coatings

Article
Encapsulation of Grapefruit Essential Oil in
Emulsion-Based Edible Film Prepared by Plum
(Pruni Domesticae Semen) Seed Protein Isolate and
Gum Acacia Conjugates
Chen Li 1 , Jiliu Pei 1 , Xiaohui Xiong 1, * and Feng Xue 2
1 College of Food Science and Light Industry, Nanjing Tech University, 30 Puzhu South Road, Pukou District,
Nanjing 211816, China; lichenfs@njtech.edu.cn (C.L.); jiliupei@njtech.edu.cn (J.P.)
2 School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210023, China; xuefeng@njucm.edu.cn
* Correspondence: sateam@njtech.edu.cn; Tel.: +86-138-13362715




Received: 14 July 2020; Accepted: 8 August 2020; Published: 12 August 2020


Abstract: A dry-heated Maillard reaction was used to prepare plum seed protein isolate and gum
acacia conjugates. Emulsion-based edible films (EBEF) were prepared by the encapsulation of
grapefruit essential oil using conjugates solution as the continuous phase. The conjugates formed
from 3 days of dry heating showed a significant improvement in emulsifying properties due to
the unfolding of protein, as confirmed by structure analysis. The droplet size, electrical charge,
and viscosity of emulsions increased with the increasing essential oil concentration, and all emulsions
exhibited ‘gel’-like behavior. The water vapor barrier property, surface hydrophobicity, mechanical
properties, and thermal stability of the films were improved as the essential oil content increased in
the range of 1–4% due to enhancement in intermolecular interaction and compatibility, as well as a
denser microstructure. Furthermore, all films exhibited an inhibitory effect against E. coli, while their
radical scavenging activity depended on the release rate from films. The results obtained in this work
confirmed that EBEF could be used as a novel food active packaging in the near future.

Keywords: Maillard conjugates; emulsion based edible films; grapefruit essential oil

1. Introduction
Proteins, polysaccharides, and lipids are the main components used to produce edible films.
However, there are some disadvantages for each polymer. Protein films, such as soy protein, corn protein,
whey protein, and caseinates, possessed better barrier properties but poorer mechanical properties than
polysaccharides films [1,2]. The films made from polysaccharides (such as chitosan, fiber, and starch)
were more transparent and oil-resistant but exhibited higher water permeabilities than protein films.
The lipids films, such as beeswax and fatty acids, were fragile and brittle, but were used for water
transmission reduction [3]. Therefore, it is important to produce hybrid films by combining hydrophilic
matrix with hydrophobic compounds in order to obtain multiple properties.
The composite films from hydrocolloids and lipids mixtures can be obtained by bilayers or
emulsions technology. The bi-layers films have shown some cracks or non-uniform surface [4].
Therefore, the emulsion-based edible films (EBEF), which can be easily obtained by film-forming
casting following drying process, have attracted increasing research interest [5]. The properties of
EBEF can be significantly affected by emulsification techniques, as well as the type and quantities
of hydrocolloids or lipids and their compatibility [6]. Therefore, the emulsifying properties of
hydrocolloids are important to properties of films.

Coatings 2020, 10, 784; doi:10.3390/coatings10080784 www.mdpi.com/journal/coatings

Coatings 2020, 10, 784 2 of 19

Protein and polysaccharide conjugates can be produced by Maillard-type reactions. It has been
reported that conjugates prepared from this method exhibited remarkable emulsifying properties [7].
Our previous studies have also suggested that grafted soy protein, peanut protein, and buckwheat
protein showed significant improvement in emulsifying properties [8–12]. Moreover, our previous
study found that grafted peanut protein showed superior film-forming abilities [13]. Therefore,
the grafted protein could be a potential material for the preparation of EBEF.
Plum (Pruni domesticae semen) is a multi-purpose crop of great industrial importance, which is
widely cultivated in China. Plum seed, due to its high content in amygdalin (20 g kg−1 ) and protein
(typically over 280 g kg−1 ), became a valuable source of Chinese medicine and food [14]. Usually,
the seed was only used for extraction of amygdalin for its anti-tumor activity, resulting in abundant
protein waste. Therefore, it is necessary to employ a technique, such as graft modification, in order to
utilize this protein preferably.
The essential oil (EO) has shown many biological activities, such as antioxidant, antimicrobial,
and insecticidal properties [15]. For this reason, incorporation of EO into edible films as food active
packaging is preferred in food industry. It was reported that EO from grapefruit exhibited antibacterial
and antifungal effects [16]. Edible films incorporated with citrus oil have also been reported to be able
to enhance the overall quality of fruits and vegetables. Therefore, grapefruit essential oil could serve as
a potential active compound in food active packaging to inhibit the growth of spoilage and protect
food against oxidation [17].
Gum acacia (GA) is a well-characterized and favorable polysaccharide, which is utilized as a
thickener, emulsifier, and film-forming agent in the food industry [9,18,19]. In this study, for the
first time, new plum seed protein isolate (PSPI) conjugates were prepared with gum acacia through
Maillard reaction, and the conjugates were applied to prepare EBEF. The emulsion behavior prepared
by PSPI-GA conjugates and grapefruit essential oil (GF-EO) was characterized; the physical, structural,
antioxidant, and antimicrobial properties as well as the release patterns of essential oil from PSPI-GA
emulsion-based films were also evaluated.

2. Results and Discussion

2.1. Physicochemical Properties of Conjugates

The contents of lysine and arginine were lower in PSPI-GA conjugates (PSPI-GA 1, PSPI-GA 3,
PSPI-GA 5) compared to the PSPI and GA mixtures (PSPI-GA) Figure 1A), which is consistent with
previous study that showed that lysine and arginine are main free amino groups taking part in the
Maillard reaction [9]. The results indicated that the conjugates were formed by covalent binding.
As shown in Figure 1B,C, both EAI and ESI of conjugates increased as the conjugation reaction
continued up to 3 days, while decreased afterwards. This increase is attributed to the conjugates
combining the emulsifying property of proteins with the solvation property of the polysaccharides [20].
The decrease might be due to the generation of polymerization products as Maillard reaction
proceeded [18].
As suggested in Figure 1D, H0 values of conjugates were significantly (p < 0.05) lower than that of
PSPI/GA mixtures. As shown in Figure 1E, λmax was shifted to longer wavelengths (bathochromic
shift) when PSPI was grafted with GA, indicating the changes of PSPI conformation. As shown in
Figure 1F, conjugates exhibited an increase of unordered coils. These changes in structure all suggested
that the attachment of GA to PSPI lead to more hydrophilic and disordered structures with greater
conformational flexibility.
Coatings 2020, 10, 784 3 of 19
Coatings 2020, 10, x FOR PEER REVIEW 3 of 21

Figure 1. Lysine and arginine content (A), emulsifying properties (B) and (C) and surface hydrophobicity
(D), intrinsic
Figure emission
1. Lysine andfluorescence (E) and circular
arginine content dichroism properties
(A), emulsifying spectrum (F) of conjugates.
(B) and (C) and PSPI/GA:
surface
mixture of plum seed protein isolate (PSPI) and gum acacia (GA); PSPI-GA 1/3/5:
hydrophobicity (D), intrinsic emission fluorescence (E) and circular dichroism spectrum (F) of the PSPI-GA
conjugates
conjugates.prepared from
PSPI/GA: 1, 3, orof5 plum
mixture days. PSPI: plum seed
seed protein protein
isolate isolates.
(PSPI) GA: gum
and gum acaciaacacia.
(GA);Different
PSPI-GA
letters in the same pattern represent significant difference (p < 0.05).
1/3/5: the PSPI-GA conjugates prepared from 1, 3, or 5 days. PSPI: plum seed protein isolates. GA:
gum acacia. Different letters in the same pattern represent significant difference (p < 0.05).
2.2. Film-Forming Emulsion Properties

2.2. Film-Forming
2.2.1. Emulsion Properties
Size Distribution
Considering
2.2.1. the negative effects of extended dry heating on emulsifying properties, the conjugates
Size Distribution
prepared from 3-day reactions were chosen for preparing the film-forming emulsion. The size
Considering
distribution the negative
of film-forming effectscontaining
emulsion of extended dry contents
different heating on emulsifying
of EO properties,
was analyzed. Only one the
conjugates prepared from 3-day reactions were chosen for preparing the film-forming
peak was observed in Figure 2A, indicating that the incorporation of EO into the emulsion of emulsion. The
size distribution
conjugates resultedof in
film-forming emulsion
a mono-modal containing
distribution different contents
of droplets. of EO
This result was analyzed.
is consistent with Only
soy
protein films containing cinnamon and ginger EO previously reported [21]. In contrast, researchersof
one peak was observed in Figure 2A, indicating that the incorporation of EO into the emulsion
conjugates
observed resulted in distribution
a multimodal a mono-modal distribution
of droplets, when ofcinnamon
droplets. or
This result
ginger EOiswas
consistent with soy
incorporated in
protein films containing cinnamon and ginger EO previously reported [21]. In contrast, researchers
observed a multimodal distribution of droplets, when cinnamon or ginger EO was incorporated in
Coatings 2020, 10, 784 4 of 19
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sodium caseinate-based films [22]. Multimodal distribution was also reported in soy protein-based
sodium caseinate-based films [22]. Multimodal distribution was also reported in soy protein-based
film emulsified with flaxseed oil [23], gelatin-based films emulsified with olive oil [24], and whey
film emulsified with flaxseed oil [23], gelatin-based films emulsified with olive oil [24], and whey
protein-based films emulsified with rapeseed oil [25]. They attributed this phenomenon to the
protein-based films emulsified with rapeseed oil [25]. They attributed this phenomenon to the
aggregation of polymers and coalescence of smaller droplets into larger ones after emulsification.
aggregation of polymers and coalescence of smaller droplets into larger ones after emulsification. In
In addition, Volume-mean diameter (D [3,4]) values of emulsion with EO concentrations ranging from
addition, Volume-mean diameter (D [3,4]) values of emulsion with EO concentrations ranging from
1% to 6% were 33.02 ± 2.01, 34.17 ± 1.94, 40.90 ± 3.04, and 63.84 ± 7.78 µm, respectively, indicating
1% to 6% were 33.02 ± 2.01, 34.17 ± 1.94, 40.90 ± 3.04, and 63.84 ± 7.78 μm, respectively, indicating that
that the droplet diameter of film-forming emulsion increased as the EO concentration increased.
the droplet diameter of film-forming emulsion increased as the EO concentration increased. This
This result might be due to the fact that with the increasing of EO concentration, insufficient adsorption
result might be due to the fact that with the increasing of EO concentration, insufficient adsorption
of conjugates at the oil–water interface might occur, leading to flocculation and coalescence after
of conjugates at the oil–water interface might occur, leading to flocculation and coalescence after
emulsification. On the other hand, the increasing of EO concentration could also resulted in thinner
emulsification. On the other hand, the increasing of EO concentration could also resulted in thinner
interfacial film
interfacial film surrounding
surrounding the
the oil
oil droplets,
droplets, which
which is
is more
more susceptible
susceptible to
to rupturing.
rupturing.

Figure
Figure 2.
2. Droplet
Droplet size
size distributions
distributions (A)
(A) and
and ζ-potential
ζ-potential (B)
(B) of
of film-forming
film-forming emulsion.
emulsion. EO EO1/2/4/6:
1/2/4/6:
PSPI-GA
PSPI-GA conjugates
conjugates emulsions
emulsions containing
containing grapefruit
grapefruit essential
essential oil
oil at
at 1%/2%/4%/6%
1%/2%/4%/6% level.level. PSPI:
PSPI: plum
plum
seed protein isolates.
seed protein isolates.GA:
GA:gumgum acacia.
acacia. EO:EO: grapefruit
grapefruit essential
essential oil. Different
oil. Different lettersletters in thepattern
in the same same
pattern represent
represent significant
significant (p < 0.05).
difference
difference (p < 0.05).

2.2.2. ζ-Potential
2.2.2. ζ-Potential
It is well known that the electrical charge of oil droplets is important in the stability of emulsion by
It is well known that the electrical charge of oil droplets is important in the stability of emulsion
affecting the electrostatic repulsion. As suggested in Figure 2B, due to the anionic nature of PSPI-GA
by affecting the electrostatic repulsion. As suggested in Figure 2B, due to the anionic nature of
conjugates absorbed to oil droplets, the negative electrical charge was observed in all emulsions.
PSPI-GA conjugates absorbed to oil droplets, the negative electrical charge was observed in all
EO concentration significantly affect the electrical charge. This is probably attributed to the ionizable
emulsions. EO concentration significantly affect the electrical charge. This is probably attributed to
compounds presented in EO. Furthermore, the difference of ζ-potential might lead to the changing of
the ionizable compounds presented in EO. Furthermore, the difference of ζ-potential might lead to
the intermolecular electrical repulsion in films, which could result in the changing of film structures.
the changing of the intermolecular electrical repulsion in films, which could result in the changing of
film
2.2.3.structures.
Rheological Behavior

2.2.3. The viscosityBehavior

Rheological of the film-forming solution are important to film properties, which could affect the
removal of air bubbles [26] and elimination of sagging [27] during the process. Therefore, the flow
The viscosity of the film-forming solution are important to film properties, which could affect
curves of film-forming emulsion loaded with different concentrations of EO were analyzed. As shown
the removal of air bubbles [26] and elimination of sagging [27] during the process. Therefore, the
in Figure 3A, the viscosity of emulsion increased as the EO concentration increased, which is probably
flow curves of film-forming emulsion loaded with different concentrations of EO were analyzed. As
due to the high viscosity of oil. In general, the film-forming emulsion showed shear thinning behavior.
shown2 in Figure 3A, the viscosity of emulsion increased as the EO concentration increased, which is
The R (coefficient of determination) values for emulsion with EO concentration ranging from 1% to 6%
probably due to the high viscosity of oil. In general, the film-forming emulsion showed shear
were 0.7331, 0.6891, 0.9456 and 0.9262, respectively. These results indicated that the fitted curves for the
thinning behavior. The R2 (coefficient of determination) values for emulsion with EO concentration
emulsions with a higher concentration of EO gave good agreement with the experimental data and that the
ranging from 1% to 6% were 0.7331, 0.6891, 0.9456 and 0.9262, respectively. These results indicated
degree of shear thinning increased as the concentration increased. The flow behavior index of emulsion
that the fitted curves for the emulsions with a higher concentration of EO gave good agreement with
with EO concentration ranging from 1% to 6% was calculated as 0.677, 0.629, 0.476, and 0.412, respectively.
the experimental data and that the degree of shear thinning increased as the concentration increased.
These results indicated that all the film-forming emulsions exhibited a pseudo plastic behavior (the flow
The flow behavior index of emulsion with EO concentration ranging from 1% to 6% was calculated
behavior index < 1) in which the viscosity decreases with the increasing of shear rate. This behavior is
as 0.677, 0.629, 0.476, and 0.412, respectively. These results indicated that all the film-forming
emulsions exhibited a pseudo plastic behavior (the flow behavior index < 1) in which the viscosity
Coatings 2020, 10, 784 5 of 19

attributed to the fact that interactions between the emulsion components were disrupted as the shear
rates increased [23]. Previous studies reported a similar result in which that shear thinning behavior for
emulsion was observed when flaxseed oil was emulsified into soy protein films at concentrations ranging
from 3% to 10% [23]. The consistency coefficient of emulsion with EO concentration ranging from 1% to
6% was calculated as 0.0093, 0.0158, 0.0352 and 0.0798 Pa sn , respectively. The value of the consistency
coefficient is related to the inter-molecular forces of emulsion according to previous study [28]. Thus,
the inter-molecular forces in emulsion increased as the EO concentration increased.
Coatings 2020, 10, x FOR PEER REVIEW 6 of 21

Figure 3. Flow
Figure curves
3. Flow curves(A),
(A), storage modulus(B),
storage modulus (B),
andand
lossloss modulus
modulus (C) of(C) of film-forming
film-forming emulsion.emulsion.
EO
1/2/4/6: PSPI-GA
EO 1/2/4/6: PSPI-GA conjugates
conjugatesemulsions
emulsionscontaining
containinggrapefruit essential
grapefruit oil atoil1%/2%/4%/6%
essential level. level.
at 1%/2%/4%/6%
PSPI:PSPI:
plumplumseedseed protein
protein isolates.GA:
isolates. GA:gum
gumacacia.
acacia. EO:
EO:grapefruit essential
grapefruit oil.oil.
essential

2.3. Film Physical Properties

2.3.1. Transparency, Whiteness Index, and Swelling Ability

Coatings 2020, 10, 784 6 of 19

Figure 3B,C shows the influence of EO concentration on the variation of the storage modulus (G0 )
and loss modulus (G0 ) of film-forming emulsion. It could be observed that the value of G0 is higher
that G” for all samples. These results indicated that all samples exhibited ‘gel’-like behavior along
the entire frequency range. The concentration of EO significantly affected the values of G0 and G” of
the film-forming emulsion. The possible reason for this phenomenon is that the concentration of EO
could change the particle size distribution, which has been proven to have dramatic effects on flow
properties [29]. On the other hand, the concentration of EO could also change the inter-molecular
forces, which have been proven to affect the formation of the gel network [30].

2.3. Film Physical Properties

2.3.1. Transparency, Whiteness Index, and Swelling Ability

As shown in Table 1, the transparency of films was significantly affected by EO concentration.
The values decreased as Coatings
EO concentration
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6% EO level. On one hand, the transparency of emulsion-based films is affected by the oil type and
As
As shown
shown in
in Table
Table 1,
1, the
the transparency
transparency of
of films
films was
was significantly
significantly affected
affected by
by EO
EO concentration.
concentration.
concentration due to the fact As
that
As shown
oil
shown in
in Table
could
Table 1,
change
1,EOthe transparency
the extent
theconcentration
transparency of
of films was
of light
films was significantly
scattering.
significantly affected
On the
affected by
by EO concentration.
other,
EO theatfilm
concentration.
The
The values
values decreased
decreased as
as EO concentration increased
increased from
from 1%
1% to
to 4%,
4%, followed
followed by
by an
an increase
increase at the
the
The
The values
values decreased
decreased as
as EO
EO concentration
concentration increased
increased from
from 1%
1% to
to 4%,
4%, followed
followed by
by an
an increase
increase at the
atand
the
surface and internal structure
6%
6%
6%
EO
EO
EO
could
level.
level.
level.
On
On
On
also one
one
one
affect
hand,
hand,
hand,
the
the
the
the
light reflection
transparency
transparency
transparency
of
of
of
and absorption.
emulsion-based
emulsion-based
emulsion-based
films
films
films
is
is
is
Therefore,
affected
affected
affected
by
by
by
the
the
the
the
oil
oil
oil
effects
type
type
type and
and
6% EO level.
concentration On
due one
to hand,
the fact the
that transparency
oil could of
change emulsion-based
the extent of films
light is affected
scattering. Onby the
the oil
other,type
the and
film
of oil concentration on the microstructure
concentration
concentration
concentration
due
due
due to offact
to the
to the
the films
fact
fact
that
that were
oil
that oil
oil studied
could
could
could
change
change
change the later.
the
the
extent
extent of light
extent of
of light
scattering. On
light scattering.
scattering. On
the other,
On the
the other,
the film
other, the
the film
film
surface and
surface internal structure
and internal could also
structure could affect the
also affect light reflection
the light and absorption.
reflection and Therefore, the
absorption. Therefore, the
surface
surface and
and internal
internal structure
structure could
could also
also affect
affect the
the light
light reflection
reflection and
and absorption.
absorption. Therefore,
Therefore, the
the
Table 1. Transparency,effects
effects of
of
whiteness
effects
effects of
oil
oil concentration
of oil
concentration on
on
index, swelling,
oil concentration
concentration on
the
the microstructure
on the water vaporof
microstructure
the microstructure
microstructure
of films
films were
were studied
ofpermeability
of films
films were
studied
were studied
later.
later.
(WVP),
studied later. contact angle,
later.
tensile strength (TS), elongation
Table 1.
Table
at break (EB),
1. Transparency, and
Transparency, whiteness appearance
whiteness index, of
index, swelling, PSPI-GA
swelling, water
water vapor films made
vapor permeability fromcontact
permeability (WVP),
(WVP), essential
contact angle,
angle,
Table
Table 1.1. Transparency,
Transparency, whiteness
whiteness index, swelling,
index,(EB),
swelling, water
water vapor
vapor permeability
permeability (WVP),
(WVP), contact
contact angle,
angle,
oil emulsions. tensile
tensile strength
tensile strength
(TS),
strength (TS),
elongation
(TS), elongation
at
elongation at
break
at break
break (EB),
and
(EB), and
appearance
and appearance
of
appearance of
PSPI-GA
of PSPI-GA
films
PSPI-GA films
made
films made
from
made from
essential
from essential
essential
tensile
oil strength (TS), elongation at break (EB), and appearance of PSPI-GA films made from essential
emulsions.
oil
oil emulsions.
emulsions.
Transparency Whitenessoil emulsions. WVP Contact
Sample Swelling (%) Whiteness
Transparency
Transparency Whiteness Swelling
Swelling WVP
WVP TS (MPa)
Contact
Contact EB (%) Appearance
mg/(cm2 ·d)
◦
(%/mm) Index WI
Sample
Sample Transparency
Transparency Whiteness
Whiteness SwellingAngleWVP
Swelling ( )2
WVP Contact
Contact° TS
TS (MPa)
(MPa) EB
EB (%)
(%) Appearance
Appearance
Sample (%/mm)
(%/mm) Index
Index WI
WI (%)
(%) mg/(cm
mg/(cm 2∙d) Angle ((°)) TS
TS (MPa) EB
EB (%) Appearance
Sample (%/mm) Index 2∙d) Angle (MPa) (%) Appearance
Index WI (%) mg/(cm2∙d)
mg/(cm Angle
Angle ((°))
°
(%/mm) WI (%) ∙d)
d 1.36
1.36 69.99
69.99 599.57
599.57
c 90.98 a
90.98 104.94
104.94 1.88
1.88 26.55a
26.55
EO 1 1.36 ± 0.06 69.99 ± EOd 1
0.68
EO 1 599.57 ±
1.36
1.3615.07 d 90.98
69.99
69.99 ± 1.80 599.57
599.57104.94 ± 1.20
90.98
90.98 1.88 ±
104.94
104.94 0.09 b 1.88
1.8826.55 ± 4.02
26.55
26.55
EO
EO 11 ± 0.06
± 0.06 dd ± 0.68
± 0.68 d
d ± 15.07
± 15.07
d
d ± 1.80
± 1.80 cc ± 1.20
± 1.20 aa ± 0.09
± 0.09 bb ± 4.02
± 4.02 aa
±± 0.06
0.06 d
d ±± 0.68
0.68 d
d ±± 15.07 d
15.07 d ±± 1.80
1.80 c
c ±± 1.20
1.20 a
a ±± 0.09
0.09 b
b ±± 4.02
4.02 a
a

0.85
0.85 64.34
64.34 567.75
567.75 87.22
87.22 108.14
108.14 2.78
2.78 30.43
30.43
EO 2 0.85 ± 0.01 c EOc 2
EO
64.34 ± 0.42 2 567.75 0.85
± 11.07 c 64.34 567.75
b 87.22 b 108.14
2.78 ± 0.17 c 2.7830.43 30.43
±±±2.99 a
EO 2 ± 0.85
± 0.01 c 0.42 c± 1.06±
±87.22
64.34 c 11.07108.14
±567.75 c ±±±87.22
0.81
1.06 bb ±108.14
0.81 b ± 2.78
0.17 c 30.43
2.99 a a
±± 0.01 ±
±± 0.42 ±± 11.07 ±± 1.06 ±
±± 0.81 ±
±± 0.17 ±± 2.99
c c b c
EO 2 0.01 c 0.42 c 11.07 c 1.06 b 0.81 b 0.17 c 2.99 a
0.01 c 0.42 c 11.07 c 1.06 b 0.81 b 0.17 c 2.99 a
0.71
0.71 59.47
59.47 466.51
466.51 78.95
78.95 114.96
114.96 3.45
3.45 45.30
45.30
EO 4
EO 4 0.71 a 59.47 466.51 b 78.95 114.96 3.45 45.30
EO 4 0.71 ± 0.02 a EOb
EO 44
59.47 ± 0.44 466.51 ±0.71
± 0.02
± 42.21 b 59.47
±78.95±2.22
0.44 bb ±a466.51
42.21114.96 78.95
±±2.222.01aaac
±2.22 114.96
± 2.01
3.45 2.01
± 0.24c 3.45
d ± 0.24 d
45.30 45.30 ccc
±±±±1ss.76
1ss.76
±± 0.02 ± 0.44 ± 42.21 ± ±± 0.24 1ss.76
a b c d
0.02
0.02
a
a ± 0.44
± 0.44
b
b ± 42.21
± 42.21
b
b ±2.22
±2.22 a ±± 2.01
2.01 c
c
± 0.24
0.24 d
d
± 1ss.76
1ss.76 c
c

0.78
0.78 56.52
56.52 388.77
388.77 80.22
80.22 110.85
110.85 1.54
1.54 39.40
39.40
EO 6
EO 6 0.78 b 56.52 a 388.77 a 80.22 110.85 1.54 39.40
EO
EOa 66 ±0.78
0.01 b a ±56.52
0.64 a ±a388.77
30.47 a 80.22aa
±1.83 ±110.85
3.11 bcbc
a ± ±1.54
0.11 aa ±39.40
2.21 bbb
EO 6 0.78 ± 0.01 b56.52 ± 0.64 388.77 ±
±± 0.01
± 30.47
0.01
0.01
b
b
±
± 0.64
80.22±1.83
0.64
± 0.64
a
a
±± 30.47
110.85
30.47
± 30.47
a
a ± 3.11 bc
±1.83
±1.83
±1.83 a
a ±
±± 3.11
1.54 ± 0.11
3.11
3.11 bc
bc ±± 0.11
39.40
0.11
0.11 a
a ±±±±2.21
2.21
2.21
2.21 b
b

EO
EO 1/2/4/6:
1/2/4/6: PSPI-GA
PSPI-GA conjugate
conjugate films
films containing
containing grapefruit
grapefruit essential
essential oil
oil at
at 1%/2%/4%/6%
1%/2%/4%/6% level.
level. PSPI:
PSPI:
EO
EO 1/2/4/6:
1/2/4/6: PSPI-GA conjugate
PSPI-GAisolates.
conjugate films
films containing
containing grapefruit
grapefruit essential
essential oil at
at 1%/2%/4%/6%
oiloil. 1%/2%/4%/6% level.
level. PSPI:
PSPI:
EO 1/2/4/6: PSPI-GA conjugate plum
plum seed
films
seed protein
containing
protein isolates. GA:
grapefruit
GA: gum
gum acacia.
essential
acacia. EO:
oil
EO: grapefruit
at essential
1%/2%/4%/6%
grapefruit essential Different
level.
oil. PSPI:
Different letters
plum
letters within
seed
within aa
plum
plum seed
seed protein
protein isolates.
isolates. GA:
GA: gum
gum acacia.
acacia. EO:
EO: grapefruit
grapefruit essential
essential oil.
oil. Different
Different letters
letters within
within aa
protein isolates. GA: gum acacia.column
column EO: represent
represent significant
grapefruit
significant difference
essential oil.
difference (p << 0.05).
0.05).
Different
(p letters within a column represent significant
column
column represent
represent significant
significant difference
difference (p
(p << 0.05).
0.05).
difference (p < 0.05).
As shown
As shown in in Table
Table 1,1, the
the concentration
concentration of of EO
EO had
had aa significant
significant effect
effect on
on the
the whiteness
whiteness index
index of of
As
As shown in
in Table
shown films Table 1,
1, the concentration
thedarker
concentration of
of EO
EO had
had aa significant
significant effect
effect on the
the whiteness
on increase whiteness index
index of of
films.
films. Overall,
Overall, films became
became darker as
as the
the EO
EO concentration
concentration increased.
increased. The
The increase in
in darkness
darkness is
is
films.
films. Overall,
Overall, films
films became
became darker
darker as
as the
the EO
EO concentration
concentration increased.
increased. The
The increase
increase in
in darkness
darkness is
is
As shown in Table 1,presumably
the concentration
presumably due due toto the of
the existence EO
existence of had
of EO,
EO, whicha significant
which contains effect
contains pigments
pigments and on
and is the whiteness
is orange
orange in in color.
color. Theindex
The result of
result isis
presumably
presumably due
due to
to the
the existence
existence of
of EO,
EO, which
which contains
contains pigments
pigments and
and is
is orange
orange in
in color.
color. The
The result
result is
is
consistent
films. Overall, films became darker
consistent with
with the visual
as the
the visualEO appearance
concentration
appearance in
in films (Table 1).
increased.
films (Table 1). Changes
Changes Thein
in whiteness
increase index affected
in darkness by the
is
consistent
consistent with
with the visual appearance in
in films (Table 1).
1). Changes in whiteness
whiteness index
index affected
affected by
by the
the
color
color of the
of the oilthe
oil
visualwere
added
added
appearance
were also observed
also observedfilms in
(Table
in previous
previous
Changes
study in
study in whiteness
in which the
which theindex affected
lightness
lightness of by
of
the
aaa soy
soy
presumably due to the existence
color
color of
of of
the
the
protein-based
EO,
oil
oil which
added
added were
were contains
also
also pigments
observed
observed in
in and
previous
previous is
study
studyorange
in
in in
which
which color.
the
the The
lightness
lightness result
of
of a is
soy
soy
protein-based emulsion-type
emulsion-type film
film decreased
decreased as
as the
the flaxseed
flaxseed oil
oil concentration
concentration increased
increased [23].
[23].
consistent with the visualprotein-based
protein-based
appearance
As suggested
As
emulsion-type
emulsion-type
suggested in in films
in Table
Table 1,
film
film
(Table decreased
decreased
1, swelling 1).
swelling ability
as
as the
the
Changes
ability of
of the
flaxseed
flaxseed
in
the films
oil
oil concentration
concentration
whiteness
films decreased
decreased along index
along with
increased
increased
with theaffected [23].
[23].
the increase by
increase of the
of EOEO
As
As suggested
suggested in
in Table
Table 1,1,inswelling
swelling ability
ability of the
of is films
films decreased
thethought decreased along
along with
with the
the increase
increase of
of EOEO
color of the oil added wereconcentration.
also observed
concentration.
concentration.
concentration.
The
The
The
The
decrease
in previous
decrease
decrease
decrease
in
in
in
swelling
study
swelling
swelling
swelling
ability
in which
ability
ability
ability
is
is
is
the
thought
thought
thought
to
to
to
be
lightness
to be
be
be
associated
of
associated
associated
associated
a with
soy
with
with
with
an
an
an
an
increasing
protein-based
increasing
increasing
increasing
of
of
of
of
hydrophobicity by
hydrophobicity by adding
adding EO,EO, which
which could
could prevent
prevent the the matrix
matrix from
from binding
binding strongly
strongly to to water
water and and
emulsion-type film decreased as the
hydrophobicity
hydrophobicity
thus reduce
flaxseed
water
by adding
by uptake.
adding EO,oil
EO,
Our
concentration
which
which
result
could
could
is
prevent increased
preventwith
consistent
the matrix
the matrix
previous
[23].
from
from binding strongly
binding strongly
researches that
to water
to water
showed that
and
and
the
thus
thus reduce
reduce water
water uptake.
uptake. Our result is consistent with
with previous researches that showed that the
As suggested in Table thus1,reduce
swelling
swellingswellingwater
ability
ability abilityOur
uptake.
of chitosan-based
of of result
Our
chitosan-based result
the
films
films
is
films consistent
isdecreased
consistent aswith
decreased
decreased as the
previous
previous
along
the virgin
virgin
researches
researches
with
coconut
coconut oil the
oil
that
that showed
increase
concentration
concentration
that
showedincreased
that
of EO
increased
the
the
swelling
swelling ability of
ability to chitosan-based
of results
chitosan-based films
films decreased
decreased as the
as the virgin
virgin coconut
coconut oil concentration increased
oil concentration increased
concentration. The decrease[31]
[31] and similar
andinsimilar
swelling
to results observed
ability
observed in
is in soy
thought protein
soy protein film
tofilm with
bewith flaxseed
associated
flaxseed oiloil [23].
with
[23]. an increasing of
[31]
[31] and
and similar
similar toto results
results observed
observed in in soy
soy protein
protein film
film with
with flaxseed
flaxseed oil oil [23].
[23].
hydrophobicity by adding EO,
2.3.2. which
Water Vapor could prevent
Permeability (WVP),the matrix
Contact from
Angle, and binding
Mechanical strongly
Properties to water and
2.3.2.
2.3.2. Water
Water Vapor
Vapor Permeability
Permeability (WVP),
(WVP), Contact
Contact Angle,
Angle, and
and Mechanical
Mechanical Properties
Properties
2.3.2.
thus reduce water uptake. Our Water Vapor
result Permeability
is consistent (WVP),
withContact Angle,
previous and Mechanical
researches Properties
that showed thatwater
the
The water
The water vapor
vapor permeability
permeability is
is directly
directly related
related to to the
the property
property of
of the
the film
film to
to restrict
restrict water
The
The water
water vapor
vapor permeability
permeability is
is directly
directly related
related to
to the
the property
property of
of the
the film
film to restrict
to vapor water
restrictbarrier
water
swelling ability of chitosan-based
migration
migration films
of
of the decreased
food.
the food. The as
most
The most the virgin
striking coconut
feature
striking feature for
for EBEF oil
EBEF isisconcentration
the excellent
the excellent increased
water [31]
migration of
of the
migration which food.
food. The
the might The most
most striking
striking feature
feature for
for EBEF
EBEF is is the
the excellent water
water vapor
vapor barrier
barrier
property,
and similar to results observed
property, in soy
which protein
might be film
be attributed
with
attributed toflaxseed
to the
the oil incorporation.
oil incorporation.
oil [23]. As excellent
As shown in
shown inwater
Tablevapor
Table 1, thebarrier
1, the WVP
WVP
property,
property, which might be attributed to the oil incorporation. As shown in Table 1,
which might be attributed to the oil incorporation. As shown in Table 1, the
the WVP
WVP
decreased
decreased as
as the
the EO
EO concentration
concentration increased
increased from
from 1%
1% to 4%. This
This is probable
probable due to the
the fact that
that the
decreased
decreasedof as the
the EO
EO concentration
as film-forming
concentration increased
increased from
from 1% to
to 4%.
4%.
4%. This
is
is
is probable
due
due to
to
to the
fact
fact
fact that
the
the
viscosity
viscosity of film-forming emulsion
emulsion increased
increased as1%
as EOtolevels
EO levelsThis probable
increased.
increased.
due been
It has
It has theproven
been proven
thatthat
the
that
2.3.2. Water Vapor Permeability
viscosity (WVP),
viscosity of
increasing
of
the
Contact
film-forming
film-forming
viscosity of
Angle,
emulsion and
increased
emulsion increased
film-forming emulsion
Mechanical
as EO
ascould levels
EO reduce Properties
increased.
levels increased.
water
It has
It has
mobility
been
been the
through
proven
proven
film
that
that
[23].
increasing
increasing the
the viscosity of film-forming emulsion could reduce water mobility through the film [23].
However, the viscosity
increasing the viscosity
WVP was
of film-forming
ofnot
film-forming
decreased
emulsion
emulsion
as EO
could
could
levels
reduce
reduce water
increased water
to 6%.
mobility
mobility
This is
through
through
probably
the
the
due
film
filmto
[23].
[23].
the
However,
The water vapor permeability
However, the
the WVP
is
WVP was
directly
was not
not decreased
related
decreased as
to
as EO
the
EO levels increased
property
levels increased of to
to 6%.
the
6%. This
film
This is
istoprobably
restrict
probably due
due to
water
to the
the
However,
disruption ofthe WVP
of structure was not
structure which decreased
which might
might provide as EO levels
provide channels increased
channels for
for water to 6%.
water migration.
migration. This is probably due to the
disruption
disruption of structure which might provide channels for water migration.
migration of the food. The most
disruption
Water
Water
striking
of
contact feature
structure which
angle are for
might EBEF
provide
important is
in the excellent
channels
the film for water
surface water vapor
migration.
wettability and barrier
moisture property,
transport.
Water contact
contact angle
angle are important
angle are
are important
in
in the
the film
film surface
surface wettability
wettability and and moisture
moisture transport.
transport.
which might be attributed toWater
Higher
Higher
Higher the
surface
surface
surface
contact
oil incorporation.
hydrophobicity
hydrophobicity
hydrophobicity
important
is
is necessary
infor
As shown
is necessary
necessary for
for
the filmwhen
EBEF
EBEF
EBEF
surface
inwhen
Table
when
used
used
used1,wettability
the
as
as
as WVP
packaging
packaging
packaging
andor
or
or
moisture
decreased transport.
as the
coatings; hence,
coatings;
coatings;
hence,
hence,
the
the
the
Higher
contact surface
angle ofhydrophobicity
edible films is
should necessary
be as for
large EBEF
as when used
possible. As as packaging
suggested in or coatings;
Table 1, filmshence,
made the
of
contact
contact angle
angle of
of edible
edible films
films should
should be
be as
as large
large as
as possible.
possible. As
As suggested
suggested in
in Table
Table 1,
1, films
films made
made of
of
contact
EO at angle
at the
the 4% of
4% level edible films
level possessed should
possessed the be
the highest as large
highest contact as possible.
contact angle.
angle. This As
This is suggested
is helpful
helpful for in Table
for explaining 1,
explaining whyfilms made
why this
this film
filmof
EO
EO at the 4% level possessed the highest contact angle. This is helpful for explaining why this film
EO at
showed the
the4% level
lowest possessed
water vapor the highest contact
permeability. angle. This is helpful for explaining why this film
showed
showed the lowest water vapor permeability.
showed thethe lowest
lowest water
water vapor
vapor permeability.
permeability.
Coatings 2020, 10, 784 7 of 19

EO concentration increased from 1% to 4%. This is probable due to the fact that the viscosity of
film-forming emulsion increased as EO levels increased. It has been proven that increasing the viscosity
of film-forming emulsion could reduce water mobility through the film [23]. However, the WVP was
not decreased as EO levels increased to 6%. This is probably due to the disruption of structure which
might provide channels for water migration.
Water contact angle are important in the film surface wettability and moisture transport.
Higher surface hydrophobicity is necessary for EBEF when used as packaging or coatings; hence,
the contact angle of edible films should be as large as possible. As suggested in Table 1, films made of
EO at the 4% level possessed the highest contact angle. This is helpful for explaining why this film
showed the lowest water vapor permeability.
Mechanical properties, which are usually measured by tensile strength (TS) and elongation at break
(EB), are key factors that determined the industrial application of films. In theory, the incorporation
of oil would disrupt the biopolymer network in the film, leading to increased flexibility (EB) and
decreased TS [32]. For instance, the incorporation of beeswax into pea-starch films decreased the
tensile strength when lipids became greater than 20% [33]; incorporation of olive oil decreased the
tensile strength of a gelatin-based film when the oil-to-protein ratio was increased from 5% to 10% [24];
the incorporation of sunflower oil decreased the tensile strength of quinoa protein-chitosan based
films when the lipids concentration was increased from 2.9% to 34.7% [34]. In contrast (as shown in
Table 1), the tensile strength of films increased as the EO concentration increased in the range of 1–4%.
This trend has been observed in previous studies in which the incorporation of flaxseed oil into soy
protein-based films increased the tensile strength when the lipids concentration was increased from
1% to 5% [23]. Ataréz et al. also reported that the increasing of cinnamon oil content resulted in an
increase in the tensile strength of soy protein-based films [21]. They attribute this phenomenon into
protein rearrangement, which resulted in a more ordered structure. The tensile strength decreased as
the EO content increased to 6%, which is probably due to disruption of the biopolymer network and
the formation of a holey microstructure. As expected in Table 1, the elongation of films was found to
be increased significantly when the EO content increased from 1% to 4%, and then it decreased with
the further increasing of EO content. The increasing of elongation was due to the increase in electrical
charge of film-forming emulsion and droplet sizes. It has been proven that repulsive forces among
molecules can increase the distance between polymers and that larger droplet sizes could decrease
chain–chain interactions, which resulted in a plasticizing effect [28]. In fact, the mechanical properties
of films are possibly dependent upon a variety of parameters, such as the type of ingredient, oil content,
properties of film-formation emulsion, microstructure of films, and so on. In this study, it is possible
that 6% EO concentration might result in a discontinuous microstructure to give lower elongation.
This is probably due to the EO migration upwards in the films and further volatilization during water
evaporation, leading to a holey structure.

2.4. Thermal Properties of Films

Differential scanning calorimetry (DSC) technology is generally employed to evaluate the thermal
transition of edible films. As shown in Figure 4A, endothermic peaks appeared in the range of
180–200 ◦ C, which can be attributed to the melting temperature (Tm) of films. The films with 2% or 4%
EO have one endothermic peak, indicating good compatibility between the film components. Multiple
peaks were observed as the EO concentration increased to 6%. All those results indicated that the
concentration of EO could affect the interactions between polymers, which could result in changes
of the Tm [35]. On the other hand, the appearance of a new peak suggested that a Maillard reaction
occurred during the film-forming process [36].
Coatings 2020, 10, 784 8 of 19

Coatings 2020, 10, x FOR PEER REVIEW 9 of 21

Figure 4. Differential scanning calorimetry (DSC) curves (A), Thermal gravimetric (TG) thermograms
(B), X-ray diffraction patterns (C) and Fourier Transform Infrared (FTIR) spectra (D) of PSPI-GA films
containing essential oil. EO 1/2/4/6: PSPI-GA conjugates films containing grapefruit essential oil at
1%/2%/4%/6% level. PSPI: plum seed protein isolates. GA: gum acacia. EO: grapefruit essential oil.
Coatings 2020, 10, 784 9 of 19

TG curves are widely applied to study the thermal decomposition of films as reflected by their
weight loss under continuous heating conditions. The higher onset decomposition temperatures of
films indicated the better thermal stability [37]. As shown in Figure 4B, the thermal stability of films
was significantly improved as the EO content increased in the range of 1–4%; then, it decreased with
the further increasing of EO concentration.

2.5. X-Ray Diffractometry

XRD was widely applied to study the compatibility of components in the films. As shown in
Figure 4C, a single peak located around 2θ = 21◦ was observed in films with 1%–4% EO, indicating
that the components in the films were in an amorphous state. The peak became broader as the
EO concentration increased from 1% to 4%, suggesting the good compatibility between the EO and
polymers in the films. A strong peak located around 2θ = 19◦ appeared as the EO content increased
to 6%, indicating the formation of new crystalline domains. This result supported the appearance of
multiple peaks in DSC analyses.

2.6. Fourier Transform Infrared Spectroscopy (FTIR)

FTIR was employed to analyze the functional groups in EBEF to evaluate the effect of EO on the
interactions between polymers. The absorption band of amide-A was commonly used to study the
hydrogen bonds, due to N–H and O–H bands participate in the formation of hydrogen bonds [38].
As shown in Figure 4D, the band intensity of amide-A was affected by the content of EO, which indicated
that the concentration of EO could affect the hydrogen bonds’ interaction between the polymers.
In addition, amide-I and amide-II are common parameters for studying Maillard reaction (C=N
stretching vibration) between proteins and polysaccharides [35]. As suggested by Figure 4D, the band
intensity of amide-I and amide-II increased with the increasing of EO concentration, which suggested
that the concentration of EO could affect the Maillard reaction during the film-forming process and
then change the physicochemical properties of films.

2.7. Microstructure
The surface (S) and cross-section (C) of films are shown in Figure 5. Generally, films containing
EO showed a rough surface. This result is consistent with previous researches that incorporation of EO
led to the increase of coarseness on film surfaces [28,39]. They attributed this result to the oil droplets
migration and volatilization upwards the films, leading to an irregular surface. Moreover, both the
surface and cross-section became denser and more compact as the EO content increased from 1% to
4%, which could help understand the improvement in TS and thermal stability. However, the holey
surface in the cross-section was observed as the EO concentration increased to 6%. The appearance
of a holey microstructure might help us understand why the film with 6% EO showed a decrease in
transparency, TS, EB, and thermal stability.
Coatings 2020, 10, 784 10 of 19
Coatings 2020, 10, x FOR PEER REVIEW 11 of 21

Figure5.5.SEM
Figure SEMimages
imagesofofthe
thesurface
surface(S)
(S)and
andcross-section (C)
cross-section ofof
(C) PSPI-GA
PSPI-GA films containing
films essential
containing oil.
essential
EO
oil.1/2/4/6: PSPI-GA
EO 1/2/4/6: conjugates
PSPI-GA filmsfilms
conjugates containing grapefruit
containing essential
grapefruit oil atoil
essential 1%/2%/4%/6% level.level.
at 1%/2%/4%/6% PSPI:
PSPI:seed
plum plum seed protein
protein isolates.
isolates. GA: gum GA:acacia.
gum acacia. EO: grapefruit
EO: grapefruit essential
essential oil. oil.

2.8.
2.8.Antioxidant
Antioxidantand
andAntimicrobial
Antimicrobial Activity
Activity
Grapefruit
Grapefruitisisregarded
regardedas ashighly
highly nutritional
nutritional because
because ofof the
the presence
presence of of various
variousphytonutrients,
phytonutrients,
such
suchasasvitamins,
vitamins,terpenes,
terpenes,and andother
other compounds.
compounds. The The essential
essential oil
oil from
from grapefruit
grapefruitwas wasreported
reportedtoto
possess antibacterial and antifungal effects [16]. A previous report suggested
possess antibacterial and antifungal effects [16]. A previous report suggested that films emulsified that films emulsified
with cinnamon essential oil could exhibit antioxidant activity, which increasing
with cinnamon essential oil could exhibit antioxidant activity, which increasing as the essential oil as the essential oil
content
contentincreased
increased[40]. However,
[40]. However, in our study,
in our all films
study, with EO
all films with exhibited radicalradical
EO exhibited scavenging activity
scavenging
but the film
activity butwith 1% EO
the film showed
with 1% EOthe best ofthe
showed antioxidant activity (Figure
best of antioxidant 6A).
activity In fact,6A).
(Figure the In
antioxidant
fact, the
activity of film
antioxidant is related
activity to the
of film is EO concentration
related to the EO released from film.
concentration Therefore,
released from film.the release kinetics
Therefore, the
ofrelease
EO from PSPI-GA
kinetics of EOfilms
fromwas also investigated
PSPI-GA films was also in this study. Furthermore,
investigated in this study.all films containing
Furthermore, EO
all films
demonstrated
containing EOantimicrobial
demonstrated effect against E.effect
antimicrobial coli (as shownE.incoli
against Figure 6B, and
(as shown in itFigure
was not 6B,significantly
and it was
affected by EO concentration
not significantly affected by EO (p <concentration
0.05)). The result is consistent
(p < 0.05)). The resultwithisprevious
consistent studies, in which
with previous
essential
studies, oil incorporated
in which in oil
essential polysaccharides
incorporated films showed antimicrobial
in polysaccharides behavior
films showed against E. coli
antimicrobial due to
behavior
against
the E. coli due
destruction to the
of the destruction
bacteria of the bacteria
cell membrane [41,42].cell membrane [41,42].
Coatings 2020, 10, 784 11 of 19

Coatings 2020, 10, x FOR PEER REVIEW 12 of 21

Figure 6. Antioxidant (A), antimicrobial (B) activity, and oil release kinetics (C) of PSPI-GA films
Figure 6. Antioxidant (A), antimicrobial (B) activity, and oil release kinetics (C) of PSPI-GA films
containing essential oil. EO 1/2/4/6: PSPI-GA conjugates films containing grapefruit essential oil at
containing essential oil. EO 1/2/4/6: PSPI-GA conjugates films containing grapefruit essential oil at
1%/2%/4%/6% level. PSPI: plum seed protein isolates. GA: gum acacia. EO: grapefruit essential oil.
1%/2%/4%/6% level. PSPI: plum seed protein isolates. GA: gum acacia. EO: grapefruit essential oil.
Different letters in the same pattern represent significant difference (p < 0.05).
Different letters in the same pattern represent significant difference (p < 0.05).

2.9. Release Kinetics of EO from Films

It can be observed from Figure 6C that the release kinetics of EO from PSPI-GA films followed a
typical exponential pattern. This result is consistent with previous study of the release behavior of
Coatings 2020, 10, 784 12 of 19

2.9. Release Kinetics of EO from Films

It can be observed from Figure 6C that the release kinetics of EO from PSPI-GA films followed a
typical exponential pattern. This result is consistent with previous study of the release behavior of
lemongrass oil from alginate film [43]. As suggested in Table 2, the release data of EO fitted better
to the Peppas model (r2 close to 1) than the Weibull model. In general, the n constant of the Peppas
model took values lower than 0.5, indicating that the release mechanism is a combination of partial
diffusion through a swollen matrix and pores filled with water [44]. Interestingly, the levels of essential
oil released from films decreased as the EO content increased in the range of 1–4%. This is probably
due to the formation of a more ordered structure as supported by TS and microstructure analysis.
The released level increased as the EO content increased to 6%. This is probably due to the formation
of a holey structure, which might benefit the release of EO from films. In addition, released levels are
positively related to their antioxidant activity.

Table 2. Kinetics parameters obtained from release curves of films containing essential oil.

Peppas Model
Sample
Regression Coefficient (r2 ) Kp n
EO 1 0.9771 0.3230 0.1643
EO 2 0.9928 0.2581 0.1986
EO 4 0.9835 0.2753 0.1777
EO 6 0.9671 0.2689 0.1938
Weibull Model
Sample
Regression Coefficient (r2 ) a b
EO 1 0.9352 0.3100 0.3027
EO 2 0.9589 0.2384 0.3347
EO 4 0.9696 0.2721 0.2878
EO 6 0.9504 0.2340 0.3455
EO 1/2/4/6: PSPI-GA conjugates films containing grapefruit essential oil at 1%/2%/4%/6% level. PSPI: plum seed
protein isolates. GA: gum acacia. EO: grapefruit essential oil.

3. Materials and Methods

3.1. Materials and Chemicals

The plum seed flour was provided by a local supplier in Nanjing (China); grapefruit essential oil
(main constituents provided in Supplementary Materials); 2,20 -Azino-bis (3-ethylbenzo-thiazoline-
6-sulfonic acid) diammonium salt (ABTS) radical; strain of Escherichia coli (CMCC 44102) were purchased
from Shanghai Yuanye biotechnology Co., Ltd. (Shanghai, China). The glycerol was obtained from
Macklin Biochemical Co., Ltd. (Shanghai, China). Gum acacia (GA) and other reagents were obtained
from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

3.2. Analysis of Grapefruit Essential Oil

Solid-phase microextraction (SPME) coupled with gas chromatography mass spectrometry
(GC-MS) was used to analyze main components in grapefruit essential oil. The volatiles were extracted
using a 50/30 µm Divinylbenzene/Carboxen/Polydimethylsiloxane (DVB/CAR/PDMS) fiber (Supelco,
Bellefonte, PA, USA). The essential oil (10 mL) was placed in a 20 mL vial, and extracted by the fiber at
40 ◦ C for 30 min. After extraction, the fiber was thermally desorbed in the GC injection port for 5 min.
The analysis was performed by An Agilent 7890 B GC coupled with an Agilent 7000 mass
spectrometer (Agilent, Santa Clara, CA, USA), equipped with an HP-5MS fused silica capillary column
(30 m length, 0.25 mm inner diameter, 0.25 µm film thickness; Agilent). The mass spectrometer ion source
temperature was at 230 ◦ C, the electron energy was at −70 eV. The oven temperature was first raised
Coatings 2020, 10, 784 13 of 19

from 60 to 210 ◦ C at 3 ◦ C/min, then raised to 240 ◦ C at 20 ◦ C/min, and held for 8 min. The compounds
were identified by comparing the mass spectra with those contained in the database (NIST11).

3.3. Preparation of Plum Seed Protein Isolates (PSPI)—Gum Acacia (GA) Conjugates
The PSPI was prepared using alkali-acid precipitation according to our previous study [45].
Conjugates of PSPI-GA were also prepared according to our previous study [18]. PSPI-GA (1:1 in
deionized water, w/w) solutions (10%, w/v) were spray-dried. The powders obtained were heated for
1, 3, or 5 days at 60 ◦ C in the presence of saturated KBr solution (79% relative humidity), and then
vacuum-dried. The resultant samples were PSPI-GA conjugates (PSPI-GA 1, PSPI-GA 3, and PSPI-GA
5). PSPI-GA powders without dry-heating were used as control (PSPI-GA).

3.4. Determination of Amino Acids, Emulsifying Properties, Surface Hydrophobicity, and Structure
of Conjugates
The lysine and arginine levels, emulsifying activity index (EAI), emulsifying stability index (ESI),
surface hydrophobicity, intrinsic emission fluorescence spectra, and circular dichroism spectrum were
measured according to our previous study [9]. The lysine and arginine contents were measured by
an Agilent 1100 high performance liquid chromatograph (Agilent technologies Co., Ltd., Santa Clara,
CA, USA) equipped with an ODS Hypersil column (5 µm, 250 × 4.6 mm). Fluorescence intensity
(FI) was determined with a Hitachi F-7000 fluorescence spectrometer (Hitachi, Ltd., Tokyo, Japan) at
excitation wavelength of 390 nm and emission wavelength of 470 nm. The initial slope of the FI versus
protein concentration plot was the index of surface hydrophobicity. Intrinsic emission fluorescence
spectra of the samples were analyzed by a Hitachi F-7000 fluorescence spectrophotometer (Hitachi,
Ltd., Tokyo, Japan). The circular dichroism spectrum of samples was obtained using a Mos-450 CD
spectropolarimeter (Biologic, Claix, France).

3.5. Preparation of Film-Forming Emulsion

Conjugates (3 days incubation) solution (5%, w/v) was stirred for at least 12 h (4 ◦ C), and then
added with glycerol (2%, w/v), stirring for another 1 h. For coarse emulsion forming, grapefruit essential
oil at proportions of 1%, 2%, 4%, and 6% (w/w) were incorporated into the dispersion by using the
FM200 homogenizer (FLUKO, Shanghai, China) at 10,000 rpm for 2 min. Then, the coarse emulsion
was treated with a pressure homogenizer (ATS, Beijing, China) at 70 MPa for 2 passes. The resulted
emulsion was then degassed for at least 30 min using vacuum oven.

3.6. Characterization of Film-Forming Emulsion

3.6.1. Particle Size and ζ-Potentials

The film-forming emulsions were diluted with distilled water (1/20, w/w) and stirred for 5 min
at 25 ◦ C. The particle sizes of samples were measured by light scattering using a Mastersizer 2000
equipped with a Hydro 2000 MU dispersion unit from Malvern Instruments Ltd. (Worcestershire, UK).
The pump speed was settled at 1800 rpm, and the refractive index and absorption parameter were
1.330 and 0.001, respectively. The ζ-potential of film-forming solutions was measured using a nanoZS
instrument (Malvern Instruments, Worcestershire, UK).

3.6.2. Rheological Behavior of Film-Forming Emulsions

An AR2000 rheometer (TA Instruments, Leatherhead, UK) fitted with parallel plates (50 mm
diameter and 1 mm gap) was employed to measure the rheological behavior of film forming emulsions.
The shear rate was increased linearly from 0 to 100 s−1 . A dynamic strain sweep was conducted
in a range for angular frequency (omega) of 0.1–100 rad/s at amplitude (gamma) = 0.1%. The flow
rheological properties of emulsion were fitted to the power law equation (log viscosity = (n−1) log shear
rate + log m) which is applied extensively to describe the rheological behavior of food emulsions [46].
Coatings 2020, 10, 784 14 of 19

The flow behavior index and the consistency coefficient were also calculated from the power law
equation, where n is the flow behavior index, and m is the consistency coefficient [47].

3.7. Film Formation

The films were prepared by casting emulsion, as outlined in Section 3.4, on leveled
polytetrafluoroethylene plates (42 × 42 cm2 ) and dried using a KBF720 ventilated chamber (Binder,
Germany) at 30 ◦ C and 43% relative humidity for 18 h.

3.8. Characterization of Films

3.8.1. Transparency, Whiteness Index (WI), Swelling Ability, Water Vapor Permeability
(WVP) Measurements
The transparency of films was determined by employing Rubilar’s method by using the Spark
10M microplate spectrophotometer (Tecan, Switzerland) [48] and calculated by Equation (1). The color
of the film was obtained by using a Hunter-Lab colorimeter (Reston, VA, USA) and the L (lightness),
a* (redness and greenness), and b* (yellowness and blueness) were recorded. The whiteness index
of films was calculated according to Equation (2). The swelling ability of films was measured by
immersing the films in water at 25 ◦ C for 5 h and then calculated the weight gain (equation (3)) [23].
The water vapor permeability (WVP) of films was tested by using the gravimetric method [49]. Films
were placed in measuring cells containing silica gel and deposited in a ventilated chamber at 25 ◦ C and
75%. The WVP value was calculated according to Equation (4).

T600
Transparency = (1)
x
where T600 is the transmittance of light through the film at 600 nm and x is the film thickness (mm),
which was measured by a micrometer with the accuracy of 0.001 mm (Jiangsu, China).
q
Whiteness index = 100 − (100 − L)2 + a∗2 + b∗2 (2)

M2 − M1
Sweeling (%) = × 100 (3)
M1
where M1 is the mass (g) of the initial film before immersion in water and M2 is the mass (g) of the film
after immersion in water for 5 h.
∆m
WVP = (4)
∆t × A
where ∆m is the weight gain (mg) of the cups during time ∆t (d) and A is the area of exposed film (cm2 ).

3.8.2. Contact Angle-Sessile Drop Method

The contact angle of EBEF was tested using an OCA15EC goniometer (Stuttgart, Germany).
Deionized (10 µL) water was released onto the EBEF, and the image was recorded after 5 s. The contact
angle was defined as the angle between the baseline and the tangent to the drop boundary.

3.8.3. Mechanical Properties

The tensile strength (TS) and elongation at break (EB) of films were determined using a TA-XT2i
texture analyzer (London, UK). The initial distance of separation and cross-head speed was fixed at
50 mm and 1 mm/s, respectively. TS was calculated according to Equation (5), and EB was calculated
according to Equation (6).
F
TS = (5)
S
Coatings 2020, 10, 784 15 of 19

where F is the maximum force at break (Kg) and S is the initial transverse section (mm2 ).

L2 − L1
EB (%) = × 100 (6)
L1

where L1 is the original length (mm) and L2 is the length at break (mm).

3.8.4. Differential Scanning Calorimetry (DSC)

The DSC was performed using a thermal analyzer (DSC214 Polyma, Netzsch, Germany).
Films (2–4 mg) was heated in an aluminum pan from 25 to 250 ◦ C at a rate 10 ◦ C min−1 under
nitrogen atmosphere. The data were analyzed with TA Universal Analysis software.

3.8.5. Thermal Gravimetric Analysis (TG)

Thermal gravimetric analysis was carried out a Q500 thermal analyzer (TA Instruments, New Castle,
USA). Film samples (7 mg) were sealed in ceramic pans, and the temperature was raised from 25 to
800 ◦ C at a heating rate of 10 ◦ C min−1 . The nitrogen was at a constant flow rate of 60 mL min−1 .

3.8.6. X-ray
X-ray diffraction patterns were performed by a Smartlab-3kw X-ray diffractometer (Rigaku, Japan)
with Cu Kα radiation at 40 kV and 30 mA. The scan rate was 10◦ min−1 and the patterns were collected
in the range of 2θ from 5◦ to 55◦ .

3.8.7. Fourier Transform Infrared Spectroscopy (FTIR)

The Fourier transform infrared spectra of samples were obtained with a FTIR-7600 (Lambda,
Australia). Scanning was carried out in the range from 4000 to 400 and 4 cm−1 resolution.

3.8.8. Scanning Electron Microscopy (SEM)

The films were sputter-coated with gold–palladium, and the microstructures were observed with
MERLIN scanning electron microscope (MERLIN SEM, ZEISS, Germany). The films were fractured by
immersing in liquid nitrogen to observe the microstructure of the cross section.

3.9. Antioxidant and Antimicrobial Activity of Films

The scavenging activity of ABTS was determined by the method of Yikling et al. [50] with
modifications. Films were mixed with ABTS (7 mM) to give a final film concentration of 0.5 mg/mL.
After incubation (10 min), the sample was centrifuged at 12,000× g, 4 ◦ C for 2 min. The absorbance
values of 200 µL supernatant were recorded using a Spark 10M microplate spectrophotometer (Tecan,
Switzerland). The solution containing films without free radicals was used as blank.
The films (1 mg) were placed into Luria–Bertani medium (2 mL) that had been previously seeded
with inoculum containing indicator bacteria in the range of 106 –108 CFU/mL, and then they were
incubated at 37 ◦ C for 24 h. The absorbance values of 200 µL samples at 600 nm were measured using a
Spark 10M microplate spectrophotometer at 0, 4, 8, 12, and 24 h. The medium containing films without
bacteria was used as a blank.

3.10. Essential Oil Release Kinetic from Films

Release kinetics of the EO from films was performed according to the previous method [44].
The solution of ethanol:water (50:50, v:v) was used as simulant to study the migration. The films
(2 × 2 cm2 ) were placed inside dialysis bags (12,000 Dalton) and submerged in 30 mL of simulant.
A calibration curve was obtained for EO under study using dilutions of EO with the simulant, and the
absorbance was measured at a wavelength of 274 nm using a Spark 10M microplate spectrophotometer
Coatings 2020, 10, 784 16 of 19

(Tecan, Switzerland). Then, the concentration of EO released in the simulant was determined. Release
data were fitted to Weibull (Equation (7)) and Peppas (Equation (8)) models.

Ln(1 − Q) = −a·tb (7)

Q = KP ·tn (8)

where Q is the fraction of EO released, a and Kp are constants, n and b are constants indicative of the
release mechanism, and t is time.

3.11. Statistical Analysis

All the tests were repeated three times and the data obtained were analyzed by one-way analysis
of variance using SPSS Windows version 17.0. Values are expressed as means ± standard deviation.
Duncan’s multiple range test was used to identify significant differences (p < 0.05) between means.

4. Conclusions
The results obtained in this research give some insights on the preparation of edible films using
emulsions of PSPI-GA/EO as film-forming solution. It was found that the emulsifying properties of
PSPI could be improved after being grafted with GA. The improvement is related to the changes in
surface hydrophobicity, secondary structure, and tertiary structure. The droplet size, surface charge,
and viscosity of emulsion increased as the EO concentration increased. However, the water vapor
barrier property, surface hydrophobicity, mechanical properties, and thermal stability of EBEF were
improved as the EO content increased in the range of 1–4%, while it decreased as the EO concentration
increased to 6% due to the formation of a holey microstructure. The release data of EO from films fitted
well to the Peppas model, and the radical scavenging activity of EBEF was significantly affected by the
different release patterns due to the variation of EO concentration.

Supplementary Materials: The following are available online at http://www.mdpi.com/2079-6412/10/8/784/s1,

Figure S1: SPME-GC-MS chromatogram of grapefruit essential oil, Table S1: Identification of volatile compounds
in grapefruit essential oil.
Author Contributions: Conceptualization, C.L. and F.X.; investigation, J.P.; writing—original draft preparation,
C.L.; writing—review and editing, X.X.; project administration, X.X. All authors have read and agreed to the
published version of the manuscript.
Funding: This work was supported by Natural Science Foundation of Jiangsu Province (BK20171066) and The
Priority Academic Program Development of Jiangsu Higher Education Institutions (035062002002A).
Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations
The following abbreviations are used in this manuscript:

EBEF emulsion-based edible films

PSPI plum seed protein isolate
GA gum acacia
EO grapefruit essential oil

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