Food Chemistry 297 (2019) 125021

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Effect of ultrasonic intensity on the conformational changes in citrus pectin T

under ultrasonic processing
⁎
Wen-Yi Qiu1, Wu-Dan Cai1, Meng Wang, Jing-Kun Yan
School of Food & Biological Engineering, Institute of Food Physical Processing, Jiangsu University, Zhenjiang 212013, China

A R T I C LE I N FO A B S T R A C T

Keywords: In this study, the effects of ultrasonic intensity on conformational changes in aqueous citrus pectin solution
Pectin under ultrasonic processing and its possible transition mechanism were investigated. The results demonstrated
Ultrasound degradation that higher ultrasonic intensity (104.7 W/cm2) caused larger alterations in the molecular and conformational
Ultrasonic intensity parameters of the semiflexible pectin (Mark–Houwink relation exponent a: 0.820, conformational parameter α:
Chain conformation
0.607, structural parameter ρ: 2.22) in aqueous solution. Meanwhile, the semiflexible chain of pectin became
Microstructure
more flexible (a: 0.804, α: 0.601, ρ: 1.75) at higher ultrasonic intensity in aqueous solution, as was verified by
atomic force microscopy. Moreover, conformational changes in pectin from semiflexible chains to flexible chains
or even flexible coils (a: 0.791, α: 0.597, ρ: 1.70) could be attributed to the decreased degree of methoxylation
and neutral sugars in side chains and the destruction of inter- and intramolecular hydrogen bonds under ul-
trasonic processing. Therefore, these results have important implications for understanding the ultrasonic
modification of pectin.

1. Introduction great significance.

Pectin is a complex structural heteropolysaccharide found ubiqui-
tously in the cell walls of fruits and vegetables and is typically isolated
from citrus, sugar beet, and apple. It is mainly composed of (1 → 4)-α-D-
galacturonic acid (GalUA). Pectin, a well-known gelling agent, stabi-
lizer, and fat replacer, has been widely used in diverse food applica-
tions, such as jams and jellies, fruit juices and acidified milk drinks, ice
creams, and emulsified meat products, and in antioxidant-fortified
foods (Maxwell, Belshaw, Waldron, & Morris, 2012). However, due to
the high molecular weight and complex structures of pectin, its prac-
tical applications in food and pharmaceutical industries have been se-
verely limited. To resolve these issues, increasingly, researchers have
paid considerable attention to pectin modification to meet different
functional and nutritional requirements (Chen et al., 2015). To date,
chemical and enzymatic methods have been widely used for pectin
modification. However, these methods are often associated with some
shortcomings, such as time-consuming operation, high cost, un-
controlled molecular weight reduction, high environmental pollution,
and complex subsequent processing (Gogate & Prajapat, 2015).
Therefore, developing simple, green, and controllable approaches for
pectin modification, which can improve its functional properties and
expand its applications in food and pharmaceutical industries, is of
In recent years, power ultrasounds, which are one of the green
nonthermal technologies, have been widely used to degrade and modify
different polysaccharides (PSs), including cellulose, starch, chitosan,
dextran, pullulan, pectin, and carrageenans, with high efficiency and
low cost (Gogate & Prajapat, 2015). Moreover, in our research, ultra-
sound irradiation was successfully employed for depolymerizations of
carboxylic curdlan and Phellinus linteus PSs to improve their functional
and biological properties (Yan, Pei, Ma, & Wang, 2015; Yan, Wang, Ma,
& Wang, 2016). It is known that cavitation is generally defined as the
generation, subsequent growth and collapse of cavities, which results in
very high local energy densities. Moreover, the spectacular effects of
cavitation phenomena, that is, very high temperature and local pres-
sure, strong acoustic streaming, high stress near the bubble wall, mi-
crojets near the solid surface due to asymmetric collapse of bubble and
turbulence, which are generated by using ultrasound (acoustic cavita-
tion) have been applied more commonly in food and in bioprocessing
industries (Gogate & Kabadi, 2009; Gogate, Sutkar, & Pandit, 2011).
Physical modifications of pectin by power ultrasound have been rea-
lized through multieffects of pectin degradation, including cavitation,
disturbance, high shear, crushing, and stirring caused by ultrasound
irradiation. Power ultrasounds have the advantages of low structural
damage, low dispersion of degradation products, and green chemistry.

⁎
Corresponding author.
E-mail address: jkyan27@ujs.edu.cn (J.-K. Yan).
1
These authors contributed equally to this work and should be considered co-first authors.

https://doi.org/10.1016/j.foodchem.2019.125021
Received 9 April 2019; Received in revised form 13 June 2019; Accepted 15 June 2019
Available online 15 June 2019
0308-8146/ © 2019 Elsevier Ltd. All rights reserved.
W.-Y. Qiu, et al.
For example, Ogutu and Mu (2017) indicated that ultrasounds offer
effective and green process for pectin transformation and the creation
of antioxidant potent pectin products. Liu’s research group system-
atically studied the ultrasonic degradation of pectin and the mechanism
thereof (Wang et al., 2018; Zhang, Ye, Ding et al., 2013, Zhang, Ye, Xue
et al., 2013, Zhang, Zhang, Liu, Ding, & Ye, 2015). Liu’s group found
that ultrasonic degradation (20 kHz) could not change the mono-
saccharide types and backbone structure of citrus pectin, but its mole-
cular weight and apparent viscosity decreased obviously with enhanced
ultrasonic intensity and a prolonged ultrasonic time, which further re-
sulted in the formation of weaker gels. Under relatively low tempera-
tures (0–20 °C), citrus pectin would attain a lower molecular weight and
degree of methyl esterification after ultrasonic treatment. Additionally,
Liu’s group also confirmed that the ultrasound effect was more efficient
when the citrus pectin was dissolved in distilled water than in 0.1 M
HCl (1.5 h, 2 s pulse on: 2 s pulse off). Based on the above findings, Liu’s
group highlighted the directional degradation and modification of
pectin by power ultrasound and revealed that the mechanical effect
caused by ultrasonic cavitation is the main cause of pectin degradation.
During ultrasonic degradation of PSs, the secondary bonds between
PS molecules are initially disrupted, and then, the higher-order struc-
tures of PSs are destroyed, causing the macromolecular structures to
loosen, which leads to the cleavage of glycosidic bonds. In the ultra-
sonic degradation process, ultrasonic operating parameters (e.g., in-
tensity, frequency, and time) and environmental factors (e.g., con-
centration, temperature, pH, and ionic strength) could cause changes in
the chain conformations of PSs from multistrand aggregates or ag-
glomerations to extended single-strands, thereby affecting their mi-
crostructures and functional properties (Chen et al., 2014; Wang,
Cheung, Leung, & Wu, 2010; Yan et al., 2015; Yang & Zhang, 2009). For
instance, our research group found that high intensity ultrasound
(20 kHz) could destroy intra- and intermolecular hydrogen bonds of
carboxylic curdlan, thus resulting in the transition from compact
random coil conformation to more flexible and even shorter extended
chains with a prolonged sonication time (Yan et al., 2015). Meanwhile,
the fracture of the PS molecule by power ultrasound is mainly asso-
ciated with high shear force produced from the rapid collapse of cavi-
tation bubbles (Wu, Zivanovic, Hayes, & Weiss, 2008). As one of the
most important factors, ultrasonic intensity significantly influences
different stages of acoustic cavitations, namely, nucleation, bubble
growth, and collapse (Guo et al., 2014). However, to the best of our
knowledge, not much information is available regarding the effects of
ultrasonic intensity on conformational changes in citrus pectin and its
possible transition mechanism under the ultrasonic degradation pro-
cess.
Therefore, this study investigated the effects of ultrasonic intensity
on chain conformational changes in citrus pectin during ultrasonic ir-
radiation by using capillary viscometry and size-exclusion chromato-
graphy combined with multiangle laser light scattering (SEC-MALLS)
and atomic force microscopy (AFM) as well as the theory of dilute
polymer solutions. The possible conformational transition mechanisms
of pectin during ultrasonic modification were also explored. This study
will establish the theoretical basis for the functional design and process
control of the pectin structure and further broaden the actual applica-
tions of pectin in the food industry.
2. Materials and methods
2.1. Materials and chemicals
Pectin from citrus peel with galacturonic acid (≥74%, dried basis)
and methoxyl groups (≥6.7%, dried basis), 1-phenyl-3-methyl-5-pyr-
azolone (PMP), trifluoroacetic acid (TFA), deuterium oxide (D2O), and
monosaccharide standards [glucose (Glc), mannose (Man), galactose
(Gal), arabinose (Ara), rhamnose (Rha), and galacturonic acid (GalUA)]
were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA).
2
Food Chemistry 297 (2019) 125021
All other chemicals and solvents were of laboratory grade and used
without further purification.
2.2. Preparation of pectin solution
The stock solution of pectin (2.0 mg/mL) was previously prepared
by dissolving 4.0 g pectin in 2.0 L deionized (DI) water under constant
stirring at 30 °C for 12 h, and the dispersion was kept at 4 °C with gentle
stirring overnight to achieve complete hydration.
2.3. Ultrasonic degradation
Ultrasonic degradation experiments were carried out by using a
probe ultrasonic processor (Scientz-IID, Ningbo Scientz Biotechnology
Co., Ningbo, China), which had 20 kHz frequency and 600 W maximum
output power and was equipped with a 6 mm-tip-diameter horn probe.
A total of 80 mL pectin solution (2.0 mg/mL) was placed into a reaction
vessel (a 100 mL glass beaker) with external circulation, and the ul-
trasonic probe was lowered into the solution to a fixed depth of 20 mm.
The pectin solution was subjected to a pulsed ultrasound (10 s on and
10 s off period) at different ultrasonic powers (100, 200, and 400 W) for
different times (5, 10, 20, 30, 40, 50, and 60 min) at 30 ± 0.5 °C under
continuous stirring. During sonication, the solution was maintained at a
constant temperature by using a low-temperature thermostatic water
bath. After sonication, the resultant solution was centrifuged at 4000g
for 30 min, dialyzed (MWCO: 6–8 kDa) against distilled water for 48 h,
and then freeze dried for further analyses. The schematic of the ex-
perimental apparatus that was used for the pectin solution under ul-
trasound irradiation is depicted in Fig. S1 (Supplementary data).
Using calorimetric power measurements (Mhetre & Gogate, 2014),
the actual power dissipations in the system were calculated as 6.0, 14.4
and 29.6 W for the rated ultrasonic powers of 100, 200, and 400 W
giving calorimetric efficiencies of 6.0%, 7.2% and 7.4%, respectively.
The ultrasonic intensity was predicted according to the following
equation (Li, Pordesimo, & Weiss, 2004):
I = P /(πr 2) (1)
where I is the ultrasound intensity, P is the actual power dissipation,
and r is the radius of the ultrasonic probe tip (3 mm). In the present
study, for the rated power levels of 100, 200 and 400 W, the calculated
ultrasonic intensities were 21.2, 51.0 and 104.7 W/cm2, respectively.
2.4. Intrinsic viscosity measurements
The intrinsic viscosity ([η]) was determined in DI water at
30 ± 0.1 °C by using a glass Ubbelohde-type capillary viscometer
(0.5–0.6 mm capillary diameter) in a thermostatic water bath. The ki-
netic energy correlation could always be ignored. The Solomon–Ciuta
approximation (Solomon & Ciuta, 1962) was used to calculate the [η]
value according to the following formula:
1
[η] = 2(ηsp − ln ηr )
c (2)
where c represents the polymer concentration, ηsp represents the spe-
cific viscosity, and ηr represents the relative viscosity.
2.5. SEC-MALLS analysis
The SEC-MALLS system (DAWN HELEOS II λ = 658 nm; Wyatt
Technologies Corporation, USA) was conducted on an Agilent 1260
Infinity series HPLC instrument (Agilent, USA) with a differential re-
fractometer (RI) detector (Agilent G136A, USA). The eluent was a 0.1 M
NaCl solution with 0.02% Na3N at a flow rate of 0.5 mL/min. Pectin and
sonicated pectins (2.0 mg) were dissolved in 2.0 mL eluent for 12 h
while stirring at 4 °C, were filtered through 0.22 μm membranes
(Millipore, USA), and then were injected (100 µL) onto the SEC-MALLS
W.-Y. Qiu, et al.
system, which consisted of an OHpak SB-G guard column (Shodex,
Japan) and two SEC columns (OHpak SB-806 M HQ and SB-805 HQ,
8 mm Φ × 30 cm, Shodex, Japan) at 25 °C. The reported value of
0.146 mL/g was used as a refractive index increment (dn/dc) (Morres
et al., 2008). The online Astra software (version 6.1.7, Wyatt Tech-
nologies, USA) attached to the MALLS system was employed for data
collection and processing.
2.6. Degree of methoxylation (DM), methoxyl (MeO), and GalUA content
The DM, MeO, and GalUA contents were determined by titration
according to a reported method (Food Chemical Codex, 1996). The DM,
MeO, and GalUA were calculated according to the equation that was
described by Shaha, Punichelvana, and Afandi (2013). Meanwhile, the
GalUA content was further confirmed by using the sulfuric acid–-
carbazole method with GalUA used as the standard (Bitter & Muir,
1962).
2.7. Monosaccharide composition analysis
The PMP-HPLC derivation method was used to determine the
monosaccharide composition of pectin and sonicated pectins according
to a previous study (Guo et al., 2014). In brief, the samples were sub-
jected to a series of pretreatments, including acid hydrolysis, PMP de-
rivation, neutralization, and chloroform extraction. The resultant aqu-
eous layer was then collected and filtered through a 0.45 μm membrane
and was applied to the HPLC analysis. The HPLC was carried out on an
Agilent 1100 instrument with an Agilent ZORBAX Eclipse XDB-C18
column (4.6 mm × 250 mm, 5 µm; Agilent, USA) at 25 °C with UV de-
tection at 250 nm. The mobile phase was potassium phosphate-buffered
saline (PBS, 0.05 M, pH 6.9) containing 15% acetonitrile (solvent A)
and 40% acetonitrile (solvent B) using a linear gradient elution of
0–15% (0–10 min) then 15–25% solvent B (10–30 min) at a flow rate of
1.0 mL/min. Monosaccharide standards were used for the identification
and quantification of the corresponding peaks.
2.8. Fourier transform infrared (FT-IR), nuclear magnetic resonance
(NMR), and circular dichroism (CD) spectroscopies
The FT-IR spectra of pectin and sonicated pectins were recorded
between 500 cm−1 and 4,000 cm−1 in attenuated total reflection mode
at a resolution of 4 cm−1 using 128 scans (Nicolet 380, Thermo
Scientific, UK). For NMR analysis, 30 mg pectin was previously dis-
solved in 0.5 mL D2O. The 1H and 13C NMR spectra were recorded on a
Bruker AVANCE III 400 MHz spectrometer (Bruker, Rheinstetten,
Germany) at 25 °C. All the chemical shifts were expressed in reference
to DDS (δ 1H 0.00 ppm, δ 13C 0.00 ppm) as an external standard. The CD
spectra of pectin and sonicated pectins were obtained with a dichroism
spectrometer (JASCO J-815, Japan) at room temperature using a 0.1 cm
path length quartz cell in the wavelength range of 190–250 nm.
2.9. Atomic force microscopy (AFM)
The molecular morphologies of original pectin and sonicated pec-
tins were observed by using a Multimode8 Nanoscope Atomic Force
Microscope (Bruker Nano Surfaces Division, Santa Barbara, CA). Prior
to observation, the sample solution (20 μL) at a concentration of 10 μg/
mL in DI water was deposited onto a freshly cleaved mica surface and
was dried at room temperature. Details of the operating conditions and
methods were described as previously reported (Cai, Qiu, Ding, Wu, &
Yan, 2019).
2.10. Statistical analysis
All the experiments were conducted in three replicates, and the
mean ± standard deviation (SD) was used in the analysis. Analysis of
3
Food Chemistry 297 (2019) 125021
variance (ANOVA) and Duncan’s multiple range tests were carried to
evaluate the differences between groups out by using OriginPro
Software Version 8.0 (OriginLab Corp., MA, USA). P < 0.05 indicated
statistically significant differences.
3. Results and discussion
3.1. Confirmation of the structure and chain conformation of original citrus
pectin
In this study, commercial pectin from citrus peel used for ultrasonic
degradation was previously characterized for structural and con-
formational features relevant to the present investigation. Chemical
analyses showed that the GalUA, MeO, and DM contents of citrus pectin
were 71.69% ± 1.7%, 12.27% ± 2.4%, and 76.63% ± 0.5%, re-
spectively, which indicated that the citrus pectin is highly methylated.
According to monosaccharide composition, FT-IR, SEC-MALLS, and
NMR measurements (Fig. S2, and Table S1, Supplementary data), the
pectin is an acidic heteropolysaccharide that is mainly composed of
arabinose, rhamnose, galactose, and GalUA, with a weight-average
molecular weight (Mw) of 173.7 kDa. Homogalacturonan is the smooth
region consisting of a linear backbone of 1,4-linked α-D-GalUA with a
variable number of methyl ester groups at the C-6 position. Rhamno-
galacturonan-I is the primary hairy region, with branched PSs con-
taining blocks of neutral monosaccharide units, galactose, rhamnose
and arabinose. The possible chemical structure model of citrus pectin is
graphically displayed in Fig. S2F (Supplementary data), which is con-
sistent with that of the previous study by Mohnen (2008).
The molecular properties, conformational parameters, and chain
conformation of original citrus pectin were characterized by using
viscometry, SEC-MALLS analysis, and the theory of dilute polymer so-
lutions, as well as the relevant empirical equations, such as the
Mark–Houwink (M−H) relation ([η] = k·Mwa) and Rg = f·Mwα, where a
and α have defined values for specific conformation types (Yang &
Zhang, 2009). The obtained results are summarized in Fig. S2D and
Table S2 (Supplementary data). As expected, the single symmetrical
peak of the pectin solution (Fig. S2D) suggested relatively high purity
and uniformity. Moreover, the [η], z-average radius of gyration (Rg),
hydrodynamic radius (RH), a, α, and structure parameter (ρ = Rg/RH) of
citrus pectin were calculated to be 457.93 cm3 g−1, 38.8 nm, 17.5 nm,
0.820, 0.607 and 2.22, respectively, which indicated that the original
citrus pectin predominantly behaved as a rigid semiflexible chain
conformation in the aqueous solution.
3.2. Effect of ultrasonic intensity on [η] and Mw
Fig. 1A presents the [η] of the pectin (2.0 mg/mL) solutions under
ultrasonic treatments as a function of the sonication time from 0 min to
60 min at various ultrasonic intensities. The [η] of pectin solution
dramatically decreased within the first 30 min but dropped slowly as
the sonication time continued to increase from 30 min to 60 min. No-
tably, the reduction of [η] was more pronounced at a stronger ultra-
sonic intensity (104.7 W/cm2) during ultrasound irradiation. For ex-
ample, after 60 min sonication, the [η] values of sonicated pectin
solutions at 21.2, 51.0, and 104.7 W/cm2 were 352.08, 278.11, and
247.90 cm3 g−1, respectively. [η], which represents the inherent visc-
osity, is one of the most important parameters that can directly reflect
the PS conformation. Usually, a more extended structure for a given
chain length is associated with a high [η] value (Lapasin & Pricl, 1995).
Acoustic cavitation is the main force that affects the glycosidic bond
breakage of pectin molecule, and ultrasonic treatments at much higher
ultrasonic intensities generate pectin molecules with a short molecular
size, which leads to the decrease of [η] during ultrasonic treatment. A
similar phenomenon was observed in a previous study (Wang et al.,
2010). Meanwhile, in the current work, a first order reaction kinetic
model that was published previously in the literature was employed for
W.-Y. Qiu, et al.
Fig. 1. Effects of ultrasonic intensities on the (A) [η], (B) Mw, (C) Rg and (D) RH of pectin under ultrasound treatments as a function of the sonication time.
better understanding of the degradation efficiency of citrus pectin
under various ultrasonic intensities (Prajapat & Gogate, 2015). Ac-
cording to the kinetic model, the rate constant for ultrasonic degrada-
tion of the pectin solution could have been derived from a plot of the ln
(A) versus sonication time (t), where A = (η0-η∞)/(ηt-η∞) for all the
experiment runs considering the initial intrinsic viscosity (η0), intrinsic
viscosity at time t (ηt) and the limiting intrinsic viscosity (η∞) of the
pectin solution, and the obtained results are depicted in Fig. S3
(Supplementary data). The higher correlation coefficient (R2 > 0.96)
indicated that the first-order reaction kinetic model was well workable
for evaluating the degradation of pectin. Moreover, it can be noted from
Fig. S3 that the rate constant for the degradation of pectin increased
from 0.0464 min−1 to 0.0535 min−1 as the ultrasonic intensity in-
creased from 21.2 W/cm2 to 104.7 W/cm2. This might be because the
increase in ultrasonic intensity led to a corresponding increase in the
number of cavitation events, which contributed to the enhancement of
cavitational intensity and resulted in an increase in the extent of de-
gradation. A similar phenomenon has also been observed in our pre-
vious study (Yan et al., 2016).
Based on the SEC elution profiles of pectin and its degradation
products (Fig. S4, Supplementary data), the SEC elution peak of pectin
shifted to a longer elution time as the ultrasound irradiation increased
from 0 min to 60 min irrespective of ultrasonic intensities, which sug-
gested that depolymerization occurred in the pectin chains. Further-
more, a higher ultrasonic intensity produced a larger shift in the SEC
elution pattern of pectin, which further indicated that the extent of
degradation is more pronounced at a higher ultrasonic intensity. As
shown in Fig. 1B, the Mw of original citrus pectin was 173.7 kDa. Sig-
nificant reductions were observed within the first 30 min, but the de-
creasing tendency slowed down in the remaining 30–60 min of ultra-
sound treatment, which was consistent with the changes in [η]
(Fig. 1A). The ultrasonic intensity significantly decreased the Mw of
pectin, and a lower Mw of pectin was obtained under high ultrasonic
intensity. Theoretically, the ultrasonic frequency and intensity (affected
by power dissipation) could significantly affect the energy dissipation
4
Food Chemistry 297 (2019) 125021
rates. In general, cavitation could not be produced in the liquid, and
degradation would not occur when there was less than a critical sound
intensity. However, beyond the critical intensity, the increase in the
intensity enhanced the degradation rate and the extent of degradation
(Lorimer, Mason, Cuthbert, & Brookfield, 1995; Mason & Lorimer,
2002). Therefore, in the present study, high ultrasonic intensity, which
was achieved by increasing the power dissipation, could promote the
energy of cavitation, decrease the threshold of cavitation, and increase
the quantity of the cavitation bubbles, thus showing a greater de-
gradation effect (Jiang, Pétrier, & David Waite, 2002). For example, the
Mw of pectin decreased from 151.5 kDa to 125.8 kDa when the ultra-
sonic intensity increased from 21.2 W/cm2 to 104.7 W/cm2 for 60 min.
3.3. Effect of ultrasonic intensity on Rg and RH
In general, Rg depicts the mass average (root mean square) distance
of each point in a molecule from the molecule’s center of gravity, which
is related to the actual space that was occupied by the PS chain (Yang &
Zhang, 2009). In practice, the RH and Mw of a biopolymer in a solution
are not independent variables, with RH typically increasing with the
increasing Mw and relying on a biopolymer shape. Despite only a small
amount of aggregates, in the dilute solution, the RH value for the bio-
polymer may have been influenced to some extent. In this context,
Einstein’s theoretical equation (Eq. (3)) on the biopolymer molecule in
the solution was applied to calibrate the RH (Smith & Raymonda, 1972):
3
[η] Mw = ν(a / b) = NA π (RH )3
4 (3)
where the number of Simha (ν(a/b)) is a function of axial ratio. For citrus
pectin, the value of ν(a/b) that was 10.5 (Masuelli, 2011). NA is Avo-
gadro’s number (6.02 × 1023 mol−1). Fig. 1C and D present the changes
in Rg and RH of pectin solutions under ultrasonic treatments at various
ultrasonic intensities as a function of the sonication time. The Rg and RH
of pectin were in the same downtrend as Mw within the measured ul-
trasonic irradiations. A higher ultrasonic intensity produced lower
W.-Y. Qiu, et al. Food Chemistry 297 (2019) 125021

values of Rg and RH; that is, after 30 min, the Rg and RH values for pectin macromolecular conformation is closely correlated with the M−H re-
at the ultrasonic intensity of 21.2 W/cm2 were 29.6 and 15.9 nm, re- lation exponent a and the conformation parameter α. Based on the
spectively, whereas lower values of Rg (24.5 nm) and RH (14.1 nm) for theory of polymer dilute solution, the exponent a values of 0–0.5,
pectin were observed at the ultrasonic intensity of 104.7 W/cm2. This 0.5–0.8, and 0.8–2.0 reflect the chain conformation as a rigid sphere,
finding suggested that the molecular sizes of pectin became shorter at random coil, and rigid or rod-like (stiff) chain, respectively (Yang &
higher ultrasonic intensities, which might be because the shearing force Zhang, 2009). The α values of 0.33, 0.5–0.6, 0.6–1.0, and 1.0 represent
produced from cavitation bubbles not only disrupted the inter- and the chain conformation of spheres, flexible chain, semiflexible chain,
intramolecular hydrogen bonds of pectin chains but also destroyed the and rigid rods, respectively (Kato et al., 1982). As shown in Table 1, the
glycosidic bonds of pectin molecules, thus leading to the depolymer- values of a and α for citrus pectin in the 0.1 M NaCl aqueous solution
ization of pectin and a decrease in the molecular size.- Increasing ul- decreased from 0.8205 and 0.6068 to 0.8078 and 0.6026, respectively,
trasonic intensity allowed enhanced acoustic cavitation to be achieved, with the increase of the sonication time from 0 min to 60 min at the
which might be a direct consequence of the rising degradation of pectin ultrasonic intensity of 21.2 W/cm2, and the extent of reduction in the
and a reduction- in the molecular sizes (e.g., Rg and RH). However, as two parameters was more pronounced at a higher ultrasonic intensity.
ultrasonic irradiation was prolonged from 30 min to 60 min, especially The results indicated that with the increase of the ultrasonic time and
at a high ultrasonic intensity (104.7 W/cm2), more cavitation bubbles intensity, the rigid semiflexible chain conformation of pectin might turn
were generated around the acoustic source, which restrained the effi- into even, flexible coils, and the pectin chain stiffness and extension
ciency of energy transmission into the reaction medium and impeded became weaker, which might be because the higher ultrasonic intensity
the propagation of the ultrasound wave (Mason & Lorimer, 2002), and longer irradiation duration could break up neutral sugars, such as
thereby resulting in no apparent changes in Rg and RH. For example, the rhamanose, galactose, and arabinose, from the side chains of pectin;
Rg and RH values of pectin slightly varied from 23.6 and 13.5 nm to 21.8 reduce the degrees of methoxylation and acetylation; and weaken the
and 12.8 nm, respectively, as the sonication time increased from 40 min rigidity of the semiflexible chain of pectin (BeMiller, 1986, ch. 1).
to 60 min at the ultrasonic intensity of 104.7 W/cm2. Therefore, based Moreover, relevant structural information of the PS could be further
on the results of [η], Mw, Rg, and RH, it could be concluded that an derived by investigating the fractal dimension (df) value, which is de-
increase in the ultrasonic intensity as a function of the sonication time fined as the inverse of the conformational exponent α; that is, df = 1/α
caused the pectin/water system to exhibit an interesting trend of the (Yang & Zhang, 2009). In general, the df values of 1, 5/3–2, and 3 re-
rigid semiflexible macromolecule to compaction (i.e., a decrease in [η] flect a rigid rod, linear polymers with a Gaussian coil nature, and a
and RH). three-dimensional object with a homogeneous density, respectively. As
seen from Table 1, the df values for pectin under ultrasonic treatments
at different ultrasonic intensities and durations varied within the range
3.4. Effect of the ultrasonic intensity on chain conformational
of approximately 1.64–1.68, which indicated that original citrus pectin
characteristics
and sonicated pectins existed as chain conformations in the aqueous
solution between the rigid rod and linear polymers with a Gaussian coil
The hydrodynamic results obtained from SEC-MALLS and viscosity
nature. Meanwhile, with the extension of the sonication time and the
measurements combined with the theory of polymer dilute solutions
increase of ultrasonic intensity, the df values for pectin generally in-
were used to study the changes in chain conformational characteristics
creased, which suggested that the rigidity of the pectin chain became
of pectin under ultrasonic treatment at various ultrasonic intensities,
weaker. Noticeably, higher ultrasonic intensity led to weaker chain ri-
and the established conformational parameters are summarized in
gidity.
Table 1. The values of [η], Mw, and Rg estimated from SEC-MALLS and
Based on the values of Rg and RH, the structural parameter ρ (Rg/
viscometry can be used to establish the relationships between Mw and
RH), which is indicative of the polymer shape in solution, could be
[η] (i.e., M−H relation, [η] = k·Mwa), and Mw and Rg (Rg = f·Mwα)
acquired. In a given polymer solution, the ρ value relies on its chain
(Kato, Okamoto, Tokuya, & Takahashi, 1982). In general, the shape of
architecture, conformation, and polydispersity, but not the molar mass.
ρ values of 0.775, 1.5–1.8, and ≥2 indicate the chain conformation as
Table 1
Molecular and conformational parameters of pectin under ultrasound treatment
compact spheres, flexible random coils, and extended chains (cylin-
at various ultrasonic intensities. ders), respectively. That is, higher ρ values indicate more rigid chains
(Yang & Zhang, 2009). Table 1 shows that the ρ values of pectin at
Samples Intensity (W/ Time a α df ρ = Rg/RH
different ultrasonic intensities decreased with the increasing sonication
cm2) (min)
time. The rate of the decrease in ρ was more pronounced at a higher
Pectin 0 0.8205 0.6068 1.6479 1.894 ultrasonic intensity. For instance, the ρ value decreased from 1.894 to
21.2 5 0.8134 0.6045 1.6543 1.866 1.798 at the ultrasonic intensity of 21.2 W/cm2 after 60 min of ultra-
10 0.8133 0.6044 1.6544 1.836
sonic irradiation, whereas the ρ value of 1.700 was observed under the
20 0.8116 0.6039 1.6560 1.857
30 0.8103 0.6034 1.6572 1.866 ultrasonic intensity of 104.7 W/cm2. The results illustrated that higher
40 0.8105 0.6035 1.6570 1.824 ultrasonic intensity and longer duration resulted in a larger reduction in
50 0.8103 0.6034 1.6572 1.811 chain rigidity and the extension of semiflexible pectin under ultrasonic
60 0.8078 0.6026 1.6594 1.798 treatments, which was consistent with the results of a, α, and df. Given
51.0 5 0.8132 0.6044 1.6545 1.842
the above findings, we could conclude that increasing the ultrasonic
10 0.8124 0.6041 1.6553 1.805
20 0.8101 0.6034 1.6574 1.790 intensity (or duration) results in decreased conformation parameters
30 0.8066 0.6022 1.6606 1.773 (i.e., a, α, and ρ) and less-extended conformation, which further implies
40 0.8023 0.6008 1.6645 1.747 that the rigid semiflexible chain conformation of pectin in aqueous
50 0.7969 0.5990 1.6695 1.746
solution becomes more flexible under higher ultrasonic intensities.
60 0.7938 0.5979 1.6724 1.735
104.7 5 0.8117 0.6039 1.6559 1.822
10 0.8092 0.6030 1.6582 1.769 3.5. Molecular morphology observation
20 0.8037 0.6012 1.6632 1.750
30 0.7973 0.5991 1.6692 1.736 AFM technology is widely used to observe and evaluate the surface
40 0.7956 0.5985 1.6707 1.717
topography of macromolecules, thereby providing direct evidence re-
50 0.7942 0.5980 1.6721 1.704
60 0.7907 0.5969 1.6753 1.700 garding the chain conformation of PS (Yang & Zhang, 2009). In the
present study, the results of the AFM analyses of pectin and sonicated

5
W.-Y. Qiu, et al.
Fig. 2. AFM topographic images of (A) original citrus pectin, and sonicated pectins at 104.7 W/cm2 for (B) 5 min, (C) 10 min, (D) 30 min and (E) 60 min in an
aqueous solution at a polysaccharide concentration of 10 μg/mL.
pectins at a constant ultrasonic intensity (104.7 W/cm2) for different
durations (5, 10, 30, and 60 min) are graphically displayed in Fig. 2.
The original citrus pectin exhibited semiflexible ordered chains and
continuous network structures (Fig. 2A), and the crossing points of
entwining chains were also observed in pure water. This might be be-
cause semiflexible pectins have more complicated structures and more
neutral sugar side chains, which can interact with each other through
negatively charged polymer chains, hydrogen bonds, or van der Waals
forces (Wang, Xu, & Zhang, 2008). Hence, the semiflexible single chains
were intertwined to form network structures in an aqueous solution.
Under the ultrasonic treatments, the network structures, which formed
via inter- and/or intramolecular entwining of the pectin chains, became
disordered, partially fractured (Fig. 2B–D), and even dissociated
(Fig. 2E) as the ultrasonic irradiation continued. Moreover, the crossing
points that were present in pectin networks were variously disrupted,
which resulted in the formation of some individual strands. The
6
Food Chemistry 297 (2019) 125021
presumable reason for this phenomenon was that ultrasound irradia-
tion, especially at higher ultrasonic intensity (104.7 W/cm2), decreased
the Mw of pectin via the breakages of glycosidic bonds and fractures of
neutral sugar side chains, shortened the length of semiflexible chain,
and reduced the stiffness of the pectin chain. Apparently, interactions
holding sonicated pectin networks together are weaker than those
holding original pectin networks, which leads to the rupture of network
structures. Taken together, according to the above discussion, the
possible degradation process, conformational changes, and micro-
structures of pectin in an aqueous solution under ultrasonic processing
are depicted schematically in Fig. 3.
3.6. Effects of structural parameters on the conformational characteristics
Fig. 4A and B present the FT-IR spectra of original citrus pectin and
pectins ultrasonicated at 21.2 and 104.7 W/cm2 for different sonication
W.-Y. Qiu, et al.
Fig. 3. Schematic diagram of the degradation process, conformation transition, and microstructures of aqueous pectin under ultrasonic processing.
durations. After ultrasonic treatments, the major IR characteristic peaks
at approximately 3440 (OeH), 2933 (CeH), 1750 (COOeCH3), 1635
(COOe), 1445 (CeOH), 1103 (CeO), 1016 (CeC), and 955 cm−1 re-
mained unchanged compared with the original pectin. These findings
indicated that ultrasonic treatment mainly led to the breakage of gly-
cosidic bonds, but there was no significant change in the primary
chemical structure of pectin. The DM of pectin could be evaluated
simply by measuring the average of the ratio of the peak area at
1750 cm−1 (COOCH3) over the sum of the peak areas of 1750
(COOCH3) and 1635 cm−1 (COOe) (Chatjigakis et al., 1998). In the
present study, the DM values of original and sonicated pectins were
estimated and are listed in Table 2. The DM values of sonicated pectins
decreased compared with those of native pectin (55.38%), with the
sonication time increasing from 5 min to 60 min, and a greater reduc-
tion in DM was found at a higher ultrasonic intensity. For example, the
DM of sonicated pectin decreased from 43.04% to 37.42% when the
ultrasonic intensity increased from 21.2 W/cm2 to 104.7 W/cm2 after
60 min. This reduced trend in DM could be explained by the hydrolysis
of ester groups by acoustic cavitation or the reaction of ester groups
with ionized groups generated during sonolysis, which was also verified
by Ogutu and Mu (2017). Moreover, the decreased DM weakened the
hindrances of pectin molecules to some extent, thus leading to the
pectin chains becoming more flexible under ultrasonic processing.
Table 2 summarizes the monosaccharide compositions of the ori-
ginal citrus pectin and sonicated pectins under different ultrasonic
treatments. In the process of the ultrasound, compared with the native
pectin, the monosaccharide types remained unchanged, but the
monosaccharide contents changed obviously. GalUA was considered to
be the predominant component in all the pectin samples studied, and
the GalUA content increased significantly (P < 0.05) with ultra-
sonication power. Higher ultrasonic intensity led to an increased GalUA
content. The increased GalUA content might be due to the scission of
the pectin side chain, which led to GalUA enrichment in the main chain,
7
Food Chemistry 297 (2019) 125021
thereby enhancing GalUA content from 59.24% in the original pectin
up to 72.55% in 104.7 W/cm2 in the sonicated pectin after 60 min.
Zhang, Ye, Xue et al. (2013) also revealed that GalUA is more resistant
to neutral sugars, which further verified the increasing GalUA content
with enhanced ultrasonic intensity. The slight variation in the ratio of
Rha/GalUA indicated that the power ultrasound, particularly at lower
ultrasonic intensity, had minor influences on the pectin main chain.
Additionally, the Rha content slightly decreased under ultrasonic pro-
cessing, which might be attributed to the breakage of Rha in the side
chain. The presence of Rha in the side chain enhanced the rigidity of the
pectin chain and restricted the aggregations, entanglements, and for-
mation of the junction zones (BeMiller, 1986, ch. 1). Hence, the de-
creased Rha could promote the flexibility of the pectin chain. Table 2
shows that the contents of neutral sugars, such as Gal and Ara, de-
creased significantly under ultrasonic processing (P < 0.05), especially
at a higher ultrasonic intensity. Moreover, the (Gal + Ara)/Rha ratio,
which reflects the amount of neutral sugars in pectin side chains, de-
creased under the effect of the ultrasound. The reduction in the
(Gal + Ara)/Rha ratio during ultrasonic treatments indicated the de-
gradation of side chains, and higher ultrasonic intensity led to a more
pronounced decrease of the pectin side chains. For example, the ratio of
(Gal + Ara)/Rha significantly dropped from 2.82 to 2.09 after ultra-
sonic treatment at 104.7 W/cm2 for 60 min (P < 0.05). Similarly, as
described by Ogutu and Mu (2017), no significant change was observed
in the main chain, whereas a decrease of the pectin side chains was
remarkably observed. Neutral sugar constituent and linkages strongly
affected the conformation of pectin (BeMiller, 1986, ch. 1). Under ul-
trasonic processing, acoustic cavitation produced by the ultrasound
fractured the pectin side chains, reduced the contents of neutral sugars,
and weakened the steric hindrances and chain rigidity of pectin chains,
thereby triggering the pectin chain to become more flexible. Therefore,
it could be concluded that the degradation of neutral sugars in the side
chains induced by ultrasonication potentially contributed to the
W.-Y. Qiu, et al. Food Chemistry 297 (2019) 125021

Fig. 4. FT-IR spectra of original citrus pectin and sonicated pectins at (A) 21.2 W/cm2 and (B) 104.7 W/cm2 for 5, 10, 30, and 60 min. CD spectra of original citrus
pectin and sonicated pectins at (C) 21.2 W/cm2 and (D) 104.7 W/cm2 for 5, 10, 30, and 60 min. (E) and (F) are the ellipticities at 202 nm of (C) and (D), respectively.

enhanced flexibility of the pectin chain. process; that is, the ellipticity at 202 nm pectin reduced from
19.34 deg cm2 g−1 to 15.72 deg cm2 g−1 at 21.2 W/cm2 and
14.88 deg cm2 g−1 at 104.7 W/cm2 after 60 min sonication. Both the
3.7. CD analysis
COOH and COOCH3 groups in pectin had equal contributions to the
molecular ellipticity. In general, carboxyl ionization causes a sharp
CD could provide a convenient method for investigating the con-
decrease in the ellipticity, and the association of pectins may cause a
formational transition of PSs with uronic acids, acetate groups, and
two-fold increase in ellipticity (Plaschina et al., 1978). Thus, in this
amide chromophores. The pectinic substances showed a characteristic
study, the decrease in molecular ellipticity at 202 nm with increase in
positive Cotton effect with a maximum at approximately 210 nm, which
ultrasonic intensity and duration might be associated with a decrease in
corresponded to an n-π* transition of the carboxyl group, and the op-
the degree of association because of the destruction of intra- and in-
tical activity of the carboxyl chromophore could be affected by intra-
termolecular hydrogen bonds that plays an important role in the pro-
and intermolecular interactions (Plaschina, Braudo, & Tolstoguzov,
cess of conformational change of pectin from semiflexible chains into
1978). In our current research, the CD technique was employed to
flexible chains, which was consistent with the results of the AFM ana-
determine conformational changes of pectin under ultrasonic proces-
lyses (Fig. 2). Moreover, a similar result was reported by Nitta et al.
sing, and the obtained results are displayed in Fig. 4C-F. Fig. 4C and D
(2003), who found that a helix-forming gellan gum showed a single
show that the original and sonicated pectins exhibited positive Cotton
peak at approximately 200 nm in the CD spectrum, and the ellipticity at
effects with maximum peaks at nearly 202 nm in the measured range of
202 nm decreased at a lower temperature and increased at a higher
190–250 nm. As the sonication time increased from 5 min to 60 min, the
temperature, which further suggested a helix-coil transition of the
positive peaks at 202 nm gradually decreased. Furthermore, higher
conformational change of gellan. Collectively, the change in the mo-
ultrasonic intensity led to a larger decrease in the characteristic positive
lecular ellipticity under ultrasonic processing was induced by the con-
peak at 202 nm, which indicated that the structure and chain con-
formational transition of pectin from a semiflexible chain into a flexible
formation of pectin were altered by ultrasonication. Fig. 4E and F show
chain.
that the ellipticity at 202 nm similarly showed a decreased trend as a
function of the sonication time, and the reduction in ellipticity was
more pronounced at a higher ultrasonic intensity in the ultrasound

8
W.-Y. Qiu, et al. Food Chemistry 297 (2019) 125021

Note: Rha/GalUA and (Gal + Ara)/Rha represent molar ratios. Values with different letters in the same row are significantly different according to Duncan’s multiple range test (P < 0.05). DM is calculated by measuring
4. Conclusion

DM (%)

55.38
46.23
45.82
45.47
43.04
45.49
43.77
42.51
37.42
Ultrasonic intensity affected the conformational changes of the rigid
semiflexible citrus pectin in an aqueous solution under ultrasonic de-
0.007 ab
(Gal + Ara)/Rha

gradation processing. Higher ultrasonic intensity led to larger varia-

0.07 ab
0.29 ab

0.07 bc

0.12 bc
0.06 cd
0.03 a

0.04b
0.10c
tions in the molecular and conformational parameters such as the
weight-average molecular weight, intrinsic viscosity, z-average radius
±
±
±
±
±
±
±
±
±
of gyration, hydrodynamic radius, Mark–Houwink relation exponent,
2.82
2.94
2.83
2.66
2.34
2.28
2.32
2.24
2.09
conformation parameter, fractal dimension, and structural parameter;
and fractured the inter- and intramolecular entanglements, which were
0.004 abc

further verified by AFM images. The conformational transition of citrus

0.004 ab

0.002 de
0.003 bc
0.009 bc

0.003 bc

0.005 cd
0.004b

pectin from semiflexible chains into flexible chains or even flexible coils
0.02 a
Rha/GalUA

in the process of ultrasonic degradation could be attributed to the de-

±
±
±
±
±
±
±
±
±

crease of DM and neutral sugars in side chains and the destruction of

0.15
0.14
0.13
0.13
0.14
0.12
0.12
0.11
0.11

inter- and intramolecular hydrogen bonds. Further investigations on the

effects of conformational changes in citrus pectin induced by ultra-
sound on rheological and emulsifying properties are underway in our
0.31abc
0.65 ab

2.28 ab
0.59 ab

0.49 ab
2.06 a

0.52 a

0.69c
0.24c

laboratory.
±
±
±
±
±
±
±
±
±

Declaration of interests
5.23
4.18
5.06
4.78
4.03
3.61
4.39
2.28
2.24
Ara

The authors declare that they have no known competing financial

the average of the ratio of the peak area at 1750 cm−1 over the sum of the peak areas of 1750 cm−1 and 1635 cm−1 in the FT-IR spectroscopy.

interests or personal relationships that could have appeared to influ-

0.03 cd
0.18 cd
0.06 cd
0.46 d
0.48 a
0.59b

0.68b

0.13b
0.59c

ence the work reported in this paper.

±
±
±
±
±
±
±
±
±

Acknowledgements
19.14
21.55
18.95
18.01
16.90
15.86
14.82
14.76
14.25
Gal

This work was supported financially by the National Natural Science

Foundation of China (31671812), the Key Research & Development
Monosaccharide composition of pectin and sonicated pectins under ultrasonic treatments at various ultrasonic intensities.

0.37 cd
0.18 cd
0.45d

0.41b
0.62c

0.20c
0.32c
0.07c

Program (Modern Agriculture) of Jiangsu Province (BE2017362),

0.7b

Jiangsu Overseas Research & Training Program for University

±
±
±
±
±
±
±
±
±

Prominent Young & Middle-aged Teachers and Presidents.

5.72
2.53
3.10
3.31
3.55
3.10
1.40
1.79
2.25
Glc

Appendix A. Supplementary data

0.76abc
0.67 ab
3.35 bc

0.09 a
3.04 a
0.95 a

0.68 a
0.46 a
0.93c

Supplementary data to this article can be found online at https://

doi.org/10.1016/j.foodchem.2019.125021.
±
±
±
±
±
±
±
±
±
GalUA

59.24
61.96
63.33
64.09
65.46
68.21
70.09
72.63
72.55

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