materials

Article
Functionalization of Polyethylene (PE) and
Polypropylene (PP) Material Using Chitosan
Nanoparticles with Incorporated Resveratrol
as Potential Active Packaging
Tjaša Kraševac Glaser 1, *, Olivija Plohl 1 , Alenka Vesel 2 , Urban Ajdnik 1 , Nataša Poklar Ulrih 3 ,
Maša Knez Hrnčič 4 , Urban Bren 4 and Lidija Fras Zemljič 1, *
1 Laboratory for Characterization and Processing of Polymers, Faculty of Mechanical Engineering,
University of Maribor, Smetanova 17, SI-2000 Maribor, Slovenia
2 Department of Surface Engineering and Optoelectronics, Jožef Stefan Institute, Teslova 30,
SI-1000 Ljubljana, Slovenia
3 Department of Food Science and Technology, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101,
SI-1000 Ljubljana, Slovenia
4 Faculty of Chemistry and Chemical Engineering, University of Maribor, Smetanova 17,
SI-2000 Maribor, Slovenia
* Correspondence: tjasa.glaser@um.si (T.K.G.); lidija.fras@um.si (L.F.Z.);
Tel.: +386-2-220-7607 (T.G.K.); +386-2-220-7909 (L.F.Z.)




Received: 21 May 2019; Accepted: 28 June 2019; Published: 1 July 2019


Abstract: The present paper reports a novel method to improve the properties of polyethylene (PE)
and polypropylene (PP) polymer foils suitable for applications in food packaging. It relates to the
adsorption of chitosan-colloidal systems onto untreated and oxygen plasma-treated foil surfaces.
It is hypothesized that the first coated layer of chitosan macromolecular solution enables excellent
antibacterial properties, while the second (uppermost) layer contains a network of polyphenol
resveratrol, embedded into chitosan nanoparticles, which enables antioxidant and antimicrobial
properties simultaneously. X-ray photon spectroscopy (XPS) and infrared spectroscopy (FTIR) showed
successful binding of both coatings onto foils as confirmed by gravimetric method. In addition, both
attached layers (chitosan macromolecular solution and dispersion of chitosan nanoparticles with
incorporated resveratrol) onto foils reduced oxygen permeability and wetting contact angle of foils;
the latter indicates good anti-fog foil properties. Reduction of both oxygen permeability and wetting
contact angle is more pronounced when foils are previously activated by O2 plasma. Moreover,
oxygen plasma treatment improves stability and adhesion of chitosan structured adsorbates onto PP
and PE foils. Foils also exhibit over 90% reduction of Staphylococcus aureus and over 77% reduction of
Escherichia coli as compared to untreated foils and increase antioxidant activity for over a factor of 10.
The present method may be useful in different packaging applications such as food (meat, vegetables,
dairy, and bakery products) and pharmaceutical packaging, where such properties of foils are desired.

Keywords: nanoparticles; chitosan-resveratrol; PE/PP functionalization; O2 plasma; active packaging

1. Introduction
Recently, novel-packaging materials have become an essential part of the global food packaging
market aiding the ever-increasing consumer awareness and importance of eco-friendly substitutes.
The demand for innovative packaging is expanding and will continue to grow as the companies
utilize packaging as a medium to protect their products and to promote environment protection [1–5].

Materials 2019, 12, 2118; doi:10.3390/ma12132118 www.mdpi.com/journal/materials

Materials 2019, 12, 2118 2 of 20

The European packaging industry has a market value of approximately 80 billion EUR and accounts
for ≈40% of the global packaging market. The future trend is oriented toward growing markets
on a global scale [2]. Major manufacturers of packaging materials are looking to differentiate their
products from those of their competitors by providing the best possible functional and/or biodegradable
packaging products as per consumer demands. To be competitive, it is important that new packaging
solutions are bio-based, multifunctional, economically viable, and easily incorporated into established
industrial manufacturing processes. Sufficient competitiveness can only be realized if the EU food
industry speeds up the pace of innovation [1–5]. Over recent decades, majority of research on the
development of innovative food packaging materials has been carried out with a view to combat
against pathogens, to reduce spoilage and waste, to optimize process efficiency, to reduce the need
for chemical preservatives, to improve the functionality of foods, and to improve the nutritional and
sensorial properties of food, responding to the demands of different consumer niches and markets,
also in terms of affordability [1–7].
However, few of these investigations were economical, eco-friendly, and healthy; thus, it is still a
great challenge to be involved in the study of a new concept of active packaging material [1–7]. Beside
increasing public health awareness, the changes in retailing practices (such as market globalization
resulting in longer distribution of food) and fact that the industry must follow the strict EU packaging
guidelines and regulations, all act as a driving force for a significant interest to incorporate natural
antimicrobial ingredients into a basic packaging material and, in this way, developing an active concept
of packaging [1–7].
Examples of prospective natural biodegradable and bioactive substances are less-employed
antimicrobial polysaccharides and their derivatives. Chitosan, obtained from chitin by partial
deacetylation, which makes it soluble in acidic aqueous solutions, has diverse, industrially important
properties, such as biocompatibility, biodegradability, and non-toxicity. It is widely known as a valuable
material for different biomedical and food applications [3,7–12]. There are numerous studies on utilizing
chitosan as coating for potential packaging material [13–21]. However, in spite of chitosan’s excellent
antimicrobial activity, it has been found that chitosan shows very poor antioxidant activity [22,23]. The
latter is required for a bioactive packaging material in order to impede the natural processes, as food
spoilage (i.e., the oxidative deterioration of meat products is caused by the degradation reactions of fats
and pigments) is diminished by reduction of oxygen and moisture. Thus, it is greatly appreciated to
increase antioxidant activity of chitosan with the natural compounds including proven high antioxidant
capacity, such as plant phenolics. Tocopherols are the only natural food grade antioxidants regulated
by the Food and Drug Administration (FDA). Spices and flavorings like rosemary and sage are
regulated only as flavorings. An ever-popular trend is the use of vegetable and essential oils as
natural antioxidants [24–26]. Some plant extracts such as grapefruit seed, cinnamon, horseradish,
clove, garlic, and many others have been previously added to a packaging system to demonstrate
effective antioxidant–antimicrobial activity against spoilage and pathogenic bacteria [27–32]. Today,
resveratrol (RES) is believed to be one of the most potent polyphenols and strongest protectors against
symptoms, associated with aging and free radical damage [33,34]. Resveratrol has already been
combined with chitosan with the aim to improve the solubility, stability, and cellular uptake of RES
by nanoencapsulation using chitosan (CS) and γ-poly (glutamic acid) (γ-PGA) [35]. Another study
suggests that chitosan–sodium tripolyphosphate (TPP) microspheres could be used as a potential
delivery system with controlled release of resveratrol [36]. Active linear low-density polyethylene
(LLDPE) composites were manufactured by immobilization of RES in the polymeric matrix and by
pre-incorporation of the RES into a food-contact-permitted montmorillonite clay prior to melt mixing
with the polymer. The resulting composites not only showed strong antioxidant activity, but also
antimicrobial activity. In addition, the thiobarbituric acid reactive substances method (TBARS) was
applied to assess the comparative oxidative behavior of fresh meat in air and direct contact with the
LLDPE film containing the active clay. The results suggest that this technology could potentially extend
the shelf-life of red meat over a few days by a mechanism of free radicals trapping and the subsequent
Materials 2019, 12, 2118 3 of 20

disabling of food oxidation processes [37]. Active, cellulose derivative-based films (hydroxyethyl
cellulose and cellulose acetate) were developed and prepared by the casting method using RES and its
inclusion into a complex with hydroxypropyl-γ-cyclodextrin as an active agent was demonstrated.
The structural, mechanical, barrier, surface free energy, optical, and release properties were analyzed.
The antimicrobial activity of these films against Campylobacter jejuni, Campylobacter coli, and Arcobacter
butzleri were evaluated by the agar diffusion method. The anti-quorum sensing (QS) activity of films
was studied using biosensor strain Chromobacterium violaceum ATCC 12472. The active films developed
in this work were transparent, inhibited the growth of the tested strains, and possessed anti-QS activity
as well. This study showed the potential of these active films as a new approach to control the growth
of Campylobacter genus [38].
However, to the best of our knowledge, until now, there was no methodologic functionalization
and detailed characterization of PE and PP foils, as the most representative packaging material,
functionalized by chitosan with incorporated resveratrol, showed clear and highly significant advances
compared to the other relevant studies. Because PE and PP materials are inert and hydrophobic, the
adsorption of chitosan-colloidal systems onto its surfaces may be improved using different activation
procedures in order to obtain carboxylic groups on its surface, that represent binding places for
chitosan amino groups (Coulomb interactions). Plasma activation can tune hydrophobic character
into hydrophilic and, thus, increase the ability of PE and PP for improved attachment of chitosan.
In our previous work, we have already shown and discussed that advanced and environmentally
friendly cold oxygen plasma treatment can be used to enhance the adhesion of chitosan [16]. This
paper introduces the development of complex, composite material based on previous PE/PP-cold
oxygen plasma activation and further attachment of chitosan colloidal systems in different structural
forms; i.e., (i) chitosan macromolecular solution was applied as a first layer onto PE and PP foils and,
(ii) RES was encapsulated into chitosan nanoparticles and further applied onto i) coated PE and PP
foils as an upper layer (layer-by-layer deposition).
The functionalized foils were physicochemical analyzed from the following point of view:
(i) Gravimetric, (ii) surface elemental composition of functionalized foils, (iii) oxygen permeability,
(iv) wettability, and (v) morphology. In order to obtain migration profile of chitosan and resveratrol
from foils surface, desorption studies were done using polyelectrolyte titration. Most importantly,
bioactive properties of foils were also examined. The antimicrobial properties were analyzed using the
standard ISO 22196, whereas the anti-oxidative properties were determined spectrophotometrically
using ABTS test.

2. Materials and Methods

2.1. Materials
Low-molecular weight chitosan (50 to 190 kDa), poly (D-glucosamine) from Sigma-Aldrich; sodium
tripolyphosphate—TPP (MW = 367.86 g/mol) from Sigma-Aldrich, St. Louis, MI, USA; Resveratrol
(MW = 228.24 g/mol), ≥99% (HPLC) from Sigma-Aldrich; acetic acid (MW = 60.05 g/mol), ≥99.8% from
Sigma-Aldrich; ethanol (99.8%, GC) from Sigma-Aldrich. Ultrapure water—Milli-Q Direct system
(Millipore, Burlington, MA, USA). Polyethylene—PE normal quality, transparent, GSM = 46.28 g/m2
(thickness 50 µm, Slippery 0.207) from Makoter d.o.o., Ljutomer, Slovenia; Polypropylene—PP normal
transparent oriented, GSM = 22.93 g/m2 (Thickness 27 µm, Slippery 0.278) from Manucor S.p.A., Sessa
Aurunca, Italy.

2.2. Solutions/Dispersions

2.2.1. Preparation of Chitosan Solutions

Different concentrations of chitosan solutions (1% and 2%, w/v), were prepared by adding chitosan
powder in ultrapure water under constant magnetic stirring, while acetic acid was added drop-wise
Materials 2019, 12, 2118 4 of 20

to enable dissolution of chitosan. The solution was left stirring overnight and pH was adjusted with
acetic acid to 4.0.

2.2.2. Preparation of TPP Solution

Sodium tripolyphosphate (TPP) powder was suspended in ultrapure water in order to prepare a
0.2% (w/v) solution.

2.2.3. Preparation of Resveratrol Solution

Resveratrol (RES) powder was suspended in absolute ethanol in order to prepare a
12.5 mg/mL solution.

2.2.4. Preparation of Resveratrol-Loaded Chitosan-TPP Nano Dispersion (i.e., Chitosan–Resveratrol

Nano Dispersion—CSNPs RES)
Chitosan nanoparticles were prepared by the ionic gelation technique. Simultaneously, 0.2%
(w/v) of TPP solution and 12.5 mg/mL resveratrol solutions were added to a fixed volume of 1% (w/v)
chitosan solution in order to obtain 5:1 chitosan to TPP weight ratio. This ratio was chosen according
to the previously published work, reporting it as an optimal ratio to obtain desirable antimicrobial
activity of nanoparticles’ dispersion [39]. Particles were formed spontaneously under constant stirring
for 1 h at room temperature. The final pH of CS nanoparticles dispersions with RES (CSNPs RES) was
adjusted to 4.0 with acetic acid.

2.3. Functionalization of Foils

2.3.1. Pre-Treatment of PE and PP with Plasma

PE and PP foils were cleaned, dried, and cut to the size of the print. Foils were activated with
oxygen microwave plasma in a surfatron mode. The forward power was set to 200 W. Oxygen pressure
in the treatment chamber made of quartz glass was set to 50 Pa. The samples were exposed to late
oxygen afterglow for 60 s.

2.3.2. Functionalization of PE and PP Surface with Chitosan, Chitosan–Resveratrol Nano Dispersion

(CSNPs RES dispersion)
For antimicrobial and anti-oxidative functionalization of PE and PP surface (untreated and oxygen
plasma treated), 2% chitosan and CSNPs RES dispersions were used. The coating was applied in two
layers (layer-by-layer deposition). The first layer was made of 2% chitosan, which serves for better
adhesion and a higher antimicrobial effect. The second (upper) layer was formed with CSNPs RES
dispersion. PE and PP foils were previously cleaned, dried, cut to the size of the print, and weighed.
For the application of functional coatings, the method of printing through the fine fabric using a magnet
was applied (roll printing). The foils were dried at each step at room temperature. Sample descriptions
and notations are given in the Table 1.
Materials 2019, 12, 2118 5 of 20

Table 1. Samples descriptions.

Sample Notation Description of Sample

A1 polyethylene (PE) foil
A2 polypropylene (PP) foil
A3 PE foil treated with O2 plasma
A4 PP foil treated with O2 plasma
B1 chitosan powder (CS)
B2 2% (w/v) chitosan (CS)
B3 chitosan nanoparticles (CSNPs) dispersion
C1 resveratrol powder (RES)
C2 Chitosan–resveratrol nano dispersion (CSNPs RES)
D1 untreated PE foil, applicate with CS-1. layer and CSNPs RES-2. layer
D2 untreated PP foil, applicate with CS-1. layer and CSNPs RES-2. layer
E1 PE foil treated with O2 plasma, applicate with CS-1. layer and CSNPs RES-2. layer
E2 PP foil treated with O2 plasma, applicate with CS-1. layer and CSNPs RES-2. layer

2.4. Methods

2.4.1. Dispersions Characterization

Particle Size, PDI, and Zeta Potential Determination

The particles hydrodynamic diameter and polydispersity index (PDI) of prepared nanoparticles
dispersion were determined by dynamic light scattering—DLS (Zetasizer Nano ZS, Malvern
Instruments Ltd., Malvern, UK)—at a temperature of 25 ◦ C. Zeta potential (ZP) was determined
by performing an electrophoresis experiments and measuring the velocity of the particles using
laser doppler velocimetry (LDV) on Zetasizer Nano ZS (Malvern Instruments Ltd.). Before analyses,
dispersion was stirred for 15 min and adjusted to pH 4 with acetic acid if it was necessary. For
carrying out the DLS measurements, the disposable cuvettes were used and for ZP determination
disposable folded capillary cell with electrodes. Data were collected using Malvern Zetasizer Software
version 7.12.

Minimal Inhibitory Concentration (MIC)

MIC of macromolecular chitosan solution, nanoparticles dispersion, and resveratrol solution
was defined as the lowest concentration that allowed no more than 20% growth of microbes. Three
methods (disk diffusion method, agar dilution method, broth microdilution method) were used for
MIC determination, while the most useful was the microdilution method. Therefore, it was chosen as
protocol for our samples.
For the test, 50 µL of each bacterial suspension in a suitable growth medium was added to
the wells of a sterile 96-well microtiter plate already containing 50 µL of two-fold serially diluted
chosen substrate in a proper growth medium. The final volume was 100 µL. Control wells were
prepared with a culture medium, bacterial suspension only, chitosan solution, chitosan nanoparticles
dispersion, and resveratrol solution only and ethanol in amounts corresponding to the highest quantity
present in resveratrol solution. The contents of each, respectively, were well mixed on a microplate
shaker at 900 rpm for 1 min prior to incubation for 24 h in the cultivation conditions described
above [40]. The MIC was the lowest concentration where no viability was observed after 24 h because
of metabolic activity [41]. To indicate respiratory activity, the presence of color was determined after
adding 10 µL/well of INT (2-p-iodophenyl-3-pnitrophenyl-5-phenyl tetrazolium chloride, Sigma) or
TTC (2,3,5-triphenyl tetrazolium chloride) dissolved in water (INT 2 mg/mL, TTC 20 mg/mL) and
incubated under appropriate cultivation conditions for 30 min in the dark [42]. To determine the ATP
activity, the bioluminescence signal was measured by a Microplate Reader after adding 100 µL/well
of BacTiter-Glo™ reagent and after 5 min incubation in the dark. Positive controls were wells with a
bacterial suspension in an appropriate growth medium and a bacterial suspension in an appropriate
Materials 2019, 12, 2118 6 of 20

growth medium with ethanol in amounts corresponding to the highest quantity present in the broth
microdilution assay. Negative controls were wells with growth medium and chitosan substrates and
resveratrol. All measurements of MIC values were repeated in triplicate [40].

2.4.2. Functionalized Foils

Gravimetric Measurements of Sample Mass

All samples were weighed (four decimal places) to compare their weights with untreated reference
foils, which were previously cleaned, dried, and cut to the size of the print. The final weight differences
between the reference foils and applied foils showed a successful application of the formulation onto
the PE and PP foils. The final weight differences between the reference foils and functionalized foils
were calculated for absolute dry samples.

Surface Elemental Composition of Functionalized Foils: ATR-FTIR Spectroscopy

The ATR-FTIR spectra were recorded on a Perkin Elmer Spectrum GX NIR FT-Raman spectrometer
(Waltham, MA, USA). The ATR accessory contained a diamond crystal. All spectra (16 scans at 4 cm−1
resolution, background, and the sample spectra were obtained in the 400–4000 cm−1 wavenumber
range) were recorded at room temperature. Spectra of samples were deconvoluted with smoothing
filter and baseline corrected.

Surface Elemental Composition of Functionalized Foils: XPS Analysis

Spectra were recorded using XPS instrument PHI TFA XPS Physical Electronics, Chanhassen,
MN, USA in order to assess the surface of the sample. The base pressure in the XPS analysis chamber
was approximately 6 × 10−8 Pa. The samples were excited with X-rays with monochromatic Al Kα1.2
radiation (1486.6 eV) operating at 200 W. Photoelectrons were detected with a hemispherical analyzer,
positioned at an angle of 45◦ with respect to the normal to the sample surface. The energy resolution
was about 0.6 eV. Spectra were recorded from at least two locations on each sample, using analysis
area of 400 µm. Surface elemental concentrations were calculated from the survey-scan spectra using
the Multipak software version 9.6.0.

Oxygen Permeability
The oxygen permeability was determined using Oxygen Transmission Rate System PERME®
OX2/230 (Labthink Instruments Co., Ltd., Jinan, China), by standard ASTM D3985. Oxygen transmission
rate (OTR) values and coefficient values are averaged results, obtained by two testing of five
measurements. All specimens were conditioned at 23 ◦ C and 50% relative humidity 24 h prior
testing (flux = 10 mL/min). Thickness of PE and PP foils were measured with calliper at five
different places.

Goniometry
Contact angles were measured to estimate the surface wettability of coated foils via goniometer
DataPhysics, Germany. Static contact angle (SCA) was measured using a drop of liquid resting on the
surface. Goniometer with SCA 20 software was used to determine CSA at room temperature with
ultrapure water. A small drop (3 µL) of water was carefully placed on the surface of the sample. For
each sample, three repetitions were made.

Morphology: Scanning Electron Microscopy

For scanning electron microscopy (SEM) imaging of surface morphology, samples of PE and
PP foils were prepared by cutting the foils into small, approximately 0.5 × 0.5 cm2 square pieces.
These pieces were attached to the aluminum sample holders with an adhesive carbon tape to ensure
conductivity. For this purpose, Carl Zeiss Supra 35VP scanning electron microscope (Jena, Germany)
Materials 2019, 12, 2118 7 of 20

was employed, where the reference foils and functionalized foils were analyzed at an accelerating
voltage of 1 kV and variable working distance using a 10 µm-sized aperture.

Migration: Desorption Experiment—Polyelectrolyte Titration

Preparation of desorption bath and samples—0.5 g of PE and PP foils were treated for 24 h with
30 mL of ultrapure water (pH = 5.8, adjusted by 0.1 M hydrochloric acid). The solution was filtered
and further used for polyelectrolyte titration to analyze the amount of desorbed chitosan from foils.
For incremental addition of the polyelectrolyte titrant (PES-Na; c = 10 mM) into 30 mL of
desorption bath, Mettler Toledo DL 53 titration unit with a 10 mL burette was used. The incremental
additions of 0.05 mL were added every 5–10 s. The absorbance was measured as a potential change in
mV, using Mettler Toledo DP5 Phototrode at a wavelength of 660 nm. Concentration of the protonated
amino groups was determined from the equivalent point of titration curve (Figure 7), as the steepest
bit of the curve, and by estimating a 1.1 binding stoichiometry of polyethylenesufonate to chitosan
amine groups.

Bioactivity
(1) Antimicrobial Activity Testing
For antimicrobial test of functionalized PE and PP foils surface, a modified version of ISO 22196
(plastics—measurement of antibacterial activity on plastics surfaces) was followed [43]. This is currently
the test protocol of choice for testing surfaces for antimicrobial effectiveness. Antibacterial activity
is described as a state, where growth of bacteria on the surfaces of products is suppressed or as the
effect of an agent, which suppresses the growth of bacteria on the surfaces of products. Gram-positive
bacteria (Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli) from the culture collection
of Laboratory for Food Microbiology at Department of Food Science and Technology, Biotechnical
Faculty, University of Ljubljana were tested. After determination of viable cells number, antimicrobial
activity was calculated using Equation (1) and percentage of reduction using Equation (2).

R = Ut − At, (1)

Reduction (%) = ((Ut − At)/Ut) × 100, (2)

where R is the antibacterial activity; Ut is the number of viable cells recovered from the untreated
material after incubation; and At is the number of viable cells recovered from the treated material
after incubation.
(2) Anti-Oxidative Activity (ABTS)
Anti-oxidative activity was determined using a biochemical reagent, ABTS
0
(2,2 -azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)). The method is based on the reduction of
the ABTS•+ radical, which is determined spectrophotometrically at a wavelength of 734 nm. The
ABTS•+ cation radical was produced by the reaction between 7 mM ABTS in ultrapure and 2.45 mM
potassium persulfate and stored in the dark at room temperature for 12 h. Then, 3.9 mL of diluted
ABTS•+ solution was added to 100 mg of functionalized foil. After 15 and 60 min, the scavenging
capability of ABTS•+ at 734 nm was calculated using Equation (3):

Inhibition = (AControl − ASample )/AControl · 100, (3)

where AControl is the absorbance, measured at the starting concentration of ABTS•+, and ASample is the
absorbance, of the remaining concentration of ABTS•+ in the presence of applicate chitosan–resveratrol
onto the foils.
Materials 2019, 12, 2118 8 of 20

2.4.3. Statistical Analysis

All the experimental data in this study are expressed as mean ± standard deviation and processed
by Microsoft Excel 2016 (Microsoft Corporation, Redmond, Washington, DC, USA). One-way ANOVA
analysis of variance was used in order to measure the statistical significance. Differences were taken to
be significant for p < 0.05. Statistically significant results are pointed out by *.

3. Results and Discussion

3.1. Dispersions Characterization

3.1.1. Particle size, PDI, and Zeta Potential Determination

PDI and zeta potential of chitosan nanoparticles with embedded resveratrol were compared to
those results of chitosan nanoparticles alone. Both types of dispersions had pH 4. The average size
by intensity of synthesized nanoparticles determined with DLS measurements was 359 ± 40 nm for
CSNPs and 950 ± 87 nm for CSNP’s RES, which is significantly different as seen from Table 2. This
size increase clearly shows that RES was incorporated inside the particles and/or on the particles
surface. PDI for CSNPs was 0.87 ± 0.09 and for CSNPs, RES was 0.58 ± 0.18, which suggests the
higher monodispersity of CSNPs with incorporated RES, although there was no significant difference.
Zeta potential measurements showed positive values and were 36 ± 5 mV for CSNPs and 48 ± 10 mV
for CSNPs RES, respectively, with no significant difference. The raise in zeta potential values can be
assigned to the presence of protonated amino groups at pH = 4 on the particles surface and also indicate
the stability of nanoparticles dispersion [44]. The smaller increase in the positive ZP for nanoparticles
dispersion with embedded resveratrol indicates that most of hydrophilic RES polyphenol was adsorbed
into inner part of chitosan nanoparticles or onto chitosan nanoparticles surface in the way to provide
Materials 2019, 12, x FOR PEER REVIEW 8 of 19
sufficient steric or electrostatic repulsive forces among particles to ensure dispersion stability.
ensure dispersion stability. Table 2. Particle size, polydispersity index (PDI) and zeta potential of
Table 2. Particle
chitosan size, polydispersity
nanoparticles (CSNPs) andindex (PDI)
CSNPs and zeta(RES).
resveratrol potential of chitosan nanoparticles (CSNPs)
and CSNPs resveratrol (RES).
Sample Z-Average (nm) by Intensity PDI ZP (mV) pH
Sample Z-Average (nm) by Intensity PDI ZP (mV) pH
CSNPs CSNPs 359 ± 40
359 ± 40 ± 0.09
0.870.87 ± 0.09 3636± ±
5 5 4.0 4.0
CSNPs RES 950 ± 87 * 0.58 ± 0.18 48 ± 10 4.0
CSNPs RES 950 ± 87* 0.58 ± 0.18 48 ± 10 4.0

Results
Resultsofofparticle
particlesize
sizeand
andzeta
zetapotential
potentialofofCSNPs
CSNPsand
andCSNPs
CSNPsRES
RESare
arepresented
presentedininFigure
Figure1.1.

Figure1.1.Particle
Figure Particlesize,
size,PDI,
PDI,and
andzeta
zetapotential
potentialofofCSNPs
CSNPsand
andCSNPs
CSNPsRES.
RES.

3.1.2. MIC
MIC values of resveratrol (Table 3) were determined as an evaluation of their antimicrobial
activity against selected bacteria. The method was used to test the ability of bacteria to produce visible
growth when certain concentrations of resveratrol was added. The minimal inhibitory concentration
of chitosan solutions and chitosan nanoparticles dispersion were determined; the results are listed in
Materials 2019, 12, 2118 9 of 20

3.1.2. MIC
MIC values of resveratrol (Table 3) were determined as an evaluation of their antimicrobial
activity against selected bacteria. The method was used to test the ability of bacteria to produce visible
growth when certain concentrations of resveratrol was added. The minimal inhibitory concentration
of chitosan solutions and chitosan nanoparticles dispersion were determined; the results are listed in
Table 4.

Table 3. Minimal inhibitory concentration (MIC) (mg/mL) of resveratrol determined by

microdilution method.

Bacteria MIC (mg/mL)

Escherichia coli 5.01
Staphylococcus aureus 0.16

Table 4. Minimal inhibitory concentrations of chitosan solutions (CS) and dispersion of chitosan
nanoparticles (CSNPs).

Bacteria MIC (mg/mL) of CS MIC (mg/mL) of CSNPs

Escherichia coli 0.0053 ± 0.0011 0.0092 ± 0.0029
Staphylococcus aureus 0.0039 ± 0.0001 0.0078 ± 0.0002

Obtained results are important in order to define the efficient concentration of resveratrol to be
added during the synthesis of nanoparticles.
As it can be seen from Table 4, no significant differences are found in MIC for both bacteria; the
gram-negative one, Escherichia coli, and the gram-positive one, Staphylococcus aureus.

3.2. Functional foils

3.2.1. Gravimetric Measurements of Sample Mass

Differences in sample weight (∆m) using the gravimetric method evidently confirms the successful
chitosan, chitosan nanoparticles-resveratrol application of the formulation onto the PE or PP foils
(Table 5). In all cases, the mass of the samples increased, insignificantly, with respect to the reference
foil. The effect of the hydrophobic foil and the application of hydrophilic colloidal systems as coatings
showed poor adhesion and inhomogeneity. When the foil is treated with O2 plasma, the coating is
much more homogenous. All these were clearly seen visually as well as by SEM as discussed later by
SEM results. In all cases, foils were transparent after coatings.

Table 5. Percentage (%) of adsorption of chitosan macromolecular solution and CSNPs RES dispersion
onto the foil.

Mass of Dry Reference Mass of Dry Functionalised

Sample ∆m [g/cm2 ] Adsorption [%]
Foil [g/cm2 ] Sample [g/cm2 ]
D1 46.36 ± 0.03 46.70 ± 0.01 0.34 ± 0.02 0.73
D2 23.20 ± 0.06 23.52 ± 0.08 0.32 ± 0.07 1.36
E1 46.32 ± 0.02 46.49 ± 0.02 0.17 ± 0.02 0.36
E2 23.23 ± 0.01 23.50 ± 0.04 0.27 ± 0.02 1.18

3.2.2. ATR-FTIR Spectroscopy

Typical peaks for PE and PP can be seen from the FTIR spectrum A1 and A2 (reference samples in
Figures 2 and 3). Details on FTIR spectrum of PE foil, labelled as A1, are shown in Figure 2. The wave
numbers of FTIR spectra of PE at 2915, 2848, 1463, and 718.2 cm−1 were assigned to CH2 asymmetric
stretching, CH2 symmetric stretching, bending deformation, and rocking deformation, respectively.
Materials 2019, 12, 2118 10 of 20

Details of FTIR spectrum of PP foil (A2), are shown in Figure 2. The wave number of FTIR spectra of
PP at 2950, 2917, 2867, 2842, 1458, 1376, 1167, 997, and 973 cm−1 were assigned to CH3 stretching, CH2
asymmetric stretching, CH2 symmetric stretching, CH2 symmetric stretching, CH2 bending vibration,
CH3 bending vibration, CH3 symmetric deformation vibration, CH3 rocking vibration, and CH2
rocking vibration, respectively. The presence of mainly C–H vibrational groups clearly shows on the
materials’ hydrophobicity.
Spectrum B1 (Figures 2 and 3) represents the chitosan powder. A strong band in the region
3303–3347 cm−1 corresponds to N–H and O–H stretching, as well as the intramolecular hydrogen
bonds. The absorption bands at around 2921 and 2877 cm−1 can be attributed to C–H symmetric and
asymmetric stretching, respectively. The presence of residual N-acetyl groups was confirmed by the
bands at around 1654 cm−1 (C=O stretching of amide I) and 1581 cm−1 (N–H bending of amide II);
1419 cm−1 belongs to the N–H stretching of the amide and the ether bonds and N–H stretching at
1375 cm−1 (amide III band), respectively. The CH2 bending and CH3 symmetrical deformations were
confirmed by the presence of bands at around 1423 and 1375 cm−1 , respectively. The absorption band
at 1150 cm−1 can be attributed to asymmetric stretching of the C–O–C bridge. The bands at 1066 and
1026 cm−1 correspond to C–O stretching [45,46].
The FTIR spectrum of resveratrol showed a typical OH stretching band at 3182 cm−1 and intense
bands at 1604 and 1583 cm-1 corresponding to C–C aromatic double bond stretching and C–C olefinic
stretching, respectively. The peaks at 1513 and 1462 cm−1 reflect the benzene skeleton vibrations, while
C–O stretching vibrations were seen by the peak at 1381 cm−1 . The peaks at 986 and 964 cm−1 were
ascribed to the bending vibration of C=C–H [47].
By comparing the peaks of reference foils (A1 and A2) with functionalized foils (D1, D2, E1, and
E2), a successful application of coatings is seen. Spectrum D1 and D2 represent untreated PE and
PP foils, which were firstly functionalized with solution of chitosan (2% w/v) and secondly with the
chitosan–resveratrol nano dispersion (layer-by-layer). There are peaks on FTIR spectrum, which could
confirm the presence of chitosan and resveratrol. The typical peaks of chitosan and resveratrol are
smaller, but there are visible bonds such as N–H and O–H bond, all three amide bonds, C=C alkene,
C=C aromatic, C–O phenols, and =C–H aromatic bonds. It must be pointed out that the coating on the
films (spectra D1 and D2) were not homogeneous, due to the hydrophobic character of foils and the
hydrophilic nature of chitosan and chitosan–resveratrol formulation. Due to the difference in polarity,
the adhesion of applied formulations is lowered and not uniform.
All these peaks can also be observed on the spectra E1 and E2, but with the significant higher
intensity. For all the coated PE and PP films, the characteristic peaks of chitosan were observed (N–H
and O–H stretching; amide I, amide II, and amide III; C–O stretching). Comparison of the FTIR spectra
between D1 and E1 (by normalizing) is shown in the Figure 2b. The bands corresponding to C–O
stretching (glycoside bonds) and C=O stretching (amide I) are shifted towards higher wavenumber
(1096 and 1675 cm−1 ) implying the linkage of CSNPs and CSPSs RES with the oxygen-rich foil with
respect to unmodified foil (plasma-non activated). In addition, the vibration frequencies of N–H and
O–H bands are also shifted towards higher wavenumber (3310 and 3383 cm−1 ) and the peak is more
pronounced with larger intensity for O2 modified foil with CSNPs RES applied formulation. The
increased band vibrations frequency of N–H and O–H bonds suggest that the interactions between the
chitosan groups and foils surface are present in larger amount. It might be concluded that with O2
plasma treatment the new binding sites (the appearance of oxygen-rich groups on PE surface such as
CO, COOH, OH) are yielded onto foils surface, which are available for chemically binding with amino
groups of chitosan and, consequently, more chitosan–resveratrol nano dispersion could be attached on
this first bounded layer during a second coating step. When FTIR spectra between D2 and E2 for PP
foils are compared (Figure 3b), a similar trend was obtained, i.e., the occurrence of oxygen containing
groups with O2 plasma treatment led to better adsorption of chitosan as well as further attachment of
chitosan nanoparticles with embedded resveratrol.
groups on PE surface such as CO, COOH, OH) are yielded onto foils surface, which are available for
groups on PE surface such as CO, COOH, OH) are yielded onto foils surface, which are available for
chemically binding with amino groups of chitosan and, consequently, more chitosan–resveratrol
chemically binding with amino groups of chitosan and, consequently, more chitosan–resveratrol
nano dispersion could be attached on this first bounded layer during a second coating step. When
nano dispersion could be attached on this first bounded layer during a second coating step. When
FTIR spectra between D2 and E2 for PP foils are compared (Figure 3b), a similar trend was obtained,
FTIR spectra between D2 and E2 for PP foils are compared (Figure 3b), a similar trend was obtained,
i.e., the 2019,
Materials
occurrence of oxygen containing groups with O2 plasma treatment led to better adsorption
12, 2118 of oxygen containing groups with O2 plasma treatment led to better adsorption
of
11 of 20
i.e., the occurrence of
chitosan as well as further attachment of chitosan nanoparticles with embedded resveratrol.
chitosan as well as further attachment of chitosan nanoparticles with embedded resveratrol.

Figure 2.2. FTIR

FTIRspectra
spectraof of reference
reference polyethylene
polyethylene (PE) (PE)
(A1), (A1), chitosan (B1), resveratrol
(C1), and(C1), and
Figure
Figure 2. FTIR spectra of reference polyethylene (PE) chitosan (B1), resveratrol
(A1), chitosan (B1), resveratrol different
(C1), and
different
treated treated
PEtreated PE
foils with foils with application (D1 and E1) (a); comparison of the FTIR spectra between
different PE application (D1 and E1)(D1
foils with application (a);and
comparison of the FTIR of
E1) (a); comparison spectra between
the FTIR D1between
spectra and E1
D1 and
(by E1 (by normalizing) (b).
D1normalizing) (b).
and E1 (by normalizing) (b).

Figure 3. FTIR spectra of reference polypropylene (PP) (A2), chitosan (B1), resveratrol (C1), and
different treated PP foils with application (D2 and E2) (a); comparison of the FTIR spectra between D2
and E2 (by normalizing) (b).

3.2.3. XPS Analysis

The surface chemical composition of the reference foils and plasma-functionalized foils determined
by XPS is presented in Table 6. For the untreated PE and PP foils (A1 and A2), mostly carbon was
detected, which is in the agreement with chemical composition of the virgin foils. Small amounts
of oxygen (approximately 1 at.%) are due to the surface contamination as often observed in XPS
spectra measured on pure polymer foils. After oxygen plasma treatment, the oxygen concentration
significantly increased to 13.7 ± 0.3 and 14.77 ± 0.1 at.% for PE and PP, respectively (samples A3 and
A4 in Table 6). Slightly higher oxygen concentration on PP sample can be explained by oxidation of the
side methyl group, which is absent on PE. Increased oxygen concentration clearly indicates successful
surface plasma activation of both polymer foils with introduction of oxygen functional groups that may
act as a binding place for chitosan. This is further proved by Figure 4 showing formation of hydroxyl,
carbonyl, and carboxyl groups on the surface of the PE and PP foils.
When untreated (plasma none activated) foils were coated with chitosan and chitosan–resveratrol
nano dispersion, nitrogen appears on the surface of the foils; i.e., 3.3 at.% for PE (D1) and 2.1 at.%
for PP (D2). According to chemical structure, only chitosan contains nitrogen whereas resveratrol
should not possess any nitrogen; therefore, nitrogen is explained by the presence of amino groups
originating from chitosan because of its attachment on the foils. It can also be observed, that the carbon
content is reduced, whereas the oxygen content is increased by 8% for PE (D1) and 7.4% for PP (D2)
Materials 2019, 12, 2118 12 of 20

in comparison to the untreated foils, which is explained by a high oxygen content in both organic
substances i.e., chitosan and resveratrol.
Functionalized foils that were previously treated with oxygen plasma show the highest nitrogen
content, indicating a higher amount of adsorbed chitosan on the surface as compared to the untreated
foils. This may be due to the chemisorption of prepared chitosan dispersion onto foils. As it has
been shown, oxygen plasma introduced carbonyl and carboxyl group that may form binding places
for chitosan amino groups [16]. Oxygen functional groups formed by plasma treatment also raised
hydrophilicity of the foils causing better wetting of the surface with chitosan solution. All these may
improve the ability for chitosan adsorption, i.e., higher amount of chitosan could be bounded, which
was clearly indicated in our case by a higher concentration of nitrogen; i.e., nitrogen concentration
significantly increased for 6.7% on PE surface (E1) and for 6.7% on PP surface (E2). The carbon content
considerably decreased by as much as 25.7% on PE (E1) and 25.8% on PP (E2), and the oxygen content
significantly increased by as much as 18% on PE (E1) and PP (E2) indicating attachment of chitosan
and resveratrol, which are both rich with oxygen. (E2). All these observations are in a good agreement
with the results, obtained from the infrared spectroscopy.
Summarizing, when all plasma-functionalized samples are compared to those that were previously
not activated by plasma via reference ones, it is clearly observed that the amount of nitrogen, originating
from the coating, is significantly increased, which means that chitosan has adsorbed onto plasma-treated
foil to a much higher extent.

Table 6. Surface chemical composition (at.%) of the reference foils and plasma-treated foils with the
two-layer coating (at.%). Statistical significance is defined as * p < 0.05 compared to control sample
(ANOVA test).

Untreated Foils O2 Treated Foils

Sample C N O Sample C N O
A1 98.9 ± 0.6 - 1.1 ± 0.3 A3 86.3 ± 0.3 * - 13.7 ± 0.3 *
A2 98.7 ± 0.3 - 1.3 ± 0.1 A4 85.3 ± 0.1 * - 14.7 ± 0.1 *
D1 87.7 ± 2.6 * 3.3 ± 0.8 9.1 ± 2.0 * E1 60.6 ± 0.0 6.7 ± 0.2 31.7 ± 0.3
D22019, 12, 89.7
Materials x FOR± PEER 2.0 ± 0.1
0.8 * REVIEW 8.3 ± 0.8 * E2 59.5 ± 0.1 6.7 ± 0.0 ± 0.5
32.7 12 of 19

Figure 4. High-resolution carbon C1s spectrum of plasma-treated (a) PE and (b) PP samples.
Figure 4. High-resolution carbon C1s spectrum of plasma-treated (a) PE and (b) PP samples.
3.2.4. Oxygen Permeability
3.2.4. Oxygen Permeability
Table 7 contains the results of oxygen permeability for the reference foils compared to the
Table 7 contains
functionalized the results
foils. Oxygen of oxygenafter
permeability permeability
coatings offor thehas
foils reference foils compared
been reduced to The
in all cases. the
functionalized foils. Oxygen
permeability decrease for the permeability afterPE
plasma untreated coatings
and PPoffoils
foils
inhas been reduced
comparison in all cases.
with reference The
samples
permeability decrease for the plasma untreated PE and PP foils in comparison with reference
was lower than for the same samples, which were previously activated by O2 plasma. This may be samples
was lower to
attributed than
thefor the same
higher samples,
and uniform whichof
amount were previously
attached chitosanactivated by O2 plasma.
macromolecular This
solution as may be
the first
attributed to the higher and uniform amount of attached chitosan macromolecular solution as the
first layer and, thus, further better affinity for chitosan-RES nanoparticles as a second layer. The
statistical analysis showed that the highest significant decrease in permeability was observed for O2
plasma-treated PE foil with a layer of chitosan and a layer of chitosan–resveratrol nano dispersion
(E1). For this sample, the oxygen permeability was reduced from 3226 ± 62 cm3/m2d by as much as
202 ± 16 cm3/m2d. Significantly different results were also obtained for the plasma-functionalized PP
Materials 2019, 12, 2118 13 of 20

layer and, thus, further better affinity for chitosan-RES nanoparticles as a second layer. The statistical
analysis showed that the highest significant decrease in permeability was observed for O2 plasma-treated
PE foil with a layer of chitosan and a layer of chitosan–resveratrol nano dispersion (E1). For this sample,
the oxygen permeability was reduced from 3226 ± 62 cm3 /m2 d by as much as 202 ± 16 cm3 /m2 d.
Significantly different results were also obtained for the plasma-functionalized PP foil (E2), as oxygen
permeability decreased from 1078 ± 36 cm3 /m2 d to 195 ± 14 cm3 /m2 d. For plasma-untreated PE and
PP foils coated with a layer of chitosan and a second layer of chitosan–resveratrol nano dispersion,
the maximum permeability of oxygen has non-significantly reduced from 3226 ± 62 cm3 /m2 d to
2417 ± 104 cm3 /m2 d for sample D1 and from 1078 ± 36 cm3 /m2 d to 968 ± 19 cm3 /m2 d for sample D2.
The latest may be attributed to the lower, and especially homogenous, adsorption of chitosan colloidal
systems onto both plasma non-activated foils, as will be discussed in Section 3.2.6.

Table 7. The oxygen permeability between the reference foils and the foils with the application.
Statistical significance is defined as * p < 0.05 compared to control sample (ANOVA test).

Sample OTR (cm3 /m2 d)

A1 3226 ± 62
A2 1078 ± 36
D1 2417 ± 104
D2 968 ± 19
E1 202 ± 16 *
E2 195 ± 14 *

3.2.5. Goniometry
Static contact angle (SCA) measurements, which provides an inverse measure of wettability, have
also been performed. SCA of various samples are also shown in Figure 5. On the basis of SCA results,
hydrophilicity/hydrophobicity of PE and PP surfaces was determined. Reduction of contact angle
is of a great importance for practical use as the hydrophilic surface of the foils reduces the potential
process of dew condensation on the surface of the foils (anti-fog efficiency) in contact with food, which
worsens the packaging conditions and thus increases the food contamination [48]. It is, thus, extremely
important to avoid this process.
The results of SCA measurements are presented in Table 8. The reference PE and PP foils had
contact angles of 108.2◦ and 109.3◦ , which pointed out foils hydrophobicity. Statistically, all samples
are significantly different compared to A1 or A2 (control sample). The treatment with oxygen plasma
was successful as expected and resulted into contact angle decrease for around three to four times (A3,
A4). With the functionalization of the foils using chitosan systems, the SCA significantly decreased in
comparison to reference foils (A1 and A2). The contact angles slightly decreased for the samples D1,
D2; however, the biggest significant difference in the decrease of the contact angle was observed for the
samples E1 and E2, i.e., contact angle decreased down to 42.4◦ and 45.3◦ . It can be concluded that when
foils (PE or PP) are previously treated with O2 plasma, the higher and more homogeneous adsorption
of chitosan macromolecular solution and chitosan–resveratrol nano dispersion occurred. Because of
the adsorbate foils polarity and hydrophilicity, it is obvious that coatings on the plasma-activated PE
and PP surface has better wettability and are thus more homogeneous after attachment.
dispersion occurred. Because of the adsorbate foils polarity and hydrophilicity, it is obvious that
coatings on the plasma-activated PE and PP surface has better wettability and are thus more
homogeneous after attachment.

Table
Materials 2019,8.12,
Values
2118 of static contact angle (SCA) measurements. Statistical significance is defined as *P 14
< of 20
0.05 compared to control sample (ANOVA) test).

Sampleangle
Table 8. Values of static contact Average angle
(SCA) (α/°) Difference
measurements. (%) significance is defined as
Statistical
* p < 0.05 compared to controlA1 108.2 ± 1.2
sample (ANOVA) test). /
A2 109.3 ± 0.7 /
Sample Average Angle (α/◦ ) Difference (%)
A3 29.70 ± 0.5* 72.6
A1 108.2 ± 1.2 /
A2 A4 37.0 ± 0.5*
109.3 ± 0.7 66.1 /
A3 D1 93.1 ± 1.2*
29.70 ± 0.5 * 14.0 72.6
A4 D2 37.0±±1.4*
84.8 0.5 * 22.4 66.1
D1 93.1 ± 1.2 * 14.0
E1 42.4 ± 1.8* 60.8
D2 84.8 ± 1.4 * 22.4
E1 E2 42.4±±0.9*
45.3 1.8 * 58.6 60.8
E2 45.3 ± 0.9 * 58.6

A1 D1 E1

A2 D2 E2

Figure 5.
Figure 5. CSA
CSA of
of samples.
samples.

3.2.6. Morphology: Scanning

3.2.6. Morphology: Scanning Electron
Electron Microscopy—SEM
Microscopy—SEM
Figure
Figure 66 presents
presents SEM
SEM images
images of of the
the surface
surface morphology
morphology and and the
the adhesion
adhesion uniformity
uniformity ofof the
the
uncoated
uncoated (reference
(reference samples
samples A1 A1 and
and A2)
A2) and
and coated
coated foils
foils with
with chitosan
chitosan in
in the
the first
first layer
layer and
and CSNPs
CSNPs
RES
RES formulation
formulation in in the
the second
second (upper)
(upper) layer. For easier
layer. For easier comparison,
comparison, allall images
images were
were taken
taken at
at the
the
comparable magnification (300×)
comparable magnification (300×)andandatatthe thesame
sameimaging
imaging parameters.
parameters. ForFor samples
samples D1 D1
andand
D2, D2,
the
the coating
coating is visible
is visible on on
thethe surface
surface of ofthe
thefoils
foilswith
withsignificantly
significantlyhigher
higher contrast
contrast together
together with
with the
the
deposited
deposited agglomerates. However, the coating was rough and did not completely cover the foil as it
agglomerates. However, the coating was rough and did not completely cover the foil as it
can
can be
be seen
seen inin comparison
comparison to to the
the reference
reference foils.
foils. This
This can
can be
be explained
explained byby the
the hydrophobicity
hydrophobicity of of the
the
two
two polymers
polymers (the
(the hydrophobicity
hydrophobicity of of the
the reference
reference PEPE and
and PPPP foils
foils were
were demonstrated
demonstrated by by measuring
measuring
the contact angle as well as surface techniques that suggested the presence of nonpolar groups) that
lowers the adhesion of applied formulations due to the difference in polarity.
With O2 plasma treatment/foils surface modification before coating, the surface of the PE and PP
foils were modified. The latter significantly improved the interaction between the two polymers (PE/PP
foil and chitosan). When the reference foils are compared with the samples E1 and E2 (Figure 6), a very
successful application is obtained as the uniform hydrophilic surface, where the coating is homogenous,
smoother, and thinner, uniformly covering the foil with less observed agglomerates. It was found
by surface analyses (i.e., infrared spectroscopy and XPS) that the occurrence of oxygen-containing
functional groups (C=O, C–O, and –OH) of the O2 plasma-treated PE and PP foils increased from
those of the untreated one, indicating that the O2 plasma enhanced hydrophilicity of the PE and
PP foils (E1 and E2). The SEM analysis also supported the oxygen permeability results, where the
homogeneous formulation coating (samples E1 and E2) also showed higher reduced permeability with
respect to samples D1 and D2. This can be in part explained by the more homogenous adhesion of the
applied layer-by-layer coating, as evident from SEM images.
 was found by surface analyses (i.e., infrared spectroscopy and XPS) that the occurrence of oxygen-
containing functional groups (C═O, C–O, and –OH) of the O2 plasma-treated PE and PP foils
increased from those of the untreated one, indicating that the O2 plasma enhanced hydrophilicity of
the PE and PP foils (E1 and E2). The SEM analysis also supported the oxygen permeability results,
where the homogeneous formulation coating (samples E1 and E2) also showed higher reduced
Materials 2019, 12, 2118with respect to samples D1 and D2. This can be in part explained by the more
permeability 15 of 20
homogenous adhesion of the applied layer-by-layer coating, as evident from SEM images.

Figure
Figure 6. SEM
6. SEM micrographs
micrographs ofof functionalized surface
functionalized surface of
ofPE
PEand
andPPPP
with resveratrol-loaded
with chitosan-
resveratrol-loaded chitosan-
sodium
sodium tripolyphosphate
tripolyphosphate (TPP)
(TPP) nanodispersion.
nano dispersion.

3.2.7.3.2.7. Migration:
Migration: Desorption
Desorption Experiment—Polyelectrolyte Titration
Experiment—Polyelectrolyte Titration
Chitosan
Chitosan maymay be be releasedfrom
released fromthe the PE
PE and
and PP
PP surface
surfaceif iffoils areare
foils treated in ainway
treated that that
a way the the
interactions between both polymers are weakening. Desorption experiments were performed at pH
interactions between both polymers are weakening. Desorption experiments were performed at
= 5.8 (mimicking the pH of most of the food). Prior to the polyelectrolyte titration, pH of bath solution
pH = 5.8 (mimicking the pH of most of the food). Prior to the polyelectrolyte titration, pH of bath
was adjusted by 0.1 M HCl to 3.6. Polyelectrolyte titration results shows how much chitosan desorbed
solution
fromwas adjusted
the surface of by
PE 0.1
andM PPHCl
afterto243.6. Polyelectrolyte
h. Table 9 shows the titration
amount ofresults
aminoshows
groupshow much that
of chitosan chitosan
desorbed from the surface of PE and PP after 24 h. Table 9 shows the amount of
were desorbed from one gram of PE or PP foil in the indicated range of five repetitions with standardamino groups of
chitosan that were desorbed from one gram of PE or PP foil in the indicated range
deviation within interval of 0.5%–3%. If the previously untreated foils (D1 and D2) are compared of five repetitions
with with
standard deviation
the treated oneswithin interval
with oxygen of 0.5%–3%.
plasma (E1 and If theitpreviously
E2), can be seenuntreated foils (D1
that less amino andare
groups D2) are
desorbed
compared withfrom
the the latterones
treated ones.with oxygen plasma (E1 and E2), it can be seen that less amino groups
are desorbedNamely,
fromforthesamples D1 and D2, 0.0189 mmol and 0.0118 mmol of amino groups per gram of
latter ones.
foil were desorbed, respectively,
Namely, for samples D1 and D2, whilst for mmol
0.0189 E1 andand
E2, 0.0019
0.0118mmolmmoland 0.0051 mmol
of amino groups amino groupsof foil
per gram
per gram of foil. This, again, suggests better adhesion of coating formulations on previously modified
were desorbed, respectively, whilst for E1 and E2, 0.0019 mmol and 0.0051 mmol amino groups per
foils with O2 plasma with stronger interactions among bonding sites (ionic interactions). The amount
gram of foil. This, again, suggests better adhesion of coating formulations on previously modified
of desorbed chitosan in mmol/g is shown in Table 9 and the example of polyelectrolyte titration in
foils with
FigureO7.2 plasma with stronger interactions among bonding sites (ionic interactions). The amount
of desorbed chitosan in mmol/g is shown in Table 9 and the example of polyelectrolyte titration in
Figure 7.

Table 9. Desorption of chitosan amino group per gram of foil.

Sample
Desorption of Chitosan
D1 D2 E1 E2
[mmol/kg] 18.90 ± 2.84 11.80 ± 1.30 1.92 ± 0.42 0.51 ± 0.31
 Table 9. Desorption of chitosan amino group per gram of foil.

Sample
Desorption of Chitosan
Materials 2019, 12, 2118 D1 D2 E1 E2 16 of 20
[mmol/kg] 18.90 ± 2.84 11.80 ± 1.30 1.92 ± 0.42 0.51 ± 0.31

Figure7.7.Polyelectrolyte
Figure Polyelectrolytetitration
titrationcurve
curveon
onthe
theexample
exampleofofsample
sampleE1
E1after
after2424h.h.

3.2.8. Bioactivity
3.2.8. Bioactivity
Antimicrobial ActivityActivity
3.2.8.1 Antimicrobial
Usingantimicrobial
Using antimicrobial testing,
testing, the
the chitosan/chitosan–resveratrol
chitosan/chitosan–resveratrolnano nanodispersion-coated
dispersion-coatedPEPE andand PP
PPfoils were
foils tested
were against
tested gram-positive
against (Staphylococcus
gram-positive aureus) and
(Staphylococcus gram-negative
aureus) bacteria (Escherichia
and gram-negative bacteria
coli). The results of antimicrobial efficacy are presented in Table
(Escherichia coli). The results of antimicrobial efficacy are presented in Table 10. 10.
ItItisisknown
knownthat thatthe
theprotonated
protonatedamino aminogroups
groupsfrom
fromchitosan
chitosanstructure
structureplayplayaacrucial
crucialrole
roleininthe
the
antibacterialactivity.
antibacterial activity. It has
It has to betopointed
be pointed outthere
out that thatare
there
two are two
layers layers ofoncoatings
of coatings the foils,on the foils,
containing
containing 2% chitosan and chitosan–resveratrol nano dispersion.
2% chitosan and chitosan–resveratrol nano dispersion. For resveratrol, which is a stilbenoid, a typeFor resveratrol, which is a
ofstilbenoid,
natural phenol,a type and
of natural phenol, and
a phytoalexin, a phytoalexin,
significant significant
and strong and strong
antioxidant antioxidant
properties properties
are known with
are known with simultaneous
simultaneous antimicrobial efficiency [33]. antimicrobial efficiency [33].
ResultsininTable
Results Table1010showshowthatthatbybyfunctionalized
functionalizedfoils
foilssurface,
surface,antimicrobial
antimicrobialefficiency
efficiencyisisachieved
achieved
ininall
allcases.
cases.As Asalready
alreadymentioned,
mentioned,the theapplication
applicationon onthethesamples
samplesD1 D1andandD2D2isisnon-homogeneous;
non-homogeneous;
therefore, the values of reduction are different. Somewhere they
therefore, the values of reduction are different. Somewhere they are lower (D2), but are lower (D2), but in in
thethe
case of D1,
case of
D1, high antimicrobial efficacy is seen. Very high and statistically significant antimicrobial activity isis
high antimicrobial efficacy is seen. Very high and statistically significant antimicrobial activity
visiblefor
visible forthe
thefoils
foilspreviously
previouslytreatedtreatedwith
withOO2plasma.
plasma.ItItcan
canbe beseen
seenforforboth
bothsamples
samplesE1 E1and
andE2,
E2,that
that
2
antimicrobial efficacy for bacteria Staphylococcus aureus is more than 90%.
antimicrobial efficacy for bacteria Staphylococcus aureus is more than 90%. The same functionalized The same functionalized
foilsalso
foils alsohavehave high
high and
and statistically
statistically significant
significant antimicrobial
antimicrobial efficiency
efficiency against
against bacteria
bacteria Escherichia
Escherichia coli;
coli; samples E1 and E2, showed reduction of 77.55 ± 2.57% and
samples E1 and E2, showed reduction of 77.55 ± 2.57% and 79.43 ± 5.86%, respectively. 79.43 ± 5.86%, respectively.

Table10.
Table 10.Antimicrobial
Antimicrobialefficacy
efficacyofofgram-positive
gram-positive(Staphylococcus
(Staphylococcusaureus)
aureus)and
andgram-negative
gram-negativebacteria
bacteria
(Escherichia
(Escherichia coli).
coli). Statistical
Statistical significance
significance is defined
is defined as * as
p <*P < 0.05
0.05 compared
compared to control
to control sample
sample (ANOVA
(ANOVA test).
test).
Bacteria Staphylococcus aureus Escherichia coli
Bacteria Staphylococcus Antimicrobial
Number of Cells
aureus Efficacy
Escherichia
Number of Cells
coli
Antimicrobial Efficacy
Sample
Number (log cfu/cm2 ) Antimicrobial(%)
of Cells efficacy Number
(log of 2)
Cells
cfu/cm Antimicrobial
(%) Efficacy
Sample
A1 4.58 ± 0.06
(log cfu/cm²) (%) - 4.58 ± 0.06
(log cfu/cm²) (%)-
A2 5.10 ± 0.05 - 5.10 ± 0.05 -
A1D1 4.58 ± 0.06
1.35 ± 0.30 -
72.91 ± 8.26 4.583.25
± 0.06
± 0.14 37.73- ± 2.77
A2D2 3.57 ± 0.33
5.10 ± 0.05 22.03
- ± 7.28 3.89 ± 0.36
5.10 ± 0.05 25.32- ± 6.87
E1 0.67 ± 0.21 * 93.54 ± 3.95 1.24 ± 0.09 * 77.55 ± 2.57
D1E2 1.35 ± 0.51
0.30± 0.08 * 72.9191.21
± 8.26
± 2.22 3.25 ± 0.14
1.07 ± 0.31 * 37.73
79.43±±2.77
5.86
D2 3.57 ± 0.33 22.03 ± 7.28 3.89 ± 0.36 25.32 ± 6.87
Anti-Oxidant
E1 activity—ABTS
0.67 ± 0.21* 93.54 ± 3.95 1.24 ± 0.09* 77.55 ± 2.57

Figure 8 shows the results of anti-oxidative activity. The test was used to confirm how
functionalized packaging influence the oxidative process inhibition. As can be seen from the results
presented in Figure 8, the lowest anti-oxidative activity is shown for the reference PE (4.00%) and PP
 E2 0.51 ± 0.08* 91.21 ± 2.22 1.07 ± 0.31* 79.43 ± 5.86

3.2.8.2. Anti-Oxidant activity—ABTS

Figure 8 shows the results of anti-oxidative activity. The test was used to confirm how
functionalized packaging
Materials 2019, 12, 2118 influence the oxidative process inhibition. As can be seen from the results
17 of 20
presented in Figure 8, the lowest anti-oxidative activity is shown for the reference PE (4.00%) and PP
(5.44%) foils. Each applied foil has a greatly increased effect, which means that resveratrol is a very
(5.44%) foils. Each applied foil has a greatly increased effect, which means that resveratrol is a very
good antioxidant. Good anti-oxidative activity results mean that resveratrol has been successfully
good antioxidant. Good anti-oxidative activity results mean that resveratrol has been successfully
loaded to chitosan nano dispersion, whilst chitosan itself does not possess antioxidant properties [30].
loaded to chitosan nano dispersion, whilst chitosan itself does not possess antioxidant properties [30].
The results for samples D1 (89.68%), D2 (95.72%), E1 (100%), and E2 (100%) after 15 min, show
The results for samples D1 (89.68%), D2 (95.72%), E1 (100%), and E2 (100%) after 15 min, show very
very good inhibition on all applied foils. Because they were applied onto the foils in layers, which
good inhibition on all applied foils. Because they were applied onto the foils in layers, which means
means that after the 2% chitosan adsorption a layer of chitosan–resveratrol nano dispersion was
that after the 2% chitosan adsorption a layer of chitosan–resveratrol nano dispersion was applied onto
applied onto dry foil. This is, therefore, the external layer of the foil surface and as such, easily
dry foil. This is, therefore, the external layer of the foil surface and as such, easily accessible for free
accessible for free radicals. In cases E1 and E2, a 100% inhibition is obtained after 15 min; in other
radicals. In cases E1 and E2, a 100% inhibition is obtained after 15 min; in other cases, (D1 and D2)
cases, (D1 and D2) after 60 min.
after 60 min.

Figure
Figure 8. Graphical
8. Graphical representation
representation of of anti-oxidative
anti-oxidative activity
activity of of
CS,CS, CSNPs
CSNPs RES-coated
RES-coated PE,
PE, and
and PPPP foils
foils
after
after thethe same
same time
time interval.
interval.
4. Conclusions
4. Conclusions
It was demonstrated that the additive effect of antimicrobial chitosan together with anti-oxidative
It was demonstrated that the additive effect of antimicrobial chitosan together with anti-
resveratrol as an adsorbate for PP and PE, introduces bioactive properties to foils surface. XPS and
oxidative resveratrol as an adsorbate for PP and PE, introduces bioactive properties to foils surface.
FTIR methods show successful binding of all chitosan and chitosan–resveratrol structured adsorbates
XPS and FTIR methods show successful binding of all chitosan and chitosan–resveratrol structured
onto foils. This is also supported by the differences in sample weight after coating application onto
adsorbates onto foils. This is also supported by the differences in sample weight after coating
foils using gravimetric method. Both attached layers (chitosan macromolecular solution and further
application onto foils using gravimetric method. Both attached layers (chitosan macromolecular
dispersion of chitosan nanoparticles with embedded resveratrol) lowered foils oxygen permeability as
solution and further dispersion of chitosan nanoparticles with embedded resveratrol) lowered foils
well as reduced contact angle, which indicates great barrier and good anti-fog foils properties.
oxygen permeability as well as reduced contact angle, which indicates great barrier and good anti-
With O2 plasma treatment, the surface of the PE and PP foils were modified, and significant
fog foils properties.
improvement of the interaction between the two polymers, PE/PP foil and chitosan, was achieved
With O2 plasma treatment, the surface of the PE and PP foils were modified, and significant
with very successful coating application. It was found by surface analyses (FTIR and XPS) that the
improvement of the interaction between the two polymers, PE/PP foil and chitosan, was achieved
occurrence of oxygen-containing functional groups (C=O, C–O, and −OH) of the O2 plasma-treated
with very successful coating application. It was found by surface analyses (FTIR and XPS) that the
PE and PP foils increased from those of the untreated one, indicating that the O2 plasma-enhanced
occurrence of oxygen-containing functional groups (C═O, C–O, and −OH) of the O2 plasma-treated
hydrophilicity of the PE and PP foils resulting into more homogeneous formulation coating as evident
PE and PP foils increased from those of the untreated one, indicating that the O2 plasma-enhanced
from SEM images.
hydrophilicity of the PE and PP foils resulting into more homogeneous formulation coating as
The coatings were smoother and thinner, uniformly covering the foils with less observed
evident from SEM images.
agglomerates as in the case of plasma non-activated foils. Moreover, plasma-activated and further
coated foils by two mentioned layers show much higher reduction of oxygen permeability as well as
much better anti-fog efficiency due to lower contact angle. Besides all mentioned and good applicable
properties, these functionalized foils also have superior antimicrobial and anti-oxidative properties. The
method of previous plasma activation of foils and further functionalization by chitosan macromolecular
Materials 2019, 12, 2118 18 of 20

solution as a first layer and chitosan nanoparticles with integrated resveratrol as a second layer, shows
great potential for different packaging applications such as active packaging in food industry.

Author Contributions: The manuscript was written through contributions of all authors. All authors have given
approval to the final version of the manuscript. T.K.G. and L.F.Z. designed the study and wrote the original draft.
L.F.Z. performed the supervision. U.A., O.P., A.V., N.P.U.; U.B., M.K.H. performed the experiments and validation
of results. All authors wrote/updated the manuscript in specific parts and prepared the specific figures. U.A.
completed and edited the paper. All authors reviewed the manuscript.
Funding: This work is a part of project Functional food for Future—F4F (Strategy of smart specialization),
which is co-financed by the Republic of Slovenia and the European Union under the European Regional
Development Fund. Funding through Slovenian Research Agency (ARRS) project grant J1-6736 and P2-0118 is
also gratefully acknowledged.
Acknowledgments: The authors acknowledge the team at Biotechnical Faculty, University of Ljubljana, for
antimicrobial test performance.
Conflicts of Interest: The authors declare no conflict of interest.

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