Food Packaging and Shelf Life 21 (2019) 100342

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Food Packaging and Shelf Life

journal homepage: www.elsevier.com/locate/fpsl

Investigation of physicochemical and antioxidant properties of gelatin T

edible film mixed with blood orange (Citrus sinensis) peel extract
⁎
Mourad Jridia,b, , Soumaya Boughribaa, Ola Abdelhedia, Hend Nciria, Rim Nasria, Hela Kchaoua,
Murat Kayac, Hichem Sebaib, Nacim Zouaria,d, Moncef Nasria
a
Laboratory of Enzyme Engineering and Microbiology, Engineering National School of Sfax (ENIS), University of Sfax, Sfax, Tunisia
b
Higher Institute of Biotechnology of Beja, University of Jendouba, Beja, Tunisia
c
Higher Institute of Applied Biology of Medenine, University of Gabes, Medenine, Tunisia
d
Department of Biotechnology and Molecular Biology, Faculty of Science and Letters Aksaray University, 68100, Aksaray, Turkey

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

Keywords: The present study aims at developing Grey triggerfish skin gelatin films containing phenolic extracts from blood
Grey triggerfish skin gelatin orange (Citrus sinensis) peel, as an alternative for the existing synthetic packaging films. The effect of drying
Blood orange (Citrus sinensis) peel pretreatment on the phenolic compounds’ profile and antioxidant and antibacterial activities of the extracts was
Release kinetics of phenolic extract investigated. Physicochemical, thermal and mechanical properties of gelatin films incorporated with orange peel
FTIR analysis
extract, at different concentrations and plasticized with glycerol were investigated. Films were also tested for
Wetting and thermal properties
their antioxidant capacity, monitored through the β-carotene bleaching, DPPH radical-scavenging and reducing
power activities. Results showed that the fresh orange peel extract (FOPE) was more effective against all bacteria
tested and exhibited higher antioxidant effect than the thermally-dried orange peel extract (DOPE). In addition,
the LC-ESI-MS analysis showed that the quinic acid was the major compound among the total poly-phenols
followed by rutin, trans-ferulic acid, naringenin and 4,5-di-O-caffeoylquinate. After its addition, FOPE modified
the mechanical and thermal properties of gelatin films, which showed more deformable texture than the control
gelatin film. This effect may be caused by the interactions between phenolic compounds of the extract and
gelatin chains, as assessed by the Fourier Transform infrared (FTIR) analysis. The FOPE-added films presented
higher antioxidant properties than the control. Furthermore, release kinetics of FOPE through gelatin film
showed controlled quasi-Fickian diffusion. The overall results emphasized the potential use of phenolic com-
pounds from blood orange by-products to produce bioactive films intended for food packaging.

1. Introduction
Natural biopolymers are attracting the scientific attention, as they
can be used to address environmental concerns and consumer demands
(Niaounakis, 2015; Sangha, Ravichandran, Prithiviraj, Critchley, &
Prithiviraj, 2010). These biopolymers can be derived from different
origins, including polysaccharides, lipids or proteins (such as gelatin)
(Abdelhedi et al., 2018; Arfat, Ahmed, Hiremath, Auras, & Joseph,
2017; Chiralt, González-Martínez, Vargas, & Atarés, 2018; Costa, Silva,
& Boccaccini, 2018).
Gelatin has valuable properties in pharmaceutical, medicinal and
industrial applications due to its transparency, film-forming ability, and
high barrier properties (Etxabide, Uranga, Guerrero, & de la Caba,
2017). In this context, edible gelatin-based coatings have been used to
preserve foods, including meat (Jridi et al., 2018) and fruits (Khan, Zill-
E-Huma, & Dangles, 2014), in order to extend their shelf-life and im-
prove their quality and safety during storage. Moreover, gelatin-based
films may be used as carriers of bioactive additives, to achieve active
packaging functions and protect, consecutively, food products from
oxidation and microbial spoilage, resulting in their quality preserva-
tion. The interactions between the bioactive and the gelatin polymer
can provide its controlled release over the storage time. Among the
most used functional molecules, emulsifiers, antioxidants and anti-
microbials, are usaually used to achieve this goal (Adilah, Jamilah,
Noranizan, & Hanani, 2018; Ramos, Valdés, Beltrán, & Garrigós, 2016;
Santoro, Tatara, & Mikos, 2014). Due to safety concerns associated to
synthetic active compounds, extensive research has been performed for
the naturally-occuring molecules, where the most famous family is the

⁎
Corresponding author at: Laboratoire de Génie Enzymatique et de Microbiologie, Université de Sfax, Ecole Nationale d’Ingénieurs de Sfax, B.P. 1173-3038 Sfax,
Tunisie.
E-mail address: jridimourad@gmail.com (M. Jridi).

https://doi.org/10.1016/j.fpsl.2019.100342
Received 18 September 2018; Received in revised form 8 April 2019; Accepted 29 May 2019
2214-2894/ © 2019 Elsevier Ltd. All rights reserved.
M. Jridi, et al.
phenolic compounds, largely present in plants.
Citrus is among the most important fruit tree in the world, with an
annual production up to 140 million tons in 2015 (FAOSTAT, 2018).
Citrus sinensis represents the largest citrus cultivar group grown around
the world, accounting for about 70% of the total annual production of
Citrus species (Abbate et al., 2012). The citrus industry generated
substantial quantities of peels and seed residues as by-products of or-
ange juice processing, accounting for up to 50% of the total fruit weight
(Balasundram, Sundram, & Samman, 2006). Considering the composi-
tional profile of Citrus peels, their transformation into value-added
products, like natural bioactive extracts rich in phenolic compounds, is
a promising option with great economic and environmental perspec-
tives (Laufenberg, Kunz, & Nystroem, 2003). In fact, recent studies have
reported the efficiency of orange peel as an excellent source of phe-
nolics and flavonoids (Ozturk, Parkinson, & Gonzalez-Miquel, 2018).
Moreover, orange peels have been studied due to their numerous bio-
logical activities (Barrales et al., 2018; Devi et al., 2015; Lee, Lee, Lee,
Baeg, & Shim, 2012; Ozturk et al., 2018).
In this context, the aim of the present study is improve the biolo-
gical properties of grey triggerfish skin gelatin films (GF) by the in-
corporation of phenolic extracts from blood orange (C. sinensis) peels.
The phenolic profiles and antioxidant and antibacterial activities of the
extracts were determined, as well as their effects on the physicochem-
ical, structural, thermal, mechanical and biological properties of the
resulting films were investigated.
2. Materials and methods
2.1. Extraction of gelatin from grey triggerfish
Skin from grey triggerfish (Balistes capriscus) was obtained from the
local fish market of Sfax City, Tunisia. Skin was cut into small pieces
(1 cm × 1 cm) and then soaked in 0.05 M NaOH (1:10 w/v). The mix-
ture was stirred for 2 h and the alkaline solution was changed every
30 min. The alkaline-treated skins were then washed with distilled
water until a neutral pH was obtained, and then soaked in 100 mM
glycin-HCl buffer (pH 2.0) with a solid/solvent ratio of 1:10 (w/v).
Pepsin was then added (5 units of pepsin /g of skin), for collagen hy-
drolysis, as described in Jellouli et al. (2011). Grey triggerfish skin
gelatin (G) obtained was freeze-dried and then used for films prepara-
tion.
2.2. Preparation of blood orange powders and their ethanolic extracts
Blood orange peels were collected on March 2018 from the area of
Beja (Tunisia), washed and dried in a convection oven at 80 °C during
2 h (Polin A511088/AL/3125, Verona, Italy). The dried peels were
ground in a spice grinder (Black & Decker CBG100S Smartgrind,
Maryland, USA), sieved through 250 μm sieve and the obtained powder,
referring to the dried blood orange peels powder was stored at 25 °C
until use. The powder (25 g) was Soxhlet-extracted using 300 ml of
ethanol during 6 h to obtain dried orange peel extract (DOPE).
Similarly, fresh peels, without drying, were used to prepare a fresh
orange peel extract (FOPE), with the same manner as DOPE. The sol-
vent (ethanol) was then evaporated under vacuum and the residual
solvent was removed by flushing with nitrogen. Thereafter, extracts
were freeze-dried to obtain DOPE and FOPE powder, kept in the dark at
4 °C until analysis.
2.3. Total phenolic and flavonoid contents
Total phenolic and flavonoid contents were measured in orange peel
extracts as previously described (Dewanto, Wu, Adom, & Liu, 2002;
Sun, Ricardo-da-Silva, & Spranger, 1998). Total phenolics content was
expressed as mg gallic acid equivalents (GAE)/g of extract. Flavonoids
content was expressed as mg quercetin equivalents (QE)/g of extract.
2
Food Packaging and Shelf Life 21 (2019) 100342
2.4. Liquid chromatography-electrospray ionization mass spectrometry (LC-
ESI-MS) analysis
Twenty mg of each extract powder were dissolved in 1 ml of 10%
methanol, and then the mixture was filtered through a 0.45 μm mem-
brane filter before injection into the HPLC system. Analysis was per-
formed using a LC–MS-2020 quadrupole mass spectrometer analyzer
(Shimadzu, Kyoto, Japan) equipped with an electro-spray ionization
source (ESI) and operated in negative ionization mode. Mass spectro-
meter was coupled online with an ultra-fast liquid chromatography
system consisted of LC-20AD XR binary pump system, SIL-20AC XR
autosampler, CTO-20AC column oven and DGU-20A 3R degasser
(Shimadzu, Kyoto, Japan). Spectra were monitored in mode SIM
(Selected Ion Monitoring) and processed using Shimadzu LabSolutions
LC–MS software. The mass spectrometer was operated in negative ion
mode with a capillary voltage of −3.5 V, a nebulizing gas flow of 1.5 l/
min, a dry gas flow rate of 12 l/min, a dissolving line temperature of
250 °C, a block source temperature of 400 °C, and a voltage detector of
1.2 V. The spectra were fully scanned from 50 to 2000 Da.
2.5. Evaluation of antioxidant activities
2.5.1. Ferric (Fe3+) reducing power
The ability of samples to reduce iron was determined according to
the method of Yıldırım, Mavi, and Kara (2001). A volume of 0.5 ml of
each sample or small pieces of each film (10 mg), was mixed with
1.25 ml of potassium phosphate buffer (0.2 M, pH 6.6) and 1.25 ml of
1% potassium ferricyanide solution. The reaction mixtures were in-
cubated for 20 min at 50 °C. After incubation, 0.5 ml of 10% tri-
chloroacetic acid (TCA) was added and the reaction mixtures were then
centrifuged for 10 min at 3000 rpm. Finally, 1.25 ml of the supernatant
solution from each sample mixture was added to 1.25 ml potassium
phosphate buffer and 0.25 ml of 0.1% ferric chloride. The absorbance of
the resulting solutions was measured at 700 nm after 10 min of in-
cubation. Three replicates were done for each test sample.
2.5.2. Antioxidant assay using the β-carotene bleaching method
The prevention of β-carotene from bleaching was determined ac-
cording to the method of Koleva, van Beek, Linssen, de Groot, and
Evstatieva (2002). First, the emulsion of β-carotene/linoleic acid was
freshly prepared by dissolving 0.5 mg of β-carotene, 25 μl of linoleic
acid and 200 μl of Tween 40 in 1 ml of chloroform. The chloroform was
then completely evaporated under vacuum in a rotatory evaporator at
50 °C and then 100 ml of distilled water were added. In the reaction
tube, 2.5 ml of the β-carotene/linoleic acid emulsion was mixed with
0.5 ml of each sample or small pieces of each film (10 mg). Control
tubes were prepared under the same conditions by adding 0.5 ml of
water to the emulsion. The absorbance of each test tube was measured
at 470 nm twice, before and after incubation for 1 to 2 h at 50 °C. Tests
were carried out in triplicate and the antioxidant activity was evaluated
in terms of β-carotene bleaching inhibition using the following equa-
tion:
β-carotene bleaching inhibition (%) = [1− (OD0 − ODt)/(OD0’−
ODt’)] × 100
Where OD0 and ODt are the absorbance of the test sample measured
before and after incubation, respectively, and OD0’ and ODt’ are the
absorbance of the control measured before and after incubation, re-
spectively.
2.5.3. Free radical scavenging activity on 1,1-diphenyl-2-picrylhydrazyl
(DPPH%)
The DPPH%-radical scavenging activity was determined as described
previously by Bersuder, Hole, and Smith (1998). A volume of 500 μl of
sample or small pieces of each film (10 mg) were allowed to react with
M. Jridi, et al.
125 μl of 0.02% DPPH% solution (in ethanol) and 375 μl of ethanol.
Blank tube was prepared in the same way, but with substituting the
sample by distilled water. The reaction mixtures were incubated for
60 min in dark at room temperature and the reduction of DPPH% radical
was measured at 517 nm. The test was carried out in triplicate and the
DPPH%-radical scavenging activity was calculated as follows:
Scavenging activity (%) = [(ODC + ODB − ODS)/ODC] × 100
Where ODC, ODB and ODS represent the absorbance of the control, the
blank and the sample reaction solution, respectively.
2.5.4. Lipid peroxidation inhibition
The inhibition of in vitro lipid peroxidation of each sample was
determined by assessing their ability to inhibit the oxidation of linoleic
acid in an emulsified model system. Briefly, the sample at different
concentrations were dissolved in 2.5 ml of 50 mM phosphate buffer
(pH = 7.0) and added to a 2.5 ml of 50 mM linoleic acid in ethanol
(95%). The final volume was then adjusted to 6.25 ml with distilled
water. The obtained mixture was incubated in a 10 ml tube with silicon
rubber caps at 45 °C for 8 days in dark and the degree of oxidation was
evaluated by measuring the ferric thiocyanate values. An aliquot of
reaction mixture (0.1 ml) was mixed with 4.7 ml of 75% ethanol fol-
lowed by the addition of 0.1 ml of 30% ammonium thiocyanate and
0.1 ml of 20 mM ferrous chloride solution in 3.5% HCl. After stirring for
3 min, the degree of color development was measured at 500 nm. α-
tocopherol was used as reference and control reaction was conducted
without sample. The percentage of oxidation inhibition was expressed
as follows:
Lipid peroxidation inhibition (%) = [1 – (ODS / ODNC)] × 100
Where ODS and ODNC represent the absorbance of the sample and the
negative control tubes, respectively.
2.6. Antibacterial activity
2.6.1. Microbial strains
Seven Gram-negative and Gram-positive pathogenic bacteria strains
were used for the assessment of antibacterial activity in the extracts:
Micrococcus luteus (ATCC 4698), Staphylococcus aureus (ATCC 25923),
Bacillus cereus (ATCC 11778) Pseudomonas aeruginosa (ATCC 27853),
Salmonella enterica (ATCC 43972), Listeria monocytogenes (ATCC 19117)
and Enterobacter sp.
2.6.2. Agar diffusion method
The antibacterial activity assay was performed referring to the
method described by vanden Berghe and Vlietinck (1991). Micro-
organism’s culture suspensions (200 μl), containing 106 colony forming
units (CFU/ml) of bacteria cells, were spread on the surface of Luria-
Bertani (LB) agar medium. Then, 60 μl of each extract (5 and 10 mg/ml)
were loaded into wells punched in the agar layer. Before incubation, all
plates were stored in the dark at 4 °C for 2 h, to allow the diffusion of
the extract. At the end of the incubation time, the antibacterial activity
was detected by the presence of measurable clear inhibition zone
around the well. Negative control was prepared using sterile water.
Antimicrobial activity was evaluated by measuring the growth inhibi-
tion zone (diameter expressed in millimeters) present around the well.
2.7. Film preparation and characterization
2.7.1. Film preparation and thickness
To prepare film-forming solution (FFS), grey triggerfish skin gelatin
was dissolved in distilled water to achieve a final concentration of 3%
(w/v). As plasticizer, glycerol was added to the gelatin solution at a
level of 15% (w/w) and then gently mixed at 40 °C for 30 min to ensure
total homogenization. For the bioactive films, selected FOPE (5 or
3
Food Packaging and Shelf Life 21 (2019) 100342
10 mg/ml) were dispersed in FFS and mixed at 25 °C until complete
solubility. Films were obtained by casting 25 ml of FFS on Petri dishes
(12 cm × 12 cm), dried at 25 °C at a relative humidity (RH) of 50% and
then peeled off manually. Prior to characterization, all films were kept
at 25 °C and 50% RH for at least 14 days.
The thickness of films was measured using a digital thickness gauge
(Schmidt, Control instrument). Ten random locations (from the center
and close to the perimeter) were taken from each film sample, and the
average was used in the calculations of transparency and mechanical
properties.
2.7.2. Color, light transmission and transparency
Color of the film samples was determined using a chromameter (CR-
200, Minolta, Japan) and expressed as L* (lightness/brightness), a*
(redness/greenness) and b* (yellowness/blueness) values. The barrier
properties of films against ultraviolet (UV) and visible light were
measured at wavelengths ranged between 200 and 800 nm, using a
UV–vis spectrophotometer. The transparency value of the film was
calculated by the following equation:
Transparency value = − log T600/e
where T600 is the fractional transmittance value at 600 nm and e is the
film thickness (mm). The greater transparency value represents the
lower transparency of the film.
2.7.3. Fourier transform infrared spectroscopy
Fourier transform infrared (FTIR) spectra of different films were
determined using a PerkinElmer infrared spectrometer equipped with
an attenuated total reflection (ATR) accessory. Films were analyzed
with a 32 scans per minute at a resolution of 4 cm−1 in the wavenumber
region between 400 cm−1 and 4000 cm−1.
2.7.4. Mechanical properties
Tensile strength (TS) and elongation at break (EAB) point of film
samples were determined using a texture analyzer (Lloyd Instrument,
Hampshire, UK). Rectangular film samples (5 cm × 2.5 cm) were pre-
pared using a precision standard cutter (Thwing-Albert JDC Precision
Sample Cutter, USA) in order to get pieces with an accurate width and
parallel sides throughout the entire length. The film samples were
clamped and deformed under tensile loading using a 300 N load cell
with a speed of 50 mm/min, until films’ breaking. TS (MPa) and EAB
(%) were determined from the stress-strain curves, analyzed six times
for each film sample.
2.7.5. Thermal properties
Prior to experiments, samples were kept at 25 °C and 0% RH (silica
gel) for 48 h to obtain the maximum dehydrated film samples.
Conditioned films (5 mg) were then hermetically sealed in specific
aluminum pans (PerkinElmer®) and scanned using a differential scan-
ning calorimeter DSC (Mettler Toledo Star). DSC measurements were
carried twice for each film.
2.7.6. Contact angle measurements and surface properties of films
Surface wettability of films was evaluated from the contact angle
measurements between films surface and water, using a goniometer
(Krüss Drop Shape Analyzer, Germany) equipped with image analysis
software (Krüss Advance, version 1.4.1.2) and via the sessile drop
method, as described by Karbowiak, Debeaufort, Champion, and
Voilley (2006).
2.7.7. Release of FOPE
The maximal extract (FOPE) absorbance value was determined from
the absorbance spectrum of the pure extract in water/ethanol solution.
To assess FOPE release through the gelatin film, a film sample of about
200 mg (˜5 × 5 cm2) was fully immersed in 100 ml of distilled water/
M. Jridi, et al. Food Packaging and Shelf Life 21 (2019) 100342

Table 1 Table 2
Yield and chemical characteristics of FOPE and DOPE. LC-ESI-MS analysis of the DOPE and FOPE.
Extraction yielda Total phenolic content *
Flavonoids content ** a
Compounds b
Retention Formula Mass Concentration
time (min) (g/ (μg/g extract)
DOPE 31.2 ± 0.24 8.68 ± 0.87 5.57 ± 0.09 mol)
FOPE 25.3 ± 0.07 10.07 ± 0.62 6.30 ± 0.42 DOPE FOPE

FOPE and DOPE represent fresh and dried orange peel extracts, respectively. 1 Quinic acid 2.308 C7H12O6 192 3813.55 4759.31
a 2 Protocatchuic acid 8.159 C7H6O4 154 9.89 28.33
g/100 g of dried mass.
* mg gallic acid equivalents (GAE)/g of extract. 3 Caffeic acid 16.658 C9H8O4 180 4.01 4.18
4 Epicatechin* 18.85 C15H14O6 290 – 0.74
** mg quercetin equivalents (QE)/g of extract.
5 p-coumaric acid 22.877 C9H8O3 164 0.69 19.59
6 trans-frulic acid 24.906 C10H10O4 194 74.52 78.09
ethanol (1:1, v/v) solution, under a constant rotation speed of 50 rpm at 7 Hyperoside 25.92 C21H20O12 464 16.21 15.33
room temperature (25 ± 1 °C) during 96 h. The amount of the extract (quercetin-3-o-
released in the liquid medium was determined at regular intervals of galactoside*
8 Rutin* 25.233 C27H30O16 610 108.25 108.70
time by UV–vis spectrophotometer. 9 Luteolin-7-O- 26.142 C21H20O11 448 2.18 1.40
glucoside*
10 3,4-di-O- 26.763 C25H24O12 516 1.56 50.45
2.8. Statistical analysis
caffeoyquinic acid
11 Apegenin-7-O- 28.11 C21H20O10 432 3.21 2.15
All analytical results were expressed in mean ± standard deviation glucoside*
(SD). One-way analysis of variance was conducted using SPSS software, 12 4,5-di-O- 28.634 C25H24O12 516 27.48 20.75
caffeoyquinic acid
ver. 17.0. A difference was considered statistically significant at p <
13 Quercetin 33.403 C15H10O7 302 4.05 25.49
0.05. 14 Naringenin* 35.374 C15H12O5 272 47.71 41.71
15 Apegenin* 35.976 C15H10O5 270 0.11 0.26
16 Luteolin* 36.402 C15H10O6 286 6.96 7.80
3. Results and discussion 17 Cirsiliol* 36.759 C17H14O7 330 8.50 30.43
18 Cirsilineol* 40.066 C18H16O7 344 1.4 2.65
3.1. Effect of blood orange peel drying pretreatment on the physic-chemical
a
and biological properties of the extracts The numbering refers to elution order of compounds from an Aquasil C18
column.
b
The extraction yields, the total phenolic and flavonoid contents are Identification was confirmed using 32 authentic commercial standards.
* Flavonoids. FOPE and DOPE represent the extracts from fresh and dried
shown in Table 1. The extraction yield of the DOPE, prepared from the
orange peel, respectively.
dried blood orange peels, was of 31.2% (w/w), which was significantly
higher than that prepared from fresh peels (FOPE) (25.3%).
and DOPE, respectively) and 4,5-di-O-caffeoylquinic acid (20.75 and
Phenolic compounds are primarily responsible for antioxidant
27.48 μg/g of FOPE and DOPE, respectively). However, FOPE contained
properties and several research have been developed to find natural
higher contents in 3,4-di-O-caffeoyquinic acid, cirsiliol, protocatchuic
antioxidants in cheap plant raw materials (Lachos-Perez et al., 2018).
acid, quercetin, and p-coumaric acid, compared to DOPE. The com-
Results of Table 1 showed that there was a significant difference be-
parison between the compositions of the two extracts prepared with
tween the total phenolic contents in both extracts, which were of about
orange peel markedly showed that the drying step affected the con-
8.68 and 10.07 mg GAE/g of DOPE and FOPE, respectively. In addition,
centrations of some phenolic compounds. Ozturk et al. (2018) and
the flavonoid content in DOPE was about 5.57 mg QE/g of extract,
Lachos-Perez et al. (2018) reported, similarly, the presence of phenolic
which was lower than that of FOPE (6.30 mg QE/g of extract), in-
acids including gallic acid, p-coumaric acid, ferulic acid, caffeic acid,
dicating that the drying pretreatment of orange peels before the ex-
trans-cinnamic acid, flavone and thymol in orange peel extract. Ad-
traction may affect the phenolics’ concentration. These results are in
ditionally, it has been reported that quantitative determination of gallic
agreement with those of M’hiri, Ioannou, Mihoubi Boudhrioua, and
acid, ferulic acid and p-coumaric acid are the most abundant phenolic
Ghoul (2015) who showed that microwave, ultrasound or thermal heat
compounds in Citrus aurantium peel (Karoui & Marzouk, 2013). How-
flow pretreatments made flavonoids unstable and easily degradable.
ever, among the identified flavonoids, hesperidin was the most abun-
Espinosa-Pardo, Nakajima, Macedo, Macedo, and Martínez (2017) ex-
dant, corresponding to the typical flavonoid composition of citrus peel
tracted phenolic compounds from orange processing by-products by
(Rafiq et al., 2016). Commonly, several authors have agreed that the
supercritical CO2 using ethanol as co-solvent and they found that the
highest contents of phenolic acids in citrus peels are the caffeic, ferulic,
total phenolic compounds content was about 0.57 mg GAE/g of dry
coumaric and sinapic acids (Rafiq et al., 2016; Robbins, 2003).
peel. In another work, Hegde, Agrawal, and Gupta (2015) used 50% (v/
The obtained phenolic profiles expected an important antioxidant
v) aqueous methanol and acidified aqueous methanol to extract poly-
potency of both extracts. In fact, the hydroxyl groups (eOH) in quinic
phenolic compounds from orange peel, and reported total phenolic
acid, found the major compound in FOPE and DOPE, is capable to
compounds content ranged from 1.4 to 2.6 mg GAE/ g. On the other
prevent or slow-down the oxidation processes, besides its antiviral,
hand, Hayat et al. (2010) and Ahmad and Langrish (2012) demon-
antibacterial and antifungal activities.
strated that drying peels at medium temperatures facilitate the release
The antioxidant activities of the orange peel extracts were in-
of phenolic compounds during extraction, while the use of temperatures
vestigated by using four methods: DPPH% radical-scavenging, Fe3+ re-
up to 80 °C will accelerate their degradation (M’hiri et al., 2015).
ducing power, antioxidant assay using the β-carotene/linoleate model
Orange peels are composed from a variety of polyphenols that in-
system and lipid oxidation inhibition activities (Fig. 1).
clude anthocyanins, flavonoids, tannins and procyanidins (Karoui &
The results of the scavenging effect of extracts on DPPH% radicals
Marzouk, 2013). Table 2 shows the results of LC-ESI-MS analysis of
(Fig. 1A) showed an increased activity with a dose-dependent manner,
DOPE and FOPE. Data revealed that the quinic acid was the major
with no significant difference between both extracts above 3 mg/ml.
compound among the total polyphenols (4759.31 and 3813.55 μg/g of
The IC50 values of FOPE and DOPE were about 0.7 and 1.05 mg/ml,
FOPE and DOPE, respectively), followed by rutin (about 108 μg/g in
respectively, which were comparable to that obtained by Barrales et al.
both extracts), trans-ferulic acid (78.09 and 74.52 μg/g extract for FOPE
(2018) for orange peel extract (IC50 = 11.18 μg/ml). The ferric
and DOPE, respectively), naringenin (41.71 and 47.71 μg/g of FOPE

4
M. Jridi, et al. Food Packaging and Shelf Life 21 (2019) 100342

Fig. 1. Antioxidant activities of extracts. (A) DPPH%-scavenging activity (%); (B) Fe3+ reducing antioxidant power (OD 700 nm); (C) β-carotene bleaching inhibition
(%); (D) Lipid oxidation inhibition (%).

reducing power measures the reducing ability of Fe3+ ions, giving idea
about the electron donation ability (Khantaphant & Benjakul, 2008).
The Fe3+ reducing power of extracts at different concentrations is
presented in Fig. 1B. Based on absorbance values, the activity increased
with increasing concentration between the range of 0.1 and 5 mg/ml
extracts. FOPE was more effective than DOPE, reaching a maximal
absorbance value of 1.2 at 5 mg/ml. In a similar study, Atrooz (2009)
demonstrated that the seed extract of C. sinensis exhibited antioxidant
activity using reducing power and DPPH% radical-scavenging assays.
Furthermore, the antioxidant activity of extracts was determined as %
of β-carotene bleaching inhibition in an emulsified linoleic acid model
system (Fig. 1C). From the obtained results, it can be clearly observed
that FOPE prevented more effectively the β-carotene from bleaching
than DOPE, by donating hydrogen atoms to the hydroxyl radicals of the
oxidized linoleic acid. Indeed, the IC50 values of FOPE and DOPE were
about 0.95 and 2.12 mg/ml, respectively. Moreover, FOPE and DOPE
showed an important ability of linoleic acid protection against oxida-
tion, which increased with increasing the extract’s concentration
(Fig. 1D). During the first 4 incubation days, FOPE showed higher li-
noleic acid protection activity than DOPE, while their activities re-
mained similar after 6 days. In this context, Oboh and Ademosun
free radicals, lowering cholesterol level, and inhibiting the low-density
lipoprotein cholesterol oxidation. On the other hand, ferulic acid de-
monstrated anticancer and antidiabetic activities and protective capa-
city from cardiovascular diseases (Srinivasan, Sudheer, & Menon,
2007).
On the other hand, the antimicrobial activities of the extracts (5 and
10 mg/ml) against seven Gram-positive and Gram-negative bacteria
strains were evaluated by determining the inhibition zones (mm) on
solid medium (Table 3). The extracts presented an interesting anti-
bacterial potential against all investigated micro-organisms. In fact, the
values of the inhibition zones of the tested strains varied between 9.0
(P. aeruginosa) and 39.5 mm (S. enterica). S. aureus was the most sen-
sitive strain against the effect of FOPE (36.0 mm) and DOPE (37.5 mm).
Overall, data demonstrated that FOPE was more effective than DOPE
against all Gram-positive and Gram-negative bacteria tested. Naila,
Table 3
Antimicrobial activities of DOPE and FOPE against Gram-positive and Gram-
negative bacteria strains.
Strains Inhibition zone diameters (mm)

(2012) reported that the phenolic extract from citrus peel showed ra- FOPE FOPE DOPE DOPE
dical DPPH% scavenging activity, %OH scavenging activity, Fe2+ che- (5 mg/ml) (10 mg/ml) (5 mg/ml) (10 mg/ml)
lating power and lipid peroxidation inhibition in pancreas, with re-
b a
spective IC50 values of 1, 4, 0.48 and 0.143 mg/ml. M. luteus 29.0 ± 0.5 34.0 ± 1.5 23.0 ± 1.0 a 24.5 ± 0.5 a

b a
Enterobacter sp. 25.0 ± 1.5 30.0 ± 0.2 26.5 ± 2.0 a 28.0 ± 2.0 a
Previous works reported positive correlation between the total S. aureus 29.0 ± 1.5 b
36.0 ± 1.0 a
25.0 ± 0.5 b 37.5 ± 2.0 a

phenolic contents and antioxidant activities in plant extracts (Shofinita, P. aeruginosa 17.0 ± 1.5 a
19.0 ± 1.0 a
9.0 ± 1.0 a 12.0 ± 1.5 a

Feng, & Langrish, 2015; Skotti, Anastasaki, Kanellou, Polissiou, & B. cereus 26.0 ± 2.0 b
32.0 ± 2.0 a
24.0 ± 2.0 a 27.0 ± 1.5 a

a a
Tarantilis, 2014). In this regard, Karoui and Marzouk (2013) found that L. monocytogenes 31.0 ± 1.5 33.5 ± 2.0 26.5 ± 2.0 a 28.0 ± 0.5 a

b a
S. enterica 26.5 ± 1.0 39.5 ± 1.0 25 ± 1.0 a 23.5 ± 2.0 a
rutin and naringenin dominate the list of identified flavonoids in Tu-
nisian bitter orange (C. aurantium L.) extracts, known by various ben- FOPE and DOPE represent extract from fresh and dried orange peel, respec-
eficial biological activities to the human health. In addition, Zang et al. tively. Different letters in the same line within different concentrations indicate
(2000) demonstrated the direct effect of p-coumaric acid in reducing significant difference (p < 0.05).

5
M. Jridi, et al.
Table 4
Thickness and thermal and mechanical properties of films.
Tg (°C) Thickness (μm) TS (MPa) EAB (%)
a a
GF 69.59 76.26 ± 0.01 6.23 ± 0.25 10.97 ± 1.10
b a
GF-FOPE (5 mg/ 70.25 75.09 ± 0.21 6.53 ± 0.12 15.36 ± 2.19
ml)
GF-FOPE (10 mg/ 71.90 76.36 ± 0.31 a
6.15 ± 0.40 a
20.25 ± 0.56
ml)
TS: Tensile strength; EAB: Elongation at break. GF and FOPE indicate gelatin
film and fresh orange peel extract. All measurements were performed at 25 °C
and RH = 50%. Different letters in the same column indicate significant dif-
ference (p < 0.05).
Nadia, and Zahoor (2014) showed that nanoparticles, synthesized by
mixing silver nitrate solution with C. sinesis extract, displayed great
antibacterial activity against B. subtilis, S. aureus and E. coli.
It is worth pointing out that orange peel extracts contain compounds
(phenolics and flavonoids) with good antioxidant and antibacterial
activities and having wide spectra of action, either by interrupting the
oxidation reaction with different ways, or by inhibiting the growth of a
great number of food spoilage bacteria. Of particular, FOPE was found
more effective than DOPE, which promotes its use as a bioactive mo-
lecule to be incorporated in food packaging materials, e.g. the grey
triggerfish skin gelatin in this study.
3.2. Films characterization
3.2.1. Mechanical, thermal, surface and structural properties
Results of thickness, tensile strength (TS), and elongation at break
(EAB) of triggerfish gelatin films (GF), incorporated or not with FOPE
are shown in Table 4. No significant variations in thickness and TS
values between all films were observed (p ≥ 0.05). However, the in-
corporation of 10 mg/ml FOPE in the gelatin film increased twice the
elongation at break (20.25 ± 0.56%), compared to the control GF
(10.97 ± 1.10%). In the same context, Bitencourt, Fávaro-Trindade,
Sobral, and Carvalho (2014) showed a significant increase in EAB after
the incorporation of diverse concentrations of ethanolic extract of
Curcuma in gelatin based films. Similarly, Bonilla and Sobral (2016)
demonstrated that the incorporation of cinnamon and guarana extracts
increased the EAB, two folds, when compared to control gelatin film.
Changes may be due to the interactions between the phenolic com-
pounds (present in the extract) and gelatin poly-peptides. On the other
hand, the addition of the extract increased slightly the glass transition
temperature (Tg) of the gelatin film to reach 70.25 and 71.90 °C, after
the addition of 5 and 10 mg/ml FOPE (Table 4), mainly due to the in-
termolecular interactions between gelatin and phenolic compounds/
floavonoids. DSC results indicated also that gelatin, glycerol and orange
peel extract were completely miscible, as revealed by the unique en-
dothermic peak recorded in the thermograms.
The surface-tension characterization through the measurement of
contact angle (θ), between water and films’ surface, is one of the key
properties of packaging materials, giving idea about their wettability.
Fig. 2 shows the water contact angle (WCA) value through 60 s. Al-
though the initial WCA value of GF was found to be slightly higher than
those of films incorporated with FOPE, all values indicated about the
hydrophobic character of films’ surface, as the WCA values were higher
than 90° in all the samples. In addition, after 60 s of water drop de-
position, the WCA values decreased in the GF, compared to the values
at t = 0 s, while this decrease was significantly reduced after FOPE
addition, indicating the enhancement of films’ resistance against wett-
ability.
For better understanding the structure of the films, FTIR analysis
was carried out to analyze the interactions among the components of
the film (Table 5). The most characteristic bands of gelatin film are
related to C]O stretching (amide I) at 1658 cm−1, NeH bending
Food Packaging and Shelf Life 21 (2019) 100342
(amide II) at 1490 cm−1, and CeN stretching (amide III) at 1041 cm−1
(Uranga et al., 2018). The main absorption bands of glycerol appear at
the 900–1050 cm−1 region and they are related to the vibrations of
CeC and CeO bands (Uranga et al., 2018). Phenolic compounds
c
showed diverse absorption bands due to the presence of a carboxylic
b
group at about 1600–1700 cm−1, which cause a modification of the
a gelatin amide I band, and the absorption bands of the hydroxyl groups.
Additionally, increasing FOPE concentration led to changes in the
bands’ position of amide I and amide II, as shown in Table 5. This might
be due to the promotion of the cross-linking reaction between gelatin,
glycerol and phenolic acids. In this context, Uranga et al. (2018) re-
ported the interactions between amine groups of fish gelatin and car-
boxylic group of gallic acid, through the FTIR analysis.
3.2.2. Color, light transmission and transparency of films
Color and light transmission parameters of films are presented in
Table 6. Results showed that the incorporation of FOPE significantly
affected the color of the film surface (p < 0.05), by decreading L*
values and inceasing a* and b* values. In fact, the addition of FOPE at 5
or 10 mg/ml significantly (p < 0.05) reduced film lightness from 90.57
to 85.02 and 62.65, respectively. High b* values of films indicated that
they got yellow color after FOPE addition. These variations of films’
color are mainly caused by the natural coloured pigments present in the
extract (orange color). In this context, Adilah et al. (2018) reported that
films incorporated with mango peel extract showed yellowish color,
comparing to control gelatin film.
The incorporation of the extract in FG did not markedly changed the
transmittance values in the UV–vis light region, showing a slight in-
crement of these values for visible light between 400 and 600 nm
(Table 6). Results of films’ opacity showed that GF was the lightest,
while the incorporation of FOPE increased the darkness of gelatin film,
due to the orange-colored pigments in the extract that absorb at this
visible range, affecting therefore the opacity of the FOPE-added films
(Bonilla & Sobral, 2016).
3.2.3. Antioxidant activity of films
Antioxidant packaging are the most favorite categories among ac-
tive packaging thanks to their promising role in extending food pro-
ducts shelf-life (Benbettaïeb, Chambin, Karbowiak, & Debeaufort, 2016;
Bonilla & Sobral, 2016; Jridi et al., 2014, 2013). The antioxidant power
of the different films was measured using three tests and the results are
presented in Table 6. The control GF (without FOPE) showed the lowest
antioxidant activities (0.12, 12.68% and 20.35% for reducing power, β-
carotene bleaching inhibition and DPPH radical-scavenging activities,
respectively). Whatever the test used, the increase of FOPE concentra-
tion significantly increased the antioxidant activity (p < 0.05). In fact,
FG incorporated with 10 mg/ml FOPE, showed the highest reducing
power, β-carotene bleaching inhibition and DPPH radical-scavenging
activities, which were of 1.38, 95.76% and 99.7%, respectively. These
results were not surprising as FOPE has demonstrated its in vitro anti-
oxidant potential as an hydrogen/electron donating agent or by che-
lating metals, resulting therefore in the interruption of the oxidation
chain reaction.
The comparison between the antioxidant activities of the FOPE-
added films and the free extract showed that the bioactivity of the ex-
tract decreased following its incorporation in the film matrix. This
could be explained by the cross-linking of phenolics and flavonoids in
the film forming gelatin network.
3.2.4. Release behavior of FOPE
The release kinetic of substances encapsulated in biomaterials is a
crucial parameter that must be controlled in industrial applications, as
it gives idea about the time attributed to completely deliver the
bioactive substance across the film (Benbettaïeb et al., 2016). To better
understand the mechanism of release of orange peel extract, the ki-
netics of release of FOPE from FG was carried out and results are
6
M. Jridi, et al. Food Packaging and Shelf Life 21 (2019) 100342

Fig. 2. Shape behavior (t = 0 s) and kinetics of water droplets deposited on the surface of films as a function of time; measurements were done at 25 °C and
RH = 50%.

Table 5
Fourier Transform infrared spectra (FTIR) of composite films.
Wavenumbers (cm−1) Amide A Amide B Amide I Amide II Amide III

GF 3297 2996 1658 1490 1041

GF-FOPE (5 mg/ml) 3269 2991 1652 1458 1040
GF-FOPE (10 mg/ml) 3278 2968 1655 1478 1040

GF: Gelatin film, FOPE indicates extract from fresh orange peel.

presented in Fig. 3. During the first 24 h, the release of extract from the
gelatin film network began slowly, which could be due to the interac-
tion between amine groups of fish gelatin and carboxylic group of gallic
acid resulting in great bounding affinity between the bioactives and the
gelatin polymer. Then, the release speed increased between 24 and 36 h
of reaction, showing a sustained drug release profile with a quasi-
Fickian diffusion mechanism (n-value less than 0.5). This mechanism Fig. 3. Cumulative release of FOPE from the gelatin film matrix. The non-added
indicated that gelatin was hydrated, swelled and then the drug diffused gelatin film (FG) was used as control.
through the swollen matrix system. Thereafter, beyond 72 h, the release
reached a stability stage, which slowed down the kinetic release. films demonstrated the presence of interactions between the gelatin
film and the phenolic molecules. These interactions affected FOPE re-
lease kinetic, which was prolonged up to 96 h. Overall, this work gives a
4. Conclusion
new strategy of Citrus peel utilization through development of edible
packaging with good biological properties.
The present research studied the effect of drying on the quality of
blood orange peel extracts and underlined the successful addition of the
active extract in Grey triggerfish gelatin film. Physicochemical, me- Declaration of interest
chanical and antioxidant properties of the FOPE-added films were sig-
nificantly enhanced, compared to FG. Structural and thermal analysis of The authors declare no conflicts of interest. The authors alone are

Table 6
Light transmission, opacity, color, and antioxidant properties of films.
GF GF-FOPE GF-FOPE
(5 mg/ml) (10 mg/ml)

Wavelength (nm) 200 0.01 0.01 0.01

280 0.01 0.00 0.01
350 28.36 26.32 25.25
400 49.7 48.6 49.2
500 69.4 67.3 68.1
600 82.6 80.2 80.0
700 88.4 89.5 89.6
800 90.5 90.4 90.6
Opacity 1.09 1.28 1.27
Color L* 90.57 ± 0.26 a 85.02 ± 0.10 b 62.65 ± 0.05 c
a* −0.36 ± 0.01 c 6.26 ± 0.52 b 8.32 ± 0.10 a
b* 0.35 ± 0.03 c 2.10 ± 0.11 a 3.68 ± 0.23 a
Antioxidant activities Reducing power (A700) 0.12 ± 0.01 c 0.89 ± 0.11 b 1.38 ± 0.11 a
β-carotene bleaching inhibition (%) 12.68 ± 1.68 c 67.95 ± 3.65 b 95.76 ± 0.19 a
DPPH radical-scavenging activity (%) 20.35 ± 0.20 c 76.9 ± 2.35 b 99.7 ± 1.74 a

Values of light transmission were measured at 25 °C and RH of 50% and expressed in %. Different letters in the same line indicate significant difference (p < 0.05).
Opacity = -log (Transmission) / Thickness. GF: Gelatin film, FOPE: extract from fresh orange peel.

7
M. Jridi, et al.
responsible for the content and writing of this paper.
Acknowledgement
This work was funded by the Ministry of Higher Education and
Scientific Research, Tunisia (Grant no. 18PJEC06-03).
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