Accepted Manuscript

Title: Characterization and functional properties of mango

peel pectin extracted by ultrasound assisted citric acid

Author: Miaomiao Wang Chuanhui Fan Kaili Zhao Bohui

Huang Hao Hu Xiaoyun Xu Siyi Pan Fengxia Liu

PII: S0141-8130(16)30537-2
DOI: http://dx.doi.org/doi:10.1016/j.ijbiomac.2016.06.011
Reference: BIOMAC 6184

To appear in: International Journal of Biological Macromolecules

Received date: 2-5-2016

Revised date: 31-5-2016
Accepted date: 5-6-2016

Please cite this article as: Miaomiao Wang, Chuanhui Fan, Kaili Zhao, Bohui Huang,
Hao Hu, Xiaoyun Xu, Siyi Pan, Fengxia Liu, Characterization and functional properties
of mango peel pectin extracted by ultrasound assisted citric acid, International Journal
of Biological Macromolecules http://dx.doi.org/10.1016/j.ijbiomac.2016.06.011

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Characterization and functional properties of mango peel pectin

extracted by ultrasound assisted citric acid

Miaomiao Wang, Chuanhui Fan, Kaili Zhao, Bohui Huang, Hao Hu, Xiaoyun Xu, Siyi Pan,

Fengxia Liu*

College of Food Science and Technology, Huazhong Agricultural University, Wuhan,

Hubei 430070, P.R. China

Key Laboratory of Environment Correlative Dietology, Ministry of Education, P.R. China

*Corresponding author

Tel.: +86 13871280854.

E-mail address: liufxia@mail.hzau.edu.cn

Abstract:
Pectin was extracted from ‘Tainong No.1’ mango peels, using a chelating agent-citric
acid as extraction medium by ultrasound-assisted extraction (UAE) and conventional
extraction (CE) at temperatures of 20 and 80 °C. Chemical structures, rheological and
emulsifying properties of mango peel pectins (MPPs) were comparatively studied
with laboratory grade citrus pectin (CP). All MPPs exhibited higher protein content
(4.74%-5.94%), degree of methoxylation (85.43-88.38%), average molecular weight
(Mw, 378.4-2858 kDa) than the CP, but lower galacuronic acid content (GalA,
52.21-53.35%). CE or UAE at 80 °C resulted in significantly higher pectin yield than
those at 20 °C, while the extraction time for UAE-80 °C (15 min) was significantly
shorter compared to CE-80 °C (2 h) with comparable pectin yield. Moreover, MPPs
extracted at 80 °C were observed with higher GalA and protein content, higher Mw,
resulting in higher viscosity, better emulsifying capacity and stability, as compared to
those extracted at 20 °C and the CP. Therefore, these results suggested that MPPs
from ‘Tainong No.1’ may become a highly promising pectin with good thickening
and emulsifying properties, using ultrasound-assisted citric acid as an efficient and
eco-friendly extraction method.

Keyword: pectin; extraction; ultrasound; structure; function

1. Introduction
Pectins, a family of complex polysaccharides present within the primary cell wall
and intercellular regions of dicotyledons, are good food additives in jams, soft drinks,
and milk products [1]. Up to the last decade, most pectin applications stemmed from
its gel-forming ability, but nowadays, pectin is gradually gaining more acceptance as
an effective emulsifier [2]. These functional properties of pectins are highly related to
their structure, including the molecular weight (Mw), degree of methylation (DM), and
protein content, etc., which depends on the plant source and the extraction method
used [3]. Currently, citrus peel and apple pomace are the main sources used for the
production of commercial pectins. However, due to their low protein content and
number of acetyl groups, citrus and apple pectins are considered less useful for
emulsification [4]. The increasing industrial demand for pectins with varying ability
to stabilize products increases the need for novel pectins sources, such as sugar beet
residues [5], tomato waste [6], papaya peel [7], cacao pod husks [8], etc.
Mango (Mangifera indica L.) is an important tropical fruit, and China is the second
largest mango producer in the world [9]. Due to high perishability of the fruit, its
value added products such as mango pulp [10] and mango nectar [11-13] are of high
commercial importance. However, the accruing by-products, mango peel, which
represent 10.0-13.7% of the fruit total weight, generate a major disposal problem [3].
They have been reported to be a potential source of pectin [14], and the cultivars used
are revealed as a significant effect on pectin qualities [3]. Previous results mainly
focused on pectin recovery from several mango cultivars, such as ‘Nam Dokmai’ [15],
‘Améliorée’ and ‘Mango’ [16], ‘Kent’ [3, 17], and ‘Keitt’ mango [18].
Commercially, pectins are extracted with mineral acids, mainly HNO3, HCl or
H2SO4, for different time periods around a pH of 1.5-3.0, but generally, this process is
time consuming and may lead to pectin degradation. Recently, some organic acids
with chelating properties, are reported as effective in pectin extraction in terms of
yield and physicochemical properties [8, 16, 19]. For example, in previous literature,
mango peel pectins extracted with ammonium oxalate were observed with high Mw,
intrinsic viscosity and a high DM, compared to those extracted with HCl, or deionised
water [16]. Besides, new technologies have also explored in pectin extraction, either
sequentially or independently, such as microwave extraction [20], ultra-high pressure
extraction [21], and enzymatic extraction [22]. Ultrasound (16-100 kHz, power in the
range 10-1000 W/cm2) is another method which has been used extensively in food
industry. It uses acoustic energy and solvents to enhance the release and diffusion of
cell material for assisting extraction [23]. Ultrasound-assisted extraction (UAE) of
pectins from different sources has been studied, with different extraction solvents
including HCl [24], ammonium oxalate/oxalic acid [6], and subcritical water [5].
Most of the published studies indicated that UAE results in increased pectin yield as
well as important reduction in extraction time. However, limited studies have been
performed on the effect of ultrasound-assisted citric acid on pectin extraction,
regardless of heating.
In summary, although mango peels have been reported to be a potential source of
pectin, the pectin qualities are quite cultivar dependent. To the best of our knowledge,
no information about pectin structure and functional properties from mango peels
harvested in China is available. On the other hand, limited studies have been
documented on the effects of ultrasound-assisted chelating agents on the rheological
and emulsifying properties of pectin as related to its chemical structure. Thus, the
objective of this study was to study the effects of ultrasound-assisted citric acid
extraction (UAE) at different temperatures (20 and 80 °C) to conventional citric acid
extraction (CE) on mango peel pectins (MPPs) from ‘Tainong No.1’ cultivated in
China, in terms of some valuable functional (rheological and emulsifying) properties
and its chemical structure. The quality of MPPs was also compared with laboratory
grade citrus pectin (CP). This study could offer a promising pectin source with good
thickening and emulsifying properties, and upgrade by-products arising from the
processing of mango and other tropical fruits in an efficient and eco-friendly way.

2. Material and methods

2.1. Chemicals
Laboratory grade citrus pectin (CP, P9135), 3-phenylphenol, and D-galacturonic
acid were purchased from Sigma-Aldrich (St. Louis, USA). All other chemicals were
of analytical grade, purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai,
China).
2.2. Sample preparation
Fresh mango peels of one of the most productive cultivars (‘Tainong No.1’) in
China were collected from Hainan FangYe Agro-Products Processing Co., Ltd
(Hainan, China), as by-product of mango pulp processing. The collected mango peels
were immediately steam blanched for 5 min to inactivate the endogenous enzymes
(including polygalacturonase and pectin methyl esterase), and then dried by a vacuum
freeze dryer (LGJ-18S, Beijing Songyuan Huaxing Technology Develop Co., Ltd.,
Beijing, China) for 48 h. The dried sample was then milled with an electric grinder
(Zhejiang Industry and Trade Co., Ltd., Zhejiang, China) and filtrated using a filter
sieve (ca. 40 meshes). Finally, the mango peel powder was vacuum-packed and stored
in a drier for subsequent extraction.
2.3. Pectin extraction and purification
2.3.1. Conventional citric acid extraction (CE)
Pectin extraction of CE was based on the method of Jamsazzadeh Kermani et al.
[17], with some modifications. A ratio of 1:40 mango peel powder to citric acid
solution was selected. Four hundred milliliters of distilled water was adjusted to pH
2.5 with 1 M citric acid at 20 and 80 °C, respectively. Thereafter, 10 g of mango peel
powder was added to the citric acid solution under constant stirring, and the pH was
re-adjusted to 2.5. After incubated at 20 and 80 °C respectively for 2 h, the extraction
solution was immediately cooled in an ice-water bath and centrifuged at 10, 000 g for
15 min at 4 °C. The supernatant was dialyzed exhaustively against distilled water for
48 h, and the dialyzed extract was then freeze-dried. To obtain pectin with minimal
concentrations of pigments, soluble sugars and lipids, the extract was washed three
times using ethanol and acetone for 5 min and was referred as CE-mango peel pectin
(CE-MPP).
2.3.2. Ultrasound-assisted citric acid extraction (UAE)
Pectin extraction of UAE was carried out using an ultrasound processor model
JY92-2D (NingBo Scientz Biotechnology Co. Ltd, Ningbo, Zhejiang, China) with a
0.636 cm diameter titanium probe. A ratio of 1:40 mango peel powder to citric acid
solution (pH 2.5) was selected, and the extraction was also performed at 20 and 80 °C,
respectively. Prepared samples were treated at 20 kHz at 500 W, with pulse duration
of on-time 5 s and off-time 5 s to keep the mixture temperature even and the material
getting equal irradiation. According to Grassino et al. [6] and our previous study, the
extraction time of 15 min was chosen, since it was observed as sufficient for pectin
extraction and further prolonged the ultrasound treatment would result in lower
galacuronic acid content. During the sonication process, the sonication probe
maintained with 3 cm under the surface of extraction solution and the beaker was kept
in a water bath to maintain the sample temperature. After the ultrasonic treatment, the
extraction solution was centrifuged and purified as described above, and the pectin
obtained is referred as UAE-mango peel pectin (UAE-MPP).
The extraction yield (Y) of pectins from different methods was calculated as
follows:
Y (%, w/w) = pectin weight/mango peel powder weight × 100 (1)
2.4 Determination of physicochemical properties of pectin
2.4.1 Determination of the galacuronic acid (GalA) content
The GalA content was determined based on the method described by Zhou et al.
[25] with some modifications. The GalA content was quantified by the colorimetric
method using a spectrophotometer at 520 nm (UV-762, Lingguang, Shanghai, China)
and expressed as D-galacturonic acid equivalents in percentage.
2.4.2 Determination of the degree of methoxylation (DM)
The DM of pectin was determined by the titrimetric method of using NaOH, based
on the free carboxyl group [26]. The DM of the pectin extract was estimated by the
ratio of the molar amount of methyl-esters to the molar amount of GalA residues.
2.4.3. Determination of total sugar content
Total sugar content was measured by the phenol-sulfuric acid method [8] using
glucose as standard.
2.4.4. Determination of the protein content
The protein content was determined using Lowry protein content assay kit
(Shanghai Labaide Biotechnology Co., Ltd, Shanghai, China), quantified by a
spectrophotometer at 750 nm (UV-762, Lingguang, Shanghai, China), and expressed
as the equivalent concentration of bovine serum albumin.
2.4.5. Determination of the average molecular weight (Mw) and molecular weight
distribution
The average molecular weight and molecular weight distribution of pectin was
determined by high performance size exclusion chromatography (HPSEC) as
described [17], with some modifications. 0.05 M NaNO2 solution containing 0.5 g/L
NaN3 was used as the eluent, and filtered (0.45 μm) pectin polymers (0.1% w/v) was
monitored by a refractive index detector (Shodex RI-101, Showa Denko K.K.,
Kawazaki, Japan) coupled with a multi-angle laser light scattering (MALLS) detector
(PN3621, Postnova analytics, Germany). The data were collected and processed by a
Wyatt Technology ASTRA program.
2.4.6. Determination of pectin color
Color assessment was conducted at room temperature using a color measurement
spectrophotometer (WSD-III, Kang Guang Instrument Co. Ltd., Beijing, China) in the
reflectance mode. The parameters measured were L* for lightness, a* for redness, and
b* for yellowness. All measurements were made in triplicate and results were
averaged.
2.5. Rheological properties of pectin and O/W emulsion
Rheological measurements of pectin solution and its emulsion were carried out
using a controlled shear rate AR 2000 rheometer (TA Instruments, New Castle, DE,
USA) with its accompanying computer software (Rheology Advantage, TA
Instruments, Waters Co., Ltd.) as described by Liu et al. [10] with some modifications.
Pectin solution of 1% (m/v) or pectin emulsion prepared was placed between
cone-plate (40 mm diameter, 2° angle) using a gap size of 52 μm. For steady flow
studies, approximately 3 mL of pectin solution were placed between the plate and
measurements were carried out at 25±1 °C with the shear rate changed from 1 to 100
s-1. Apparent viscosity and shear stress of pectin solution and its emulsion at different
shear rate were obtained using the software. Moreover, values at shear rate of 50 s−1
were chosen to compare the viscosity differences between different samples, since it
has been reported that the value of shear rate could represent approximately the
sensation perceived in the mouth [27].
2.6. Emulsifying properties of pectin
2.6.1. Preparation of emulsions
O/W emulsion was prepared based on the procedure of Jamsazzadeh Kermani et al.
[18] with some modifications. MPPs or CP was dissolved into citric acid-sodium
citrate buffer solution over-night, at the concentration of 1% (w/v). To prevent the
spoilage of all the solutions during storage, 0.05% of sodium azide was added as a
preservative. The pH of the resulting solution was adjusted to 3.5, and 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. The mixture was
emulsified with a high-speed homogenizer (Ningbo Xinzhi Biotechnology Co., China)
at a speed of 10, 000 rpm for 3 min.
2.6.2. Determination of emulsion droplet-size distribution
According to Shanmugam & Ashokkumar [28], the droplet-size distribution of
emulsions immediately after preparation and after storage of 1 week was analyzed by
a computer-controlled laser diffraction apparatus (Mastersizer 2000, Malvern
Instruments Ltd., UK), with the measurement range from 0.04 to 2000 μm. During
measurement, a few droplets of the emulsions were suspended directly in recirculating
water, and the volume mean diameter D4,3 and surface mean diameter D3,2 were
recorded, both of which are mostly used in the analysis of particle size of samples.
2.6.3. Light microscopy
To elucidate the microstructure of emulsions prepared by different pectins, the
photomicrographs of all emulsions at pH of 3.5 were examined and photographed
immediately after preparation under bright field illumination with a 40 × objective
lens on an inverted microscope BIO 200-PH (Jingbaizhouxian technology Co. Ltd.,
Beijing, China) [29]. Microphotographs were taken using a Nikon D5000 digital
camera (Nikon Corporation, Sendai, Japan) equipped with the microscope.
2.6.4 Determination of emulsion stability
The emulsions stability was evaluated using a combination of centrifugation and
storage assessment [29], with some modifications. Approximately 10 g of each
emulsion was placed into 15 mL plastic centrifugal tube and centrifuged at 3000 g for
15 min at room temperature. The centrifugation treatment resulted in an aqueous
phase separation, with an emulsion phase on the top and water dispersion on the
bottom. The height of the emulsion phase after centrifugation of 15 min was measured
and recorded as H1, and the initial height of the emulsion before the centrifugation
was recorded as H0. The emulsion stability (ES15, %) was then calculated as a
percentage using equation (2):
ES15 (%)=H1/H0×100 (2)
Another portion of emulsions were used to investigate the stability of emulsions
during storage, for 1 week at chill storage condition (4 °C). These conditions are
generally considered as acceptable shelf-life for perishable market products [28].
Each emulsion (10 g) was placed into 15 mL plastic centrifugal tube, tightly sealed,
and then stored at 4 °C for about 7 days. During the storage, the emulsions may
separate into an optically opaque cream layer at the top and a transparent layer at the
bottom. The initial height of the emulsions before storage was recorded as H0. The
height of the emulsions after storage of 7 days were measured and recorded as H2.
The emulsion stability (ESsorage, %) was calculated using equation (3):
ESstroage (%)=H2/H0×100 (3)
2.7. Statistical analysis
All experiments were performed in triplicate, and the results were expressed as
means ± standard deviation (SD). Analysis of variance was carried out using Microcal
Origin 9.0 (Microcal Software, Inc., Northampton, MA, USA). Significance was set at
p<0.05.

3. Results and discussion

3.1. Extraction yield of pectin
As shown in Fig.1, the extraction yield of pectins varied greatly depending on the
extraction conditions used, particularly temperature. The extraction yields at 80 °C
(16.70-17.15%) were observed around ten times higher than those at 20 °C
(1.55-2.09%), both for CE and UAE. The high extractability of pectins with hot dilute
acid was commonly found in previous literature, either chemical acid [3, 8] or
chelating agents [16, 17], which could be attributed to the increased solubility of
pectin with increasing extraction temperature, giving a higher yield of pectin [21].
Moreover, in this study, the extraction yield of UAE at lower temperature (20 °C) was
significantly higher than that of CE, indicating that ultrasound could significantly
facilitate the extractability of pectin which may be due to the rupture of mango cell
wall, while the effect is not significant when extracted at higher temperature (80 °C).
However, Wang et al. [30] showed synergistic effects between ultrasound and heating
on the yield of pectin when HCl was used for extraction.
Many studies have proved that pectin yield could be highly related to different
extraction conditions. Rehman et al. [31] revealed that when using mineral acids
(H2SO4, HCl, and HNO3) for pectin extraction from mango peel, the highest yield is
obtained when using H2SO4, and Jamsazzadeh Kermani et al. [17] showed that under
the same conditions (pH 2.5, 80 °C, 2 h) citric acid yielded an even higher amount of
pectin compared to H2SO4 extraction. In this study, CE treatment was also carried out
under the same condition using citric acid (pH 2.5, 80 °C, 2 h), and similar pectin
yield was obtained compared to UAE at the same temperature. However, the
extraction time was only 15 min for UAE while 2 h for CE, indicating that ultrasound
assist heating citric acid could considerably shorten extraction procedure with high
pectin yield. Similarly, Grassino et al. [6] reported that ultrasound had small effect on
pectin yield from tomato waste compared with conventional extraction method using
ammonium oxalate/oxalic acid as extraction solvent at 60 and 80 °C, but the
extraction time is significantly shorter.
3.2. Physicochemical properties of pectin
Physicochemical properties of pectin, influenced by the source and the method of
extraction, determine its functionality. As shown in Table 1, GalA content of MPPs
varied from 29.35% to 53.35% depending on the extraction condition used, with the
highest percent obtained in UAE at 80 °C. Both for CE and UAE, MPPs extracted
with heat acid (80 °C) exhibited significantly higher GalA content compared to those
extracted at 20 °C, indicating that temperature had a significant effect on the GalA
content of pectins. Similarly, Vriesmann et al. [8] reported that the GalA content of
pectin from cacao pod husks increased significantly with increasing citric acid
temperature. The amount of GalA in MPPs extracted at 80 °C in this study was
significantly higher than that extracted from ‘Keitt’ mango (41%, [17]) with citric
acid at the same temperature, while still lower than that in CP (71.29%), and lower
than the 65% limit required to be classified as commercial food grade pectin.
Moreover, all MPPs fraction in this study presented high total sugar content
(57.76-76.98%), of which GalA was the main type of monosaccharide. These results
were in agreement with previous studies by Wang et al. [30], with total sugar content
observed as about 78% in grapefruit peel pectin extracted by UAE. From the results
of GalA and total sugar content, it can be concluded that besides GalA, MPPs may
also have considerable pectin related neutral sugars. Nagel et al. [15] reported that hot
acid extracted mango peel pectin from ‘Nam Dokmai’ and ‘Maha Chanok’ cultivars
exhibited comparatively poor GalA (38-47 g/hg) content in favor of the neutral sugars
(23 g/hg) content, chiefly galactose. Koubala et al. [16] reported that glucose
(probably of cellulosic origin) was the main neutral sugar followed by galactose and
arabinose in MPPs from ‘Améliorée’ and ‘Mango’ varieties, and mannose, xylose, and
rhamnose were also detected. In addition, the content of starch, low-molecular pectin
degradation products, and the arabinogalactan impurity could also contribute to the
lower GalA content observed in MPPs [3]. Despite the lower GalA content, mango
peel of ‘Tainong No.1’ still showed a promising pectin source. According to EU
regulation 68/2013/EU, the pectin could be considered for feed use at its current state,
while to meet the legal specification of commercial food-grade pectins (EU regulation
231/2012/EU), more modifications of extraction procedure, precipitation, and
especially purification are still needed.
All MPPs appeared to be highly esterified (DM>50%), and the DM values of MPPs
extracted by CE (85.43%-86.77%) and UAE (86.83%-88.38%) showed no significant
differences (Table 1), but significantly higher than that of CP (78.29%). These results
were in agreement with previous studies. A screening of 14 mango cultivars carried
out by Berardini et al. [14] has demonstrated high DM of MPPs to range from 56-66%.
Koubala et al. [16] also reported that MPPs from ‘Améliorée’ and ‘Mango’ varieties
exhibited high DM (64-86%). Wang et al. [30] found that UAE pectin possessed
lower DM than CE pectin from grapefruit peel, while no effects of ultrasound on DM
were found in this study.
 The protein content can be suggested to be highly correlated to the emulsifying
properties of pectin [29]. In this study, the protein content of MPPs was observed
significantly affected by extraction temperature, ranging from 4.74-4.85% at 20 °C
and 5.24-5.94% at 80 °C. No significant effects were found for ultrasound treatment.
All MPPs exhibited higher pectin content than CP (3.44%), therefore they have a
great potential to be the alternative pectins with higher emulsifying capacity and
stability. However, in previous literature, MPP was reported with very low content of
protein, being <2% from ‘Améliorée’ and ‘Mango’ mango [16] and ‘Keitt’ mango
[18], revealing that protein content of MPPs may also be variety dependent.
From the overlaid chromatograms shown in Fig. 2, it can be clearly concluded that
the profiles of the pectins exhibited significant differences in the molecular weight
distribution. MPPs had apparent narrower molecular weight distribution, with
retention times from 10-15 min, compared to CP (10-20 min). Based on the results,
MPPs had higher percentage of high molecular weight constituents than CP, and the
extraction method had a significant effect on the Mw value of pectins. The Mw of CE-
and UAE-extracted MPPs was 664.5 and 378.4 kDa at 20 °C, while 2858 and 2320
kDa at 80 °C, respectively (Table 1). The results indicated that high temperature may
help to isolate pectin with higher Mw. It has also been reported in previous literature,
that Mw of pectin is related to the extraction temperature and the highest Mw was
observed at 75 °C [32], which is similar to the condition used in this study. Moreover,
UAE-MPPs exhibited lower Mw compared to CE-MPPs at the same temperature.
Similar results were also reported elsewhere [30]. Even though, both CE- and
UAE-MPPs extracted from ‘Tainong No.1’ mango peel at 80 °C in this study showed
significantly higher Mw in comparison with pectin extracted from ‘Keitt’ mango [18],
‘Kent’ mango [17], ‘Améliorée’ and ‘Mango’ mango [16]. One reason for the results
could be due to cultivar difference. And another reason may be the extraction medium
difference, as citric acid with a chelating property was used as extraction medium in
this study. Applying chelating agents in the extraction medium has already been
reported to help isolate pectin with higher Mw, compared to those extracted with HCl,
or deionised water [16].
 Color of pectin is another important parameter which could affect the appearance of
the products. As shown in Table 1, different extraction conditions had significant
effects on the pectin color. L* values of MPPs were observed decreased whereas a*
and b* values increased with extraction temperature, indicating that pectins extracted
at higher temperature (80 °C) were highly colored than those extracted at lower
temperature (20 °C). As reported in previous studies, the color of pectin could be
negatively affected by non-enzymatic browning, which is favored by heating and
includes a wide number of reactions including caramelization and Maillard reaction
[33]. In the current study, pectin extracted at 80 °C by UAE showed significantly
higher L* value than that by CE, indicating that ultrasound could help to inhibit the
color browning of pectin resulted from high temperature. On the other hand, colored
pectins may contain polyphenols or other water soluble pigments trapped in the pectin
during extraction [6]. Both CE- and UAE-MPPs extracted at 80 °C exhibited smaller
L* values than CP, and the higher colored production may be attributed to the
co-extraction of polyphenols and carotenoids, in which there is considerable amounts
existed in mango peels [11]. Although later used ethanol/acetone washing of pectin
isolates reduced the content of these polyphenols and carotenoids, they may still be
partially associated to the extracted pectin.
3.3. Rheological properties of pectin
Flow curves of different pectin solutions are shown in Fig. 3A, and apparent
viscosities at 50 s−1 are shown in Table 2 to compare the viscosity differences. The
results showed that rheological behavior of different pectin solutions varied widely
depending on the extraction condition. Both for CE and UAE, MPPs extracted at
higher temperature exhibited higher viscosity, which corresponded to the higher GalA
content. Moreover, when compared at the same extraction temperature, MPPs
extracted from CE showed higher viscosity than those from UAE, which is correlated
to their higher Mw. According to the above results, it can be deduced that the
viscosities of MPPs may be affected by both Mw and GalA content, with higher
viscosity corresponding to higher Mw and GalA content. Similar results were also
found in pectins extracted with different conditions by subcritical water [34]. Besides,
the viscosity may also be influenced by the molecular conformation, the number of
reactive side groups, ionic groups and aggregation conditions [25]. In this study, MPP
extracted by UAE at 20 °C was of fairly high Mw but of extremely low viscosity, as
compared to CP and other MPPs. Moreover, the viscosity of UAE-MPP at 80 °C was
lower than that of CE-MPP at 20 °C, when the former possessed both high GalA
content and Mw. These results indicated that a less extended conformation or more
aggregation may be exhibited in UAE-MPP. These results were in accordance with
the differences on Z-average diameter of pectins observed in this study (Table 1).
Similar results were found in previous literature [35], showing that the effect of
ultrasound on the viscosity reduction of aqueous polysaccharides was influenced by
the changes of polymer coil conformation. Koubala et al. [16] also reported that
extraction process used could influence the macromolecular parameter values of
MPPs, with ammonium oxalate-extracted MPP exhibited a more extended
conformation as revealed by its very high viscosity compared with water-extracted
MPP.
Moreover, various rheological flow models based on the shear stress–shear rate
relation were tested quantitatively against the empirical results from pectin solutions:
Newtonian: σ=η*γ (1)
Herschel-Bulkley: σ=σ0HB+KHB * γnHB (2)
where σ is the shear stress (Pa), η is the apparent viscosity (Pa∙s), σ0HB is the yield
stress (Pa), γ is the shear rate (s-1), KHB is the consistency coefficient (Pa∙sn) and nHB is
the flow behavior index. From the evaluation parameters in Table 2, MPP solution
extracted by CE fitted Herschel-Bulkley model well (R2=0.999), showing typical
shear-thinning property. For CP and MPP extracted by UAE, Herschel-Bulkley model
was not a quite fitted model with σ0HB being minus from the fitting results, and
however, they fitted Newtonian model well (R2>0.976), exhibiting nearly Newtonian
fluid. These results may be attributed to the less extended conformation of
UAE-MPPs as discussed above, leading to low viscosity of the pectin solution at the
chosen concentration in this study. All these results indicated that different extraction
conditions used in this study may allow the recovery of different kinds of pectins with
respect to different structural properties, which could further influence the rheological
properties.

3.4. Emulsifying properties of pectin

3.4.1. Emulsion viscosity
Emulsion viscosity is one of the important parameters that determine the stability of
O/W emulsion. The flow curves of pectin emulsions prepared by different pectins at
the concentration of 1% (w/v) are represented in Fig. 3B, and the apparent viscosities
at 50-1 shear rate are shown in Table 2. From the results, it was obviously found that
all emulsions had a higher viscosity than their pectin solutions as shown in Fig. 3A.
Higher viscosities of emulsions prepared by citrus pectin were also reported by Guo et
al. [29], compared to those of their pectin solutions. Although different extraction
conditions resulted in the recovery of different kinds of MPPs with respect to GalA,
pectin content, Mw and conformation, there were no significant differences on the
viscosities of CE- and UAE-MPP emulsions. However, all the MPP emulsions had
significantly higher viscosity than the emulsion from CP. As has been reported, the
emulsifying properties of pectin can be correlated to the protein content [29], which is
in agreement with the protein content in different pectins observed in Table 1.
Similarly, in previous results by Jamsazzadeh Kermani et al. [18], apple pectin and
‘Keitt’ MPPs were observed as structurally different, with MPPs having a much
higher Mw, while no statistically significant differences in viscosity of their emulsions
were noted since they had comparable protein contents. Compared to CP, the higher
viscosities of MPP emulsions observed in this study could decrease the particle
movement and coalescence effect, and thus may effectively increase the stability of
the emulsions.
3.4.2. Light microscopy
To compare the microstructure of emulsions prepared by different pectins, the
photomicrographs of all emulsions were taken immediately after preparation with a
40 × objective lens. The typical micrographs for emulsions prepared by CP and CE-,
UAE-extracted MPPs at different temperatures with the concentration of 1% are
presented in Fig. 4 (A)-(E), respectively. Results showed that the O/W emulsions
prepared by different pectins varied significantly on particle diameters and volumes.
Emulsion particles prepared by CE- and UAE-MPPs extracted at 80 °C had a
relatively smaller droplet diameter, while the emulsions prepared by CP exhibited the
largest particle constitutes in diameters. The formation of larger oil droplets may be
attributed to the less sufficient interfacial resistance on the oil droplet surfaces, which
lead to the aggregation of small droplets [18]. Previous studies have shown that
protein contents and Mw of pectin have significant impact on the interfacial resistance
and emulsion stability, and however, their effects on those emulsions need further
investigations [18, 29].
3.4.3. Emulsion droplet size distribution
Droplet size and droplet size distribution of emulsions are important characteristics
defining their stability [18]. The droplet size distribution of O/W emulsions over 0
and 1 week of storage are presented in Fig. 5, and the particle mean diameter (D3,2 and
D4,3 in μm) of all emulsions were calculated and shown in Table 3. As shown in Fig. 5,
emulsions prepared with CP exhibited larger droplet size, compared to those prepared
with CE- and UAE-MPP, indicating that MPPs could form O/W emulsions with
smaller droplet diameter, which was in accordance with the results from light
microscopy of emulsions discussed above. These results were also evidenced by the
D3,2 and D4,3 values, with emulsions prepared with CP having significantly larger
values than MPPs emulsions. During the 1 week of storage, the emulsion droplet size
and droplet size distribution of CE- and UAE-MPPs extracted at 80 °C showed the
smallest increase, and those of CE- and UCE-MPPs extracted at 20 °C had a slight
larger increase, with droplet size distribution shift towards the larger droplet sizes.
However, all the emulsions prepared by MPPs from different extraction conditions
showed significantly smaller increase in their droplet size and droplet size distribution,
compared to that made of CP. Among all the emulsions, the emulsion prepared with
CP had the largest particle mean diameters and the largest droplet distribution during
the storage. Therefore, MPPs could be a potent emulsion stabilizer, since they gave
smaller droplet size, and showed more stable condition during storage.
3.4.4 Emulsion stability against creaming
In the present study, the emulsions stability against creaming was evaluated using a
combination of centrifugation assay (Table 3) and storage assessment (Fig. 6). From
the results of Table 3, it was clearly revealed that MPPs extracted at higher
temperature (80 °C) had better ES15 (100%), both for CE and UAE, compared to CP
(50.0%) and MPPs extracted at 20 °C (52.5-57.5%). These results indicated that
MPPs extracted at 80 °C exhibited better emulsifying stability with the centrifugation
assay, as compared to CP and other MPP used in this study. These results were
consistent with the droplet size and droplet size distribution of emulsions prepared by
different pectins. Guo et al. [29] reported that emulsions with smaller droplet size and
higher viscosity were expected with better stability. With regard to the primary
reasons, Mw and protein contents of pectins have been shown to have significant
impact on emulsions stability [36], which can partially explain the better emulsifying
of MPPs. In this study, MPPs were observed with significantly higher Mw and protein
content, as compared to CP, especially for MPPs extracted at higher temperature.
Similarly, the emulsification properties of sugar beet pectin is well studied and
frequently ascribed to its high protein content [37]. Ngouémazong et al. [2] concluded
that following a decrease of pectin protein content and average molecular weight, the
pectin loses most of its emulsifying activity and its emulsion-stabilizing effectiveness.
From the results of storage assessment, emulsions prepared with MPPs extracted at
20 °C and CP was unstable during the storage time. After one week of storage, a
transparent layer at the bottom observed for them. Jamsazzadeh Kermani et al. [18]
studied the stability of emulsions prepared with 0.8% MPP extracted with heat citric
acid from ‘Keitt’ mango, and a cream layer was observed to appear after 1 week
storage. However, in this study, emulsions prepared using MPPs extracted at 80 °C
remained uniform and stable during 1 week storage. Besides Mw and protein contents
discussed above, the improvement of the stability against creaming could be due to
the molecular conformation [21] and the viscosity [18] of pectin in the continuous
aqueous phase. Combined with the results of molecular weight and rheological
property of pectin solution, it could be concluded that higher Mw and viscosity could
increase the emulsion stabilities significantly in MPPs extracted CE or UAE.

4. Conclusions
In the present study, conventional citric acid extraction (CE) and
ultrasound-assisted citric acid extraction (UAE) were comparatively studied for the
recovery of pectins from ‘Tainong No. 1’ mango peels. Results showed that extraction
temperature had a significant effect on the quality of pectin, for both extraction
techniques. MPPs extracted at 80 °C were observed with higher GalA and protein
content, higher Mw, resulting in higher viscosity, better emulsifying capacity and
stability, as compared to those extracted at 20 °C and CP. From the comparison of the
pectin yields obtained at 80 °C, CE with a duration of 2 h resulted in similar pectin
yields in comparison with 15 min of UAE processing treatment. Thus, it can be
concluded that UAE at higher temperature could considerably shorten the extraction
procedure with high pectin quality.
Although with lower GalA content, mango peel of ‘Tainong No.1’ still showed a
promising pectin source, with acceptable thickening and emulsifying properties
mainly due to their high content of protein content and high Mw. Moreover, extraction
procedures and purification steps are needed to be further optimized in order to obtain
MPPs meeting the legal specification of commercial food-grade pectins.

Acknowledgements
This work was supported by the National Natural Science Foundation of China (No.
31501498), Natural Science Foundation of Hubei Province (No. 2015CFB390) and
the Fundamental Research Funds for the Central Universities (No. 2662014BQ055,
No. 2662015QC021). We also thank Hainan FangYe Agro-Products Processing Co.,
Ltd (Hainan, China) for providing mango peels.
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 20

a
18 a

16
Extraction yield (%)

14

3
b
2 c

0
CE-20 °C UAE-20 °C CE-80 °C UAE-80 °C

Fig. 1. Comparison of pectin yields of conventional citric acid extraction (CE) and
ultrasound assisted extraction (UAE) methods
 0.14 CP
CE-20°C
0.12 UAE-20°C
CE-80°C
0.10 UAE-80°C
Detector voltage (V)

0.08

0.06

0.04

0.02

0.00
5 10 15 20 25
Retention time (min)

Fig. 2. Chromatograms of citrus pectin (CP) and mango peel pectins obtained by
conventional extraction (CE) and ultrasound assisted extraction (UAE) by
HPSEC-MALLS
 0.12
CP
A
CE-20°C
0.10 UAE-20°C
Apparent viscosity (Pa.s) CE-80°C
UAE-80°C
0.08

0.06

0.04

0.02

0.00
0 20 40 60 80 100
Shear rate (1/s)
1.4
B
1.2
Apparent vsicosity (Pa.s)

1.0

0.8

0.6

0.4

0.2

0.0

0 20 40 60 80 100
Shear rate (1/s)

Fig. 3. Flow curves of different pectin solutions (A) and their O/W emulsions (B)
(CP, citrus pectin; CE, conventionally extracted mango peel pectin; UAE, ultrasound
assisted extracted mango peel pectin)
 A

B C

D E

Fig. 4. Light micrographs of the O/W emulsions prepared by different pectins (A,
citrus pectin; B, mango peel pectin obtained by conventional extraction at 20 °C; C,
mango peel pectin obtained by ultrasound assisted extraction at 20 °C; D, mango peel
pectin obtained by conventional extraction at 80 °C; E, mango peel pectin obtained by
ultrasound assisted extraction at 80 °C) (40×)
 A 8 B 10 CP
CE-20°C
UAE-20°C
8 CE-80°C
6 UAE-80°C

Volume (%)
6
Volume (%)

4
4

2
2

0 0
1 10 100 1000 10 100 1000
Particle diameter (m) Particle diameter (m)

Fig. 5. Droplet size distribution of the O/W emulsions prepared by citrus pectin (CP) and mango peel pectins obtained by conventional
extraction (CE) and ultrasound assisted extraction (UAE) over 0 (A) and 1 week (B) of storage
 A
CP CE-20 °C UAE-20 °C CE-80 °C UAE-80 °C

Fig. 6. Stability of the O/W emulsions prepared by citrus pectin (CP) and mango peel
pectins obtained by conventional extraction (CE) and ultrasound assisted extraction (UAE)
over 0 (A) and 1 week (B) storage
Table 1 Physico-chemical properties of CP and MPPs obtained by conventional citric acid extraction (CE) and ultrasound assisted extraction
(UAE)

Different GalA DM Total sugar Protein Mw Color parameter

Dz (d.nm)
pectins (%) (%) content (%) content (%) (kDa) L* a* b*
a b dc c e c b c
CP 71.29±0.81 78.29±0.34 80.56±2.33 3.44±0.50 271.5± 2.3 748.56±30.75 60.86±0.16 1.07±0.00 6.17±0.02a
CE-20 °C 35.16±0.98c 85.43±2.07a 57.76±1.33d 4.74±0.33b 664.5±7.2c 507.04±40.97d 67.91±0.52a 0.86±0.01d 0.96±0.01e
UAE-20 °C 29.35±0.74d 88.38±0.88a 61.30±1.82c 4.85±0.80b 378.4±5.0d 1989.5±81.58a 58.28±0.15c 1.10±0.01c 2.60±0.04d
CE-80 °C 52.21±1.01b 86.77±1.71a 66.43±1.99b 5.24±0.16ab 2858±17a 841.73±34.11b 52.03±1.16d 1.60±0.01a 5.44±0.13b
UAE-80 °C 53.35±1.59b 86.83±0.57a 76.98±0.52a 5.94±0.48a 2320±23b 714..43±21.37c 56.75±0.05c 1.47±0.01b 4.57±0.01c

Values are means ± SD, n=3.

a-d
Different letters in the same column indicate a significant difference (p<0.05).
CP: citrus pectin, MPP: mango peel pectin.
GalA: galacturonic acid, DM: degree of methoxylation, Mw: average molecular weight.
Dz: Z average diameter.
Table 2 Results of parameter estimation for the shear stress-shear rate flow curves of pectin solutions based on Newtonian and Herschel-Bulkley
model
Newtonian Herschel-Bulkley model Apparent viscosity Apparent viscosity
Different Apparent Consistency Flow at 50 s-1 for pectin at 50 s-1 for pectin
Yield stress
pectins viscosity (Pa R2 coefficient behavior R2 solution emulsion
σ0HB(Pa)
s) KHB (Pa sn) index nHB (mPa s) (mPa s)
d
CP 0.016±0.001 0.976 -0.217±0.012 0.214±0.049 0.503±0.040 0.998 25.87±2.13 46.11±0.25b
CE-20 °C 0.022±0.001 0.790 0.559±0.041 0.112±0.012 0.672±0.022 0.999 41.95±3.78b 216.65±5.66a
UAE-20 °C 0.006±0.000 0.999 0.000±0.002 0.009±0.001 0.902±0.008 0.999 6.18±0.51e 223.12±8.73a
CE-80 °C 0.045±0.001 0.896 0.140±0.013 0.130±0.003 0.786±0.005 0.999 59.19±5.03a 208.75±4.78a
UAE-80 °C 0.030±0.001 0.998 -0.017±0.007 0.065±0.001 0.842±0.004 0.999 34.55±2.45c 234.40±6.34a
All data were the means ± SD, n = 3.
Table 3 Particle mean diameter and stability of emulsions prepared by CP and MPPs obtained by conventional citric acid extraction (CE) and
ultrasound assisted extraction (UAE)

Particle mean diameter (μm) Stability of emulsions (%)

Different pectins
D3,2 D4,3 Centrifugation assay ES15 Storage over 1 week

CP 106.59±2.03a 154.94±3.08a 50.0 59.5

CE-20 °C 26.81±0.04b 48.85±0.62b 57.5 67.0

UAE-20 °C 26.11±0.28b 46.47±2.89b 52.5 62.5

CE-80 °C 25.03±0.07b 37.44±0.31c 100 100

UAE-80 °C 20.65±0.22c 33.74±1.32c 100 100

Values are means ± SD, n=3.

a-c
Different letters in the same row indicate a significant difference (p<0.05).
CP: citrus pectin, MPP: mango peel pectin.