molecules

Article
Development of Antimicrobial Biocomposite Films to
Preserve the Quality of Bread
Kelly J. Figueroa-Lopez 1 , Margarita María Andrade-Mahecha 2, * ID

and Olga Lucía Torres-Vargas 3

1 Optoelectronics Group, Interdisciplinary Science Institute, Faculty of Basic Science and Technologies,
Universidad del Quindío, Carrera 15 Calle 12 Norte, Armenia 630004, Colombia; kjfigueroal@iata.csic.es
2 Group of Research on Agroindustrial Processes (GIPA), Universidad Nacional de Colombia,
Palmira 763533, Colombia
3 Group of Agro-industrial Sciences, Faculty of Agro-industrial Sciences, Universidad del Quindío,
Carrera 15 Calle 12 Norte, Armenia 630004, Colombia; oltorres@uniquindio.edu.co
* Correspondence: mmandradem@unal.edu.co

Received: 12 December 2017; Accepted: 11 January 2018; Published: 19 January 2018

Abstract: This study focused on the development of gelatin-based films with incorporation of
microcrystalline cellulose as reinforcement material. Clove (Syzygium aromaticum), nutmeg (Myristica
fragrans), and black pepper (Piper nigrum) oleoresins containing antimicrobial compounds of natural
origin were incorporated into the films. The mechanical, thermal, optical, and structural properties,
as well as color, seal strength and permeability to water vapor, light, and oil of the films were
determined. Adding oleoresins to the gelatin matrix increased the elongation of the material and
significantly diminished its permeability to water vapor and oil. Evaluation of the potential use
of films containing different oleoresins as bread packaging material was influenced by the film
properties. The biocomposite film containing oleoresin from black pepper was the most effective
packaging material for maintaining bread’s quality characteristics.

Keywords: biocomposite films; gelatin; oleoresins; antimicrobial compounds; food quality

1. Introduction
Biocomposite materials are structures made up of at least one phase (matrix or reinforcement).
One of these phases is obtained from a renewable source [1,2]. Proteins, polysaccharides, or a mixture
of these which can provide mechanical and/or barrier properties, are some of those commonly used to
make up the polymer structure [3,4]. Gelatin is one of the proteins with greatest industrial application
due to its low melting point; in addition, it has a unique amino acid sequence, with high contents
of proline, glycine, and hydroxyproline, which help in the formation of flexible films [5]. Gelatin is
a good barrier against oxygen and carbon dioxide, but a poor barrier against water vapor [6]. To obtain
materials with better flexibility, manageability, and extension ability, it is necessary to incorporate
substances of low molecular weight, like plasticizers [7]. Also, compounds with antimicrobial
and antioxidant activity can be incorporated, which help to improve the physical-chemical and
microbiological properties of polymer materials and may improve the quality of the food they cover
and/or increase its shelf life [8]. Different lipids (oleoresins and essential oils) have shown antimicrobial
or antioxidant properties, which may be used for the development of biocomposite materials. Lipids
are a good barrier against water vapor. Additionally, they may decrease film transparency, which is
important to preserve the quality of food exposed to light [9,10]. Thus, the formation of active
biocomposite materials consists of incorporating compounds with antioxidant or antimicrobial
properties to the polymer matrix, which permit, through the migration of the active compounds onto
the food surface, the conservation and extension of its shelf life [11,12]. Nevertheless, film properties

Molecules 2018, 23, 212; doi:10.3390/molecules23010212 www.mdpi.com/journal/molecules

Molecules 2018, 23, 212 2 of 18

are mainly influenced by the polymers used, active compounds, and preparation method. The use
of biocomposite materials as food packaging or wrapping will depend on the mechanical properties
and the permeability to water vapor [13]. The aim of this work was to investigate the effect of adding
natural antimicrobial compounds (clove, nutmeg, and black pepper oleoresins) incorporated into
gelatin based biocomposite films, on their moisture content, water vapor permeability, oil permeability,
tensile properties, structure, optical and thermal properties. Biocomposite films were evaluated as
packaging material on bread slices stored at ambient conditions (around 25 ◦ C and 75% relative
humidity). The physical-chemical, microbiology, and sensory characteristics of bread were evaluated
for nine days.

2. Materials and Methods

2.1. Materials
To prepare the films, type B food-grade gelatin was used with a bloom of 220–240 g (Gelco S.A.,
Barranquilla, Colombia), along with pharmaceutical-grade microcrystalline cellulose, Avicel® PC
105 (FMC Biopolymer, Campinas, Brazil); 99% purity glycerol (Sigma Aldrich, St. Louis, MO, USA);
Tween 80 for synthesis (Merck, Darmstadt, Germany); and clove, nutmeg, and black pepper oleoresins
(TECNAS S.A., Antioquia, Colombia).

2.2. Minimum Inhibitory Concentration of the Oleoresins

The minimum inhibitory concentration (MIC) of the oleoresins against S. aureus and E. coli was
determined by using the classic plate micro-dilution method by the National Committee of Laboratory
Safety and Standards (NCLSS) [14]. Triplicate experiments were performed for each concentration
of natural antimicrobials (4%, 2%, 1%, 0.5%, and 0.25%). The lowest concentration of the natural
antimicrobial that showed growth inhibition was considered the MIC.

2.3. Physical-Chemical Analysis

Gelatin and microcrystalline cellulose (MCC) particle size were determined by using a Zetasizer
nano ZS90 (Malvern Instruments, Ltd., Malvern, UK). Gelatin and MCC crystallinity were determined
by using an X-ray diffractometer (Siemens, Karlsruhe, Germany) equipped with a Cu anode tube
(which emits radiation of λ = 0.154 nm) with a voltage of 40 kV and current of 30 mA. The diffractograms
were analyzed by using the Origin 8 program and crystallinity (% Icr ) was determined by using
Equation (1):
I200 − Inon−cr
Icr = × 100 (1)
I200
where I200 : Maximum intensity value of the crystalline peak, Inon-cr : Intensity value that separates both
diffraction peaks
Identification of the principal functional groups of the MCC and the gelatin was made through
Fourier transformed infrared spectroscopy (Perkin Elmer, Waltham, MA, USA). The spectra were
obtained within a range from 4000 to 400 cm−1 and analyzed by using the Origin 8 program. Thermal
characterization of the MCC, the gelatin, and the oleoresins (nutmeg, black pepper, and clove) was
carried out through differential scanning calorimetry (DSC 214 Polyma, Netzsch, Selb, Germany);
scanning was from −20 to 140 ◦ C at a rate of 5 ◦ C/min.
The chemical composition of the oleoresins (nutmeg, clove, and black pepper) was determined by
using a gas chromatograph AT 6890 Series Plus (Agilent Technologies, Palo Alto, CA, USA), coupled
to a selective mass detector (Agilent Technologies, MSD 5973) operating in full scan mode. The column
used in the analysis was DB-5MS (5%-phenylpoly(methylsiloxane), 60 m × 0.25 mm × 0.25 µm) and
the injection was conducted in split mode (30:1) with Viny = 2 µL.
Molecules 2018, 23, 212 3 of 18

2.4. Preparation of Films

Films were prepared through the “casting” method following the methodology proposed by
Andrade-Mahecha et al. [15] and by Mondragón et al. [16] with some modifications. The film-forming
solution (FFS) was obtained from an aqueous suspension of microcrystalline cellulose (0.15 g/100 g of
FFS), which was magnetically agitated for 30 min at 35 ◦ C. Simultaneously, two aqueous suspensions
were also prepared; one containing gelatin (3 g/100 g of FFS) stirred at 60 ◦ C for 30 min and another
containing glycerol (0.45 g/100 g of FFS) stirred at 35 ◦ C for 15 min. Thereafter, all the components were
mixed and kept at 60 ◦ C for 15 min under constant stirring. Next, the respective oleoresin containing
the natural antimicrobial compounds (10 g (500 µL of oleoresin/500 µL Tween 80 at 2%)/100 g FFS)
was added. The FFS was kept at 45 ◦ C for 15 min, prior to being poured into nonstick molds to be
subjected to convection drying (45 ◦ C during 4 h) on a stove with forced-air circulation (FD-53, Binder,
Tuttlingen, Germany).

2.5. Film Characterization

Film thickness was measured at five different points by using a digital micrometer (Mitutoyo,
Corp., Kawasaki, Japan).

2.5.1. Humidity Content

Film humidity content was determined immediately after drying and after a conditioning period
(58% RH during 48 h), according to the gravimetric method by ASTM D644-99 [17].

2.5.2. Water Vapor Permeability

Water vapor permeability (WVP) was determined by following the ASTM E96-05 [18]. The films
were placed on stainless steel cells subjected to external environments with different relative humidity
(RH 2–33%, 33–66%, and 64–90%). Weight loss of the cells was monitored every hour for 9 h.

2.5.3. Mechanical Properties

Mechanical tests were carried out on a texture analyzer (TA-XT plus, Stable Micro Systems, Surrey,
UK), at an operating rate of 1 mm/s and separation of 40 mm between clamps. The mechanical
properties evaluated were: tensile strength (MPa), elongation (%), and young’s modulus (MPa),
according to ASTM D882-02 [19].

2.5.4. Oil Permeability

Oil Permeability was determined by following the methodology proposed by Yan et al. (2012) [20],
using the following equation:
∆W × FT
Po =
A×T
where ∆W is the weight variation of the filter paper (g), FT is the film thickness (mm), A is the area of
effective contact (m2 ), and T is the storage period (days).

2.5.5. Measurement of Seal Strength

Film samples (7.62 × 2.54 cm) were thermally sealed for two seconds, one on top of another
with an area of 2.54 cm × 0.2 cm, using a manual thrust sealer (Hongzhan, KS-100, Wenzhou, China).
Seal strength was determined through the ASTM F-88 [21], using a texture analyzer (TA-XT plus,
Stable Micro Systems). Seal strength was calculated with the maximum force on the film width [22].

2.5.6. X-ray Diffraction (XRD)

Film samples (4 × 4 cm) were analyzed in an X-ray diffractometer (Bruker D8-Advance,
Rheinstetten, Germany) equipped with a tube with Cu anode (radiation of λ = 0.154 nm) with voltage
Molecules 2018, 23, 212 4 of 18

of 30 Kv, current of 35 Ma, filter for the Kβ line, and a scintillation detector. The diffractograms were
analyzed by using the Origin 8 program.

2.5.7. Fourier Transform Infrared (FTIR) Spectroscopy

Identification of the principal functional groups was conducted via spectroscopy equipment
(IR Prestige 21 Shimadzu, Kyoto, Japan). Spectra were obtained within a wave number range of 4000
to 400 cm−1 and analyzed by using the Origin 8 program.

2.5.8. UV-Vis Light Transmittance Values

Film light barrier (50 × 30 mm samples) was evaluated in wavelengths from 200 to 800 nm
in a spectrophotometer (Thermo Scientific Genesys-10S-UV-Vis, Waltham, MA, USA) according to
Leceta et al. [23].

2.5.9. Color
Film color was determined by using a spectrocolorimeter (Hunter Associates Laboratory, Inc.,
Reston, VA, USA), according to Arfat et al. [13]. The color difference (∆E*) was calculated by using the
following equation:
2 0.5
h i
∆E∗ = (∆L∗ )2 + (∆a∗ )2 + (∆b∗ )

where ∆E*, ∆a* and ∆b* corresponded to the differences between the color parameters of films
containing oleoresins and the values of the reference film (without oleoresin) (L* = 2.094, a* = −1.46,
b* = 4.73).

2.5.10. Thermogravimetric Analysis (TGA)

Non-isometric degradation measurements were conducted on a Thermo Microbalance (TG 209 F1
Iris, Netzsch, Selb, Germany). Tests were run from 25 ◦ C to 600 ◦ C at a heating rate of 10 ◦ C/min in
a nitrogen atmosphere (10 mL/min) [23].

2.6. Influence of Biocomposite Films on Bread Quality

The formulation of the artisan bread was carried out according to Colombian standard NTC
1363 [24]. Two lots of bread manufactured on different days were evaluated. In both lots, bread samples
were packaged in reference films (without oleoresin) and with oleoresins (clove, nutmeg, and black
pepper). The bread samples (8 cm × 8 cm) were wrapped with two sheets of the biocomposite material
(10 × 10 cm), which were thermally sealed on four sides. The physical-chemical stability (humidity,
pH, aw, and weight loss) was evaluated on days 1, 3, 5, and 9 of the storage period at 25 ◦ C and 75%
RH. The microbiological and sensory quality of the bread was evaluated at days 1, 4, and 9.

2.7. Physical-Chemical Evaluation of the Bread

Humidity was determined by using the Colombian standard NTC 282 [25]. Five-gram amounts
of the sample were taken in a metallic capsule, which was introduced with the samples into a stove
with forced-air circulation (FD-53, Binder) between 100 and 110 ◦ C and their weight was evaluated
until constant weight. The pH was determined through NTC 1363. Water activity was determined by
using a portable PawKit water activity (aw) meter (Aqualab, Pullman, WA, USA). Weight loss was
calculated as the difference between the initial weight and the final weight of the bread sample.

2.8. Microbiological Evaluation of the Bread

A microbiological count was conducted of Escherichia coli [26], Staphylococcus aureus positive
coagulase [27], Bacillus cereus [28], along with detection of Salmonella spp. [29], mold and yeast [30],
according to that indicated in the technical standard for bread.
Molecules 2018, 23, 212 5 of 18

2.9. Sensory Evaluation of the Bread

A descriptive sensory analysis was conducted with 10 untrained panelists following the NTC
3925 [31]. Attributes of aroma, flavor, hardness, and general acceptability were evaluated in a scale
from 1 to 5.

2.10. Statistical Analysis

Each treatment was done in triplicate and the results of the properties were evaluated with 5%
significance level (p ≤ 0.05). Analysis of variance (ANOVA) and a multiple comparison test (Tukey)
permitted identifying significant differences among the treatments. The results were analyzed through
the SPSS Statistics 22.0 software for Windows (SPSS Statistical software, Inc., Chicago, IL, USA).

3. Results and Discussion

3.1. Physical-Chemical and Microbiological Study of the Components of the Biocomposite Material
Black pepper showed antimicrobial activity against both bacterial strains, starting at 0.5%
(0.5 µL/100 µL), while clove started at 1% (1 µL/100 µL) against Staphylococcus aureus and as of
2% (2 µL/100 µL) against Escherichia coli. Likewise, nutmeg inhibited the growth of Staphylococcus
aureus as of 0.5% (0.5 µL/100 µL) and as of 1% (1 µL/100 µL) against Escherichia coli.
The type B gelatin used in this research had a Bloom of 220–240 g, which is related to the
gel’s mechanical elasticity, an important parameter in its capacity and gelling force, for provoking
deformation at a certain concentration and temperature.
Figure 1a shows the gelatin’s particle size distribution, which had unimodal distribution with
a broad range of sizes comprised between 3.8 and 1905 µm. A large peak was observed where there are
most of the particles with approximate sizes of 549.5 µm with 10.58% volume, this particle diameter is
close to the mean diameter (D(4.3) = 476.987 µm).
Figure 1b displays the gelatin’s X-ray diffractogram, showing a dominant peak amply widened at
20.08◦ , which is characteristic of amorphous materials, indicating that gelatin does not have a periodic
structure, given that it comprised of proteins with different structures [32].
Figure 1c shows the gelatin’s FTIR spectrum. The type A amide band at 3425 cm−1 corresponds
to stretching of N-H groups with hydrogen bonds; at 2928 cm−1 there are type B amide bands that
correspond to stretching of CH2 ; the band at 1638 cm−1 is attributed to type I amides; at 1543 cm−1 ,
we find the type II amides and at 1232 cm−1 the type III amides [33,34].
Figure 1d shows three thermal degradation stages. The first stage at 191 ◦ C with 14.15% mass
loss, corresponding to water elimination; the second stage at 434 ◦ C has the highest mass loss (55.55%),
which was associated to degradation of proteins of high molecular weight and a residual mass (ashes)
of 22.97%.
Figure 2a illustrates a unimodal distribution with size range between 0.41 and 138 µm. A large
peak was observed where there is the majority of particles with approximate sizes of 26.3 µm with
9.06% volume; this particle diameter was close to the mean diameter (D(4,3) = 25.9 µm).
Figure 2b presents the microcrystalline cellulose diffractogram, with a peak in the 2θ angle at
22.59◦ , corresponding to the crystalline and a peak on the 2θ angle at 18.8◦ , corresponding to the
amorphous material. The microcrystalline cellulose had 79.87% crystallinity, which coincides with the
values reported in other studies [35–37].
 Molecules 2018, 23, 212 6 of 18

Molecules 2018,23,
Molecules2018, 23,212
212 120 66ofof18
18
Gelatin type B
100

Intensity (a.u.)
120 80
Gelatin type B
100 60

Intensity (a.u.)
80 40

60 20

40 0
0 10 20 30 40 50 60 70 80 90
20
2θ
(a) 0 (b)
0 10 20 30 40 50 60 70 80 90

72 110 2θ
100 Gelatin type B
70
(a) Gelatin type B (b)
90
68
80
%Transmitance

72 66 110 70

%Mass
64 100 Gelatin type B
70 Gelatin type B 60
62 90 50
68
80
%Transmitance

60 40
66
70 30

%Mass
64 58
60 20
62 56 50 10
60 54 40 0
30
58 4000 3500 3000 2500 2000 1500 1000 500 0 100 200 300 400 500 600
-1 20
56 1/λ (cm ) 10
Temperature (°C )
54 0
(c) (d)
4000 3500 3000 2500 2000 1500 1000 500 0 100 200 300 400 500 600
-1
1/λ (cm ) Temperature (°C )

(c) thermal characterization of type B gelatin. (a) Particle

Figure 1. Structural and (d) size distribution; (b)
X-ray diffractogram; (c) FTIR spectrum; and (d) TGA thermogram.
Figure 1. Structural and thermal characterization of type B gelatin. (a) Particle size distribution; (b)
Figure 1. Structural and thermal characterization of type B gelatin. (a) Particle size distribution; (b)
X-ray diffractogram; (c) FTIR spectrum; and (d) TGA thermogram.
X-ray diffractogram; (c) FTIR spectrum; and (d) TGA thermogram.
350

300
MCC

250
Intensity (a.u.)

350
200
300
MCC
150
250
Intensity (a.u.)

100
200
50
150
0
100
0 10 20 30 40 50 60 70 80 90
50 2θ
0
(a) (b)
0 10 20 30 40 50 60 70 80 90

2θ

(a) 100 (b) MCC

90 MCC
80
80
%Transmitance

60
%Mass

100 MCC
90 70 MCC
80 40
80 60
%Transmitance

60 20
%Mass

70 50

40 0
60 40
0 500 1000 1500 2000 2500 3000 3500 4000 4500 0 100 200 300 400 500 600
20
50 1 /λ (c m
-1
) Temperature (°C )
0
40
(c) (d)
0 500 1000 1500 2000 2500 3000 3500 4000 4500 0 100 200 300 400 500 600
1 /λ (c m
-1
) Temperature (°C )
Figure 2. Structural and thermal characterization of MCC. (a) Particle size distribution; (b) X-ray
diffractogram; (c) spectrum;
Figure 2. Structural
(c) FTIR and thermal characterization
and (d) of MCC. (a) Particle (d)
TGA thermogram. size distribution; (b) X-ray
diffractogram; (c) FTIR spectrum; and (d) TGA thermogram.

Figure 2. Structural and thermal characterization of MCC. (a) Particle size distribution; (b) X-ray
diffractogram; (c) FTIR spectrum; and (d) TGA thermogram.
Molecules 2018, 23, 212 7 of 18

Figure 2c shows the principal functional groups of the microcrystalline cellulose. At 3399 cm−1,
Molecules 2018,
stretching of the23, -OH
212 groups was observed; the CH and CH2 groups are found at 2900 cm−17 ;ofthe 18 band

at 1427 cm−1 is associated to the presence of HCH groups and OCH vibrations; the band at 1374 cm−1
is related to the
Figure 2cvibration
shows theof CH groups;
principal the band
functional groupsat of
1217
the cm −1 is attributed to C-C groups and
microcrystalline cellulose. At 3399 cm−1at , 1039
cm−1 to stretching
stretching of the of
-OHC-O groups
groups observed; the CH and CH2 groups are found at 2900 cm−1 ; the band
was [38,39].
atFigure
1427 cm2d −1 is associated to the presence cm−1
shows that the first stage of HCH thermal degradation
groups corresponds
and OCH vibrations; the to water
band elimination
at 1374 at
is related to the vibration of CH groups; the band at 1217 cm − 1 is attributed to C-C groups and at
108 °C. Dehydration, decarboxylation, depolymerization, and glycosyl decomposition take place in
the1039 cm−stage
second
1 to stretching of C-O groups [38,39].
between 363 and 589 °C [37,40,41].
Figure 2d shows that the firstidentified
The main active compounds stage of thermal
in thedegradation corresponds
nutmeg oleoresin were:tomyristic
water elimination at
acid, miristicine,
108 ◦ C. Dehydration, decarboxylation, depolymerization, and glycosyl decomposition take place in
elemicine, sabinene and α-pinene. In the clove oleoresin, eugenol and eugenol acetate were
the second stage between 363 and 589 ◦ C [37,40,41].
identified. The black pepper oleoresin presented piperine as its main component, followed by
The main active compounds identified in the nutmeg oleoresin were: myristic acid, miristicine,
trans-β-cariophylene
elemicine, sabinene and andα-pinene.
limonene. All
In the these
clove compounds
oleoresin, have
eugenol and antimicrobial
eugenol acetate wereand antioxidant
identified.
properties,
The blackas reported
pepper by other
oleoresin authors
presented [42–44].
piperine as its main component, followed by trans-β-cariophylene
and limonene. All these compounds have antimicrobial and antioxidant properties, as reported by
3.2.other
Characterization of Films
authors [42–44].
ToCharacterization
3.2. obtain the films, 0.22 g of solution per each cm2 of support plate permitted obtaining a
of Films
continuous cohesive matrix with thicknesses of 0.058 ± 0.0013 mm containing clove oleoresin, 0.06 ±
To obtain the films, 0.22 g of solution per each cm2 of support plate permitted obtaining
0.0021 mm containing nutmeg oleoresin, and 0.062 ± 0.0018 mm containing black pepper oleoresin.
a continuous cohesive matrix with thicknesses of 0.058 ± 0.0013 mm containing clove oleoresin, 0.06 ±
0.0021 mm containing nutmeg oleoresin, and 0.062 ± 0.0018 mm containing black pepper oleoresin.
3.2.1. Humidity
3.2.1. Humidity
Figure 3 presents the humidity of films after drying (Humidity 1) and after the conditioning
period (Humidity 2). The
Figure 3 presents thehumidity
humiditycontent
of films of thedrying
after biocomposite
(Humidity films containing
1) and after the oleoresins
conditioningdid not
period
have (Humidity
significant 2). The humidity
differences content
after drying, of the biocomposite
obtaining films containing
humidity values of 10.4 ± oleoresins did not g of
0.1 g of water/100
have significant
biocomposite film.differences after drying,the
After conditioning, obtaining humidity their
films increased of 10.4 ± 0.1
valueshumidity g of water/100
content to 21.73 ±g0.32
of g of
biocomposite
water/100 film. After conditioning,
g of biocomposite the films increased
film. Additionally, their humidity
the reference content to 21.73
films (biocomposites ± 0.32 gadded
without
of water/100 g of biocomposite film. Additionally, the reference films (biocomposites without
oleoresin) had humidity contents of 8 ± 0.18 g of water/100 g of biocomposed film after drying and
added oleoresin) had humidity contents of 8 ± 0.18 g of water/100 g of biocomposed film after
12.9 ± 0.19 g of water/100 g of biocomposite film upon completing the conditioning period. The
drying and 12.9 ± 0.19 g of water/100 g of biocomposite film upon completing the conditioning
reference films
period. The had low
reference films humidity after drying
had low humidity andand
after drying after
afterconditioning
conditioning withwith respect
respect to theto the
biocomposite
biocomposite films
filmswith
witholeoresins presentingsignificant
oleoresins presenting significant differences
differences (p <(p < 0.05),
0.05), whichwhich indicates
indicates that that
oleoresins would be creating bonds with the OH groups of the plasticizer and
oleoresins would be creating bonds with the OH groups of the plasticizer and its polyphenolic groups, its polyphenolic
groups, thus, maintaining
thus, maintaining the structure
the structure hydratedhydrated
[45]. [45].

24
b b
Humidity 1 b
22
Humidity 2
20

18

16
Humidity (%)

14 a
12 b
b b
10
a
8

0
Reference Clove Nutmeg Black pepper
Biocomposite Films

Figure 3. 3.Humidity
Figure Humiditycontent
content of of biocomposite filmswith
biocomposite films with oleoresins
oleoresins andand reference
reference beforebefore and after
and after
conditioning. Different letters in the same column indicate significant differences (p
conditioning. Different letters the same column indicate significant differences (p < 0.05).< 0.05).

3.2.2. Water Vapor Permeability

Upon adding nutmeg, clove, and black pepper oleoresins to the gelatin matrix, microcrystalline
cellulose and glycerol (reference film), the permeability gradient diminished significantly, from 4 ×
Molecules 2018, 23, 212 8 of 18

3.2.2. Water Vapor Permeability

Molecules
Upon 2018, 23,nutmeg,
adding 212 clove, and black pepper oleoresins to the gelatin matrix, microcrystalline8 of 18
cellulose and 2glycerol (reference film), the permeability gradient diminished significantly,
10−7 g·m/m
−
·h·Pa 2to 4 × 10−9 g·m/m
−
2·h·Pa (Figure 4). Likewise, upon varying the relative humidity,
from 4 × 10 g·m/m ·h·Pa to 4 × 10 g·m/m2 ·h·Pa (Figure 4). Likewise, upon varying the relative
7 9
slight permeability changes were observed among the same films. This permeability decrease with
humidity, slight permeability changes were observed among the same films. This permeability decrease
respect to reference films is due to the hydrophobic nature of the oleoresins, which avoids
with respect to reference films is due to the hydrophobic nature of the oleoresins, which avoids
absorption and desorption of water molecules, increasing film hydrophobicity due to the
absorption and desorption of water molecules, increasing film hydrophobicity due to the interactions
interactions between the hydrophobic compounds in oleoresins and the other film components,
between the hydrophobic compounds in oleoresins and the other film components, which diminishes
which diminishes the availability of the hydrophilic groups. The water molecules diffuse into the
the availability of the hydrophilic groups. The water molecules diffuse into the continuous polymer
continuous polymer phase, where the presence of oily molecules introduces interruptions that
phase, where the presence of oily molecules introduces interruptions that increase the tortuosity for
increase the tortuosity for transference or mobility of water molecules [46].
transference or mobility of water molecules [46].

Figure 4. Water vapor permeability of biocomposite films containing oleoresins and reference (without
Figure 4. Water vapor permeability of biocomposite films containing oleoresins and reference
oleoresin). Different letters in the same column indicate significant differences (p < 0.05).
(without oleoresin). Different letters in the same column indicate significant differences (p < 0.05).

3.2.3.3.2.3.
Mechanical Properties
Mechanical Properties
The films without
The films oleoresin
without (reference)
oleoresin were more
(reference) were rigid,
more possibly due to due
rigid, possibly the restricted mobilitymobility
to the restricted of
the molecules present in the structure, which could limit their manipulation when
of the molecules present in the structure, which could limit their manipulation when used to wrap or used to wrap or
package foods.foods.
package In comparison to these films,
In comparison biocomposites
to these containing oleoresins
films, biocomposites containing showed a reduction
oleoresins showed a
in tensile strength (TS) and elastic modulus (EM), as well as a significant increase in
reduction in tensile strength (TS) and elastic modulus (EM), as well as a significant increase in theirtheir elongation
values (Figure 5).
elongation This(Figure
values typical5). behavior of plasticized
This typical behavior of films has beenfilms
plasticized reported
has beenin several
reportedstudies
in several
regarding
studiesthe additionthe
regarding of addition
lipid components to the biocomposite
of lipid components films. Thisfilms.
to the biocomposite is attributed to the to
This is attributed
inability of lipidsoftolipids
the inability formto continuous and cohesive
form continuous matrices
and cohesive when when
matrices incorporated
incorporatedto biocomposite
to biocomposite
filmsfilms
[47,48]. As noted in Figure 5, when adding black pepper oleoresin, higher
[47,48]. As noted in Figure 5, when adding black pepper oleoresin, higher elongation elongation values values
werewere
obtained. Some authors have indicated that the addition of oily substances such
obtained. Some authors have indicated that the addition of oily substances such as essential as essential oils
(added
oilsto(added
the matrix)
to thehas a plasticizing
matrix) role, which
has a plasticizing gives
role, the material
which gives thegreater
materialflexibility
greater[6,49,50].
flexibilityThus,
[6,49,50].
the incorporation of black pepper
Thus, the incorporation of oleoresin to the oleoresin
black pepper polymer gelatin
to thematrix
polymer broadens
gelatinthematrix
potential use of the
broadens
this material
potentialasuse food
of packaging.
this material as food packaging.
 Molecules 2018, 23, 212 9 of 18
Molecules 2018, 23, 212 9 of 18
Molecules 2018, 23, 212 45
9 of 18
a
TS (MPa)

Tensile Strength (MPa) - Elongation (%)

45 40
Elongation (%)
a
EM (MPa)
TS (MPa)

Tensile Strength (MPa) - Elongation (%)

40 35
Elongation (%)

Elastic Modulus (MPa)

EM (MPa) b
35 30 b

Elastic Modulus (MPa)

30 25 c b
b
a
25 20 c
a
20 15

15 10
b b
c c
10 5 b b
a b b
c c
5 b b
a
Reference Clove Nutmeg Black pepper
Biocomposite Films
Reference Clove Nutmeg Black pepper
Biocomposite Films
Figure 5. Mechanical properties of biocomposite films with oleoresins and reference. Different letters
Figure
in the
Figure 5. Mechanical
same column
5. Mechanical properties
indicate
properties of biocomposite
significant
of films
differences
biocomposite films (p <with
with oleoresins
0.05).
oleoresins andand reference.
reference. Different
Different letters
letters
in the same column indicate significant differences (p < 0.05).
in the same column indicate significant differences (p < 0.05).
3.2.4. Oil Permeability
3.2.4.
3.2.4. Oil
OilAs Permeability
Permeability
shown in Figure 6, oil permeability values of biocomposite films containing oleoresins
(clove,
As As nutmeg
shown
shown or
ininFigureblack
Figure 6,6, pepper) were between
oil permeability
oil permeability values
valuesof 25% and 30%, films
ofbiocomposite
biocomposite smaller in comparison
containing
films containing with
oleoresins the
(clove,
oleoresins
reference
nutmeg
(clove, orfilm
nutmeg or(0.36
black ± 0.0017
pepper)
black were
pepper) g·mm·mwere ·d
between −2 −1). This
25%
between result
and25% may
30%,and be due
smaller
30%, to a greater
insmaller
comparison finalthe
with
in comparison humidity
with content
referencethefilm
in
(0.36 ±
films0.0017 g ·
containinig
mm · m −2 ·d−1 ). This
oleoresin (Section
result 3.2.1)
may increased
be due to athe total
greater number
final humidityof hydroxyl
content groups
in films
reference film (0.36 ± 0.0017 g·mm·m ·d ). This result may be due to a greater final humidity content
−2 −1

(oleophobic)
in containinig in the oleoresin
oleoresin
films containinig film network,
(Section 3.2.1)which
(Section could
increased
3.2.1) theprevent the
thepassage
total number
increased of oil molecules
of hydroxyl
total number groups through
(oleophobic)
of hydroxyl theinfilm
groups the
(oleophobic) in the film network, which could prevent the passage of oil molecules through the film in
as
filmreported
network, by [22,51].
which Oil
could permeability
prevent the values
passage ofvaried
oil significantly
molecules through in function
the film asof the differences
reported by [22,51].
oleoresin
Oil
as reported composition.
permeability
by [22,51].values
Oil varied significantly
permeability valuesinvaried
function of the differences
significantly in functionin oleoresin composition.
of the differences in
oleoresin composition.
0.40
a
0.40 0.35
Oil Permeability (g·mm·m ·d )
-1

a
0.35 0.30
-2
Oil Permeability (g·mm·m ·d )
-1

0.30 0.25
-2

0.25 0.20

0.20 0.15
c
b
0.15 0.10
c
b d
0.10 0.05

0.05 0.00 d
Reference Clove Nutmeg Black pepper
0.00 Biocomposite Films
Reference Clove Nutmeg Black pepper
Biocomposite Films
Figure 6.
Figure 6. Oil
Oil permeability
permeability of
of biocomposite
biocomposite films
films with
with oleoresins
oleoresins and
and reference.
reference. Different
Different letters
letters in
in the
the
same
same
Figure column
6.column indicatesignificant
indicate
Oil permeability significant differences
differences
of biocomposite (p<<0.05).
films (p
with0.05).
oleoresins and reference. Different letters in the
same column indicate significant differences (p < 0.05).
3.2.5. UV-Vis
3.2.5. UV-VisLight
LightTransmittance
TransmittanceValues Values
3.2.5. UV-Vis Light in
As noted
noted Transmittance
Figure Values
7, capacity
the capacity
As in Figure 7, the UV-VisUV-Vis light transmission
light transmission of the
of the gelatin filmsgelatin films was
was significantly
significantly
reduced
As noted reduced
withinthe with the
incorporation
Figure incorporation
of oleoresins
7, the capacity of
UV-Vis oleoresins
(p light
< 0.05). (p < 0.05). This
This indicated
transmission indicated
of the that
thatgelatin biocomposite
biocomposite
films was films
films containing
containing
significantly reduced oleoresins
oleoresins
with (clove, (clove, nutmeg
nutmeg
the incorporation or black
orof black pepper)
pepper)
oleoresins enhance
enhance
(p < 0.05). theirlight
This their
indicatedlight barrier
barrier
that properties.
properties.
biocomposite
filmsThe
The referencefilm
reference
containing filmhadhad
oleoresins a higher
a higher
(clove, transmittance
transmittance
nutmeg value value
or black at 600 nm
pepper) at enhance
600 nm (75.39%),
(75.39%), lightfollowed
followed
their by the films
barrier bycontaining
the films
properties.
containing
Theblack
reference black
pepperfilm pepper
(73.04%),
had aclove(73.04%),
higher(62.81%),clove (62.81%),
and nutmeg
transmittance valueand nutmeg
(51.34%) (51.34%)
at 600 oleoresins.
nm (75.39%), oleoresins.
Thisfollowed This
result couldby be result could
theattributed
films
belight
to attributed
containing black to
scattering light
pepperduescattering
to the presence
(73.04%), due to
clove ofthe presence
oleoresin
(62.81%), of oleoresin
andinnutmeg
the form in the
of tiny
(51.34%) form
drops of tiny
Thisdrops
dispersed
oleoresins. dispersed
into polymeric
result could
into
be matrix. polymeric
attributed Thus, matrix. Thus,
the scattering
to light light barrier the light
dueproperty barrier property
of the biocomposites
to the presence of the
of oleoresin in is biocomposites
theimportant
form of tiny is important
for drops for food
food preservation,
dispersed
preservation,
intogiven given
that it matrix.
polymeric can avoid that
Thus, it can avoid
photo-oxidation
the light barrierphoto-oxidation
of property of
organic compounds organic compounds
and degradation
of the biocomposites and degradation
of vitamins
is important for foodand of
vitamins and
pigments
preservation, pigments
[9,10].
given that it[9,10].
can avoid photo-oxidation of organic compounds and degradation of
vitamins and pigments [9,10].
Molecules 2018, 23, 212 10 of 18
Molecules 2018, 23, 212 10 of 18
Molecules 2018, 23, 212 10 of 18
Reference
80 Clove
Reference
Nutmeg
80 Clove
Black Pepper
Nutmeg
60 Black Pepper

% Transmitance
60

% Transmitance
40

40

20

20

0
200 300 400 500 600 700 800

200 300 Wavelength

400 500 (nm)
600 700 800

Wavelength (nm)
Figure 7. Light transmission of biocomposite films with clove, nutmeg, and black pepper oleoresins
Figure 7. Light transmission of biocomposite films with clove, nutmeg, and black pepper oleoresins
and reference
Figure 7. Light(without oleoresin).
transmission of biocomposite films with clove, nutmeg, and black pepper oleoresins
and reference (without oleoresin).
and reference (without oleoresin).
3.2.6. Measurement of Seal Strength
3.2.6. Measurement
3.2.6. Measurement ofof
Seal
SealStrength
Strength
As shown in Figure 8, films containing oleoresins had significant changes with respect to the
AsAs shown
reference shown
filmsin(without
Figure
in Figure8,oleoresin)
8,films
filmscontaining
(p < 0.05).oleoresins
containing oleoresins had significant
hadfilms
The reference significant changeswith
changes
had a resistance with
of respect
respect
4.93 to
thethe
± 0.05toMPa.
reference
referencefilms
On average,films(without
(without
oleoresins oleoresin)
increased(p(p
oleoresin) <<0.05).
seal 0.05). The
The reference
strength reference
by films
films
8% with had aato
had
respect resistance
resistance ofof4.93
that of the 4.93 ±± 0.05
0.05
reference MPa.MPa.
film,
OnOnaverage, oleoresins
average, increased
oleoresins increased seal strength
seal by
strength 8% with
by 8% respect
with to that
respect of
to
achieving greater fusion between the components when heat was applied through the sealer.the
that reference
of the film,
reference achieving
film,
greater fusion
achieving between
greater thebetween
fusion components when heat when
the components was applied
heat was through
appliedthe sealer.the sealer.
through
7
c c
7 b c
6
c
b
6
(MPa)

5 a
(MPa)

5 a
Strength

4
Strength

4
3
SealSeal

3
2

2
1

1
0
Reference Clove Nutmeg. Black Pepper
0
Reference Biocomposite
Clove Films
Nutmeg. Black Pepper
Biocomposite Films
Figure 8. Seal strength of biocomposite films with oleoresins and reference. Different letters in the
same8.column
Figure
Figure 8. Seal
Seal indicate
strength significant
strengthof differences
ofbiocomposite
biocomposite films(p
films < 0.05).
with
with oleoresins and
oleoresins and reference.
reference.Different
Differentletters inin
letters thethe
same column indicate significant differences (p
same column indicate significant differences (p < 0.05).< 0.05).
3.2.7. X-ray Diffraction (XRD)
3.2.7. X-ray Diffraction (XRD)
3.2.7. X-ray
FigureDiffraction
9a shows(XRD) the diffractogram of the reference film, which in the 2θ angle presented two
peaks Figure 9a shows
at 19.61° andthe diffractogram
7.09°, with 54.18% of thecrystallinity.
reference film, Thewhich in the 2θ angle
diffractogram (Figure presented
9b), whichtwo
Figure 9a shows the diffractogram of the reference film, which in the 2θ angle presented two
peaks at 19.61°
corresponds to and
the 7.09°,
biocomposite with 54.18%
film with crystallinity.
clove oleoresin The diffractogram
presented two peaks(Figure
in the 9b),
2θ which
angle at
peaks at 19.61◦ and 7.09◦ , with 54.18% crystallinity. The diffractogram (Figure 9b), which corresponds
corresponds to thewith
21.33° and 7.76°, biocomposite film with clove
27.83% crystallinity. The oleoresin
diffractogrampresented
(Figure two9c),
peaks in the 2θ◦ angle
corresponding to theat ◦
to the biocomposite
21.33° and 7.76°,
film with clove oleoresin presented two peaks in the 2θ angle at 21.33 and 7.76 ,
biocomposite filmwith
with27.83%
nutmegcrystallinity.
oleoresin had Thetwo diffractogram
peaks in the (Figure
2θ angle9c), corresponding
at 20.28° and 6.71°,towith the
with 27.83%
biocomposite crystallinity.
film with
34.68% crystallinity. The
Thenutmeg diffractogram
oleoresin(Figure
diffractogram (Figure
had two 9c),
9d),peaks corresponding
in the 2θ angle
corresponding to the biocomposite
to theatbiocomposite
20.28° and 6.71°, film with
withfilm
with nutmeg oleoresin had two ◦ and 6.71◦ , with 34.68% crystallinity.
34.68%
black crystallinity.
pepper oleoresin Thehad twopeaks
peaks in
diffractogram the 2θ
the 2θ angle
in (Figure angle
9d), at at19.95°
20.28and
corresponding to the
7.09°, withbiocomposite
crystallinity of film with
42.29%.
Theblack
diffractogram
When pepper
oleoresins (Figure
oleoresin
were had 9d),
addedtwocorresponding
topeaks in the 2θtoangle
the polymeric the biocomposite
matrix,at 19.95° film
and 7.09°,
crystallinity withcrystallinity
with
diminished blackwith pepper oleoresin
of 42.29%.
respect to the
hadWhen
two peaks in This
the 2θ angle atto19.95 ◦ ◦
and 7.09tomatrix,
, the
withfact
crystallinity of 42.29%. When oleoresins were
oleoresins
reference film. were added
result could theattributed
be polymeric crystallinity diminished
that the intermolecular with respectamong
interactions to the
added to thefilm.
reference polymeric
the components This matrix,
result
of the crystallinity
could be attributed
biocomposite diminished
containing to the factwith
oleoresin that respect tomovements
the reference
the intermolecular
limited the of film.
interactions This result
among
the molecular
could
the be
chain attributed
components
segments ofto
and thethe fact that
the the
biocomposite
restrained intermolecular
containing
crystallisation oleoresin
process interactions
limited
[52]. theamong
In the the
movements
present work, components
ofthethevalue ofofthe
molecular the
chain segments and restrained the crystallisation process [52]. In the present work, the value of the
Molecules 2018, 23, 212 11 of 18

biocomposite containing oleoresin limited the movements of the molecular chain segments and
Molecules 2018, 23, 212 11 of 18
restrained the crystallisation process [52]. In the present work, the value of the crystallinity obtained
Molecules 2018, 23, 212 11 of 18
crystallinity
for the film containingobtained forpepper
black the film containing
oleoresin couldblack pepper
indicate goodoleoresin couldand
compatibility indicate good with
interaction
compatibility
the other components.
crystallinity and interaction with the other components.
obtained for the film containing black pepper oleoresin could indicate good
compatibility and interaction with the other components.
70 (a) 70 (b)
Reference Clove
60 60
(a) ICr=54.18% (b) ICr=27.83%

(a.u.) (a.u.)
70 70
(a.u.) (a.u.) 50 Reference 50
Clove
60 60
ICr=54.18% ICr=27.83%

Intensity
40 40
Intensity

50 50
30 30

Intensity
40 40
Intensity

20 20
30 30
10 10
20 20
0 0
10 0 20 40 60 80 100 10 0 20 40 60 80 100
2θ 2θ
0 0
70 (c)0 20 40 60 80 100 70 (d)0 20 40 60 80 100
2θ Nutmeg 2θ Black pepper
60 60
70 ICr=34.68% ICr=42.29%
(a.u.) (a.u.)

(c) 70 (d)

(a.u.) (a.u.)
50 Nutmeg 50 Black pepper
60 60
40 ICr=34.68% ICr=42.29%
Intensity

40

Intensity
50 50
30 30
40
Intensity

40
20 Intensity 20
30 30
10 10
20 20
0 0
10 0 20 40 60 80 100 10 0 20 40 60 80 100
2θ 2θ
0 0
0 20 40 60 80 100 0 20 40 60 80 100
Figure 9. Diffractogram 2θ
biocomposite 2θ oleoresin); (b) with clove
Figure 9. Diffractogram biocomposite filmsfilms (a) Reference
(a) Reference (without
(without oleoresin); (b) with clove oleoresin;
oleoresin;
(c) with nutmeg (c) with nutmeg
oleoresin; oleoresin;
(d) with (d)
black with
pepperblack pepper
oleoresin.oleoresin.
Figure 9. Diffractogram biocomposite films (a) Reference (without oleoresin); (b) with clove
oleoresin; (c) with nutmeg oleoresin; (d) with black pepper oleoresin.
3.2.8.3.2.8. Fourier Transformed Infrared (FTIR) Spectroscopy
Fourier Transformed Infrared (FTIR) Spectroscopy
3.2.8.InFourier
the IR Transformed Infrared
spectrum obtained for(FTIR) Spectroscopy
the reference films (Figure 10a), it can be noted that the band at
In the IR spectrum obtained for the reference films (Figure 10a), it can be noted that the band at
3422 cm
In thehas
−1
IR OH and N-H
spectrum groupsfor
obtained characteristic
the reference offilms
glycerol and gelatin
(Figure (type
10a), it can beAnoted
amide),
thatrespectively.
the band at
3422 At − 1
cm2887has OH andareN-H groups characteristic of glycerol and gelatin (type A amide), respectively.
3422 cm cm
−1
−1, there CH and CH 2 groups; the band at 1428 cm−1 has the HCH groups and OCH
has OH and N-H groups characteristic of glycerol and gelatin (type A amide), respectively.
At 2887 cm − 1 , there are CH and CH groups; the band at 1428 cm − 1 has the HCH groups and
−1 isOCH
vibrations;
At 2887 cmthe bandare
−1, there at 1347
CH and cm CH
−1 is2 2related
groups;tothe
theband
vibration of CH
at 1428 cm−1groups;
has thetheHCH band at 1219
groups andcmOCH
vibrations; the band at 1347 cm − 1 cm−1 is
attributed
vibrations;tothe C-C groups;
band andcm
at 1347 at−1is1045
related
cm to
is related
−1 to the
to C-O
vibration of CH groups;
group stretching.
the vibration Most of
of CH groups;
the
thethe
band at 1219
functional
band at 1219 groups
cm−1 is
attributed to with
C-C groups; and at at
1045 cm − 1
coincide
attributed to C-Cthatgroups;
reported andfor 1045 cm−1 to
microcrystalline to C-O
C-O groupstretching.
cellulose
group stretching.
and Most
gelatin.Most
The of the
spectra
of the infunctional
Figuregroups
functional groups
10b–d
corresponding
coincide with that to films
reported with
for clove, nutmeg,
microcrystalline and black pepper
cellulose and oleoresins,
gelatin.
coincide with that reported for microcrystalline cellulose and gelatin. The spectra in Figure 10b–d respectively,
The spectra were
in quite
Figure 10b–d
similar
corresponding amongst
corresponding themselves,
to films with
to films with but
clove, different
clove,nutmeg, from
nutmeg, and the reference
and black film.
pepperoleoresins,
black pepper oleoresins, respectively,
respectively, werewere
quitequite
similar
similar amongstamongst themselves,
themselves, butbut differentfrom
different fromthe the reference
reference film.
film.
(Normalized)

(b) Clove
(Normalized)

1.0 (a) Reference N-H

1.0 C-H C=O
(Normalized)

C-O
(b) NH2
Clove
(Normalized)

0.8
1.0 (a) Reference 0.8 N-H N-H
C-C 1.0 C-H C=O
O-H
0.6 NH2 C-O
0.8 0.8 N-H
OCH CH
C-C 0.6
%Transmitance

%Transmitance

HCH O-H
0.4
0.6 CH 0.4
CH2 OCH CH C-O 0.6
(Normalized) %Transmitance

(Normalized) %Transmitance

O-H N-H HCH

0.2
0.4
CH
C-O 0.2
0.4
CH2
0.0
0.2
O-H N-H
0.0
0.2
4000 3500 3000 2500 2000 1500 1000 500 4000 3500 3000 2500 2000 1500 1000 500
0.0 -1 -1
1/λ (cm ) 0.0 1/λ (cm )
4000 3500 3000 2500 2000 1500 1000 500 4000 3500 3000 2500 2000 1500 1000 500
1.0 (c) N-H -1 Nutmeg 1.0 (d) N-H -1
Black pepper
C-H 1/λ (cm ) C=O C-H
1/λ (cm ) C=O C-O
C-O
(Normalized)

NH2 NH
(Normalized)

1.0
0.8 (c) N-H N-H Nutmeg 0.8
1.0 (d) N-H Black
2
N-H pepper
C-H C=O O-H C-H C=O
C-O C-O
O-H NH2 NH2
0.6
0.8 N-H 0.6
0.8 N-H
O-H
%Transmitance

%Transmitance

O-H
0.6
0.4 0.4
0.6
%Transmitance

%Transmitance

0.2
0.4 0.2
0.4

0.0
0.2 0.0
0.2
4000 3500 3000 2500 2000 1500 1000 500 4000 3500 3000 2500 2000 1500 1000 500
-1 -1
0.0 1/λ (cm ) 0.0 1/λ (cm )
4000 3500 3000 2500 2000 1500 1000 500 4000 3500 3000 2500 2000 1500 1000 500
-1 -1
1/λ (cm ) 1/λ (cm )
Figure 10. FTIR spectra of biocomposite films. (a) Reference (without oleoresin); (b) with clove
oleoresin;
Figure (c) with
10. FTIR nutmeg
spectra oleoresin and (d) with black pepper oleoresin. oleoresin); (b) with clove
Figure 10. FTIR spectra of of biocompositefilms.
biocomposite films. (a)
(a) Reference
Reference(without
(without oleoresin); (b) with clove
oleoresin;
oleoresin; (c) with
(c) with nutmeg
nutmeg oleoresin
oleoresin and(d)
and (d)with
withblack
black pepper
pepperoleoresin.
oleoresin.
Molecules 2018, 23, 212 12 of 18

Hydrogen bonds of O-H and N-H groups gave way to a broad band at 3383 and 3199 cm−1
because of the compounds in the polymer matrix (gelatin, cellulose, and glycerol); C-H groups were
found at 2946 cm−1 . The band at 1638 cm−1 is attributed to C=O bonds; at 1597 and 1479 cm−1 NH2
primary amines and N-H secondary amines, respectively, and at 1065 cm−1 C-O groups. These results
indicate that adding oily molecules propitiated a structural change of the polymer matrix material.

3.2.9. Color
The color parameters of the biocomposite films are shown in Table 1. Visually, the films had
a slightly yellow aspect and their transparency was reduced upon incorporating the oleoresins.
The films containing clove, nutmeg, and black pepper oleoresins had ∆E of 4.66, 1.05, and 2.65,
respectively, with respect to the reference film.

Table 1. CIELab color parameters obtained for biocomposite films.

Color Parameters
Biocomposite Films
L* a* b* ∆E *
Reference 92.09 ± 0.094 a −1.46 ± 0.029 a 4.73 ± 0.33 a —
Clove 89.59 ± 0.22 b −1.16 ± 0.039 a 8.65 ± 0.47 b 4.66 ± 0.51 a
Nutmeg 91.30 ± 0.099 a −1.42 ± 0.014 a 5.44 ± 0.12 a 1.05 ± 0.15 b
Black pepper 90.45 ± 0.77 b −1.91 ± 0.10 a 6.73 ± 0.79 c 2.65 ± 0.99 c
Reported values correspond to the mean ± standard deviation. Different letters in the same column indicate
significant differences (p < 0.05). ∆E * calculated on the basis of the L *, a *, b * values of the reference film.

3.2.10. Thermogravimetric Analysis (TGA)

All the thermograms (not shown) detected four changes of temperature and mass; the first stage
revealed a weight loss from 12.73% to 14.37% at temperatures ranging from 150 to 161 ◦ C for all films:
This change is related to the loss of free water present in the films and volatile compounds of the
oleoresins. On the second stage, the reference biocomposite films and clove films, at temperatures
from 273 and 276 ◦ C lost 15.52% and 16.75% mass, respectively; while the films with nutmeg and black
pepper at temperatures from 284 and 274 ◦ C lost 20.42% and 20.61% mass, respectively. Within these
temperature ranges, degradation occurred of low molecular weight proteins of the polymer (gelatin)
and other compounds present in the plasticizer (glycerol). During the third stage, the temperature
changes for the biocomposite films ranged between 386 and 402 ◦ C with weight loss between 42.44%
and 46.45%, where decomposition took place of high molecular weight proteins present in the gelatin,
as well as the degradation of the microcrystalline cellulose due to the rupture of the glycosidic bonds.
On the fourth stage, the temperature changes occurred between 598.5 and 598.7 ◦ C for all the films
with weight loss between 15.73% and 16.81%, where degradation possibly occurred of the nonvolatile
phase of the oleoresins and other compounds of high molecular weight present in the films.

3.3. Physical-Chemical Evaluation of the Bread

3.3.1. Moisture
Table 2 shows that, during the first three days of storage, the bread samples packaged in films
containing oleoresins maintained desirable moisture contents according to the norm, while the bread
samples without film and those packaged in reference films (without oleoresin) had a significant
decrease (p < 0.05) in moisture content. After the nine-day storage period, it was noted that the
bread samples packaged in films containing black pepper oleoresin achieved a lower loss of moisture
compared to the rest, which is desirable to preserve the product’s texture characteristics.
Molecules 2018, 23, 212 13 of 18

Table 2. Physical-chemical characteristics of bread packaged in biocomposite films and stored during nine days at 25 ◦ C and 75% RH.

Bread Packed in Humidity pH aw % Weight loss

Biocomposite Films Day 1 Day 3 Day 5 Day 9 Day 1 Day 3 Day 5 Day 9 Day 1 Day 3 Day 5 Day 9 Day 3 Day 5 Day 9
Reference 26.34 ± 0.79 a 17.94 ± 0.55 a 9.61 ± 0.29 a 7.11 ± 0.2 a 5.73 ± 0.03 a 5.71 ± 0.02 a 5.67 ± 0.02 a 5.64 ± 0.02 a 0.84 ± 0.006 a 0.75 ± 0.006 a 0.58 ± 0.01 a 0.44 ± 0.01 a 45 ± 1.4 a 54 ± 1.5 a 34 ± 1.5 a
Clove 25.69 ± 0.95 b 21.09 ± 0.21 b 11.61 ± 0.9 b 8.86 ± 0.39 b 5.73 ± 0.03 a 5.65 ± 0.02 a 5.63 ± 0.02 a 5.59 ± 0.01 a 0.84 ± 0.006 a 0.79 ± 0.005 a 0.63 ± 0 a 0.54 ± 0.006 b 27 ± 1.3 b 29 ± 1.5 b 17 ± 0.9 b
Nutmeg 26.00 ± 0.55 a 21.05 ± 0.53 b 12.47 ± 0.9 c 8.55 ± 0.59 b 5.73 ± 0.03 a 5.63 ± 0.02 a 5.61 ± 0.02 a 5.57 ± 0.02 a 0.84 ± 0.006 a 0.79 ± 0.01 a 0.67 ± 0.047 a 0.51 ± 0.01 a 28 ± 1.5 b 32 ± 1.5 b 23 ± 1.7 b
Black pepper 25.33 ± 0.49 b 22.21 ± 0.85 c 11.37 ± 0.67 b 9.77 ± 0.45 c 5.73 ± 0.03 a 5.64 ± 0.02 a 5.62 ± 0.02 a 5.57 ± 0.02 a 0.84 ± 0.006 a 0.8 ± 0.01 a 0.67 ± 0.01 a 0.59 ± 0.006 b 20 ± 0.8 c 17 ± 0.9 c 13 ± 1.3 c
Without film 25.59 ± 0.3 b 8.45 ± 0.69 d 5.87 ± 0.25 d 4.71 ± 0.81 d 5.73 ± 0.03 a 5.59 ± 0.02 a 5.56 ± 0.01 a 5.55 ± 0.04 a 0.84 ± 0.006 a 0.52 ± 0.01 b 0.52 ± 0.006 b 0.4 ± 0.01 c 69 ± 1.8 d 41 ± 1.5 d 24 ± 1.8 b

Reported values correspond to the mean ± standard deviation. Different letters in the same column indicate significant differences (p < 0.05).
Molecules 2018, 23, 212 14 of 18

3.3.2. pH
The pH values obtained for all the samples evaluated during the storage period remained within
the quality range established by the norm [24]. At nine days of storage, the bread samples packaged
in reference films had higher pH values compared to those packaged in films containing oleoresins
(Table 2). This result was associated to the presence of acid compounds in the oleoresins. Additionally,
bread samples stored without film had the lowest pH values, which could be caused by enzymatic
and microbiological reactions catalyzed by the product’s exposure to the environment.

3.3.3. Water Activity (aw)

On the third day of storage, the bread samples packaged in films containing oleoresins maintained
a higher water activity as compared to the samples without film and those packaged in reference films.
The latter had the lowest values of aw due to the direct exposure of the product to the environment
(Table 2). Among the bread samples packaged in films containing oleoresins, those containing black
pepper had the highest values of aw. This behavior coincides with the results on humidity content of
the bread and permeability to water vapor of said films.

3.3.4. Weight Loss (%wl)

After three days of storage, the bread samples stored in films containing oleoresins had the lowest
weight loss values (20–28%). Upon completing the storage period, the bread samples packaged with
films containing clove and nutmeg oleoresins had statistically similar weight loss values, while the
samples packaged in films containing black pepper oleoresin had the lowest weight loss values (13%).
These results confirm the greatest water vapor barrier exerted by the films when incorporating black
pepper oleoresin in their formulation.

3.4. Microbiological Evaluation of the Bread

Table 3 presents the microbiological quality of bread samples packaged in biocomposite films
on days 1, 4, and 9 of storage. Upon completing the storage period evaluated, higher growth of mold
and yeast occurred on bread samples without film (2440 CFU/g) and in those packaged in reference
films (460 CFU/g). Similar behavior occurred in the S. aureus count (440 and 50 CFU/g, respectively).
In addition, the microbiological analyses evidenced no growth of S. aureus and lower growth of mold
and yeast occurred in the bread samples packaged in films containing oleoresins. During the storage
period, it was noted that using biocomposite films containing black pepper oleoresin was the most
effective packaging material in terms of the product’s microbiological quality.

Table 3. Microbiological analysis of the bread packaged in biocomposite films and stored during nine
days at 25 ◦ C and 75% RH.

Bread Packed in Count S. aureus (CFU/g) * Count Molds and Yeasts (CFU/g) *
Biocomposite Films Day 1 Day 4 Day 9 Day 1 Day 4 Day 9
Reference <10 <10 50 <10 100 460
Clove <10 <10 <10 <10 10 50
Nutmeg <10 <10 <10 <10 10 40
Black pepper <10 <10 <10 <10 <10 20
Without film <10 <10 440 <10 80 2440
* Colony-forming units per gram of sample.

3.5. Sensory Analysis of the Bread

Figure 11a displays the evolution of the aroma attribute of bread in Lot 2 during storage time.
Note that on day 1, the maximum score was obtained (I like it a lot) for all the bread samples. On day
4, perceptions were different where the highest score was obtained by the sample packaged in the film
with clove and reference (I like it) and the lowest scores were obtained by the samples packaged in
Molecules 2018, 23, 212 15 of 18

films with black

Molecules pepper
2018, 23, 212 and without film (I neither like or dislike it). Figure 11b presents 15 ofthe
18 flavor
evolution of the bread packaged in biocomposite films through nine days of storage. It was observed
in films with black pepper and without film (I neither like or dislike it). Figure 11b presents the
that on flavor
day 1 evolution
the samples had the same score (I like it a lot); on day 4, the samples packaged in films
of the bread packaged in biocomposite films through nine days of storage. It was
with clove
observed that on day 1were
and black pepper better perceived
the samples had the same(I like it) than
score (I likethose packaged
it a lot); on day in 4, films with nutmeg,
the samples
reference,
packaged in films with clove and black pepper were better perceived (I like it) than those packaged of the
and without film (I neither like or dislike it). Figure 11c shows the hardness evolution
in films with
bread packaged in nutmeg, reference,
biocomposite and during
films without the
film storage
(I neitherperiod
like or dislike it). Figure
evaluated. On 11c
dayshows
1, thethepanelists
assignedhardness
the same evolution
score to of all
the the
bread packaged
bread samples in biocomposite
(soft). On day films 4, during the storage
the samples periodin films
packaged
evaluated. On day 1, the panelists assigned the same score to all the bread samples (soft). On day 4,
with black pepper were scored as soft; the samples packaged in films with nutmeg and clove were
the samples packaged in films with black pepper were scored as soft; the samples packaged in films
perceived
withasnutmeg
firm and andthe bread
clove weresamples
perceived packaged
as firm andin the
reference films and
bread samples without
packaged film were
in reference perceived
films
as veryand
hard.without film were perceived as very hard. The general acceptability of the bread packaged shown
The general acceptability of the bread packaged in biocomposite films is in in
Figure 11d. The sample
biocomposite films with
is shownthe in
highest
Figure acceptability
11d. The sample during
with thethehighest
storage period was
acceptability that the
during packaged
in filmsstorage periodblack
containing was that packaged
pepper, in films by
followed containing black pepper,
those packaged followed
in films by those packaged
containing clove orinnutmeg.
films containing
The samples with the best cloveacceptability
or nutmeg. The samples
were thosewith the best acceptability
packaged in referencewere filmsthose
andpackaged
withoutinfilm.
reference films and without film.

(a) (b)

(c) (d)

Figure 11. Evolution of sensory attributes during nine days of storage (25 ◦ C and 75% RH) of bread
Figure 11. Evolution of sensory attributes during nine days of storage (25 °C and 75% RH) of bread
packaged in biocomposite
packaged films.
in biocomposite (a) (a)
films. Aroma;
Aroma;(b)
(b)flavor; (c)hardness;
flavor; (c) hardness;(d)(d) general
general acceptability.
acceptability.

4. Conclusions
4. Conclusions
This study showed that clove, nutmeg, and black pepper oleoresins inhibited S. aureus and E.
This study showed that clove, nutmeg, and black pepper oleoresins inhibited S. aureus and E. coli.
coli. Black pepper oleoresin inhibited both strains evaluated, at the lowest concentration studied (0.5
Black pepper
g/100 goleoresin inhibited
of film-forming both Incorporation
solution). strains evaluated, at the lowest concentration
of the aforementioned oleoresins into studied (0.5 g/100 g
biocomposite
of film-forming
films basedsolution). Incorporation
on gelatin of the aforementioned
and microcrystalline cellulose managedoleoresins
to decreaseinto biocomposite
the oil films based
and water vapor
on gelatin and microcrystalline
permeability values by 20 tocellulose managedthe
30%. Furthermore, to decrease theseal
elasticity and oil and water
strength vapor
of the permeability
polymeric
values bymatrix were
20 to 30%.increased by 5%. Upon
Furthermore, theexploring
elasticitytheand
potential use of films
seal strength of containing the different
the polymeric matrix were
oleoresins, it was evident that the incorporation of black pepper oleoresin was the most effective
increased by 5%. Upon exploring the potential use of films containing the different oleoresins, it was
packaging material for maintaining the physical-chemical, microbiological, and sensory quality of
evidentbread
that the incorporation of black pepper oleoresin was the most effective packaging material
without adding preservatives during nine days of storage.
for maintaining the physical-chemical, microbiological, and sensory quality of bread without adding
preservatives during nine days of storage.

Acknowledgments: The authors thank the Administrative Department on Science, Technology, and Innovation
(COLCIENCIAS) for funding the project ‘Biodegradable, intelligent, and active packaging for the preservation
Molecules 2018, 23, 212 16 of 18

of beef loins “Longissimus dorsi” with code (111366945140). Gratitude is also expressed to the Interdisciplinary
Science Institute at Universidad del Quindío and to the Faculty of Engineering and Administration at Universidad
Nacional de Colombia (at Palmira) for the different contributions during the development of this research.
Author Contributions: Kelly J. Figueroa-Lopez, Margarita María Andrade-Mahecha and Olga Lucía Torres-Vargas
conceived and designed the experiments; Kelly J. Figueroa-Lopez developed the experimental analysis and wrote
the manuscript; Margarita María Andrade-Mahecha supervised the data analyses and co-wrote the manuscript.
All authors reviewed the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.

References
1. Fowler, P.A.; Hughes, J.M.; Elias, R.M. Review Biocomposites: Technology, environmental credentials and
market forces. J. Sci. Food Agric. 2006, 86, 1781–1789. [CrossRef]
2. Zini, E.; Scandola, M. Green composites: An overview. Polym. Compos. 2011, 32, 1905–1915. [CrossRef]
3. Yates, M.; Barlow, C. Life cycle assessments of biodegradable, commercial biopolymers—A critical review.
Resour. Conserv. Recycl. 2013, 78, 54–66. [CrossRef]
4. Andrade, R.; Skurtys, O.; Osorio, F. Drop impact of gelatin coating for mulated with cellulose nanofibers on
banana and eggplant epicarps. LWT-Food Sci. Technol. 2015, 61, 422–429. [CrossRef]
5. Clarke, D.; Molinaro, S.; Tyuftin, A.; Bolton, D.; Fanning, S.; Kerry, J.P. Incorporation of commercially-derived
antimicrobials into gelatin-based films and assessment of their antimicrobial activity and impact on physical
film properties. Food Control 2016, 64, 202–211. [CrossRef]
6. Hosseini, S.F.; Rezaei, M.; Zandi, M.; Farahmandghavi, F. Development of bioactive fish gelatin/chitosan
nanoparticles composite films with antimicrobial properties. Food Chem. 2016, 194, 1266–1274. [CrossRef]
[PubMed]
7. Isotton, F.; Bernardo, G.; Baldasso, C.; Rosa, L.; Zeni, M. The plasticizer effect on preparation and properties
of etherified corn starchs films. Ind. Crop. Prod. 2015, 76, 717–724. [CrossRef]
8. Alves, J.; dos Reis, K.; Menezes, E.; Pereira, F.; Pereira, J. Effect of cellulose nanocrystals and gelatin in corn
starch plasticized films. Carbohydr. Polym. 2015, 115, 215–222. [CrossRef] [PubMed]
9. Alexandre, E.M.C.; Lourenço, R.V.; Habittante, A.M.Q.B.; Moraes, I.C.F.; Sobral, P.J.A. Gelatin-based films
reinforced with montmorillonite and activated with nanoemulsion of ginger essential oil for food packaging
applications. Food Packag. Shelf Life 2016, 10, 87–96. [CrossRef]
10. Farris, S.; Introzzi, L.; Piergiovanni, L. Evaluation of a Bio-coating as a Solution to Improve Barrier, Friction
and Optical Properties of Plastic Films. Packag. Technol. Sci. 2009, 22, 69–83. [CrossRef]
11. Kechichian, V.; Ditchfield, C.; Veiga-Santos, P.; Tadini, C. Natural antimicrobial ingredients incorporated in
biodegradable films based on cassava starch. LWT-Food Sci. Technol. 2010, 43, 1088–1094. [CrossRef]
12. Woranuch, S.; Yoksan, R.; Akashi, M. Ferulic acid-coupled chitosan: Thermal stability and utilization as
an antioxidant for biodegradable active packaging film. Carbohydr. Polym. 2015, 115, 744–751. [CrossRef]
[PubMed]
13. Arfat, Y.A.; Ahmed, J.; Hiremath, N.; Auras, R.; Joseph, A. Thermo-mechanical, rheological, structural and
antimicrobial properties of bionanocomposite films based on fish skin gelatin and silver-copper nanoparticles.
Food Hydrocoll. 2017, 62, 191–202. [CrossRef]
14. Lorian, V. Antibiotics in Laboratory Medicine; Williams and Wilkins: Amsterdam, The Netherlands, 1996.
15. Andrade-Mahecha, M.M.; Tapia-Blácido, D.R.; Menegalli, F.C. Development and optimization of
biodegradable film based on achira flour. Carbohydr. Polym. 2012, 88, 449–458. [CrossRef]
16. Mondragon, G.; Peña-Rodriguez, C.; González, A.; Eceiza, A.; Arbelaiz, A. Bionanocomposites based on
gelatin matrix and nanocellulose. Eur. Polym. J. 2015, 62, 1–9. [CrossRef]
17. ASTM. Designation D 644-99: Standard Test Method for Moisture Content of Paper and Paperboard by Oven
Drying. In Annual Book of ASTM Standards; American Society for Testing and Materials: West Conshohocken,
PA, USA, 1999; p. 2.
18. ASTM. Designation E 96-05: Standard Test Methods for Water Vapor Transmission of Materials. In Annual
Book of ASTM Standards; American Society for Testing and Materials: West Conshohocken, PA, USA, 2005.
19. ASTM. Designation D 882-02: Standard test method for tensile properties of thin plastic sheeting. In Annual
Book of ASTM Standards; American Society for Testing and Materials: West Conshohocken, PA, USA, 2002.
Molecules 2018, 23, 212 17 of 18

20. Yan, Q.; Hou, H.; Guo, P.; Dong, H. Effects of extrusion and glycerol content on properties of oxidized and
acetylated corn starch-based films. Carbohydr. Polym. 2012, 87, 707–712. [CrossRef]
21. ASTM. Designation F88M-15: Standard Test Method for Seal Strength of Flexible Barrier Materials. In Annual
Book of ASTM Standards; American Society for Testing and Materials: West Conshohocken, PA, USA,
2015; p. 11.
22. Farhan, A.; Hani, N.M. Characterization of edible packaging films based on semi-refined kappa-carrageenan
plasticized with glycerol and sorbitol. Food Hydrocoll. 2017, 64, 48–58. [CrossRef]
23. Leceta, I.; Peñalba, M.; Arana, P.; Guerrero, P.; de la Caba, K. Ageing of chitosan films: Effect of storage time
on structure and optical, barrier and mechanical properties. Eur. Polym. J. 2015, 66, 170–179. [CrossRef]
24. ICONTEC. Pan. Requisitos Generales. Norma Técnica Colombiana 1363; ICONTEC: Bogotá, Colombia,
2005; p. 11.
25. ICONTEC. Industrias Alimentarias. Harina de Trigo. Métodos de ensayo. Normas Técnicas Colombianas 282;
ICONTEC: Bogotá, Colombia, 1986; p. 28.
26. ICONTEC. Microbiología de Alimentos y de Alimentos Para Animales. Método Horizontal Para el Recuento
de Coliformes o Escherichia coli o Ambos. Técnica de Recuento de Colonias Utilizando Medios Fluorogénicos o
Cromogénicos. Normas Tecnicas Colombianas 4458; ICONTEC: Bogotá, Colombia, 2007; p. 12.
27. ICONTEC. Microbiología de Alimentos y Alimentos Para Animales. Método Horizontal Para el Recuento de
Estafilococos Coagulasa Positiva (Staphylococcus aureus y otras Especies). Normas Tecnicas Colombianas 4779;
ICONTEC: Bogotá, Colombia, 2007; p. 21.
28. ICONTEC. Microbiología. Método Horizontal Para el Recuento de Bacillus Cereus. Técnica de Recuento de Colonias.
Normas Tecnicas Colombianas 4679; ICONTEC: Bogotá, Colombia, 2006; p. 27.
29. ICONTEC. Microbiología de Alimentos y de Alimentos Para Animales. Método Horizontal Para la Detección de
Salmonella spp. Normas Tecnicas Colombianas 4574; ICONTEC: Bogotá, Colombia, 2007; p. 26.
30. ICONTEC. Microbiología. Guia General Para el Recuento de Mohos y Levaduras. Técnica de Recuento de Colonias a
25 ◦ C. Normas Tecnicas Colombianas 4132; ICONTEC: Bogotá, Colombia, 1997; p. 12.
31. ICONTEC. Análisis sensorial. Metodología. Guía General. Normas Tecnicas Colombianas 3925; ICONTEC: Bogotá,
Colombia, 1996; p. 25.
32. Jayabrata, M.; Ray, S.K. Removal of Cu (II) ion from water using sugar cane bagasse cellulose and gelatin
based composite hydrogels. Int. J. Biol. Macromol. 2017, 97, 238–248. [CrossRef]
33. Li, Z.-R.; Wang, B.; Chi, C.-F.; Zhang, Q.-H.; Gong, Y.-D.; Tang, J.-J.; Luo, H.-Y.; Ding, G.-F. Isolation and
characterization of acid soluble collagens and pepsin soluble collagens from the skin and bone of Spanish
mackerel (Scomberomorous niphonius). Food Hydrocoll. 2013, 31, 103–113. [CrossRef]
34. Yuan, Y.-Y.; Fan, D.-D. Coordination study of recombinant human-like collagen and zinc (II). Spectrochim.
Acta Part A 2011, 81, 412–416. [CrossRef]
35. Nada, A.-A.M.A.; El Kady, M.Y.; El Sayed, E.S.A.; Amine, F.H. Preparation and characterizacion of
microcrystalline cellulose (MCC). BioResources 2009, 4, 1359–1371.
36. García-García, L.; Bordallo-López, E.; Dopico-Ramírez, D.; Cordero-Fernández, D. Obtención de celulosa
microcristalina a partir del bagazo de la caña de azúcar. ICIDCA Sobre los Derivados de la Caña de Azúcar 2013,
47, 57–63.
37. Trache, D.; Hussin, M.H.; Chuin, C.T.H.; Sabar, S.; Fazita, M.R.N.; Taiwo, O.F.A.; Hassan, T.M.; Haafiz, M.K.M.
Microcrystalline cellulose: Isolation, characterization and bio-composites application—A review. Int. J.
Biol. Macromol. 2016, 93, 789–804. [CrossRef] [PubMed]
38. Shankar, S.; Rhim, J.-W. Preparation of nanocellulose from micro-crystalline cellulose: The effect on the
performance and properties of agar-based composite films. Carbohydr. Polym. 2016, 135, 18–26. [CrossRef]
[PubMed]
39. Zhang, M.-F.; Qin, Y.-H.; Ma, J.-Y.; Yang, L.; Wu, Z.-K.; Wang, T.-L.; Wang, W.-G.; Wang, C.-W.
Depolymerization of microcrystalline cellulose by the combination of ultrasound and Fenton reagent.
Ultrason. Sonochem. 2016, 31, 404–408. [CrossRef] [PubMed]
40. Kim, U.-J.; Eom, S.H.; Wada, M. Thermal decomposition of native cellulose: Influence on crystallite size.
Polym. Degrad. Stab. 2010, 95, 778–781. [CrossRef]
41. Trache, D.; Donnot, A.; Khimeche, K.; Benelmir, R.; Brosse, N. Physico-chemical properties and thermal
stability of microcrystalline cellulose isolated from Alfa fibres. Carbohydr. Polym. 2014, 104, 223–230.
[CrossRef] [PubMed]
Molecules 2018, 23, 212 18 of 18

42. Rodianawati, I.; Hastuti, P.; Nur Cahyanto, M. Nutmeg’s (Myristica fragrans Houtt) Oleoresin: Effect of
Heating to Chemical Compositions and Antifungal Properties. Procedia Food Sci. 2015, 3, 244–254. [CrossRef]
43. Chaieb, I.; Hajlaoui, H.; Zmantar, T.; Kahla-Nakbi, A.; Rouabhia, M.; Mahdouani, K.; Bakhrouf, A.
The chemical composition and biological activity of clove essential oil, Eugenia caryophyllata (Syzigium
aromaticum L. Myrtaceae): A short review. Phytother. Res. 2007, 21, 501–506. [CrossRef] [PubMed]
44. Dutta, S.; Bhattacharjee, P. Enzyme-assisted supercritical carbon dioxide extraction of black pepper oleoresin
for enhanced yield of piperine-rich extract. J. Biosci. Bioeng. 2015, 120, 17–23. [CrossRef] [PubMed]
45. Ojagh, S.M.; Rezaei, M.; Razavi, S.H.; Hosseini, S.M.H. Development and evaluation of a novel biodegradable
film made from chitosan and cinnamon essential oil with low affinity toward water. Food Chem. 2010, 122,
161–166. [CrossRef]
46. Acosta, S.; Chiralt, A.; Santamarina, P.; Rosello, J.; Gonzalez-Martínez, C.; Chafer, M. Antifungal films based
on starch-gelatin blend, containing essential oils. Food Hydrocoll. 2016, 61, 233–240. [CrossRef]
47. Pérez-Córdoba, L.J.; Norton, I.T.; Batchelor, H.K.; Gkatzionis, K.; Spyropoulos, F.; Sobral, P.J.A.
Physico-chemical, antimicrobial and antioxidant properties of gelatin chitosan based films loaded with
nanoemulsions encapsulating active compounds. Food Hydrocoll. 2017. [CrossRef]
48. Tongnuanchan, P.; Benjakul, S.; Prodpran, T. Structural, morphological and thermal behaviour
characterisations of fi sh gelatin fi lm incorporated with basil and citronella essential oils as affected by
surfactants. Food Hydrocoll. 2014, 41, 33–43. [CrossRef]
49. Fabra, M.J.; Talens, P.; Chiralt, A. Tensile properties and water vapor permeability of sodium caseinate films
containing oleic acid–beeswax mixtures. J. Food Eng. 2008, 85, 393–400. [CrossRef]
50. Tongnuanchan, P.; Benjakul, S.; Prodpran, T. Properties and antioxidant activity of fish skin gelatin film
incorporated with citrus essential oils. Food Chem. 2012, 134, 1571–1579. [CrossRef] [PubMed]
51. Hu, G.; Chen, J.; Gao, J. Preparation and characteristics of oxidized potato starch films. Carbohydr. Polym.
2009, 76, 291–298. [CrossRef]
52. Valenzuela, C.; Abugoch, L.; Tapia, C. Quinoa protein-chitosan-sunflower oil edible film: Mechanical, barrier
and structural properties. LWT-Food Sci. Technol. 2013, 50, 531–537. [CrossRef]

© 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).