100 98
in viscosity and imply the emulsions prepared by HHP, HSHE and 4 60 40 100 100 100
5 85 75 100 100 100
THE extracted pectin have a better stability than those prepared by
6 40 30 85 85 85
AP and SP. SP 3 15 10 75 75 75
4 50 45 80 80 80
3.6. Emulsion droplet-size distribution 5 55 50 80 80 80
6 45 45 85 83 82
AP 3 15 10 85 85 85
In emulsions, particle size distribution is generally selected as a
4 45 35 75 75 75
characterization tool and is chosen to be a quality response 5 60 45 75 75 75
parameter (Nii & Ishii, 2004). Stoke’s law states that the velocity at 6 45 40 45 45 40
which a droplet moves is proportional to the square of its radius,
the stability of an emulsion to gravitational separation or aggre-
gation can therefore be enhanced by reducing the size of the sharply to 25% with the pH increased to 6. This result was
droplets (Huang et al., 2001). The particle mean diameter (d4,3 in consistent with the viscosity and droplet-size distribution of
mm) of all emulsions with the pH of 3e6 and its change (once a emulsion at pH of 6 prepared by HHP extracted pectin and further
week) in the 3 weeks of storage are present in Fig. 5. Fig. 5(A)e(E) exhibited its sensitivity to the pH from 5 to 6. The pectins extracted
clearly revealed that all the emulsions with a pH of 3 had the by HSHE and THE have a similar ES5 (40e85%) and ES20 (30e80%)
smallest particle mean diameters and the particle mean diameter of properties with the pH in the range of 3e6 and exhibited well
emulsions increased by varying degrees and the HSHE and THE emulsifying properties compared with AP and SP. From the
extracted pectin increased sharply than others with the pH investigation of the emulsifying properties, we could found the
increased to 4, while, the particle mean diameter of emulsions emulsion stability of pectin was closely associated with the
made with HSHE and THE extraction pectins was decreased emulsion droplet-size distribution and the viscosity of emulsion.
significantly with the pH further increased to 5. From these results, Smaller droplet size and higher viscosity can efficiently enhance
we can conclude that pH has a significant influence on the particle the stability of emulsion. Combined with the results of molecular
mean diameter of emulsions. pH as a measure of the activity of the weight distribution and AFM analysis, we could conclude that the
hydrogen ion can significantly influence the molecular morphol- differences in molecular parameters should be the primary reasons
ogies and structures of pectins in solution and change their results in the differences in the emulsion stabilizing properties of
behavior in emulsions. During the 3 weeks of storage, the emulsion these pectins. Pectins with higher average molecular weight and
particle mean diameters of HHP extracted pectin also have the viscosity could increase the emulsion stabilities significantly. Pre-
smallest increase than other with pH in the range of 3e5. The vious studies have shown that DE and protein contents of pectin
emulsion particle mean diameters of HSHE and THE extracted also have significant impact on emulsions stability, however, their
pectin have a slight greater increase compared with the HHP effect in those emulsions need further investigations (Nakamura
extracted pectin. However, all the emulsions prepared by pectins et al., 2012; Surh et al., 2006).
extracted by HHP, HSHE and THE have a smaller particle mean di-
ameters increase during the storage than those made with AP and 4. Conclusions
SP. Among all the emulsion, the emulsions prepared by AP and SP
have the largest particle mean diameters and the largest diameters Emulsifying properties of pectin extracted by high hydrostatic
increase during the storage. Therefore, it can be concluded that pressure and high-speed shearing homogenization were investi-
extraction methods have significant influence on the droplet-size gated and compared with traditional thermal extracted pectin and
distributions. The results clearly revealed that HHP, HSHE and two commercial pectins. The emulsion prepared by HHP extracted
THE extracted pectins can define as potent emulsion stabilizer, pectin has the largest viscosity, narrowest particle size distribution,
because they give small particle size and temporal change in the smallest particle mean diameter and best emulsion stability
storage period. compared with other emulsions at the pH from 3 to 5. However, the
emulsion properties of HHP extracted pectin was decreased sharply
3.7. Emulsion stability with the pH increased to 6 because of its high sensitivity to pH
around 6. Compared with AP and SP, HSHE and THE extracted
In the present study, the emulsions stability was evaluated pectins also have a larger viscosity, narrower particle size distri-
using a combination of centrifugation assay and storage assess- bution, smaller particle mean diameter and better emulsion sta-
ment. From the results of Table 3, it is clearly revealed that HHP bility at the pH from 3 to 6. The physicochemical properties and
extracted pectin has the best ES5 (100%) and ES20 (100%) with pH AFM images of these pectins showed that pectins with a larger
in the range of 3e5, however, its ES5 and ES20 were decreased molecules and higher viscosity could result in a preferable

Please cite this article in press as: Guo, X., et al., Emulsion stabilizing properties of pectins extracted by high and traditional thermal methods: A
comparative study, Food Hydrocolloids (2013), http://dx.doi.org/10.1016/j.foodhyd.2013.05.010
 X. Guo et al. / Food Hydrocolloids xxx (2013) 1e9
emulsion stabilizing properties. From the above results, we can
conclude that pectins extracted by HHP and HSHE were excellent
thickening and emulsion stabilizing agent than pectins extracted by
traditional methods, especially the industrialized ones.
Acknowledgments
Financial support from the National Key Technology R&D Pro-
gram of P.R.C (2012BAD31B00), National High-tech Research
and Development (863) Program of P.R.C (2011AA100801), and
Chinese Universities Scientific Fund (2012QT012) are gratefully
acknowledged.
References
Akhtar, M., Dickinson, E., Mazoyer, J., & Langendorff, V. (2002). Emulsion stabilizing
properties of depolymerized pectin. Food Hydrocolloids, 16(3), 249e256.
Al-Hakkak, J., & Al-Hakkak, F. (2010). Functional egg whiteepectin conjugates
prepared by controlled Maillard reaction. Journal of Food Engineering, 100(1),
152e159.
Blumenkrantz, N., & Asboe-Hansen, G. (1973). New method for quantitative
determination of uronic acids. Analytical Biochemistry, 54(2), 484e489.
Christiaens, S., Van Buggenhout, S., Houben, K., Fraeye, I., Van Loey, A. M., &
Hendrickx, M. E. (2011). Towards a better understanding of the pectin struc-
tureefunction relationship in broccoli during processing: part Idmacroscopic
and molecular analyses. Food Research International, 44(6), 1604e1612.
Dalgleish, D. G. (2006). Food emulsionsdtheir structures and structure-forming
properties. Food Hydrocolloids, 20(4), 415e422.
Dickinson, E. (2003). Hydrocolloids at interfaces and the influence on the properties
of dispersed systems. Food Hydrocolloids, 17(1), 25e39.
Dickinson, E. (2009). Hydrocolloids as emulsifiers and emulsion stabilizers. Food
Hydrocolloids, 23(6), 1473e1482.
Drusch, S. (2007). Sugar beet pectin: a novel emulsifying wall component for
microencapsulation of lipophilic food ingredients by spray-drying. Food Hy-
drocolloids, 21(7), 1223e1228.
FCC. (1981). Food chemical codex (pp. 283e286). Washington, DC: National Academy
of Sciences.
Fishman, M. L., Chau, H. K., Cooke, P. H., & Hotchkiss, A. T. (2008). Global structure of
microwave-assisted flash-extracted sugar beet pectin. Journal of Agricultural and
Food Chemistry, 56(4), 1471e1478.
Fishman, M. L., Chau, H. K., Hoagland, P., & Ayyad, K. (2000). Characterization of
pectin, flash-extracted from orange albedo by microwave heating, under
pressure. Carbohydrate Research, 323(1e4), 126e138.
Guo, X., Han, D., Xi, H., Rao, L., Liao, X., Hu, X., et al. (2012). Extraction of pectin from
navel orange peel assisted by ultra-high pressure, microwave or traditional
heating: a comparison. Carbohydrate Polymers, 88(2), 441e448.
Guzey, D., Kim, H. J., & McClements, D. J. (2004). Factors influencing the production
of o/w emulsions stabilized by b-lactoglobulinepectin membranes. Food Hy-
drocolloids, 18(6), 967e975.
Huang, X., Kakuda, Y., & Cui, W. (2001). Hydrocolloids in emulsions: particle size
distribution and interfacial activity. Food Hydrocolloids, 15(4e6), 533e542.
Jolie, R. P., Christiaens, S., De Roeck, A., Fraeye, I., Houben, K., Van Buggenhout, S.,
et al. (2012). Pectin conversions under high pressure: Implications for the
structure-related quality characteristics of plant-based foods. Trends in Food
Science & Technology, 24(2), 103e118.
Please cite this article in press as: Guo, X., et al., Emulsion stabilizing properties of pectins extracted by high and traditional thermal methods: A
comparative study, Food Hydrocolloids (2013), http://dx.doi.org/10.1016/j.foodhyd.2013.05.010
9
Koubala, B. B., Mbome, L. I., Kansci, G., Mbiapo, F. T., Crepeau, M. J., Thibault, J. F.,
et al. (2008). Physicochemical properties of pectins from ambarella peels
(Spondias cytherea) obtained using different extraction conditions. Food
Chemistry, 106(3), 1202e1207.
Kratchanova, M., Panchev, I., Pavlova, E., & Shtereva, L. (1994). Extraction of pectin
from fruit materials pretreated in an electromagnetic field of super-high fre-
quency. Carbohydrate Polymers, 25(3), 141e144.
Kratchanova, M., Pavlova, E., & Panchev, I. (2004). The effect of microwave heating
of fresh orange peels on the fruit tissue and quality of extracted pectin. Car-
bohydrate Polymers, 56(2), 181e185.
Leroux, J., Langendorff, V., Schick, G., Vaishnav, V., & Mazoyer, J. (2003). Emulsion
stabilizing properties of pectin. Food Hydrocolloids, 17(4), 455e462.
Liu, Y., Shi, J., & Langrish, T. (2006). Water-based extraction of pectin from flavedo
and albedo of orange peels. Chemical Engineering Journal, 120(3), 203e209.
Masmoudi, M., Besbes, S., Chaabouni, M., Robert, C., Paquot, M., Blecker, C., et al.
(2008). Optimization of pectin extraction from lemon by-product with acidified
date juice using response surface methodology. Carbohydrate Polymers, 74(2),
185e192.
McClements, D. J. (2004). Protein-stabilized emulsions. Current Opinion in Colloid &
Interface Science, 9(5), 305e313.
Mohnen, D. (2008). Pectin structure and biosynthesis. Current Opinion in Plant
Biology, 11(3), 266e277.
Nakamura, A., Fujii, N., Tobe, J., Adachi, N., & Hirotsuka, M. (2012). Characterization
and functional properties of soybean high-molecular-mass polysaccharide
complex. Food Hydrocolloids, 29(1), 75e84.
Nakauma, M., Funami, T., Noda, S., Ishihara, S., Al-Assaf, S., Nishinari, K., et al. (2008).
Comparison of sugar beet pectin, soybean soluble polysaccharide, and gum
arabic as food emulsifiers. 1. Effect of concentration, pH, and salts on the
emulsifying properties. Food Hydrocolloids, 22(7), 1254e1267.
Neirynck, N., Van der Meeren, P., Bayarri Gorbe, S., Dierckx, S., & Dewettinck, K.
(2004). Improved emulsion stabilizing properties of whey protein isolate by
conjugation with pectins. Food Hydrocolloids, 18(6), 949e957.
Nii, T., & Ishii, F. (2004). Properties of various phosphatidylcholines as emulsifiers or
dispersing agents in microparticle preparations for drug carriers. Colloids and
Surfaces B: Biointerfaces, 39(1e2), 57e63.
Ridley, B. L., O’Neill, M. A., & Mohnen, D. A. (2001). Pectins: structure, biosynthesis,
and oligogalacturonide-related signaling. Phytochemistry, 57(6), 929e967.
Sedmak, J. J., & Grossberg, S. E. (1977). A rapid, sensitive, and versatile assay for
protein using Coomassie brilliant blue G250. Analytical Biochemistry, 79(1e2),
544e552.
Surh, J., Decker, E. A., & McClements, D. J. (2006). Influence of pH and pectin type on
properties and stability of sodium-caseinate stabilized oil-in-water emulsions.
Food Hydrocolloids, 20(5), 607e618.
Thakur, B. R., Singh, R. K., & Handa, A. K. (1997). Chemistry and uses of pectin e a
review. Critical Reviews in Food Science and Nutrition, 37(1), 47e73.
Thanasukarn, P., Pongsawatmanit, R., & McClements, D. J. (2004). Influence of
emulsifier type on freeze-thaw stability of hydrogenated palm oil-in-water
emulsions. Food Hydrocolloids, 18(6), 1033e1043.
Wang, S. J., Chen, F., Wu, J. H., Wang, Z. F., Liao, X. J., & Hu, X. S. (2007). Optimization
of pectin extraction assisted by microwave from apple pomace using response
surface methodology. Journal of Food Engineering, 78(2), 693e700.
Yang, H., Lai, S., An, H., & Li, Y. (2006). Atomic force microscopy study of the ul-
trastructural changes of chelate-soluble pectin in peaches under controlled
atmosphere storage. Postharvest Biology and Technology, 39(1), 75e83.
Yeoh, S., Shi, J., & Langrish, T. (2008). Comparisons between different techniques for
water-based extraction of pectin from orange peels. Desalination, 218(1e3),
229e237.
Zhang, L., Chen, F., Yang, H., Ye, X., Sun, X., Liu, D., et al. (2012). Effects of temper-
ature and cultivar on nanostructural changes of water-soluble pectin and
chelate-soluble pectin in peaches. Carbohydrate Polymers, 87(1), 816e821.