Food Hydrocolloids 131 (2022) 107727

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

Emulsification mechanism of persimmon pectin with promising

emulsification capability and stability
Yangyang Jia a, b, Jing Du a, b, c, Kaikai Li a, b, Chunmei Li a, b, *
a
College of Food Science and Technology, Huazhong Agricultural University, Wuhan, 430070, China
b
Environment Correlative Food Science (Huazhong Agricultural University), Ministry of Education, Wuhan, 430070, China
c
College of Food Science and Engineering, Wuhan Polytechnic University, Wuhan 430023, China

A R T I C L E I N F O A B S T R A C T

Keywords: The influence of processing factors and surrounding environment stress on persimmon pectin (PP) prepared
Persimmon pectin emulsion was compared with commercial sugar beet and citrus pectins. The results showed that although PP-
Emulsion stabilized emulsion was sensitive to salt ions, it had stronger resistance against high temperature, freeze-
Mechanism
thawing, thermal cycling, and accelerated centrifugation than commercial sugar beet and citrus pectins. To
Multi-layer adsorption
“3D-network” structure
reveal its potential emulsifying mechanism, multi-techniques including triple-phase contact angle evaluation,
adsorption layer characterization, and super-resolution imaging of emulsions were employed. Results showed
that PP had a high triple-phase contact angle of 83◦ , endowing it with balanced hydrophobicity and interfacial
adsorption. The polystyrene latex particles (PS) model analysis showed that PP exhibited the highest adsorption
layer thickness. Moreover, the quartz crystal microbalance with dissipation monitoring (QCM-D) analysis sug­
gested that PP formed a thicker interfacial film and higher adsorption mass and viscoelasticity. The cryogenic-
scanning electron micromorphology (Cryo-SEM) revealed a “3D-network” structure around the oil droplets
was formed by PP, further enhancing elastic interface film and effectively preventing the flow of the oil droplets.
Overall, PP exerted excellent emulsifying capability and stability through multi-layer adsorption. The results
suggested that PP exhibited a promising emulsification potential and could be developed as a novel poly­
saccharide emulsifier in the food industry.

1. Introduction
Emulsion-based products are thermodynamically unstable systems,
which are composed of immiscible phases (usually oil and water), in
which one phase is dispersed as droplets into the other (continuous
phase) (Wei et al., 2020). Therefore, it is desirable to make emulsions
kinetically stable over a period (from several days to years) using
emulsifiers, typically of synthetic origin (Alba & Kontogiorgos, 2017).
However, ‘natural’ products which have no synthetic additives are
increasingly attracting interest from consumers (McClements & Gumus,
2016). Typical food-grade ‘natural’ emulsifiers mainly include proteins,
polysaccharides, and phospholipids. At present, natural emulsifiers with
excellent emulsifying properties are still in short supply, and the
development of new natural emulsifiers is still in great demand.
In the past few decades, pectin has been increasingly gaining
acceptance as a natural emulsifying agent in the food industry (Alba,
Sagis, & Kontogiorgos, 2016). At present, citrus pectin and sugar beet
pectin (SBP) have been commercialized and applied in the food industry
(Gashua, Williams, & Baldwin, 2016). Among the commercial pectins,
citrus pectin is one of the most important because of its abundance in
citrus (Zhao et al., 2020). Which is one of the fruits with the largest
planting scale and yield in the world (Dranca & Oroian, 2018). Citrus
pectin could be used to prepare emulsions since it can aggregate at the
oil-water interface and increase the viscosity to promote the emulsion
stability, but it may not effectively stabilize the emulsion during storage
(Yang, Mu, & Ma, 2018). Compared to other types of pectin commonly
applied in the food industry, sugar beet pectin (SBP) has a higher content
of acetyl groups at O-2 and O-3 positions within the galacturonic
backbone, a higher content of phenolic esters in the lateral chains, and a
higher amount of covalently bound proteins in the lateral chains (Chen,
Qiu, et al., 2016). The presence of the phenolic, acetyl, and protein
groups on SBP indicated that it could adsorb to oil-water interfaces and
decrease the interfacial tension (Liu, Pi, Guo, Guo, & Yu, 2019). In
addition, the complex interactions between the protein and

* Corresponding author. College of Food Science and Technology, Huazhong Agricultural University, Wuhan, 430070, China.
E-mail address: lichmyl@mail.hzau.edu.cn (C. Li).

https://doi.org/10.1016/j.foodhyd.2022.107727
Received 7 November 2021; Received in revised form 5 April 2022; Accepted 6 April 2022
Available online 9 April 2022
0268-005X/© 2022 Elsevier Ltd. All rights reserved.
Y. Jia et al.
polysaccharide combinations in SBP can prevent droplet aggregation
(Nakauma et al., 2008). The SBP-layer formed around the oil droplets
provided electrostatic and steric repulsion, preventing droplets from
flocculation, aggregation, and coalescence. A prominent advantage of
SBP is that it could prepare emulsions at a lower dosage than other
polysaccharide emulsifiers such as gum arabic, guar gum, soybean sol­
uble polysaccharide, and corn fiber gum (Bai, Huan, Li, & McClements,
2017; Burapapadh, Kumpugdee-Vollrath, Chantasart, & Sriamornsak,
2010). Besides, SBP exhibited superior emulsification properties than
citrus pectins, but it may not provide sufficient steric effects due to its
relatively thin hydration layer (Maravić et al., 2019). As noted, in
addition to the methyl ester, acetyl groups, and ferulic acid moieties,
protein is also one of the important ingredients that help SBP to exert its
emulsifying properties, but it is not resistant to high-temperature
treatment (Chen, Qiu, et al., 2016; Ma et al., 2013). In addition,
pectin itself is not stable under heat due to beta-elimination or acid
hydrolysis depending on the pH (De Roeck, Mols, Duvetter, Van Loey, &
Hendrickx, 2010). Moreover, different structural characteristics of
pectins also have an impact on the emulsifying and emulsion stabilizing
potential. Verkempinck et al. (2018) found that the hydrodynamic
volume and adsorbed layer thickness of CP10 (citrus pectin with DM of
10%) were larger compared to CP38 (citrus pectin with DM of 38%) and
CP82 (citrus pectin with DM of 82%) at neutral pH due to the presence of
more chargeable carboxylic groups. Schmidt et al. (2015) showed that
citrus pectins with different esterification degrees exhibited excellent
emulsifying properties. Notably, pectin with a high DM was character­
ized by a smaller hydrodynamic radius and faster adsorption kinetics,
leading to the stabilization of smaller droplet sizes. Therefore, the sta­
bility of emulsions prepared from pectins required multiple consider­
ations. These studies are of great significance in meeting the market
demand for pectin as an emulsifier and enriching the types of pectin raw
materials. Therefore, exploiting pectin with potential commercial ap­
plications from other plant resources is necessary.
Persimmon (Diospyros kaki L.) is widely cultured in many Asian
countries, especially with China as the top producer and cultivation
worldwide (FAO, 2020). Studies have shown that persimmon is an
abundant source of pectin (Chen, Qiu, et al., 2016; Du et al., 2020). In
recent years, most studies on persimmon pectin have been an extraction
optimization design (Méndez et al., 2022; Nguyễn & Savage 2013),
while less research focused on its physicochemical and functional
properties. Jiang, Xu, Li, Li, and Huang (2020) optimized the extraction
conditions of persimmon peel pectin and found that persimmon peel
pectin exhibited stronger emulsifying behavior than commercial citrus
and apple pectin, but the emulsification mechanism was lacking. Our
preliminary experimental results indicated that persimmon pectin (PP)
exerts excellent emulsifying properties and is likely to be a superior
source of emerging natural emulsifiers. Structural elucidation indicated
that structural characters including high methyl-esterified (71.93 ±
1.23%), lower molecular weight (4.789 × 104 g/mol), and abundant
hydrophobic substances including acetyl groups (1.21 ± 0.09%), pro­
tein (11.54 ± 0.75 mg/g), and ferulic-acid moieties (2.47 ± 0.13 mg/g)
were found in PP. Additionally, PP was enriched in both HG (62.43 ±
1.12%) and RG-I (17.75 ± 0.48%) domains, favoring PP fractions
adsorbed at the oil-water interface.
Since emulsions need to remain stable during preparation, trans­
portation, and storage, the responsiveness of the emulsifier to the
environment during actual application is crucial. For instance, the
emulsion prepared from SBP is unstable at high temperatures, mainly
due to protein denaturation in sugar beet pectin that exerts emulsion
stability (Funami et al., 2011). Therefore, the study aimed to further
study the responsiveness of PP-stabilized emulsion to the environment
(presence of salt-ions, temperature conditions including heat treatment,
thermal cycle treatment, freeze-thaw treatment and accelerated centri­
fugation process). The emulsification mechanism of PP was also
explored by multiscale characterization methods including triple-phase
contact angle evaluation and adsorption layer characterization of pectin
2
Food Hydrocolloids 131 (2022) 107727
samples and super-resolution imaging of pectin-stabilized emulsions.
This work will provide scientific evidence for understanding the mech­
anisms of the emulsifying property of PP and its promising industrial
uses in the future.
2. Materials and methods
2.1. Materials
Fresh persimmon fruit (Diospyros kaki) was obtained from a local
market (Baishazhou Market, Wuhan city, China). Three commercial
pectins, including sugar beet pectin (SBP, Mw: 4.613 × 105 g/mol; DM:
68.29 ± 1.38%; DA: 1.44 ± 0.14%; Protein content: 21.32 ± 1.41 mg/g)
and citrus pectin of low methyl-esterification (LM, Mw: 1.179 × 106 g/
mol; DM:30.25 ± 0.39%; DA: 0.67 ± 0.13%; Protein content: 8.79 ±
0.63 mg/g), were provided from CP Kelco (Lille Skensved, Denmark),
and commercial citrus pectin of high methyl-esterification (HM, Mw:
6.443 × 105 g/mol; DM: 59.38 ± 1.01%; DA: 0.33 ± 0.05%; Protein
content: 6.42 ± 0.37 mg/g) was obtained from Aladdin (Shanghai,
China) for comparative research. Calcofluor white dye was purchased
from Sigma-Aldrich (Shanghai, China). Nile red dye was obtained from
Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Medium-
chain triglyceride (MCT) (Biosharp, Hefei, China) was used as the
emulsions’ oil phase. All the other chemicals were of analytical grade
and were purchased from Sinopharm Chemical Reagent Co., Ltd.
(Shanghai, China). The QCM-D sensors with a gold electrode (QSX 301,
5 MHz) were obtained from Q-Sense (Biolin Scientific AB, Sweden). The
polystyrene latex particles (Concentration: 5.0%, w/v; Preservation so­
lution: pure water (containing 0.05% w/v Proclin 300); ζ-potential
(diluted with MilliQ water (0.5 mL PS + 9 mL of water)): − 53.76 ± 1.70
mV; Z-average: 150 nm) were obtained from Suzhou Nanomicro Tech­
nology Co., Ltd. (Suzhou, China). Ultrapure water purified by a Milli-Q
system (resistivity of 18.2 MΩ).
The pectin extraction from persimmon fruit was conducted as fol­
lows. Briefly, persimmons were lyophilized and ground into powder
form and mixed with a 30-fold solution of pH 1.90 adjusted with 1 M
hydrochloric acid. Next, the mixture was placed in a water-bath at
91.9 ◦ C for 150 min with constant stirring at 500 rpm. The supernatants
were centrifuged (8000×g, 30 min) and concentrated by rotary evapo­
ration until achieving one-fifth of their original volume. Then, 2-fold
anhydrous ethanol was added to the pectin precipitation solution and
kept overnight at 4 ◦ C. Subsequently, the pectin precipitate was
centrifuged at 8000×g for 30 min and consecutively washed with
ethanol and acetone several times to remove low molecular weight and
polar compounds including soluble sugars, tannins and pigments (Wil­
lemsen et al., 2017; Faravash & Ashtiani, 2008). The removing effect
was confirmed by DNS and Folin-Ciocalteu methods, respectively.
Finally, the precipitate was freeze-dried at − 50 ◦ C for 48 h and then
milled, sieved, and stored in an airtight desiccator until further analysis.
2.2. Preparation of pectin-stabilized emulsions
2.2.1. The preparation of emulsions without treatment
80 mL of pectin aqueous solutions (1.0%, w/v) were mixed with 20
mL of MCT to obtain the oil to solution ratio of 1:4 (v/v) and pre-
homogenized using a T18 digital Ultra Turrax mixer (IKA, Staufen,
Germany) at 10000 rpm for 2 min, followed by passing through a high-
pressure microfluidizer processor (Microfluidics, Newton, USA) at
14000 psi for 3 passes. Sodium azide (0.02%) was added as the micro­
bial preservative. The pH values of the pectin solutions (1.0%, w/v) and
pectin-stabilized emulsions (initial and diluted system) are supple­
mented in Table S1.
2.2.2. The preparation of samples modified by the salt addition
The freshly prepared emulsions (10 mL) were placed in individual
test tubes. 6 mol/L NaCl and CaCl2 stock solutions were prepared for the
Y. Jia et al.
subsequent experiments. 2 mL salt solution was added to the test tubes
to obtain emulsions with a range of salt concentrations (0, 100, 200,
500, and 1000 mM NaCl or CaCl2), and the final oil to water phase ratio
was 1:5 (v/v). After salt-addition treatment, the average droplet size, Z-
average, zeta-potential, microscopic observation, turbidity, and SRI in­
dicators of emulsions were measured. Notably, the zeta-potential and Z-
average were determined for the samples diluted with MilliQ water
(1:100, v/v), i.e., the salt concentration was 0, 1, 2, 5, and 10 mM,
respectively. In addition, the turbidity and stability ratio index (SRI)
were measured for the samples diluted with MilliQ water (1:400, v/v), i.
e., the salt concentration was 0, 0.25, 0.4, 1.25, and 2.5 mM,
respectively.
2.3. Physical treatments applied to destabilize the emulsions
2.3.1. Effect of temperature
2.3.1.1. Effect of heat treatment. The freshly prepared emulsions were
transferred into individual test tubes and then incubated in a water bath
for 60 min at a fixed temperature ranging from 50 to 100 ◦ C. Subse­
quently, the samples were immediately cooled to ambient temperature
by submerging them within ice water. After thermal treatment, the
average droplet size, zeta-potential, and microscopic observation of
emulsions were measured.
2.3.1.2. Thermal cycle treatment. Emulsions were sampled into the
closed cell of Rheolaser Crystal (Formulaction, Toulouse, France) for
determination. Rheolaser Crystal measurement took 2 h. After a 10 ◦ C/
min ramp from 25 to 60 ◦ C with a holding time of 2.5 min, the same
temperature ramp decreased from 60 to 25 ◦ C with a holding time of 2.5
min was conducted. For a quantitative treatment of the data, the area Ai
under the peaks was calculated and then normalized using the following
equation:
Relative area (%) = [-(1-Ai)/A1)] × 100
where A is the integrated area under the peak, i is the cycle number.
2.3.1.3. Effect of freeze-thaw treatment. Emulsion samples were frozen
at − 20 ◦ C overnight and thawed at room temperature. After freeze-thaw
treatment, the average droplet size of emulsions was measured.
2.3.2. Effect of accelerated centrifugation on the stability of emulsions
The stabilization behaviors (creaming stability) of pectin emulsions
were evaluated by the LUMisizer (LUM GmbH, Berlin, Germany)
employing centrifugal separation to accelerate the occurrence of insta­
bility phenomenon (Ma et al., 2017). Emulsions were subjected to a
rotational speed of 1000×g at 25 ◦ C. About 1000 transmission profiles
were recorded at time intervals of 15 s. Normalized transmission (%)
versus position (mm) in the scale range (110 mm–130 mm) graphs were
obtained. The instability index, a factor calculated by the software, was
given to quantify transmission clarification and evaluate emulsions’
instability.
2.4. Evaluation of the emulsion potential
2.4.1. Measurement of average droplet size
The droplet size (distribution) of emulsions was evaluated using a
Mastersizer 2000 (Malvern Instruments, Worcestershire, UK). The par­
ticle size was calculated as the surface-weighted mean diameter (D32)
and volume-weighted mean diameter (D43). The refractive indexes for
the oil and water were 1.45 and 1.33, respectively.
2.4.2. Zeta-potential and Z-average measurements
The Z-average, Polydispersity Index (PdI), and zeta-potential values
of emulsions were measured using the Zetasizer Nano ZS (Malvern
3
Food Hydrocolloids 131 (2022) 107727
Instruments, Worcestershire, UK) at 25 ◦ C. Before analysis, emulsion
samples were diluted in MilliQ water at a ratio of 1:100 (v/v) to prevent
multiple scattering effects. Notably, the concentrations of salts after
dilution were 1, 2, 5, and 10 mmol/L. An individual measurement was
conducted at least 10 sub-readings and 3 readings from an individual
sample. The zeta-potential and Z-average measurements were imple­
mented in triplicate and the results were reported as mean and standard
deviation.
2.4.3. Micromorphology observation
A confocal scanning laser microscope (FV3000, Olympus, Japan)
was used to visualize the microstructure of the emulsions stabilized by
pectins. The oil droplets were stained with Nile red fluorescent dye
(excitation, 543 nm; emission, 605 nm), and Calcofluor white dye
(excitation, 405 nm; emission, 407–492 nm) was used to stain the pectin
fraction. In detail, 5 μL of Nile red dye (1 mg/mL) and 10 μL Calcofluor
white dye were added to 1 mL emulsion samples (10-fold dilution) (Zhu
et al., 2020). Before CLSM observation, a drop of the dyed emulsion was
placed on a confocal microscope slide, covered with a coverslip, and
immediately examined with a 100 × oil magnification objective lens.
2.4.4. Turbidity and stability ratio index (SRI) measurement
The turbidity of emulsions (400-fold dilution) was measured for
absorbance at 600 nm (Absλ = 600) with a UV-1800 spectrophotometer
(Shimadzu Corporation, Kyoto, Japan). Turbidity evaluation is a simple
yet indirect study of emulsifying properties. The higher the turbidity
indicated the greater emulsifying property. The stability ratio index
(SRI) is one of the indicators to calculate emulsion stability by testing the
absorbance of diluted samples at 800 and 400 nm:
SRI = Absλ = 800/Absλ = 400
The larger the SRI value, the more unstable the emulsion. According
to previous studies (Hu et al., 2020; Klein, Aserin, Svitov, & Garti, 2010),
0.3 is used as the dividing line to describe the emulsion stability
changes: when the SRI value < 0.3, the emulsion is relatively stable;
while the SRI value > 0.3, the emulsion tends to be unstable.
2.5. Mechanism exploration on the emulsifying properties of persimmon
pectin
2.5.1. Triple-phase contact angle measurement
The triple-phase contact angle of pectin samples was evaluated by a
dataphysics (OCA 20, Dataphysics Instruments GmbH, Germany) using
the method of Zhang, Liang, Li, Wei, and Li (2020) with slight modifi­
cation. The freeze-dried samples (150 mg) were compressed to thin
tablets with a thickness of 1 mm and a diameter of 13 mm. The tablets
were immersed in MCT (15 mL), and a drop of water (2 μL) was
deposited to the surface of the tablets. After about 3 s to reach equilib­
rium, the droplets were photographed, and the triple-phase contact
angle was calculated by OCA 20 software. Measurements were averaged
over at least three droplets. The measurement was conducted at room
temperature.
2.5.2. Determination of adsorption thickness
The adsorbed layer thickness of the pectins onto the polystyrene
latex particles (PS microsphere) was determined to investigate the
possible pectin organization at the droplet surface by using the Zetasizer
Nano ZS (Malvern Instruments, Worcestershire, UK). A 1.0% v/w pectin
stock solution was prepared and left overnight on a roller-mixer at room
temperature. 9.0 mL of pectin solutions at 0.01%–0.5% concentrations
were added to 0.5 mL polystyrene latex dispersion and equilibrated at a
quiescent condition for 30 min. Although the particle size of the naked
latex bead was 150 nm according to the information provided by the
distributor, the z-average size measured using the Zetasizer was 151.43
± 3.43 nm (PdI 0.018, PdI width 19.06 nm). The thickness of the
Y. Jia et al.
adsorbed pectin layer on the polystyrene microspheres was calculated
based on the difference in average diameter before and after adsorption.
To calculate the number of adsorbed layers, the hydrodynamic diameter
of polysaccharide was determined using the dependence of the Z-
average on the pectin concentration and the extrapolation to zero
(Verkempinck et al., 2018).
2.5.3. Characterization of the adsorbed layer
The adsorption behaviors of pectin samples on the oil surface were
investigated using a quartz crystal microbalance with dissipation
monitoring (QCM-D) model from Q-Sense (Biolin Scientific AB, Swe­
den). The oil film was introduced on the gold surface through the spin
coating to endow it with hydrophobicity. In brief, 0.5% w/v MCT in
chloroform was prepared, and 300 μL of the solution was spread on a
sensor and then spin-coated at 2000 rpm for 30 s. Then, the sensors were
dried at 40 ◦ C for 12 h. QCM-D assessments were conducted at 25 ◦ C at a
flow rate of 80 μL/min. The flow schedule settings were set as follows: (i)
the ultrapure water was employed to establish a signal baseline. (ii)
pectin solutions (1.0%, w/v) were used for adsorption measurements,
frequency (Δf) and energy dissipation (ΔD) changes were recorded with
time for about 60 min (iii) The solution was replaced by ultrapure water
to rinse away unbound or weakly bound pectins.
2.5.4. Microstructure observation by cryogenic-scanning electron
microscopy (Cryo-SEM)
The microstructure of emulsions was visualized using cryo-scanning
electron microscopy (SU8000, Hitachi, Japan). First, emulsion samples
were selected and placed in the slots of a stub, plunge-frozen in liquid
nitrogen, and transferred into the cryo-preparation chamber and frac­
tured. Then, the samples were sublimated at − 70 ◦ C for 5 min under
controlled vacuum conditions and sputtered with platinum using a
current of 10 mA for 60 s. The samples were scanned by Cryo-SEM at the
stage temperature of − 140 ◦ C and an accelerated voltage of 3 kV.
2.6. Statistical analysis
All experiments and measurements were implemented in triplicate
and the results were reported as mean and standard deviation. The
variance (ANOVA) of data was further analyzed using a Duncan test
(significance level at p < 0.05) by SPSS software (Version 18.0, IBM
Corp., Armonk, NY, USA).
3. Results and discussion
3.1. Characterization of pectin solutions
The pH levels of pectin solutions (0.5% and 1.0, w/v) were shown in
Table S1. For the pectin solutions (1.0, w/v), except for LM, other three
pectin samples (PP, HM, and SBP) were maintained in a range between
(3.2–3.4). Except for the structure, the pH of pectin was affected by the
extraction procedure. According to the manufacturer, SBP and HM in the
study were obtained by the hot-acid procedure. PP was also obtained by
the hot-acid method in the present study. Therefore, they exerted similar
low pH values. LM had higher numbers of carboxyl groups, but showed
higher pH value, possibly being due to the formation of intra- and
intermolecular hydrogen bonds and preventing release of H+ (Yang,
Nisar, Hou, et al., 2018; Yang, Nisar, Liang, et al., 2018). Viscosity is one
of the important characteristics of pectin, which might be related to
emulsification stability (Humerez-Flores et al., 2022). Generally,
increased viscosity of the continuous phase could suppress oil droplet
mobility, thus impeding droplet coalescence and slowing down cream­
ing. As revealed in Supplementary Fig. S1, PP (1.0%, w/v) solution
exhibited the lowest viscosity, while commercial pectin dispersions
(1.0%, w/v) showed higher viscosity than PP within the measured
shear-rate range. The viscosity of the PP showed to be less dependent on
the shear rate (close to Newton fluids), while the commercial pectins had
4
Food Hydrocolloids 131 (2022) 107727
a more pseudoplastic behavior (shear thinning). Jiang et al. (2019)
investigated the viscosity of pectin extracted from persimmon peel and
compared with commercial citrus pectin, and found that persimmon
peel pectin (1.0%, w/v; around 17 mPa s at the shear rate of 10 s− 1)
exhibited lower viscosity than that of commercial citrus pectin (around
85 mPa s at 10 s− 1), but it exhibited good emulsifying potential and
could stabilize 50% (v/v) oil fraction. Similarly, The viscosity of PP
solution (1.0%, w/v) in our study at a shear rate of 10 s− 1 was 11.29
mPa s.
To understand the influence of viscosity during the production of
emulsion droplets, the relationship between the critical Weber number
and the ratio of the dispersed phase’s viscosity (ηd) to the continuous
phase’s viscosity (ηc) is an effective tool (Humerez-Flores et al., 2022;
Abbott, 2015). Weber number is a dimensionless number used in fluid
dynamics (such as emulsion and atomization process) which is given by
the following Equation (1):
G⋅R⋅η
We = (1)
γ
where We is the Weber number, G is the shear rate, R is the radius of the
desired oil droplet, η indicates the viscosity of pectin solution (contin­
uous phase), and γ reflects the interfacial tension. As described by
Abbott (2015) and Berton-Carabin, Sagis, and Schroën (2018), the
Weber number could determine whether oil droplets were created and if
they could be of small size. A diagram exhibiting the relation between
We and ηd/ηc is shown in Supplementary Fig. S2. If the viscosity of the
dispersed phase is much lower than the continuous phase (ηd/ηc < 0.01),
the We number would be considerably high (3-10), signifying that the
process is not efficient (McClements, 2015). Additionally, if the viscosity
of the dispersed phase is significantly higher than the continuous phase
(ηd/ηc > 4), the We number becomes an infinite number, and it is hard to
form emulsion droplets (Abbott, 2015). In order to gain insight into the
viscosity results, the ηd/ηc ratio was calculated for pectin samples, as
shown in Table S2. The viscosity of MCT used (ηd) was 30 mPa s (Van
Aken, Vingerhoeds, & De Wijk, 2011). But the ηd/ηc values for PP, SBP,
HM, and LM are 2.66 ± 0.08, 1.25 ± 0.10, 0.80 ± 0.07, and 1.74 ±
0.20, respectively, all in the ηd/ηc range of 0.01 and 4, indicating that the
emulsion droplets could be created (Humerez-Flores et al., 2022).
Therefore, the effect of viscosity on the emulsifying properties of PP is
relatively small. Further, as shown in Equation (1), a reduction in
droplet size is also achieved by reducing the interfacial tension γ (Kaltsa,
Yanniotis, & Mandala, 2016). As shown in Table S2, the descending
ability order of pectins to reduce interfacial tension (γ) was PP > SBP >
LM > HM, suggesting that PP could diffuse and adsorb to the oil-water
interface more quickly than commercial SBP, HM, and LM.
3.2. Characterization of the freshly pectin-prepared emulsions
Particle size, Z-average, and turbidity of emulsions are important
indexes in evaluating the emulsifying behavior of pectins. The particle
size, including surface-weighted mean diameter (D32) and volume-
weighted mean diameter (D43), could indicate the emulsifying poten­
tial of pectin. As shown in Table S3, the D32 (0.55 ± 0.00 μm) and D43
(0.60 ± 0.00 μm) scales of the PP-prepared emulsion were significantly
smaller than commercial pectin emulsions (p < 0.05), signifying the
higher emulsifying potential of PP. The Z-average and PdI scales char­
acterizing the droplet’s hydrodynamic diameter and distribution width
(uniformity) could be determined by the Zetasizer (Shao, Ma, Zhu, &
Qiu, 2017). The PdI scale <0.2 indicated the uniformity and homoge­
neity of the emulsion (Wang et al. 2018). As shown in Table S3, the
Z-average (1054.83 ± 140.42 nm) and PdI scales (0.16 ± 0.08) of
PP-prepared emulsion were significantly smaller than commercial
pectin emulsions (p < 0.05), indicating the higher uniformity and
emulsifying potential of PP.
Turbidity evaluation is a simple yet indirect study of emulsifying
Y. Jia et al.
properties (Yadav, Johnston, Hotchkiss Jr, & Hicks, 2007). The higher
the turbidity indicated the greater the emulsifying property. As pre­
sented in Table S3, PP (Abs600 = 0.37 ± 0.00) presented significantly
higher turbidity than all commercial pectin emulsions (SBP: 0.34 ±
0.00; HM: 0.30 ± 0.00; LM: 0.13 ± 0.00), signifying the highest emul­
sifying potential.
In summary, the analysis of particle size, Z-average, and turbidity
evaluation indicated the superior emulsifying properties of PP than the
commercial pectins (HM, SBP, and LM).
3.3. The impact of surrounding environment on the stability of pectin-
prepared emulsions
Commercial emulsion-based food products are inevitably exposed to
a broad spectrum of environmental conditions during processing, stor­
age, and utilization (Jin et al., 2017; Zhu et al., 2020). Therefore,
investigating the influence of salt concentration, heating, and
freeze-thaw treatments on the physical stability of pectin-prepared
emulsions was necessary. The pH values of PP, SBP, HM, and LM sta­
bilized emulsions are 3.27 ± 0.03, 3.29 ± 0.03, 3.27 ± 0.03, and 4.69
± 0.04, as shown in Table S1. In addition, the pH values of the
pectin-stabilized emulsions diluted by Milli-Q water (1:100, v/v) are in
the range of 6.0–7.0.
3.3.1. The impact of salt concentrations on the stability of pectin-prepared
emulsions
Different salt concentrations might be encountered during food
Fig. 1. Effect of NaCl concentration on the properties of pectin-stabilized oil-in-water emulsions including (A) volume-weighted mean diameter D43, (B) turbidity,
(C) SRI value, and (D) ζ-potential value. Note: PP: Persimmon pectin; SBP: Sugar beet pectin; HM: High methyl-esterified citrus pectin; LM: Low methyl-esterified
citrus pectin. Before ζ-potential measurements, emulsion samples were diluted in MilliQ water at a ratio of 1:100 (v/v) to prevent multiple scattering effects (Fig. 1D).
5
Food Hydrocolloids 131 (2022) 107727
production; therefore, the impact of salt concentrations on the stability
of pectin-prepared emulsions must be investigated (Hosseini-Parvar,
Osano, & Matia-Merino, 2016). It is crucial that emulsion droplets are
made as small as possible to minimize gravity creaming effects (Pi, Liu,
Guo, Guo, & Meng, 2019). The droplet size could indicate the emulsi­
fying behavior of pectin. D43 was more sensitive to the change in droplet
size in the process of flocculation or aggregation (Wang, Meng, Zhang, &
Guo, 2021). Accordingly, in this study, D43 was reported as the average
droplet size of emulsions. Generally, a smaller D43 value represented
better emulsifying performance (Pi et al., 2019).
The dependence of D43 on the salt concentration (NaCl and CaCl2) for
pectin-stabilized emulsions was shown in Figs. 1A and 2A, respectively.
The droplet size of four formulated pectin emulsions increased with
increasing salt levels, signifying the reduction of emulsifying stability.
The pectin-prepared emulsions are unstable in the presence of electro­
lytes, consistent with previous research (Nakauma et al., 2008). For
PP-stabilized emulsion, the D43-value augmented with increasing salt
concentration from 0.60 to 4.17 μm and 9.10 μm upon 1 mol/L CaCl2 or
NaCl treatment, respectively, indicating that droplet aggregation and
coalescence might occur as the salt concentration increased. Compared
with PP, the oil droplets covering SBP were almost equivalent to PP
upon 1 mol/L CaCl2 and NaCl (from 0.76 to 3.38 μm and 10.21 μm,
respectively). It is interesting that for the emulsions stabilized with PP
and SBP at the highest concentration of salt, the D43 for CaCl2 treatment
was lower than for NaCl treatment. In the presence of CaCl2 the floc­
culation process may have occurred. However, the flow of samples
through the measuring cell might lead to disaggregation. Nakauma et al.
Y. Jia et al.
Fig. 2. Effect of CaCl2 concentration on the properties of pectin-stabilized oil-in-water emulsions including (A) volume-weighted mean diameter D43, (B) turbidity,
(C) SRI value, and (D) ζ-potential value. Note: PP: Persimmon pectin; SBP: Sugar beet pectin; HM: High methyl-esterified citrus pectin. Before ζ-potential mea­
surements, emulsion samples were diluted in MilliQ water at a ratio of 1:100 (v/v) to prevent multiple scattering effects (Fig. 2D). It was not possible to obtain the
data for LM pectin under CaCl2 treatment (strong gel formation).
(2008) reported the salt sensitivity (NaCl and CaCl2) of SBP-prepared
emulsions and thought that the effect of salt ions on the stability of
the emulsions was due to the increase in droplet size caused by charge
screening.
The droplet size distribution curves combining CLSM images of
emulsions were further used to elucidate what extent the flocculation,
aggregation, and coalescence of emulsion droplets induced by salt ions
(Figs. 3 and 4). Compared with the freshly prepared emulsion (Fig. S3),
increasing the salt concentration shifted the size distribution to the right
axis (large droplet size) and larger round-shaped droplets were observed
by CLSM, indicating that droplet aggregation and coalescence in emul­
sions occurred. In the absence of salt ions (freshly prepared emulsions),
the mono-modal particle size distribution of PP-prepared emulsion was
mainly located in a narrow range of 0.1–1 μm, implying a trend of high
stability (Fig. S3A). However, the droplets in PP-stabilized emulsion
coalesced into large oil droplets in the presence of salt ions, agreeing
with the change in the particle size distribution well (Figs. 3A and 4A).
Interestingly, several small droplets of SBP-prepared emulsion floccu­
lated together in the existence of Ca2+, showing a destabilizing mech­
anism different from other emulsions (Fig. 4B). As for the HM and LM
emulsions, the droplets were clearly visible in CLSM images because of
their sizable D43 value and wide particle distribution in the presence of
salt ions (Fig. 3C, D, and 4C), indicating the trend of high instability.
The effect of NaCl and CaCl2 concentration (1–10 mM, the diluted
system) on the Z-Average and PdI value of pectin-stabilized oil-in-water
emulsions were also investigated. Fig. S4 represented the evolution of
6
Food Hydrocolloids 131 (2022) 107727
the Z-average and Polydispersity Index (PdI) at different salt concen­
trations (A and B - NaCl, C and D - CaCl2). With an increase of NaCl and
CaCl2 concentration to 10 mmol/L, an augment in Z-average of PP-
stabilized emulsion from 1054 to 1355 nm and 2329 nm was
observed. As a comparison, the Z-average of SBP-prepared emulsion
changed from 1688 to 2061 nm upon 10 mmol/L NaCl and 1868 nm
upon 10 mmol/L CaCl2. Interestingly, consistent with the emulsion
particle size (D43) results determined by the diffraction method, the Z-
Average change of PP-prepared emulsion after NaCl treatment was
smaller than that of the SBP-stabilized emulsion while the opposite trend
was observed in the presence of CaCl2. In addition, the PdI of PP
emulsions was increased from 0.16 to 0.42 upon 10 mmol/L NaCl and
0.75 upon 10 mmol/L CaCl2 treatment. The observed increase in PdI of
PP-stabilized emulsion under salt treatment might be associated with
the formation of mono-populations due to the growth of larger droplets
and droplet coalesce.
Pectin is an anionic cell wall polysaccharide that is known to interact
with divalent cations through its non-methyl esterified galacturonic acid
units (Celus, Lombardo, Kyomugasho, Thielemans, & Hendrickx, 2018).
The divalent cation-binding capacity of pectin was affected by several
factors, including DM, DB, chain length, acetylation, etc (Celus, Kyo­
mugasho, Van Loey, Grauwet, & Hendrickx, 2018). Among them, DM
has received widespread attention as it is considered the leading factor
affecting the cation-binding ability. Compared with higher DM pectin,
lower DM pectins exhibited a higher charge density because of the larger
number of negatively charged carboxyl groups (non-methyl esterified
Y. Jia et al. Food Hydrocolloids 131 (2022) 107727

Fig. 3. Confocal laser scanning microscopic images combined with particle size distribution curves of pectin emulsions processed by 1000 mM NaCl: (A) PP; (B) SBP;
(C) HM; (D) LM (Subindex 1: Pectin phase; Subindex 2: Oil phase; Subindex 3: Merged image of pectin and oil phase; Subindex 4: Particle size distribution curves;
Scale bar: 10 μm). Note: PP: Persimmon pectin; SBP: Sugar beet pectin; HM: High methyl-esterified citrus pectin; LM: Low methyl-esterified citrus pectin.

carboxyl groups), which indicated that the cation-binding ability of
pectin increased with decreasing DM (Celus et al., 2017). Moreover, the
relatively severe coalescence observed for PP-prepared emulsion under
Ca2+ treatment compared to SBP might be interpreted by its lower DA
value, because the lower the DA value (Table S4) the stronger the
electrostatic binding force between Ca2+ and dissociated carboxyl
groups of GalA (Pi et al., 2019; Ralet, Crépeau, Buchholt, & Thibault,
2003). Therefore, it was inferred that PP with a relatively higher DM
(71.93%) and lower DA (1.21%) value exhibited a weaker ability to bind
divalent cations (Ca2+), resulting in worse D43, turbidity, and SRI values
of the emulsion than that of SBP (Fig. 2A, B, and C).
With increasing salt concentrations, the electrostatic repulsion
decreased due to the electrostatic screening effects, further leading to
the accumulation of droplets (Hosseini-Parvar et al., 2016). The ζ-po­
tential could reflect the electrostatic repulsion between droplets, and the
large absolute ζ-potential value indicated high electrostatic stabilization
(Zhao et al., 2020). Before ζ-potential measurements, emulsion samples
were diluted in MilliQ water at a ratio of 1:100 (v/v) to prevent multiple
scattering effects. Therefore, the actual values of salt concentrations in
the ζ-potential measurement were 1–10 mmol/L. As portrayed in
Fig. 1D, the ζ-potential of tested emulsions had less negative value with
increasing salt concentration. As for PP-stabilized emulsion, the absolute
value of ζ-potential decreased from 36.18 to 31.41 mV upon 10 mM
NaCl treatment (100-fold dilution in ζ-potential detection). Although
the absolute ζ-potential of PP-prepared emulsion was lower than that of
SBP-prepared emulsion upon adding NaCl, it was still higher than 30
mV, showing the high electrostatic stability. In addition, the absolute
ζ-potential values of HM and LM emulsions were less than 30 mV,
signifying the medium electrostatic stability (Ngouémazong, Chris­
tiaens, Shpigelman, Van Loey, & Hendrickx, 2015). As depicted in
Fig. 2D, in the case of multivalent counterions (Ca2+), the ζ-potential of
emulsions showed fewer negative values, which might be due to the
ion-binding effect. A higher decrease in the surface charge of Ca2+
compared to Na+ was observed in all emulsion samples without signif­
icant differences (Yao, Wang, Liu, Hou, & Wang, 2021). Furthermore,
monovalent ions only screen the electrostatic forces, while multivalent

7
Y. Jia et al.
Fig. 4. Confocal laser scanning microscopic images combined with particle size distribution curves of pectin emulsions processed by 1000 mM CaCl2: (A) PP; (B) SBP;
(C) HM (Subindex 1: Pectin phase; Subindex 2: Oil phase; Subindex 3: Merged image of pectin and oil phase; Subindex 4: Particle size distribution curves; Scale bar:
10 μm). Note: PP: Persimmon pectin; SBP: Sugar beet pectin; HM: High methyl-esterified citrus pectin.
ions might additionally adsorb to the surface of the emulsion droplets
and consequently neutralize the surface potential (Ralla et al., 2018).
Our study is well consistent with the study of Shao et al. (2017) and X.
Yang et al. (2018), who suggested that a high salt-ion could decrease the
electrostatic repulsion between emulsion droplets, and therefore emul­
sions tended to be less stable. In addition, the formation of bridging
between pectins and the Ca2+ cross-linking effect might occur under
Ca2+ treatment (Chan, Choo, Young, & Loh, 2017). According to Ai,
Meng, Lin, Tang, and Guo (2022) and J. Yang et al. (2018), the acetyl
groups might weaken the calcium-induced emulsion flocculation, thus
minimizing pectin flocculation and allowing free pectin to react with oil
particles to stabilize the emulsion. Thus, the PP-stabilized emulsion
experienced a higher increase in the mean droplet size than that of
SBP-prepared emulsion under Ca2+ treatment, which might be ascribed
to the steric hindrance difference from the acetyl groups (1.44 ± 0.14%
of SBP and 1.21 ± 0.09% of PP, shown in Table S4).
Turbidity and stability ratio index (SRI) evaluation of emulsions are
simple yet indirect indexes of emulsifying properties, and higher
turbidity and lower SRI value implicate higher emulsion stability
(Yadav, Johnston, Hotchkiss Jr, & Hicks, 2007). The turbidity and SRI
value of emulsions as a function of salt concentration were shown in
Fig. 1B and C and Fig. 2B and C, respectively. As the salt concentration
increased, the turbidity of all emulsions reduced, and the SRI value
increased significantly (p < 0.05). Notably, the SRI values of SBP and PP
emulsions were higher than 0.3 when salt concentrations were above
0.25 mM (the diluted system), implying that the emulsions became
unstable. The SRI values of the HM and LM emulsions without salt ion
treatment were higher than 0.8, indicating high instability. Although the
8
Food Hydrocolloids 131 (2022) 107727
turbidity and SRI value changed considerably in the presence of salt
ions, the PP-stabilized emulsion exhibited relatively higher turbidity
and lower SRI value compared with commercial pectins (SBP, HM, and
LM) prepared emulsions, indicating relatively high emulsifying stability.
Taken together, increasing the salt concentration inevitably led to an
augment in the particle size (D43) and Z-average and a decrease in ab­
solute ζ-potential and turbidity of PP-stabilized emulsion. However, PP-
prepared emulsion exhibited better stability under high concentrations
of salt ions, showing high absolute potential value, turbidity, and low
SRI value. Therefore, electrostatic repulsion might be one of the expla­
nations for the emulsification stability of PP.
3.3.2. The impact of heat treatment on the stability of pectin-prepared
emulsions
The thermal stability of the pectin-prepared emulsions could be
attributed to the thermal resistance of pectin (Hua et al. 2019). The
knowledge of the thermal behavior of pectins is crucial for their prac­
tical industrial application (Zhang et al., 2021). TG and DTG were used
to evaluate the thermodynamic properties of the pectin samples, as
illustrated in Fig. 5. The TG curve records the weight loss of pectins
during heating, and the samples revealed three stages of the weight loss
process (Fig. 5A). The first stage, ranging from 30 to 150 ◦ C, was
ascribed to water loss. The second stage, which indicated rapid mass loss
ranged from 130 to 350 ◦ C, was associated with the de-polymerization
and decomposition of the pectin molecules. Notably, PP exhibited the
highest temperature (312.38 ◦ C) at 50% weight loss, which was higher
than that of commercial pectins (268.78–289.06 ◦ C), indicating PP
exhibited better thermal stability than commercial pectins. LM had the
Y. Jia et al.
Fig. 5. Thermograms of pectins: (A) TG curves; (B and C) DTG curves. Note: PP: Persimmon pectin; SBP: Sugar beet pectin; HM: High methyl-esterified citrus pectin;
LM: Low methyl-esterified citrus pectin.
lowest temperature at 50% weight loss, indicating LM was the least
stable among the four samples. And the third stage was in the range of
350–600 ◦ C, relating to the thermal decomposition of carbon (Ai et al.,
2022). The DTG curve is the first derivation of the TG curve and reflects
the thermal degradation velocity of detected samples (Qin, Liu, Cheng,
& Wang, 2019). Two notable inflection points were observed in the DTG
curves of all pectin samples (Fig. 5B). The inflection point occurring
around 100 ◦ C was related to water loss. The second inflection point,
which occurred close to 200 ◦ C, was caused by pectin degradation
(Huang, Zong, & Lou, 2022). PP had the highest temperature
(240.40 ◦ C) corresponding to the maximum weight loss rate compared
with commercial pectins (228.23–235.33 ◦ C), and the residual mass
ratio of PP (26.28%) after the three-stage process was higher than that of
commercial pectins (SBP: 25.25%; HM: 22.12%; and LM: 22.79%),
suggesting that PP has higher thermal stability compared with com­
mercial pectins (Zhang et al., 2021). Overall, PP exhibited good thermal
stability, which might be related to its abundant RG-I content (17.75 ±
0.48%, shown in Supplementary Table S4) (Deng et al., 2020). In
addition, there was a certain potential relationship between thermal
stability and DM of pectins, and pectin with higher DM values might
possess stronger thermal stability (Liang, Liao, Qi, Jiang, & Yang, 2022).
As shown in the Supplementary Table S4, the DM value of PP (71.93 ±
1.23%) is higher than that of commercial pectins, which might
contribute to its thermal stability. Thermogravimetric analysis indicated
that PP could be used for processing involving high-temperature pro­
cesses in the food industry.
In many commercial applications, emulsions must withstand heat
9
Food Hydrocolloids 131 (2022) 107727
treatments during manufacturing (Ralla et al., 2018). The heat treat­
ment process is simulated by heating at 50–100 ◦ C for 1 h to investigate
the impact of heat treatment on the stability of pectin-prepared emul­
sions. As shown in Figs. S5A and B, the droplet size of the emulsions
changed slightly within the medium temperature range (50–70 ◦ C),
while it was increased significantly at 90 and 100 ◦ C.
The D43 value of PP-stabilized emulsion increased from 0.60 to 3.40
μm as the treating temperature progressed, exhibiting a certain degree of
droplet aggregation in the CLSM image (Fig. S6A). Similarly, Ralla et al.
(2018) found that the droplet size (D43) of emulsions stabilized by oat
bran extract increased from 0.83 μm (5 ◦ C) to 2.30 μm at 90 ◦ C.
Compared with the PP-stabilized emulsion, thermal treatment substan­
tially increased the mean droplet size (D43) of the SBP-prepared emul­
sion from 0.76 to 8.53 μm, which might be due to protein denaturation
at high temperature (Ma et al., 2013). H. Chen, Qiu, et al. (2016) found
that the droplet size of SBP-prepared emulsions changed slightly within
50–80 ◦ C treatment while increasing significantly at 90 ◦ C. Ralla et al.
(2017) indicated that the droplet size of the SBP-prepared emulsions was
unstable when heated up to 90 ◦ C, as indicated by the change of particle
size (D43: 0.18–39 μm). Meanwhile, the size distribution of
SBP-emulsion droplets drastically changed to a large group after heating
treatment, appearing as flocculation and coalescence in the CLSM image
(Fig. S6B). In addition, citrus pectins HM and LM emulsions also
exhibited an increase in the particle size after heat treatment, which was
clearly visible in the CLSM image (Fig. S6C and D). In addition, the size
change of emulsions after heat treatment was determined by Z-average
in diluted systems. As shown in Fig. S7, the Z-average of the
Y. Jia et al.
PP-stabilized emulsion exhibited an insignificant increase after heat
treatment. Notably, the Z-average and PdI value of the SBP-prepared
emulsion increased significantly after 90 and 100 ◦ C treatments (p <
0.05), corresponding to the D43 changes well.
The electrostatic stabilization of the emulsions after heat treatment
was further evaluated. As charted in Fig. S5C, the absolute ζ-potential of
PP-stabilized emulsion (in diluted systems) showed an insignificant
change with temperature progressed (˃ ±30 mV), demonstrating high
electrostatic stability. By contrast, the absolute ζ-potential of SBP-
prepared emulsion under high-temperature conditions was signifi­
cantly reduced from 36.32 to 28.97 mV (p < 0.05), manifesting the
decreases in the electrostatic stability (±20–30 mV). This may be
because one of the emulsifying components of SBP, protein, was dena­
tured under high-temperature treatment (Funami et al., 2011). Addi­
tionally, the absolute ζ-potential of HM and LM emulsions decreased
significantly as the heating temperature increased. The electrostatic
stability of citrus pectin emulsions might be compromised as the citrus
pectin emulsions underwent some flocculation and coalescence with
increasing temperature. The flocculation of emulsion droplets mainly
relied on the balance of electrostatic repulsion and attraction between
oil droplets. Therefore, the increasing heating temperature might reduce
the electrostatic repulsion of the citrus pectin emulsions, resulting in less
negative ζ-potential values. Therefore, the PP-prepared emulsion
exhibited better stability to heat treatment, while the SBP-stabilized
emulsion was extremely sensitive to the thermal process. In addition,
further investigations of chemical and physical changes in the compo­
nents of emulsions are needed to explain the observed changes in
physicochemical properties of emulsions. These results suggested that
PP could be a potential emulsion stabilizer when heat is involved in the
production process, probably due to its rich HG and RG-I content, fa­
voring in forming a structured interfacial adsorption layer (as indicated
in the QCM-D analysis).
3.3.3. The impact of freeze-thaw treatment on the stability of pectin-
prepared emulsions
Freeze-thaw processing is an inevitable process in the transportation
and storage of some kinds of food such as ice cream, emulsion condi­
ment, yogurt, and mayonnaise, etc (Shi et al., 2020). Therefore, evalu­
ating the impact of freeze-thaw treatment on the stability of
pectin-prepared emulsions would be favorable to fully understand the
potential of PP as an emulsifying agent in frozen food. The particle sizes
(D32 and D43) of the emulsions were measured after freeze-thaw treat­
ment. As shown in Fig. S8, the particle sizes D32 and D43 of the
PP-stabilized emulsion substantially increased to 20 and 40 μm after
freeze-thawing treatment, while they were significantly lower than the
particle size values of the three commercial pectins stabilized emulsions
(D32 ˃ 30 μm and D43 ˃ 40 μm). The result indicated that PP had better
stability to freeze-thaw treatment compared with commercial pectins,
but it was also in a state of instability. Similarly, Zhuang et al. (2019)
found that the particle size of pomegranate pectin increased signifi­
cantly after freeze-thaw cycles, resulting in droplet aggregation. Ralla
et al. (2018) found that the droplet size (D43) of the emulsion prepared
by oat bran extract increased from 0.83 μm to approximately 40 μm after
freeze-thaw treatment. The thickness of the interfacial adsorption layer
played an important role in emulsion stability. Usually, the thicker the
interfacial adsorption layer, the better the freeze-thaw stability (Ghosh
& Coupland, 2008). Compared with commercial pectins, the interfacial
layer of PP is thicker and stronger (as suggested in PS simulation,
QCM-D, and Cryo-SEM analysis), which might contribute to its emulsion
stability.
3.3.4. The impact of thermal cycle and accelerated centrifugation
treatments on the stability of pectin-prepared emulsions
Rheolaser Crystal, which combined the sensitivity of diffusion wave
spectroscopy with the possibility of running thermal cycles, could be
used to monitor the change process of the product’s microstructure over
10
Food Hydrocolloids 131 (2022) 107727
time or temperature (Wan et al., 2018). The laser was scattered multiple
times by the sample’s microstructure, forming mutual interference op­
tical signals. Mathematical analysis of the signal variation generated
microscopic Dynamic (mD) curves as a function of temperature or time.
Generally, stable samples show slight variations in signal height,
whereas the signal height of unstable samples will increase or decrease
significantly due to the microstructure changes. Fig. 6A showed the mD
evolution of the pectin emulsions as a function of time over ten detection
cycles. The peak height of PP-prepared emulsion exhibited slight
changes within 10 testing cycles, indicating high stability. In contrast,
commercial pectin SBP, HM, and LM emulsions showed significant
changes in signal height, indicating the microstructure variations
occurred.
To further quantify these signal variations, the area changes were
evaluated to illustrate the thermal cycle stability of emulsions. Sub­
stantial changes in the peak area indicated low stability. As charted in
Fig. 6B, the relative area change of PP-prepared emulsion was small
(2.74%), indicating the high stability of PP-prepared emulsion after 10
heating up-cooling down cycles. Significant area variations were found
in SBP (− 20.46%) and HM (− 13.81%) emulsions, signifying that the
microstructures of SBP and HM emulsions did not return to their original
state after thermal cycle detection. In addition, LM-prepared emulsion
could be clearly distinguished from the considerable signal evolution
(− 79.97%), implying the weakest stability. Therefore, the pectin-
prepared emulsions could be ranked according to their stability after
2 h of rapid thermal-cycling evaluation in the order of PP > SBP > HM >
LM.
Because LUMiSizer analytical photo-centrifuge technique could
provide information about the separation kinetics during centrifugation
(Wei et al., 2020), this study further used this method to confirm the
physical stability difference of emulsions stabilized by different pectins.
The emulsion sample was less stable if the transmission changed more
frequently (Ma et al., 2017). As shown in Fig. 7, the slightest trans­
mission change was observed in the PP-stabilized emulsion, followed by
SBP-prepared emulsion, HM, and LM emulsions showed the greatest
transmission change. To quantify these transmission changes, the
instability index was calculated. The low instability index value was
referred to the high stability (Jin et al., 2017). As charted in Fig. 7E and
F, the order of instability index of the emulsion sample was LM > HM >
SBP > PP, consisting with the results of Rheolaser Crystal determination
well.
3.4. Mechanisms elucidation on the excellent emulsifying properties of PP
Our preliminary research revealed that quick adsorption to the oil-
water interface and forming a strong elasticity interfacial film might
be one of the crucial explanations for the unique emulsifying properties
of persimmon pectin. Therefore, this study further explored the detailed
interfacial characters of persimmon pectin by multi-techniques.
The evaluation of the contact angle provided helpful information on
predicting the capability of hydrocolloid-based materials (such as
polysaccharides and proteins) to adsorb onto the water-oil interface
(Jiang et al., 2019). Generally, within 90◦ , the higher the contact angle
values, the better adsorption capability on the water-oil interface. The
near-neutral wettability with a contact angle of around 90◦ endowed the
samples the balanced amphiphilicity with maximum desorption energy,
which will promote interfacial adsorption, thereby creating steric hin­
drance against the accumulation of oil droplets. Therefore, the contact
angle of the four pectins was firstly measured to judge their adsorption
performance. As shown in Fig. 8, the contact angle of PP was about 83◦ ,
indicating a strong desorption capacity and could produce steric hin­
drance to the droplets (Zhang et al., 2020). The high contact angle of PP
might be related to its high methyl-esterification degree (71.93%) and
relatively higher amounts of hydrophobic substances like acetyl group
(1.21%), protein (11.54 mg/g), and ferulic acid content (2.47 mg/g),
etc. (Table S4). The contact angle of SBP showed a similar contact angle
Y. Jia et al. Food Hydrocolloids 131 (2022) 107727

Fig. 6. Evolution of mD as a function of time (A) and evolution of the relative area for emulsion as a function of cycle number (B). Note: PP: Persimmon pectin; SBP:
Sugar beet pectin; HM: High methyl-esterified citrus pectin; LM: Low methyl-esterified citrus pectin.

Fig. 7. Evolution of transmission profiles of pectin-stabilized emulsions (A: PP; B: SBP; C: HM; E: LM) and instability index (E and F) measured by the LUMiSizer.
Note: PP: Persimmon pectin; SBP: Sugar beet pectin; HM: High methyl-esterified citrus pectin; LM: Low methyl-esterified citrus pectin.

of around 87◦ , mainly ascribing to its rich protein content (Z. Wang,
Feng, et al., 2021). Notably, if the contact angle of samples is too far
away from 90◦ , they will be completely immersed in the oil or water
phase rather than adsorbed at the oil-water interface (Jiang et al., 2019).
The contact angle values of citrus pectin HM and LM were 77.4◦ and
58.6◦ , indicating an unfavorable to the adsorption of citrus pectin
molecules to the water-oil interface, especially for LM (Zhang et al.,
2020). The high contact angle of PP endowed it with balanced amphi­
philicity and interfacial adsorption, which may be the critical basis for
its rapid adsorption at the oil-water interface and the formation of an
elastic interface film.
It was known that the thickness of the interfacial layer strongly

11
Y. Jia et al.
Fig. 8. Images from measurements of trip-phase contact angles of pectin
samples. Note: PP: Persimmon pectin; SBP: Sugar beet pectin; HM: High methyl-
esterified citrus pectin; LM: Low methyl-esterified citrus pectin.
influenced the strength of the steric interaction between droplets (Ralla
et al., 2018). A thick interface layer indicated robust emulsion stability
(Jin et al., 2017). Currently, it is difficult to directly observe the thick­
ness of the emulsifier adsorption layer on the oil-water interface.
Interestingly, polystyrene microsphere (PS) could be used as a template
to indirectly reflect the adsorption thickness of macromolecular emul­
sifiers at the interface because PS possessed a hydrophobic surface like
oil (Ralla et al., 2018). The PS model can simulate the oil interface (MCT
in the study), reflecting the thickness of the hydrocolloid adsorbed on
the oil-water interface. For example, Gashua et al. (2016) used this
method to compare the adsorption behaviors of two gums and found
that the emulsions produced by Acacia senegal gum exhibited smaller
droplet size and higher emulsifying stability than A. seyal gum, due to
the adsorption layer thickness of A. senegal gum being five times than
that of A. seyal gum. Verkempinck et al. (2018) used the PS model to
determine the adsorbed layer of citrus pectins (Mw: 5.41-5.87 × 104
g/mol; protein content: 1.44–1.72%; hydrodynamic diameter:
Fig. 9. Adsorbed layer thickness of pectin emulsifiers adsorbed onto the polystyrene (PS) surface as a function of pectin dosage (0.01%–0.50%, w/v). Note: PP:
Persimmon pectin; SBP: Sugar beet pectin; HM: High methyl-esterified citrus pectin; LM: Low methyl-esterified citrus pectin.
12
Food Hydrocolloids 131 (2022) 107727
227.0–269.8 nm; pH: 3.0) and found that the adsorbed layer of citrus
pectins had a size of 989–1064 nm, which is around 4 times the hy­
drodynamic diameter of pectin samples. And citrus pectins adsorbed at
the oil-water interface in a multilayered way. Therefore, the PS model
could be used to reflect the adsorption thickness of pectin samples at the
oil-water interface. The hydrodynamic diameter of the pectin samples
(0.5%, w/v) in the study is shown in Supplementary Table S5. The
adsorbed layer thickness of the pectins onto the PS model was presented
as a function of the pectin concentration (Fig. 9). The thickness of the
adsorbed layer increased with the increasing amount of pectin added but
not tend to plateau. In the aqueous dispersions of pectin, an increase in
the pectin concentration causes a self-organization of macromolecules
and an increase in the value of the hydrodynamic diameter. Thus, it is
necessary to estimate one macromolecule’s size, which could be deter­
mined from the dependence of Z-average on the pectin concentration in
the aqueous dispersion by extrapolating to a concentration of 0%. As
shown in Table S7, the hydrodynamic diameter of pectin obtained using
an extrapolation to the concentration of 0% of PP, SBP, HM, and LM was
113, 208, 165, and 326 nm, respectively. In addition, the thickness of
the adsorbed layer of PP, SBP, HM, and LM at 0.5% concentration was
284, 176, 151, and 204 nm, respectively. Notably, the number of layers
of molecules adsorbed on the PS surface could be calculated by dividing
the thickness of the adsorbed layer by the hydrodynamic diameter of the
molecule. The values were calculated around be 3, 1, 1, and 1 of PP, SBP,
HM, and LM at the concentration of 0.5%. Therefore, the results of the
performed calculations at the pectin concentration of 0.5% reflected the
multilayer adsorption for PP and the monolayer adsorption for other
polysaccharides (SBP, HM, and LM). Similar multi-layer adsorption was
observed in okra pectin, which mighty be attributed to its lower hy­
drodynamic diameter, allowing it to adsorb at the interface in a multi­
layered way (Alba et al., 2016; Verkempinck et al., 2018). Siew and
Williams (2008) found that SBP mainly formed a monolayer at the
droplet interface. Nakauma et al. (2008) also indicated that the
adsorption layer thickness of SBP was not enough to obtain sufficient
steric effects. Notably, the multilayered organization was also affected
by the pH of the system (Verkempinck et al., 2018). Therefore, the pH
values of the system need to be mentioned when studying the structure
of the pectin adsorption layer adsorbed on the PS model. As shown in
Table S1, the pH values of PP, SBP, and HM were around 3.30 without
significant differences (p ≥ 0.05), while LM showed a higher pH value
(4.85 ± 0.10). Moreover, the low pH of pectin solutions should also be
considered because of the difference between the ζ-potential of PS
dispersed in the water and the pectin solutions. There is an insignificant
difference between the ζ-potential of pectin solutions and PS dispersion
in these solutions (p ≥ 0.05) (Table S6). In summary, PP exhibited
multi-layer adsorption at the oil-water interface, which may exert suf­
ficient steric effect to stabilize the emulsion. By contrast, the mono-layer
adsorption exhibited by commercial pectins might be unfavorable to
Y. Jia et al. Food Hydrocolloids 131 (2022) 107727

emulsion stability.
Because the PS model is an auxiliary method to predict the thickness
of the adsorption layer, more advanced approaches for quantitatively
measuring the adsorption layer properties of the pectins at the oil-water
interface were needed. Therefore, QCM-D was further employed to study
the adsorption kinetics and adsorbed mass/thickness of pectin emulsi­
fiers at the oil-water interface.
Representative QCM-D Δf and ΔD curves for the adsorption behav­
iors of pectin emulsifiers on oil-coated sensors are shown in Fig. 10A and
B, respectively. The negative Δf and positive ΔD values were observed
during adsorption, indicating mass deposition of the pectins onto the oil-
coated sensors. For all cases in the study, the sharp decrease in Δf value
indicated the fast adsorption process of the pectin emulsifiers and the
formation of adsorption films. Next, the specific adsorption
characteristics of different pectin samples are illustrated in Fig. 10C. In
detail, PP showed the highest response in frequency change (f1, 97.23
Hz) upon adsorption, followed by SBP (86.16 Hz), LM (65.20 Hz), and
HM (55.03 Hz). The change in f1 values indicated that the adsorbed
amount of PP at the interface was higher than that of commercial pec­
tins. Then, a gradual and linear change in Δf and ΔD was observed as the
adsorption proceeded, signifying adsorption equilibrium of pectin
samples was almost achieved after the 70-min adsorption period.
When the adsorption reached equilibrium, the sensors were rinsed
with ultrapure water to study the desorption behavior of the pectin
emulsifiers. Upon water rinsing, the Δf value (f2) initially changed
sharply due to the rapid desorption of the unbound or loosely bound
emulsifiers. The f2 value can indirectly reflect the amount of emulsifiers
being washed off during the rinsing process. As viewed in Fig. 10C, the f2

Fig. 10. (A) Δf and (B) ΔD from the third overtone as a function of time for pectin samples during adsorption and rinsing at the oil-coated quartz crystal surface; (C)
Change of f value during adsorption (f1) and desorption (f2); (D) elastic modulus μf, (E) adsorption thickness hf, and (F) adsorption mass mf as a function of time
during adsorption and rinsing processes estimated from the Q-Sense Defind software. The concentration of all the emulsifier aqueous solutions is 1.0% (w/v). Note:
PP: Persimmon pectin; SBP: Sugar beet pectin; HM: High methyl-esterified citrus pectin; LM: Low methyl-esterified citrus pectin.

13
Y. Jia et al.
value of PP was relatively higher compared with commercial pectins,
implying that part of the PP was not tightly bound during the rinsing
process. In addition, the "f1-f2" value represented the residual amount of
pectin samples on the sensor after the rinsing process. Although the
amount of PP washed away was the largest, the "f1-f2" value of PP was
the highest compared with commercial pectins due to its sizeable initial
adsorption response. Therefore, PP exhibited the largest amount of
adsorption and residual after desorption at the oil-water interface.
ΔD provided a measure of the viscoelasticity or rigidity of the
adsorbed films (Wang, Ruengruglikit, Wang, & Huang, 2007). As char­
ted in Fig. 10B, the corresponding ΔD increased sharply during
adsorption, indicating that the viscoelasticity of the adsorption films
increased. PP exhibited the highest ΔD value (37.08) upon adsorption
compared with commercial pectins, implying the strongest viscoelas­
ticity of the adsorption layer was formed. Additionally, the significant
drop of ΔD upon rinsing indicated that the structure of adsorption films
became rigid (Wei et al., 2020). Like the change of the Δf value, PP
exhibited the largest D value after rinsing, indicating that PP still
maintained a robust viscoelastic film after desorption. The change in Δf
and ΔD upon rinsing indicated that all pectin emulsifiers could interact
with the oil surfaces and cannot be thoroughly washed from the sensors.
In addition, the elastic modulus (μf) of the adsorbed films was
quantitatively evaluated by the Q-Sense Defind software. As shown in
Fig. 10D, the elastic modulus (μf) increased with adsorption evolution,
indicating that the elastic properties of the adsorbed films were
enhanced, consistent with the interfacial rheological results. PP
exhibited the highest elastic modulus (μf), which helped resist inter­
ference from external factors. The Sauerbrey model of Q-Sense Defind
software was further used to analyze the adsorbed mass and adsorption
thickness. As charted in Fig. 10E and F, the adsorption thickness (hf) and
adsorption mass (mf) of the pectin samples increased with the adsorp­
tion progress. Upon rinsing, the decrease in hf and mf of the adsorption
film might be due to the loosely bound emulsifier being washed away.
PP displayed the largest hf and mf during the adsorption and desorption
process, while the hf and mf values formed by commercial pectins were
relatively lower. The adsorption thickness measured by QCM-D was
consistent with the result of the PS model simulation, further confirming
that PP formed multi-layer adsorption.
A representation of the interfacial adsorption behaviors of PP at the
oil-water interface studied by QCM-D was shown in Fig. 11. Persimmon
pectin was largely adsorbed on the oil-water interface in a multi-layer
adsorption manner, and formed a viscoelastic interface film; after
water rinsing, the loosely bound persimmon pectin was washed away,
and the interface film became rigid and compact.
The effect of pectin on the interfacial adsorption and viscoelasticity
of the adsorption film studied by QCM-D agreed with the results of
interfacial shear rheology well. Generally, the results of the QCM-D
evaluation indicated that the adsorption film formed by PP possessed
the largest adsorption thickness (hf), adsorption mass (mf), and elastic
modulus (μf), which might help resist the factors that cause emulsion
instability. Alba and Kontogiorgos (2017) indicated that neutral sugar
Fig. 11. Schematic illustration of the interfacial adsorption behaviors of persimmon pectin at the oil/water interface studied by QCM-D.
14
Food Hydrocolloids 131 (2022) 107727
sidechains of pectins contributed to the long-term emulsion stability due
to the formation of thick interfacial layers, thus providing effective steric
stabilization that impedes emulsion coarsening. Accordingly, the
abundant HG (62.43 ± 1.12%) and RG-I (17.75 ± 0.48%) components,
as listed in Table S8, of PP may be one of the potential reasons for the
formation of the thick interfacial film. Based on the high adsorption
characteristics of PP, it is inferred that PP might form a dense and thick
interfacial adsorption layer surrounding droplets, providing robust ste­
ric stabilization for oil droplets and avoiding permeation of the oil
phase.
The super-resolution imaging technique Cryo-SEM was further used
to acquire the distribution characteristics of pectin emulsifiers on the
droplet surface (Zhu et al., 2020). As depicted in Fig. 12, the emulsion
microstructure of PP showed a coherent "3D-network". The filament and
sheet-like PP adsorbed tightly on the surface film and simultaneously
entangled the adjacent droplets, forming a strong entanglement and
junction zone for the emulsified droplets (Fig. 12A). The "3D-network"
adsorption of PP could form a stable surface film to coat the oil droplets,
functioning as a steric barrier to protect emulsion droplets against
flocculation and coalescence (Zhu et al., 2020). In addition, the forma­
tion of the "3D-network" structure of PP-stabilized emulsion might
correspond to its strong elastic interface film (as discussed in the elastic
modulus (μf) of QCM-D assessment), and the high elasticity of the
network effectively reduced the coalescence rate of emulsions. As a
comparison, the frozen oil droplets of SBP and HM emulsions presented
a near-spherical shape and an uneven surface, which might be attributed
to the non-uniform particle stack of SBP (Fig. 12B) and HM (Fig. 12C).
Interestingly, the particle morphology of SBP and HM emulsions was
observed, suggesting that SBP and HM might stabilize the emulsion by
anchoring on the surface of the oil droplets (Jiang et al., 2019). How­
ever, no "network" and particle stack were observed in the LM micro­
structure (Fig. 12D), which could explain why the LM emulsion
exhibited the worst stability. Cryo-SEM micromorphology revealed a
robust "entanglement and junction" network around the PP emulsified
droplets, which could prevent the flow of droplets, while the
near-spherical shape particle stack of oil droplets like Pickering emul­
sion was found in SBP and HM emulsions.
The abundant hydrophobic components such as protein, ferulic acid,
methyl ester, and acetyl groups (presented in Supplementary Table S4)
of PP enabled it to be adsorbed on the oil-water interface. In addition,
the relatively lower Mw of PP (4.789 × 104 g/mol) facilitated the
accessibility of these hydrophobic components at the oil-water interface,
as evidenced by its strong tension-decreasing ability (Table S2). PP with
strong tension-reducing capacity favored its adsorption on the oil-water
interface and promoted the breakup of droplets during the homogeni­
zation process. In addition, the higher DA and rich neutral sugar side
chains of the RG-I domain contributed to the emulsifying stability (Yang
et al., 2018; Ngouémazong et al., 2015). Although the abundance of
neutral sugar side chains might hinder the emulsifying process, it could
improve the emulsification stability of pectins by forming a thick
interfacial layer on the oil droplets, thus providing effective steric
Y. Jia et al.
Fig. 12. Cryo-scanning electron microscope morphology of pectin stabilized emulsions: (A) PP; (B) SBP; (C) HM; (D) LM (The Subindex 1, 2 to 3 indicated an increase
in the magnification). Note: PP: Persimmon pectin; SBP: Sugar beet pectin; HM: High methyl-esterified citrus pectin; LM: Low methyl-esterified citrus pectin.
stabilization that impedes emulsion coarsening (Funami et al., 2011;
Alba & Kontogiorgos, 2017). Therefore, the relatively lower Mw of PP
facilitated the accessibility of these hydrophobic components at the
oil-water interface and the abundant neutral sugar side chains of the
RG-I domain (17.75 ± 0.48%) of PP enhanced the formation of a thicker
interfacial layer. Interestingly, the HG and RG-I contents of PP were
closed to those of SBP (HG: 62.70 ± 1.09%) and RG-I: (18.46 ± 0.78%).
It is speculated that the relatively large molecular weight of SBP, how­
ever, may lead to a low amount of adsorbed fraction (Pi et al., 2019), and
thus forming a thin interfacial layer. The higher Mw and lack of hy­
drophobic components and RG-I domains of citrus pectins (HM and LM)
may make it difficult for them to adequately adsorb to the oil-water
interface and form a thick interfacial layer (Wang, Feng, et al., 2021).
4. Conclusion
In summary, the effect of processing factors and surrounding envi­
ronment stress of persimmon pectins (PP) stabilized emulsion was
compared with commercial sugar beet and citrus pectins. The results
15
Food Hydrocolloids 131 (2022) 107727
showed that although PP-stabilized emulsion was sensitive to salt ions, it
had stronger resistance to extreme temperature treatments such as high
temperature and freeze-thawing, thermal cycling, and accelerated
centrifugation, than commercial pectin prepared emulsions. The
detailed interfacial features of PP were explored by multi-techniques to
reveal its potential emulsifying mechanisms. PP had a high contact angle
value of 83◦ , endowing it with balanced hydrophobicity and interfacial
adsorption. PS model evaluation showed that PP exhibited the highest
adsorption layer thickness among the four pectins tested. QCM-D results
further suggested that PP formed a thicker interfacial film and higher
adsorption mass and viscoelasticity, which may be attributed to the
multi-layer adsorption. Cryo-SEM micromorphology revealed a “3D-
network” structure around the oil droplets was formed by PP, which
contributed to enhancing strong elastic interface film and effectively
preventing the flow of oil droplets. This research provided strong sup­
port that PP can be developed as a novel polysaccharide emulsifier in the
food industry.
Y. Jia et al.
Author statement
Yangyang Jia performed the experiment, the data analyses and wrote
the manuscript; Jing Du and Kaikai Li contributed to the conception of
the study; Chunmei Li helped perform the analysis with constructive
discussions, manuscript modification and supervision.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
This work was supported by the National Key R&D Program of China
(2019YFD1000600) and Fundamental Research Funds for the Central
Universities (2662020SPPY013).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.foodhyd.2022.107727.
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