Food Hydrocolloids 103 (2020) 105675

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

A peppermint oil emulsion stabilized by resveratrol-zein-pectin complex

particles: Enhancing the chemical stability and antimicrobial activity in
combination with the synergistic effect
Hao Cheng a, b, Muhammad Aslam Khan a, b, Zhenfeng Xie a, b, Shengnan Tao c, Yunxing Li c,
Li Liang a, b, *
a
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, China
b
School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, China
c
Key Laboratory of Synthetic and Biological Colloids, Ministry of Education, School of Chemical and Material Engineering, Jiangnan University, Wuxi, Jiangsu, China

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

Keywords: The combination of different antimicrobial agents might produce synergistic effects and has gained increasing
Complex particle interest. Interfacial engineering of emulsion systems has been developed to co-encapsulate and protect bioactive
Peppermint oil components with different solubility. In this study, peppermint oil and resveratrol display synergistic effect
Resveratrol
against the Gram-positive bacteria Staphylococcus aureus and the Gram-negative bacteria Salmonella Typhimu­
Co-encapsulation
rium. Partially wettable resveratrol-loaded zein-pectin complex particles with a three-phase contact angle of
Antimicrobial activity
Chemical stability ~78� were fabricated via a desolvation method. Peppermint oil emulsions with the co-inclusion of resveratrol
were successfully prepared by zein-pectin complex particles, showing a high encapsulation efficiency for
peppermint oil (~88%) and resveratrol (~99%). Addition of pectin decreased size distribution of the emulsions,
improved antimicrobial activity, physical and chemical stability and prolonged antimicrobial efficiency against
Staphylococcus aureus and Salmonella Typhimurium. Overall, the current study may have a valuable contribution
to develop an efficient antimicrobial system based on the synergistic effect of combined agents and a single
emulsion stabilized by protein-polysaccharide complex particles.

1. Introduction
Essential oils (EOs) are naturally-derived aroma compounds ob­
tained from various parts of edible and medicinal plants and exert strong
antibacterial and antifungal activity (Donsi & Ferrari, 2016; Seow, Yeo,
Chung, & Yuk, 2014). When two or more agents work together, syner­
gism occurs to produce an effect greater than the sum of individual ef­
fects, due to their function on one or more different targets in a
metabolic pathway (Seow et al., 2014). For example, the combination of
cinnamaldehyde with carvacrol showed synergistic antibacterial effects
against both Escherichia coli and Staphylococcus aureus (S. aureus) (Ye
et al., 2013). A synergistic antibacterial activity against S. aureus was
also observed in nisin combined with cinnamaldehyde in pasteurized
milk (Shi et al., 2017). The combination of multiple antibacterial agents
has become an important approach to enhance the efficiency of anti­
bacterial therapy and overcome resistance to antibacterial agents.
However, poor hydro-solubility and high volatility of EOs limit their
application in the pharmaceutical, food and cosmetic industries. It is
thus necessary to develop the carriers not only to overcome the limita­
tions but also to enhance the antibacterial activity based on the syner­
gistic effect.
Oil-in-water (O/W) emulsions have been considered to be efficient
delivery systems for improving water dispersibility of EOs and pre­
venting their interactions with other food ingredients (Donsi et al.,
2016; McClements & Li, 2010). However, the oxidative and physical
stability of conventional emulsions is limited due to the high interfacial
area and a characteristic porous thin interfacial layer (Berton-Carabin,
Sagis, & Schroen, 2018; McClements & Decker, 2018). Recently, inter­
facial engineering of emulsion systems has been developed to improve
the oxidative stability by minimizing interactions between pro-oxidants
and bioactive lipids (Berton-Carabin, Ropers, & Genot, 2014; McCle­
ments & Decker, 2018). A safflower oil emulsion stabilized by lipid

* Corresponding author. State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu,
214122, China.
E-mail address: liliang@jiangnan.edu.cn (L. Liang).

https://doi.org/10.1016/j.foodhyd.2020.105675
Received 14 June 2019; Received in revised form 14 January 2020; Accepted 16 January 2020
Available online 21 January 2020
0268-005X/© 2020 Elsevier Ltd. All rights reserved.
H. Cheng et al.
droplets coated by milk protein concentrate (MPC) was prepared,
showing slower lipid oxidation than conventional emulsions stabilized
by MPC alone. (Okubanjo, Loveday, Ye, Wilde, & Singh, 2019).
Resveratrol, as an antioxidant co-emulsifier, can be accumulated at the
oil-water interface by interacting with proteins to enhance the oxidative
stability (Wan, Wang, Wang, Yuan, & Yang, 2014; Wang, Gao et al.,
2016). These provide an opportunity not only to improve the oxidative
stability of O/W emulsions but also to co-encapsulate bioactive com­
ponents with different solubility in the single emulsions.
Zein, a major storage protein in corn, contains more than 50% hy­
drophobic amino acid residues and is soluble in concentrated aqueous
ethanol solutions (60–90%) but not in pure water (Shukla & Cheryan,
2001). This property makes zein a suitable material for the encapsula­
tion of bioactive components, such as α-tocopherol, resveratrol and
epigallocatechin gallate (Davidov-Pardo, Joye, & McClements, 2015;
Donsi, Voudouris, Veen, & Velikov, 2017; Luo, Zhang, Whent, Yu, &
Wang, 2011). A Pickering O/W emulsion was successfully produced by
bare zein colloidal particles with droplet size in the range of 10–200 μm
(de Folter, van Ruijven, & Velikov, 2012). However, the resulting
emulsions were unstable against coalescence at low pH due to the poor
wettability of the protein. Surface-modified zein particles with
water-soluble biopolymers have been utilized to regulate the surface
wettability of zein particles and form stable O/W emulsions (Chen et al.,
2018; Dai, Sun, Wei, Mao, & Gao, 2018; Feng & Lee, 2016). Pectin, an
anionic polysaccharide, belongs to a family of heterogeneous poly­
saccharides containing mainly α-(1 → 4)-linked partially methyl ester­
ified D-galacturonic acid and rhamnogalacturonan units (Synytsya,
Copikova, Matejka, & Machovic, 2003). Zein-pectin core-shell nano­
particles overcome the aggregation problem of bare zein particles and
provide better protection for encapsulated molecules than bare zein
particles did (Hu et al., 2015; Huang et al., 2017). Additionally, high
methoxyl pectin could strongly absorb to the interface of mandarin or
lemongrass oil emulsion and improve physical stability against Ostwald
ripening (Guerra-Rosas, Morales-Castro, Ochoa-Martinez,
Salvia-Trujillo, & Martin-Belloso, 2016).
Peppermint (Mentha piperita) oil is one of the most widely produced
and used essential oils in food, flavorings, and pharmaceutical products.
Peppermint oil possesses antimicrobial, antiviral and antifungal activ­
ities against various types of bacteria and yeasts (Iscan, Kirimer, Kurk­
cuoglu, Baser, & Demirci, 2002; Mahboubi & Haghi, 2008). Resveratrol,
a natural polyphenol, is produced in plants in response to injury and
fungal attack (Summerlin et al., 2015). Resveratrol exhibits a broad
spectrum of antimicrobial activity across a wide range of microorgan­
isms (Ma et al., 2018), mainly due to the generation of reactive oxygen
species causing DNA damage (Subramanian, Soundar, & Mangoli,
2016), oxidative membrane damage (Subramanian, Goswami, Chakra­
borty, & Jawali, 2014), and metabolic enzyme inhibition (Dadi, Ahmad,
& Ahmad, 2009). In this study, resveratrol-fortified zein-pectin particles
were prepared to stabilize a peppermint oil emulsion. Physiochemical
property of the colloidal particles and emulsions was characterized.
Furthermore, antimicrobial efficiency of peppermint oil and resveratrol
combination was evaluated against food-borne pathogens.
2. Materials and methods
2.1. Materials
Zein (~98%) was purchased from J&K Chemical Co., Ltd. (Shanghai,
China). Pectin (50–300 kDa, degree of esterification �47.9%) and
resveratrol (trans-isomer, �98%) were purchased from Sango Biotech
Co. (Shanghai, China). Peppermint (M. piperita) oil was obtained from
Shanghai Orinno International Business Co., Ltd. (Shanghai, China). The
composition of peppermint oil was reported in Table S1. Menthol
(�98%, GC), menthone (�97%, GC) and Nile red dye were obtained
from Sigma-Aldrich Co. (St. Louis, MO, USA). Other reagents of
analytical grade were purchased from SinoPharm CNCM Ltd. (Shanghai,
2
Food Hydrocolloids 103 (2020) 105675
China).
2.2. Preparation of resveratrol-loaded zein-pectin complex particles
Zein colloidal nanoparticles were prepared following the desolvation
method (Zhong & Jin, 2009). Briefly, 5% (w/v) zein and/or resveratrol
at various concentrations [0.10%, 0.25% and 0.50% (w/v)] were dis­
solved in 80% (v/v) ethanol aqueous solution under stirring at 600 rpm
for 30 min and then added into the anti-solvent (aqueous phase) at a
volume ratio of 1:9 under stirring at 1200 rpm for 5 min. Ethanol in the
dispersions was removed using a RE-52C rotary evaporator (Shanghai
Tianheng Instrument Co. Ltd, Shanghai, China) at 40 � C for 30 min. The
pH of the resulting dispersion was 4.0 (�0.1). Pectin at 0.5% (w/v) was
dispersed in Milli-Q water under stirring for 6 h to allow complete hy­
dration. The pH of the polysaccharide solution was adjusted to 4.0 with
0.6 M HCl or NaOH. Then zein (Z) and resveratrol-loaded zein (R/Z)
nanoparticle dispersions were poured into the pectin solution under
stirring at 700 rpm for 10 min. Freshly-prepared zein-pectin (ZP) and
resveratrol-loaded zein-pectin (R/ZP) nanoparticles contained 0.20%
(w/v) zein and 0.05%, 0.10% and 0.20% (w/v) pectin. The final con­
centration of resveratrol in R/ZP particles was 0.02% (w/v).
2.3. Preparation of peppermint oil emulsions
Emulsions were prepared according to our previous method with
some modifications (Fan et al., 2017). Peppermint oil was slowly added
to the aqueous phase of Z, R/Z, ZP or R/ZP colloidal particles at pH 4.0
to obtain coarse emulsions with a final weight of 100 g by using a
high-speed blender (Ultra Turrax T25, IKA, Germany) operating at 8000
rpm for 2 min. Oil droplet size was further reduced by passing the coarse
emulsions for four times through an ATSAH2100 high-pressure ho­
mogenizer (ATS Engineering Ltd., ON, Canada) at a pressure of 25 MPa
and 10 � C. The final concentration of zein was 0.20% (w/w), while the
concentrations of pectin were 0, 0.05%, 0.10% and 0.20% (w/w). The
final content of peppermint oil was 5% (w/w).
2.4. Size and zeta-potential measurements
Size distribution and ζ-potential of zein-based colloidal particles and
peppermint oil emulsions were analyzed by a NanoBrooker Omni Par­
ticle Sizer and ζ-Potential Analyzer (Brookhaven Instruments Ltd, New
York, USA) with a He/Ne laser (λ ¼ 633 nm). Samples were diluted with
water at pH 4.0 by 100 times before measurement at 25 � C and at a
scattering angle of 173� . Size distribution by the intensity and ζ-poten­
tial were obtained by using a NNLS mode analysis and the Smo­
luchowski model, respectively.
2.5. Encapsulation efficiency of the whole peppermint oil, menthol, and
menthone
Fresh emulsions were centrifuged at 13,000 � g for 30 min at 4 � C
using a 5804 R centrifuge (Eppendorf Co. Ltd, Hamburg, Germany). The
amount of the whole peppermint oil, menthol, and menthone in the
whole emulsion (Aw) and in the subnatant (Asub) was determined by
using a method of liquid-liquid extraction (Donsi, Annunziata, Vincensi,
& Ferrari, 2012; Wang, Gao et al., 2016). In a brief, 1 mL of emulsion or
subnatant was mixed with 1 mL ethanol under vortexing for 1 min.
Peppermint oil was then extracted by adding 4-mL hexane under vor­
texing for 90 s followed by centrifuging at 3500 � g at 4 � C for 5 min.
The content of the whole peppermint oil in hexane supernatants was
determined by a UV–visible spectrophotometer (Shimadzu Co., Tokyo,
Japan) based on a standard curve (absorbance at 240 nm) of peppermint
oil at 0.025–0.250 mg/mL in hexane (Chen & Zhong, 2015). The content
of menthol and menthone in hexane supernatants was determined by gas
chromatograph (Shimadzu Co., Kyoto, Japan) according to the proced­
ure described in section 2.13. Encapsulation efficiency was calculated
H. Cheng et al.
from the difference between Aw and Asub divided by Aw.
2.6. Quantitation of resveratrol using high performance liquid
chromatography (HPLC)
Exactly 0.2 mL sample was added into 1.6 mL polydatin (0.01 mg/
mL, internal standard) in methanol under vortexing and centrifuged at
13,000 � g for 10 min. The methanol extract was passed through a 0.22-
μm filter and then analyzed by an HPLC system (Waters, Milford, MA,
USA) according to our previous method (Cheng, Fang, Liu, Gao, & Liang,
2018).
2.7. Encapsulation efficiency of resveratrol within zein-based
nanoparticles and partition of resveratrol within peppermint oil emulsions
Freshly-prepared colloidal dispersions were centrifuged at 150,000
� g at 4 � C for 30 min by using a CP70ME ultra-centrifuge (Hitachi Co.
Ltd, Tokyo, Japan) to separate any particles (Fan et al., 2017). The
amounts of resveratrol in the whole dispersions (Rd) and the superna­
tants (Rsup) were determined by using HPLC. Encapsulation efficiency
was calculated from the difference between Rd and Rsup divided by Rd.
Partition of resveratrol in emulsions was determined according to the
procedure described by Fan et al. (2017). Emulsions were centrifuged at
13,000 � g for 30 min at 4 � C. The amount of resveratrol in the whole
emulsion (Rw), in the subnatant (Rsub) and in the precipitation (Rp) was
determined using HPLC. The percentage of resveratrol in the emulsified
oil droplets (Pe), encapsulated (Pp) and free (Pf) in the aqueous phase
was calculated using the formula as follow,
Pe (%) ¼ (Rw - Rsub - Rp)/Rw � 100 (1)
Pp (%) ¼ Rp/Rw � 100 (2)
Pf (%) ¼ Rsub/Rw � 100 (3)
2.8. Determination of zein and pectin at the oil-water interface
The content of zein and pectin at the oil-water interface was deter­
mined according to the method described by Wan, Wang, Wang, Yang, &
Yuan. (2013) and Fan et al. (2017) with some modifications.
Freshly-prepared emulsions were centrifuged using a 5804 R centrifuge
(Eppendorf Co. Ltd, Hamburg, Germany) at 13,000 � g for 30 min at 4
�
C. Content of zein and pectin in the subnatant (Csub), precipitation (Cp)
and in the whole emulsion (Cw) was determined using the Kjeldahl
method and phenol-sulfuric method (Dubois, Gilles, Hamilton, Rebers,
& Smith, 1956; Ye, Flanagan, & Singh, 2006), respectively. The inter­
facial protein and polysaccharide percentage (%) was calculated by Eq.
(4).
� each tube with a final concentration of 1 � 105 CFU/mL and incubated
Percentage ð%Þ ¼
Cw Cs Cp
� 100 (4)
Cw
2.9. Morphology
The morphological structure of zein-based particles and emulsions
was visualized on a scan electron microscopy (SEM) SIGMA HD (ZEISS,
Jena, Germany) or SU8020 (Hitachi, Tokyo, Japan). Zein colloidal
particles and emulsions were freeze-dried using Benchtop Freeze Dryers
(Freezone, 2.5, Labconco, MO, USA). Emulsions were also spray-dried
using a commercial Buchi B-290 mini Spray-dryer (Buchi Labortechnik
AG, Flawil, Switzerland). The emulsion was fed into the spray-dryer at
an inlet temperature of 150 � C, aspiration 100% and %pump of 15%,
which was equivalent to a mass flow of emulsion of 5.0 mL/min. The
outlet temperature was 80 � C. The emulsion was sprayed through a
nozzle with an inner diameter of 0.7 mm and dried under an airflow of
3
Food Hydrocolloids 103 (2020) 105675
35 m3/h. Samples were gold-sputtered and observed with magnifica­
tions of 50,000 � for freeze-dried particles, 10,000 � for freeze-dried
emulsions and 2000 � for spray-dried emulsions.
2.10. Wettability measurement
The oil-water three-phase contact angle of zein-based particles and
pectin was measured using an OCA 15 EC (Dataphysics Instruments
GmbH, Germany). Freeze-dried powders of zein-based particles and
pectin were compressed into thin tablets, which were then immersed in
peppermint oil. A droplet (2 μL) of Milli-Q water was placed on the
surface of the tablets. After equilibrium was reached, the shape of
droplets was recorded by a camera, and contact angles were calculated
using the instrumental software based on the LaPlace-Young equation
(Dai et al., 2018).
2.11. Fluorescence microscopy
Fluorescence microscopy images of emulsions were recorded on a
Zeiss Axio Vert.A1 inverted microscope (Leica, Heidelberg, Germany) at
a magnification of 40 � . Exactly 20 μL of Nile red (1 mg/mL) was added
into 1 mL of emulsions in order to stain the oil phase. The stained
emulsions were placed on concave slides and covered with coverslips
without trapping air bubbles. Nile red was excited at 634 nm.
2.12. Antimicrobial assays
2.12.1. Microorganisms and growth conditions
The Gram-positive bacteria Staphylococcus aureus (S. aureus) CGMCC
1.1861 and the Gram-negative bacterial Salmonella Typhimurium
(S. Typhimurium) CMCC 50115 were obtained from China General
Microbiological Culture Collection Center (Beijing, China) and National
Center Medical Culture Collections (Beijing China), respectively. Both of
the strains were kept at 4 � C on their appropriate slant. S. aureus and
S. Typhimurium were incubated in nutrient broth (beef extract 5 g/L,
NaCl 5 g/L, peptone 10 g/L, pH7.4) at 37 � C for 20 h, in BPY broth (beef
extract 5 g/L, NaCl 5 g/L, yeast extract 5 g/L, glucose 5 g/L, peptone 10
g/L, pH7.0) at 37 � C for 10 h, respectively, to retain early stationary
growth phase. The culture solutions with a final cell concentration of
~108 CFU/mL for S. aureus and ~109 CFU/mL for S. Typhimurium were
used as working solutions.
2.12.2. Determination of minimal inhibitory concentration
The minimal inhibitory concentration (MIC) values of peppermint
oil, resveratrol, zein particles, and pectin were determined by a broth
dilution method as described in previous studies with some modifica­
tions (Shi et al., 2017; Zhao et al., 2014). A 100 μL of antimicrobial
agents after being serially diluted was added in each tube containing 9.8
mL of broth. A 100-μL suspension of tested microorganism was added to
at 37 � C for 24 h. The MIC was defined as the lowest concentration of
antimicrobials that inhibited >90% of the microorganism’s growth by
visual reading and optical density (OD) at 600 nm using a UV-1800
UV–Vis spectrophotometer (Shimadzu Co., Tokyo, Japan) (Andrews,
2001; Oo, Cole, Garthwaite, Willcox, & Zhu, 2010).
2.12.3. Checkerboard synergy testing
In vitro interactive inhibition between resveratrol and peppermint oil
was measured with the broth dilution checkerboard assay with some
modifications (Oo et al., 2010; Shi et al., 2017). Briefly, peppermint oil
was diluted two-fold in vertical orientation, while resveratrol was
diluted two-fold in horizontal orientation. Their concentrations corre­
spond to 1/2, 1/4 and 1/8 of the MIC values, respectively. Subsequently,
100-μL suspension of the indicator strain was added to each tube with a
final concentration of 1 � 105 CFU/mL. The inoculated tubes were
incubated overnight at 37 � C for the evaluation of microbial growth.
H. Cheng et al. Food Hydrocolloids 103 (2020) 105675

Fractional inhibitory concentration index (FICI) was then calculated to 3. Results and discussion
assess the antimicrobial effects of combinations. FICI was calculated
using the following equations: 3.1. Characterization of resveratrol/zein-pectin complex particles

FICI ¼ FICIA þ FICIB (5) 3.1.1. Size and ζ-potential

FICIA ¼ MICA of the combination/MICA alone (6)
FICIB ¼ MICB of the combination/MICB alone (7)
The results were interpreted as synergistic effects (FICI < 0.9),
addictive effects (0.9 < FICI < 1.1), and antagonistic effects (FICI > 1.1)
(Romano, Abadi, Repetto, Vojnov, & Moreno, 2009; Santiesteban-Lopez,
Palou, & Lopez-Malo, 2007).
2.12.4. Antimicrobial activity during storage
Peppermint oil emulsions were stored in an incubator at 25 � C and
antimicrobial activity was characterized by standard plate count method
after storage for 7, 14, 28 and 42 days (Donsi et al., 2012). In brief,
100-μL emulsion was added into 9.8 mL 0.85% physical saline and then
mixed with 100 μL of culture solution containing 1 � 104–1 � 108
CFU/ml S. aureus or S. Typhimurium. Exactly 1.0 mL of the mixture was
plated onto nutrient agar and BPY agar for S. aureus or S. Typhimurium,
respectively, and incubated at 37 � C for 24 h for enumeration. Antimi­
crobial activity of emulsions was calculated by the following equation:
Reduction ¼ log (N/N0) log CFU/mL (8)
Where, N0 and N were the initial and final viable colony counts,
respectively.
2.13. Quantitation of menthol and menthone using gas chromatography
The size distribution of bare zein particles had a peak around 80 nm
(Fig. S1A), which was consistent with a previous report that zein
nanoparticles prepared by desolvation method had an average size of
50–200 nm (Kasaai, 2018). The presence of resveratrol at 0.004%,
0.010%, and 0.020% had no impact on the size of zein particles
(Fig. S1A). However, a further increase of resveratrol concentration to
0.040% resulted in precipitation to naked eyes. The polyphenol con­
centration of 0.020% was thus used for further study. The size of the
resveratrol-loaded zein particles gradually increased from about 140 to
240 nm as the concentration of pectin increased from 0.05 to 0.20%
(Fig. 1A).
Zein has an isoelectric point around 6.2 (Shukla et al., 2001). The
ζ-potential of bare zein particles was about þ33 mV at pH 4.0 (Fig. S1B).
The ζ-potential was similar in the absence and presence of resveratrol.
Together with size distribution (Fig. S1A), these results suggest that the
polyphenol did not affect the formation and surface property of zein
particles. The carboxyl groups on pectin have a dissociation constant
(pKa) around 3.5 (Jones, Lesmes, Dubin, & McClements, 2010). The
ζ-potential of resveratrol-loaded zein particles changed to 27 mV upon
addition of 0.05% pectin (Fig. 1B). Absolute values of ζ-potential
increased as the polysaccharide concentration increased, reaching a
constant of 35 mV at 0.10% and 0.20% pectin. These results suggest
that anionic pectin molecules absorbed by electrostatic attraction onto
the surface of cationic zein nanoparticles to form core-shell particles (Hu

Menthol and menthone in peppermint oil emulsion during storage

were determined by using Shimadzu capillary gas chromatograph model
GC-2010 (Kyoto, Japan) fitted with a Rtx-Wax silica capillary column
(30 m � 0.32 mm, 0.25 μm film thickness) and a flame ionization de­
tector (FID). Exactly 1.0 mL of the emulsion was mixed with 1 mL
ethanol under vortexing. Exactly 4-mL hexane was added to extract
menthol and menthone. After centrifuging at 3500 � g and 4 � C for 5
min, the supernatants were collected and passed through a 0.22-μm
filter. The temperature program began with 5 min at 40 � C and ramped
at 10 � C/5 min to 150 � C. The injector temperature was 200 � C, injection
volume was 1 μL, injection time was 0.04 min, split mode (1:1) was used,
and the detector temperature was 250 � C. The carrier gas was nitrogen at
a flow rate of 1.5 mL/min and the assisting gas (air) flow rate was 400
mL/min. Quantification of menthol and menthone was based on their
standard curve.

2.14. Chemical stability during storage

Samples were stored in an incubator at 25 � C and analyzed after

storage for 7, 14, 28 and 42 days. The retention of resveratrol, menthol,
and menthone in the whole emulsion during storage was expressed as a
percentage relative to the initial concentration.

2.15. Statistical analysis

All the measurements were conducted at least in triplicate and data

were presented as mean � standard deviation (SD). The significant
differences were evaluated by the one-way analysis of variance
(ANOVA), and the subsequent Duncan’s test was set at the 5% level (p <
0.05) to compare the means (SPSS 20.0 statistical software, SPSS Inc.,
Chicago, USA).

Fig. 1. Size distribution by the intensity (A) and ζ-potential (B) of resveratrol-
loaded zein particles in the absence and presence of pectin at various concen­
trations. The concentration of zein was 0.2% (w/v).

4
H. Cheng et al. Food Hydrocolloids 103 (2020) 105675

et al., 2015). The formation of pectin shell structure contributes to the

increase in the zein particle size (Fig. 1A).

3.1.2. Morphological observation

Resveratrol-loaded zein particles were observed as tightly packing of
spherical particles with the diameter smaller than 100 nm (Fig. 2A). Zein
particles coated with pectin at 0.05% also produced spherical particles
(Fig. 2B). However, irregular geometry shape particles were observed at
the pectin concentrations of 0.10% and 0.20% (Fig. 2C and D). It has
been reported that zein particles coated by gum Arabic (GA) showed an
irregular geometry shape and larger size than zein particles and was
hard to see individual particles (Dai et al., 2018). The SEM images
(Fig. 2) confirmed the results of size distribution based on dynamic light
scattering (Fig. 1A).

3.1.3. Wettability
The intermediate wettability [oil-water three-phase contact angles
(θow) close to 90� ] of particulate emulsifier is necessary to stabilize O/W
emulsion against coalescence. The wetting property of zein-based par­
ticles was detected through investigating the θow of particle tablets
immersed in peppermint oil (Fig. 3). The θow of bare zein particles was
132.07� , which was similar with the θow ~134� for medium-chain tri­
glyceride oil (Dai et al., 2018) but different from the θow ~ 107� for corn
oil (Zou, Guo, Yin, Wang, & Yang, 2015). The presence of resveratrol
had no impact on the wettability of the particles (Fig. 3). In the presence
of 0.05% pectin, R/ZP particles had near-neutral wettability (θow ~ Fig. 3. Three-phase contact angles of bare zein (Z) particles, resveratrol-loaded
78.30� ), suggesting that the particles could favor interfacial particle zein (R/Z) particles, resveratrol-loaded zein-pectin (R/ZP) particles with
adsorption and formation of O/W Pickering emulsion (Wang, Yin et al., various concentrations of pectin and pure pectin (P). The concentration of zein
2016). Further increase in the pectin concentration resulted in a gradual was 0.2% (w/v).
decrease in the θow of R/ZP particles, with 68.27� and 63.53� at the
polysaccharide concentrations of 0.10% and 0.20%, respectively. These 3.1.4. Encapsulation efficiency of resveratrol
results proved that the surface coating with hydrophilic pectin (θow ~ The encapsulation efficiency of resveratrol in zein particles was
58.65� ) through electrostatic interaction can tune the wettability of ~70%, which was independent of the polyphenol concentrations
R/ZP particles (Zhou et al., 2018). (Table 1). The similar result was previously observed for the encapsu­
lation of resveratrol in sodium caseinate-coated zein particles (Davi­
dov-Pardo et al., 2015). The encapsulation efficiency of resveratrol was

Fig. 2. SEM images of resveratrol-loaded zein particles (A) and zein-pectin particles at the polysaccharide concentrations of 0.05% (B), 0.10% (C) and 0.20% (w/v)
(D). The concentration of zein was 0.2% (w/v).

5
H. Cheng et al. Food Hydrocolloids 103 (2020) 105675

Table 1
Encapsulation efficiency of resveratrol at various concentrations in zein
colloidal particles and of resveratrol at 0.020% in zein-pectin colloidal particles
at various pectin concentrations.
Resveratrol concentration Pectin concentration Encapsulation efficiency
(w/v%) (w/v%) (%)

0.004 70.8 � 1.1a

0.010 69.0 � 1.5a
0.020 69.0 � 2.6a
0.020 0.05 72.4 � 1.8ab
0.020 0.10 74.5 � 1.2b
0.020 0.20 75.6 � 2.2b

Different letters in the same column represent statistically significant differences

(p < 0.05).

about 74% in zein-pectin complex particles (Table 1). The increase in

the encapsulation efficiency might be due to the fact that pectin could
interact with resveratrol through hydrogen bonding (Buchweitz, Speth,
Kammerer, & Carle, 2013; Cheng et al., 2018; Davidov-Pardo et al.,
2015). Loading efficiency of resveratrol was greater in β-lactoglobu­
lin-pectin complex particles than in β-lactoglobulin alone (Cheng et al.,
2018).
3.2. Particle-stabilized peppermint oil emulsions
3.2.1. Emulsion formation
In the emulsions stabilized by resveratrol-loaded zein and zein-
pectin particles, peppermint oil droplets were visualized using an
inverted fluorescence microscope. In the emulsions stabilized by R/Z
particles, both large (~1–2 μm) and small (<0.5 μm) oil droplets were
observed and some droplets aggregated together (Fig. 4A). The per­
centage of zein adsorbed at the surface of peppermint oil droplets was
only 45% (Fig. 5). The emulsions in the presence of pectin at 0.05%
showed small (<0.5 μm) and homogeneous oil droplets with no sign of
flocculation (Fig. 4B). The interfacial protein percentage increased to
73%, while the interfacial pectin percentage was 21% (Fig. 5). These
Fig. 5. Interfacial zein or pectin percentages of the emulsions stabilized by
resveratrol-loaded zein particles at various pectin concentrations. The concen­
tration of zein was 0.2% (w/w).
results might be attributed to the fact that the near-neutral wettability
(Fig. 3) could promote R/ZP particles to absorb on the oil-water inter­
face against aggregation and coalescence by electrostatic repulsion and
steric hindrance (Dai et al., 2018; Feng & Lee, 2016). Most oil droplets
were smaller than 0.2 μm at higher pectin concentrations (Fig. 4C and
D). The sum of the interfacial protein and polysaccharide percentages
kept constant as the pectin concentration increased (Fig. 5). However,
the interfacial protein percentage decreased to 70% and 64%, while the
interfacial pectin percentage increased to 24% and 30% at 0.10% and
0.20% pectin, respectively. In the case of canola oil emulsion stabilized
by zein-sodium caseinate (SC) particles, surface protein concentration
decreased when the zein/SC mass ratio decreased from 10:2 to 10:4, due
to that the adsorption of SC occupied the interface and hindered the
further adsorption of zein-SC particles (Feng & Lee, 2016). Methyl

Fig. 4. Fluorescent microscopic images of the emulsions stabilized by resveratrol-loaded zein particles (A) and zein-pectin particles at pectin concentrations of 0.05%
(B), 0.10% (C) and 0.20% (w/w) (D). The concentration of zein was 0.2% (w/w).

6
H. Cheng et al.
groups and/or acetyl groups in pectin molecules enhance the hydro­
phobic nature, make it exhibit surface activity (Dickinson, 2009).
Competitive adsorption between zein-pectin particles and excessive
pectin molecules occurred when the polysaccharide concentration was
above 0.10% (Fig. 5), contributing to a zein-pectin particles and pectin
molecules mixed layer formation.
3.2.2. Emulsion characterization
3.2.2.1. Size distribution and ζ-potential. The emulsions stabilized by
resveratrol-loaded zein particles were distributed in three peaks around
80, 434 and 1890 nm (Fig. 6A). The inclusion of resveratrol had no
impact on the emulsion size distribution (Fig. S2). After centrifugation,
size distribution in the subnatant was similar to that of corresponding
colloidal particles (Fig. S1A) and the smallest peak of corresponding
emulsions (Fig. S2). These results indicate that the peak around 80 nm
was attributed to zein-based colloidal particles, while the larger peaks
were for the particle-stabilized peppermint oil droplets. Similar results
were observed in sunflower oil emulsions stabilized by whey protein
microgel particles (Fan et al., 2017; Sarkar et al., 2016). Addition of
pectin resulted in a uniform size distribution around 709, 583 and 529
nm at the polysaccharide concentrations of 0.05%, 0.10%, and 0.20%
pectin, respectively (Fig. 6A), which shows a similar trend with the
inverted fluorescence microscope (Fig. 4). The sizes of the emulsified oil
droplets are approximately equal to the sum of two-fold zein-pectin
particle size (Figs. 1 and 2) and oil droplet size (Fig. 4). At pH 4.0,
ζ-potential of freshly-prepared emulsions stabilized by R/Z particles was
þ61 mV and changed to 38, 41 and 44 mV in the presence of
0.05%, 0.10% and 0.20% pectin (Fig. 6B).
Fig. 6. Size distribution by the intensity [( ), storage for 0 day; (- - -), storage
for 42 days] and ζ-potential (B) of the emulsions stabilized by resveratrol-
loaded zein particles at various pectin concentrations. The concentration of
zein was 0.2% (w/w).
7
Food Hydrocolloids 103 (2020) 105675
3.2.2.2. Morphology. Surface morphology of freeze-dried and spray-
dried powders was observed using SEM (Fig. 7). The zein-stabilized
emulsions after freeze-drying showed irregular shape with a porous
network structure, and zein particles agglomerated and formed clusters
(Fig. 7A). A similar structure was also observed in the cyclohexane
emulsion stabilized by melamine-based microporous organic polymer
particles after freeze-drying (Lee & Chang, 2018). The porous structure
formation might be due to the fact that essential oils are susceptible to
volatilization and loss during the freeze-drying process. The addition of
pectin resulted in the formation of film (Fig. 7B), which became denser
as pectin concentration increased (Fig. 7C and D). A sheet form with an
irregular geometry has been reported for freeze-dried powders of
thymol-carvacrol or chia-essential oil emulsions stabilized by SC-lactose
mixture (Gursul, Karabulut, & Durmaz, 2019; Rodriguez et al., 2019)
and fish oil emulsion stabilized by octenyl-succinic-anhydride modified
starch (OSA-starch) (Melgosa, Benito-Roman, Sanz, de Paz, & Beltran,
2019).
The powder prepared from zein-stabilized emulsions displayed
irregular shapes (Fig. 7E). It seems that the microcapsules formed
agglomeration after the spray-drying process, possibly due to the high
surface hydrophobicity of zein (Zhang, Luo, & Wang, 2011). A wide
range of microcapsules with a dimension from 0.5 to 5 μm was observed
for the emulsion stabilized by zein plus pectin at 0.05% (Fig. 7F).
Fig. 7. SEM images of the emulsions stabilized by resveratrol-loaded zein
particles (A, E) and zein-pectin particles at pectin concentrations of 0.05% (B,
F), 0.10% (C, G) and 0.20% (w/w) (D, H). A-D, powder obtained by freeze-
drying; E-G, powder obtained by spray-drying. The concentration of zein was
0.2% (w/w).
H. Cheng et al. Food Hydrocolloids 103 (2020) 105675

Further increase in the pectin concentration resulted in the formation of
bigger microcapsules (5–10 μm, Fig. 7G and H), possibly due to an in­
crease in the viscosity at such high concentrations of pectin (Bai et al.,
2017; Fernandes et al., 2016). All the dimensions of spray-dried powders
are bigger than those of individual zein particles and oil droplets
(Figs. 2, 4 and 6), possibly due to the fact that multiple particles and oil
droplets are present in one atomized droplet during spraying (Hogan,
McNamee, O’Riordan, & O’Sullivan, 2001). It is noteworthy that more
and more holes on the surface of microcapsules were observed as the
pectin concentration increased, which is consistent with the structure of
OSA-starch-stabilized caraway essential oil emulsions (Baranauskiene,
Rutkaite, Peciulyte, Kazernaviciute, & Venskutonis, 2016). Similarly,
some holes were observed at the surface of spray-dried microcapsules
from caprylic capric glycerid oil emulsions stabilized by pectin (Benja­
sirimongkol, Piriyaprasarth, Moribe, & Sriamornsak, 2019). The for­
mation of holes might be associated with competitive adsorption
between zein-pectin particles and pectin molecules (Fig. 5) and the
interstitial space between particles coverage by excessive pectin mole­
cules. Fig. 8 illustrates the formation and interfacial structures of
peppermint oil emulsions at various concentrations of pectin.
the emulsions against aggregation, creaming and Ostwald ripening
during storage, which is essentially dependent on the polysaccharide
concentration. Pectin could reportedly improve physical stability of
orange beverage emulsions containing GA (Mirhosseini et al., 2008),
lemongrass and mandarin essential oil emulsions stabilized by Tween 80
(Guerra-Rosas et al., 2016) against cream and phase separation, due to
strong adsorption of pectin at the oil surface to provide effectively
repulsive forces.
3.3. Co-encapsulation and protection of peppermint oil and resveratrol
3.3.1. Encapsulation
3.3.1.1. Encapsulation of peppermint oil. The encapsulation efficiency of
peppermint oil in zein-stabilized emulsions was 74% (Table 2). The
encapsulation efficiency of peppermint oil increased gradually as pectin
concentration increased, reaching about 88% at 0.10% and 0.20%
pectin. The enhanced encapsulation efficiency might be due to efficient
adsorption of partially wettable R/ZP particles and packing of both the

3.2.2.3. Physical stability. The emulsions stabilized by R/Z particles had Table 2
the peaks around 74, 324 and 3085 nm after storage for 42 days Encapsulation efficiency of the whole peppermint oil (PO), menthol, and men­
thone and partition of resveratrol in the emulsions stabilized by resveratrol-
(Fig. 6A). ζ-Potential of the emulsions stabilized by R/Z particles grad­
loaded zein and zein-pectin particles at various pectin concentrations.
ually decreased during storage and was þ20 mV after 42 days (Fig. 6B).
The decrease in the magnitude of the ζ-potential may destabilize zein- Pectin PO Menthol Menthone Resveratrol (%)
(%) (%) (%) (%)
stabilized emulsions during storage, due to the lack of strongly repul­ Aqueous Emulsified Total
sive forces against aggregation. Additionally, a cream layer also phase oil droplet
appeared during storage, in agreement with a previous report on zein- Particle
particle-stabilized soybean oil emulsions (de Folter et al., 2012). It is
0.00 74.1 72.2 � 73.7 � 33.3 � 65.7 � 2.9a 99.0
thus suggested that a significant increase in the largest peak (Fig. 6A) is � 3.0a 2.9a 1.8a �
due to Ostwald ripening and flocculation (Dickinson, 2009). Creaming 2.1a 0.9a
was not observed in the presence of pectin. The size distribution of the 0.05 83.1 84.5 � 82.8 � 17.7 � 81.5 � 2.1b 99.2
2.6b 2.0b 1.8b
emulsions stabilized by R/ZP particles at 0.05% pectin remained un­ � �
1.3b 0.3a
changed for 14 days (data not shown) but changed to two peaks around 0.10 88.4 89.9 � 88.3 � 19.2 � 79.2 � 1.8b 98.4
434 and 1714 nm after 42 days (Fig. 6A). These results suggest that � 1.1c 2.1c 2.2b �
pectin at 0.05% could not maintain long-term stability of the emulsions. 1.8c 0.8a
At 0.10% and 0.20% pectin, the size distribution of the emulsions 0.20 87.4 89.1 � 88.9 � 26.2 � 72.3 � 2.5c 98.5
1.9c 2.8c 1.7c
increased to 709 and 643 nm after 42 days, respectively (Fig. 6A). At
� �
1.8c 0.7a
0.05%, 0.10% and 0.20% pectin, ζ-potential of the emulsions gradually
changed to 33, 36 and 40 mV after 42 days, respectively (Fig. 6B). Different letters in the same column represent statistically significant differences
On the whole, the addition of pectin could improve physical stability of (p < 0.05).

Fig. 8. Schematic diagram of peppermint oil emulsions formation and interfacial structures at various concentrations of pectin. The concentration of zein was 0.2%
(w/w).

8
H. Cheng et al. Food Hydrocolloids 103 (2020) 105675

particles and pectin molecules on the surface of oil droplets (Figs. 3 and
5). The encapsulation efficiency of peppermint oil in zein-GA nano­
particles reportedly varied from 89% to 54% as the zein/peppermint oil
mass ratio decreased from 4:1 to 1:1 (Chen et al., 2015). The encapsu­
lation efficiency of menthol and menthone was consistent with that of
the whole peppermint oil (Table 2). The efficiency of thymol oil
encapsulated in SC-stabilized emulsions decreased from 98% to 72%
with the protein/oil mass ratio from 10:1 to 10:4 (Pan, Chen, Davidson,
& Zhong, 2014). The efficiency of clove bud oil encapsulated by whey
protein concentrate-GA mixtures was about 80% at the emulsifier/oil
mass ratio of 1:1 (Luo et al., 2014). In the present study, the zein-pectin
particles showed similar encapsulation efficiency for peppermint oil
even though at the emulsifier/oil mass ratio lower than 1:10.

3.3.1.2. Partition of resveratrol. The total encapsulation efficiency of

resveratrol in all the emulsions stabilized by zein and zein-pectin par­
ticles was about 99% (Table 2). The polyphenol encapsulation efficiency
in the peppermint oil emulsions was significantly greater than that in the
corresponding lipid-free system of colloidal particles (Table 1). When
the concentration of pectin increased from 0.05% to 0.10%, the per­
centage of resveratrol encapsulated by zein colloidal particles in the
aqueous phase decreased from 33% to 18%, while the polyphenol con­
tent in the emulsified oil droplets increased from 66% to 80%. However,
the percentage of resveratrol in zein colloidal particles and in the
emulsified oil droplets was 26% and 72% at 0.20% pectin, respectively.
A significant positive correlation (r ¼ 0.963, p ¼ 0.037) between the
percentage of resveratrol in the emulsified oil droplets and the interfa­
cial zein percentage was observed, while the percentage of encapsulated
resveratrol in the aqueous phase was negatively correlated with the
protein interfacial percentage (r ¼ 0.971, p ¼ 0.029). Similar trends
were also reported in sunflower oil emulsions stabilized by whey protein
isolate-resveratrol complexes (Fan et al., 2017) and corn oil emulsions
stabilized by soy protein isolate-resveratrol complexes (Wan et al.,
2014). It has been reported that resveratrol has limited solubility both in
aqueous solution (30 μg/mL) (Summerlin et al., 2015) and vegetable oils
(e.g. ~ 86 μg/mL in corn oil and below 0.1 μg/mL in sunflower oil) (Filip
et al., 2003; Hung, Chen, Liao, Lo, & Fang, 2006). The total encapsu­
lation efficiencies of resveratrol in peppermint oil emulsions (Table 2)
Fig. 9. Retention of menthol (A) and menthone (B) in emulsions prepared by
were greater than that (~70%–80%) in sunflower oil emulsions (Fan
bare zein particles (Z), resveratrol-loaded zein (R/Z) and zein-pectin (R/ZP)
et al., 2017). Matos et al. (2018) reported that about 98% resveratrol
particles at various pectin concentrations and stored up to 42 days. The con­
was encapsulated in miglyol and orange oil emulsions stabilized by centration of zein was 0.2% (w/w).
OSA-starch particles, as a result of the polyphenol high solubility in
flavor oils. It is thus speculated that resveratrol co-existed in the inner oil
3.3.2.2. Resveratrol. Resveratrol is labile to isomerization, oxidation,
phase and at the surface of emulsified peppermint oil droplets. The less
and degradation, which was dependent on light, pH, and temperature
than 2% of free resveratrol (~4 μg/mL) in the aqueous phase was lower
(Zupancic, Lavric, & Kristl, 2015). The content of resveratrol in the
than its solubility (30 μg/mL) (Summerlin et al., 2015). Therefore,
whole zein-stabilized emulsions decreased slowly to 93% after storage
peppermint essential oil emulsions stabilized by zein-based particles
for 14 days, which then accelerated to 52% remaining after 42 days
could be a suitable carrier for the encapsulation of resveratrol.
(Fig. 10A). The decomposition was faster than that in
soy-lecithin-stabilized peanut oil emulsions with about 80% remaining
3.3.2. Storage stability of bioactive compounds
after 30 days (Sessa, Tsao, Liu, Ferrari, & Donsi, 2011). The stability of
resveratrol was significantly improved in the whole emulsions with
3.3.2.1. Menthol and menthone. Peppermint oil contained about 27% pectin, with 95% of the polyphenol remaining after 28 days. Then, the
menthol and 29% menthone as shown in the composition reported in resveratrol decomposition was dependent on the concentration of
Table S1. Loss of menthol and menthone in zein-stabilized emulsions pectin, with 70%, 82% and 83% remaining at the polysaccharide con­
was fast, with 18% and 26% remaining after 42 days, respectively centrations of 0.05%, 0.10% and 0.20% after 42 days, respectively
(Fig. 9A and B). Inclusion of resveratrol in zein-stabilized emulsions (Fig. 10A). Similar degradation patterns were observed in the aqueous
significantly delayed the decomposition of both menthol and menthone, phase (Fig. 10B) and the emulsified oil droplets (Fig. 10C). However,
with 37% and 51% remaining after 42 days, respectively. This is in loss of resveratrol was faster in the emulsified oil droplets than in the
agreement with a previous report that oxidative stability of corn oil continuous phase.
emulsion was enhanced by using soy protein-resveratrol complexes as Addition of pectin facilitated higher surface coverage of both oil
stabilizers with reduced lipid hydroperoxides and volatile hexanal (Wan droplets and resveratrol-loaded zein particles (Figs. 2 and 5) to further
et al., 2014). A further decrease in the peppermint oil decomposition shield peppermint oil and resveratrol from environmental factors, thus
was observed as the concentration of pectin increased. At 0.2% pectin, improving their storage stability (Figs. 9–10). It has been reported that
approximately 70% of menthol and 76% of menthone remained after 42 resveratrol at the oil-water interface could provide the protective effect
days. on α-tocopherol dissolved in the inner oil phase of sunflower oil

9
H. Cheng et al. Food Hydrocolloids 103 (2020) 105675

Table 3
MIC and fractional inhibitory concentration index (FICI) of resveratrol and
peppermint oil (PO) against the two microorganisms tested.
Strains MIC (μg/mL) FICI

Alone Combination

Resveratrol PO Resveratrol PO

S. aureus 100 1000 25 500 0.750

S. Typhimurium 150 1250 18.75 625 0.625

from the lower MIC value against S. aureus (100 μg/mL) and
S. Typhimurium (150 μg/mL). It has been reported that the MIC of
resveratrol against Gram-positive and Gram-negative bacteria was
16.5–260 μg/mL and 0.625–521 μg/mL, respectively (Ma et al., 2018).
The more effective antimicrobial activity against S. aureus than
S. Typhimurium was due to the difference in the outer layers of
Gram-negative and Gram-positive bacteria (Seow et al., 2014).
Peppermint oil and resveratrol displayed a synergistic effect with the
FICI values of 0.750 and 0.625 for S. aureus and S. Typhimurium,
respectively (Table 3). Phenolic compounds exhibit the highest antimi­
crobial activity among the plant phytochemicals (Burt, 2004). Menthol
and menthone were reportedly the major compounds contributing to
antimicrobial activity of peppermint oil (Iscan et al., 2002). Synergistic
effect in antimicrobial activity is believed to be the fact that the com­
bined compounds with distinct molecular structures simultaneously
challenge the resistance of target microorganisms at multiple different
sites (Burt, 2004; Seow et al., 2014; Shi et al., 2017).

3.4.2. Effect of resveratrol on antimicrobial activity of peppermint oil

emulsions
The MIC values of zein and pectin were higher than 5000 μg/mL
(data not shown), indicating that both materials had no significant
antimicrobial activity. The MIC values of peppermint oil emulsions
stabilized by bare zein particles for S. aureus and S. Typhimurium in
Table 4 were similar to those of bulk peppermint oil. Liang et al. (2012)
reported that peppermint oil emulsions stabilized by modified starch
had the same value of MIC as bulk oil for L. monocytogenes and S. aureus.
A significant enhancement in the antimicrobial activity of
zein-stabilized emulsions was observed upon the addition of pectin. In
the case of 0.05% pectin, the MIC values against S. aureus and
S. Typhimurium were 750 and 875 μg/mL, respectively. However, a
further increase in pectin concentration resulted in a decrease in anti­
microbial activity. The co-inclusion of resveratrol further improved the
antimicrobial activity of all the emulsions.
The emulsion-based delivery of EOs is likely to improve antimicro­
bial activity (Zahi, El Hattab, Liang, & Yuan, 2017), which is related to
oil droplet size and surface charge (Donsi et al., 2016). This influences
Fig. 10. Retention of resveratrol in peppermint oil emulsions made with zein-
based particles at various pectin concentrations and stored up to 42 days; (A),
the transport of EOs to the cell membrane, as well as their interaction
the whole emulsion; (B), the aqueous phase; (C), the emulsified oil droplets. The with the multiple molecular sites at the microbial cell membrane. A
concentration of zein was 0.2% (w/w). significant decrease in droplet size as a result of pectin addition (Fig. 6)
might contribute to bringing the peppermint oil molecules in contact
emulsions against decomposition by loss of the polyphenol itself (Wang, with their action sites for Gram-positive bacteria (Nazzaro, Fratianni, De
Gao et al., 2016). Therefore, resveratrol in the emulsified oil droplets Martino, Coppola, & De Feo, 2013). Furthermore, the emulsion droplets
could provide better protection of peppermint oil against decomposition
compared to the polyphenol in the aqueous phase. Table 4
Effect of resveratrol (Res) on the MIC values of peppermint oil emulsions sta­
bilized by bare zein (Z) and zein-pectin (ZP) particles at various pectin con­
3.4. Antimicrobial activity
centrations against S. aureus and S. Typhimurium. The MIC values were expressed
as real contents of Res and peppermint oil on the emulsion.
3.4.1. Antimicrobial activity and synergism testing
Emulsion MIC (μg/mL, S. aureus) MIC (μg/mL, S. Typhimurium)
The MIC value of peppermint oil against S. aureus and S. Typhimurium
was 1000 and 1250 μg/mL, respectively (Table 3). These results are in PO PO, Res PO PO, Res
agreement with the finding of Iscan et al. (2002) that peppermint oil Z 1000 875, 3 1250 1000, 4
showed moderate inhibitory effects for S. aureus (MIC 625–2500 μg/mL) ZP (0.05%) 750 625, 2 875 625, 2.5
and S. Typhimurium (MIC 1250–2500 μg/mL). Resveratrol displayed a ZP (0.10%) 875 750, 2.5 1000 750, 3
ZP (0.20%) 875 750, 2.5 1000 750, 3
higher inhibitory effect compared to peppermint oil, which is evident

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H. Cheng et al. Food Hydrocolloids 103 (2020) 105675

with hydrophilic surfaces might pass through the cell membrane of

Gram-negative bacteria (Majeed et al., 2016). However, the decrease in
the antimicrobial activity at higher pectin concentrations (Table 4)
might be due to high encapsulation efficiency (Table 2), leading to less
content of antimicrobial components dispersed into the aqueous phase
(Donsi et al., 2012).

3.4.3. Antimicrobial activity of peppermint oil emulsions during storage

The reductions of S. aureus and S. Typhimurium with regard to
freshly-prepared emulsions stabilized by bare zein particles was 3.33
and 3.17 Log CFU/mL (Fig. 11A and B), respectively. The emulsions
stabilized by resveratrol-loaded zein-pectin particles exhibited the
highest inhibitory effect against both targeted microorganisms when the
pectin concentration was 0.05%. A decrease in the inhibitory effect was
observed at higher pectin concentrations. The results were consistent
with those obtained by MIC determination (Table 4). Resveratrol-loaded
zein and zein-pectin colloidal particles reduced about 0.38 and 0.22 Log
CFU/mL for S. aureus and S. Typhimurium, respectively (Table S2). The
peppermint oil emulsions stabilized by R/Z and R/ZP particles had a
greater reduction of both targeted microorganisms than the sum of
corresponding colloidal particles and peppermint oil emulsions. These
results indicate that the combination of peppermint oil and resveratrol
showed a synergistic antimicrobial effect against the two tested
microorganisms.
Antimicrobial activity of all peppermint oil emulsions decreased
significantly over time (Fig. 11). The reductions of S. aureus and
S. Typhimurium always ranked in the order R/ZP-stabilized emulsions >
R/Z-stabilized emulsions > Z-stabilized emulsions. After storage for 42
days, the reduction of S. aureus was 0.68, 1.00 and 1.78 Log CFU/mL,
while the reductions of S. Typhimurium were 0.54, 0.78 and 1.54 Log
CFU/mL, respectively. Further increases in the pectin concentration
resulted in the improvement of retention of antimicrobial activity during
storage, with about 2.92 Log CFU/mL reduction for S. aureus and 2.75
Log CFU/mL reduction for S. Typhimurium remaining after 42 days at
0.20% pectin. In general, the higher the pectin concentration, the higher
was the retention of antimicrobial activity. Although pectin at 0.10%
and 0.20% impaired antimicrobial activity of freshly-prepared emul­
sions slightly (Fig. 11 and Table 4), pectin at such concentrations could Fig. 11. Reductions of S. aureus (A) and S. Typhimurium (B) treated with
significantly improve chemical stability of both resveratrol and emulsions stabilized by bare zein particles (Z), resveratrol-loaded zein (R/Z)
peppermint oil (Figs. 9–10), contributing to a long-term antimicrobial and zein-pectin (R/ZP) particles at various pectin concentrations and stored up
activity in the whole emulsions during storage (Fig. 11). Additionally, to 42 days. The concentration of zein was 0.2% (w/w).
the sustained release over time of the EOs from the emulsion droplets to
the aqueous phase may contribute to prolonged antimicrobial activity CRediT authorship contribution statement
(Donsi et al., 2016). An increase in the pectin concentration resulted in
the formation of more and more holes on the surface of oil droplets Hao Cheng: Conceptualization, Investigation, Writing - original
(Fig. 7F–H), which may facilitate the release of antimicrobial compo­ draft, Writing - review & editing. Muhammad Aslam Khan: Investi­
nents from the emulsified oil droplets to the aqueous phase during gation. Zhenfeng Xie: Investigation. Shengnan Tao: Resources.
storage. Yunxing Li: Resources. Li Liang: Conceptualization, Resources, Writing
- review & editing, Supervision.
4. Conclusions
Acknowledgements
In the present study, peppermint oil emulsions stabilized by
resveratrol-zein-pectin ternary complex particles have been successfully This work received supports from the National Natural Science
prepared, showing a good encapsulation performance for both resver­ Foundation of China (NSFC Project 31571781), the Fundamental
atrol and peppermint oil. This system has combined the synergistic ef­ Research Funds for the Central Universities (JUSRP51711B) and the
fect of two antibacterial agents and emulsion-based carrier, which Postgraduate Research & Practice Innovation Program of Jiangsu
contributes to the improvement of antimicrobial efficiency and chemical Province (KYCX17_1411).
stability. These results obtained here should provide the possibility of
co-encapsulating multiple antimicrobial agents with different physico­ Appendix A. Supplementary data
chemical properties within single-emulsion-based carrier systems.
Supplementary data to this article can be found online at https://doi.
Declaration of competing interest org/10.1016/j.foodhyd.2020.105675.

We wish to confirm that there are no known conflicts of interest

associated with this publication and there has been no significant
financial support for this work that could have influenced its outcome.

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H. Cheng et al. Food Hydrocolloids 103 (2020) 105675

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