Accelerat ing t he world's research.

Microstructure and physico-chemical

evaluation of nano-emulsion-based
antimicrobial peptides embedded in
bioacti...
m imran
Food Hydrocolloids

Cite this paper Downloaded from Academia.edu 

Get the citation in MLA, APA, or Chicago styles

Related papers Download a PDF Pack of t he best relat ed papers 

A Novel Funct ional Wrapping Design by Complexat ion of ε-Polylysine wit h Liposomes Ent rappi…
Begoña Ferrari

Physical Charact erist ics, Release Propert ies, and Ant ioxidant and Ant imicrobial Act ivit ies of Whey Pro…
sara gh

Humect abilit y and physical propert ies of hydroxypropyl met hylcellulose coat ings wit h liposome-cellul…
Marcela Zamorano
 Food Hydrocolloids 29 (2012) 407e419

Contents lists available at SciVerse ScienceDirect

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

Microstructure and physico-chemical evaluation of nano-emulsion-based

antimicrobial peptides embedded in bioactive packaging films
Muhammad Imran, Anne-Marie Revol-Junelles, Noémie René, Majid Jamshidian,
Muhammad Javeed Akhtar, Elmira Arab-Tehrany, Muriel Jacquot, Stéphane Desobry*
Laboratoire d’Ingénierie des Biomolécules (LIBio), université de Lorraine, École nationale supérieure d’agronomie et des industries alimentaires (ENSAIA),
2 avenue de la Forêt de Haye, 54505 Vandoeuvre-lès-Nancy Cedex, France

a r t i c l e i n f o a b s t r a c t

Article history: Customized application of antimicrobial peptide (AMP) ‘nisin’ directly into food (neither in active
Received 2 March 2011 packaging nor encapsulated form) is expensive and associated with loss of activity due to deactivation in
Accepted 11 April 2012 complex food systems. The purpose of the present study was to fusion the two concepts for improved
bioavailability i.e. AMP nanoencapsulation and biopolymer immobilizing to formulate the next genera-
Keywords: tion biodegradable films embedded with either active agent, nano-encapsulated active agent or both of
Biodegradable polymer
them. Nanoliposomes were prepared using soy-lecithin by microfluidizer at 2000 bar with 5 cycles to
Bioactive peptide
generate an average size of 151  4 nm with 50  3% encapsulation efficiency. For active films, nisin had
Emulsion
Controlled release
demonstrated no negative impact on transparency, thickness and water sorption behavior obtained by
Nanoencapsulation GAB model (25  C, 0e0.95 aw). For nano-active films, the results clearly illustrated that different physico-
chemical properties including barrier (oxygen and water vapor permeability), color and transparency
(200e900 nm) remained comparable to native hydroxypropyl methylcellulose (HPMC) films and were
significantly improved than using lecithin directly without nano-scale restructuring. The microstructure
studies (topography and morphology) by scanning and transmission electron microscopes (SEM/TEM)
revealed different (pore, lamellar, fusion) modes of nisin release from nanoliposomes embedded in
HPMC matrix. As microbiological worth, nisin nano-emulsion (encapsulated and free nisin) films were
effective against potential foodborne pathogen Listeria monocytogenes. This innovative concept of
biodegradable nano-active films may thus be a preventive system toward improved food safety.
Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction (O’Sullivan, Ross, & Hill, 2002). It is the only antimicrobial bacte-
riocin with the status of generally recognized as safe (GRAS)
‘Green consumerism’, a trend since the beginning of the approved by the Food and Drug Administration (FDA) (Sanjurjo,
previous decade, had emerged due to increasing consumer demand Flores, Gerschenson, & Jagus, 2006). Nisin offers effective control
for natural antimicrobial compounds. Such biomolecules are of against broad spectrum of Gram positive bacteria especially against
natural origin, non-toxic for humans, environmentally safe and the foodborne pathogens Listeria monocytogenes, Staphylococcus
effective in preserving foods by controlling microorganism’s aureus and Bacillus cereus (Joerger, 2007). This AMP kills susceptible
activity (Mastromatteo, Conte, & Del Nobile, 2010). Antimicrobial bacteria through a multi-step process that destabilize the phos-
peptides (AMPs) are widely recognized as promising alternatives to pholipids bilayer of the cell and creates transient pores (Breukink &
the current use of antibiotics (Marcos & Gandia, 2009). Nisin is de Kruijff, 2006).
probably the most utilized AMP in the food industry as food Stability issues like proteolytic degradation and the potential
biopreservative world-wide (Ercolini et al., 2010). Nisin is a 3.5 kDa interaction of the AMP with food components might result in
cationic polypeptide produced from Lactococcus lactis strains decreased antimicrobial activity. Use of nisin in its free form
approved for specific uses in foods in more than 40 countries (unpackaged or unencapsulated) is expensive and is associated
with loss of activity due to degradation or deactivation and
emergence of nisin-resistant bacteria strains (Benech, Kheadr,
* Corresponding author. Tel.: þ33 (0) 3 83 59 58 80; fax: þ33 (0) 3 83 59 57 72.
Lacroix, & Fliss, 2002; Laridi et al., 2003). Significant loss of nisin
E-mail addresses: [email protected] (M. Imran), stephane.desobry@ activity in different foods due to its interactions with complex food
univ-lorraine.fr (S. Desobry). components such as divalent cations, enzymes, fat and other

0268-005X/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodhyd.2012.04.010
408 M. Imran et al. / Food Hydrocolloids 29 (2012) 407e419

components have already been reported (Davies et al., 1999; through the liposome (simple model cell membrane) by pore
Roberts & Zottola, 1993; Taylor, Gaysinsky, Davidson, Bruce, & formation as it was demonstrated against bacteria (Breukink & De
Weiss, 2007). Kruijff, 1999). Further it can diffuse from the network of film to
In the food industry, liposomes have been investigated to deliver reach the food in-contact, thus present strategy may guarantee the
proteins, enzymes, vitamins, antioxidants and flavors (Khosravi- availability of fresh/active nisin protected from its inhibitors in
Darani, Pardakhty, Honarpisheh, Rao, & Mozafari, 2007; Laridi complex food systems (Fig. 1).
et al., 2003; Mozafari, Johnson, Hatziantoniou, & Demetzos, 2008; Nanoencapsulation of nisin in soybean-lecithin by an innovative,
Taylor, Bruce, Weiss, & Davidson, 2008). The entrapment of AMP into rapid, without organic solvents, efficient and industrially applicable
liposomes might represent an alternative to overcome the problems method of microfluidic format was accomplished in the present
related to the direct application of these AMP in food. Due to the study. The incorporation of nano-structures in HPMC film forming
presence of both lipid and aqueous phases in the structure of lipid solution (FFS) may change the native microstructure (topography
vesicles, recently studied nanoliposomes can be utilized in the and morphology), barrier (O2, H2O), mechanical, color, light trans-
entrapment, homogenous dispersion, protection, delivery and mission and water sorption properties of formulated packaging
release of water-soluble and lipid-soluble materials (Taylor et al., films. To analyze the suitability of the composite films, in the present
2008). As nisin is amphiphilic in nature (Breukink, Ganz, De project we had studied the above-mentioned significant attributes
Kruijff, & Seelig, 2000), it is entrapped simultaneously in the core to judge and compare the biodegradable non-active, active and
and bilayers of liposome. Thus, it generates the possibility for nano-active packaging films. At the same time, the physico-chemical
superior bioavailability of nisin by phospholipid-based nano- characters of films were studied in the presence of hydrophilic and
delivery systems. Long term stability of liposome-encapsulated hydrophobic plasticizers. Moreover, microbiological study of
AMP (nisin, pediocin) had been demonstrated, which correlates to different treatments was performed to ensure the antimicrobial
its better bioavailability during storage period (Degnan, Buyong, & efficacy against potential food pathogen L. monocytogenes.
Luchansky, 1993; Laridi et al., 2003).
To control the bacterial load at food surface in different food 2. Materials and methods
products, immobilized AMP can be applied for development of
bioactive food packaging. Currently, environment conservation 2.1. Materials
awareness has led to a paradigm shift to look for packaging films
and processes that are biodegradable. Thus, the concept of Nisin Z was purchased from Honghao Chemical Co. (Shanghai,
biodegradability enjoys both user-friendly and eco-friendly attri- China). Nisin used in this study contained >90% pure nisin
butes. Biodegradable polymer hydroxypropyl methylcellulose (3.84  106 IU per gram and 6.88% moisture content). HPMC powder
(HPMC) edible films are attractive for food applications because with hydroxypropoxyl content w9% and viscosity w15 mPa s (2% in
this edible plant derivative had shown to form transparent, H2O, 25  C) was obtained from Fluka-Biochemika, Japan. Commer-
odorless, tasteless, oil-resistant, water-soluble films with very cial soybean lecithin (SL) was used, extracted according to method
efficient oxygen, carbon dioxide, aroma and lipid barriers, but with describe previously (Wu & Wang, 2003). Glycerol (>97% purity) was
moderate resistance to water vapor transport (Imran, El-Fahmy, used as a plasticizer and was purchased from Merck (Darmstadt,
Revol-Junelles, & Desobry, 2010; Villalobos, Chanona, Hernandez, Germany). Millipore nylon filters (0.2 mm) were obtained from
Gutierrez, & Chiralt, 2005). To realize the objectives of preserva- Millipore (Cork, Ireland). The liposomal ingredients were kept
tive slow release into food and its surface contamination preven- under nitrogen atmosphere at the recommended storage temper-
tion, the incorporation of nisin in sodium caseinate films has been atures (0e4  C). Ammonium molybdate was purchased from Fisher
reported in recent work (Cao-Hoang, Chaine, Grégoire, & Waché, Scientific (France). Bicinchoninic acid (BCA) reagents were obtained
2010). from Sigma Chemical Co. (Lyon, France).
An improvement of antimicrobial packaging systems is needed in Microorganism and culture media: L. monocytogenes CIP 82110T
order to prevent the growth of spoilage related microbial species for strain was purchased from collection of institute Pasteur. The
longer conservation period to enhance the shelf-life and quality of bacteria were cultivated in trypticase soybean broth (Biokar Diag-
food. In this regard, the aim of the present project was to fusion the nostics, Beauvais, France) supplemented with 6 g L 1 of bacto-yeast
above-mentioned two concepts i.e. AMP nanoencapsulation and extract (Biokar) (TSB-YE). Incubation was performed at 37  C over-
biopolymer immobilizing to formulate the next generation biode- night. The strain was stored in appropriate culture medium sup-
gradable films embedded with either active agent, nano-encapsulated plemented with glycerol (10%) at 30  C and propagated twice
active agent or both of them. Thus, nisin could diffuse/migrate before use.

Fig. 1. The diffusion/release of liposome-encapsulated nisin embedded in HPMC nano-active films.

 M. Imran et al. / Food Hydrocolloids 29 (2012) 407e419 409

2.2. Nisin encapsulation in nanoliposome 2.4. Film preparation

High pressure homogenization method (constant cell disruptor Film forming solutions (FFS) were prepared by dissolving HPMC
system e CCDS, Northants, UK) was preliminary optimized using in distilled water (final concentration 6% w/v). The solutions were
soybean-lecithin (SL) with different concentrations, number of mixed for 40 min at 65  C using a heating magnetic stirrer (Fisher
cycles and pressure. From these, optimum concentration (5% w/v), Bio-block scientific). Composition of the HPMC biodegradable
pressure (2000 bar) and number of passes (5 cycles) were used for active film formulations are shown in Table 1. For non-active and
preparing nanoliposomes. These values were selected by consid- active films, selected amounts of glycerol (30% w/w D.M.), lecithin
ering the nisin encapsulation efficiency, size and polydispersity (2.5% w/v) and nisin (1 mg mL 1) were added during heating and
index (PDI) of vesicles. Thus, five percent SL was mixed with an stirring. To obtain the active FFS solutions with nisin, it was dis-
aqueous solution containing nisin [3.8  104 IU]. The mixture was solved in 10 mL of distilled water at 65  C and FFS was prepared
stirred (50e60 rpm) for 15 min, diluted with deionized water to with the remaining 90 mL. For the nano-active treatments, initially
obtain 1, 2 or 4 mg mL 1 of nisin Z concentration (to obtain a final emulsion and liposomes were obtained as described above, and
concentration of 1 mg mL 1 in different film forming solutions) and then nano-vesicles solution was added to double concentrated
re-stirred for 15 min by magnetic stirring. Further it was thoroughly HPMC FFS (1:1) to obtain a final concentration of 6% for HPMC. The
mixed by Ultraturax T-25 (Avantek, Strasbourg, France) at encapsulation efficiency of the CCDS method for nisin entrapment
13,500 rpm for 3 min. The phospholipid dispersion was then passed was found 50  3% with different concentration of nisin (1, 2 or
through CCDS with a vertical interaction chamber for 5 cycles at 4 mg mL 1). Thus in case of nano-emulsion (encapsulated and free
given pressure. The homogenization temperature was kept below nisin) approximately half of the given nisin concentration is
10  C by using a flow of cold water flowing (4  C) through a cooling encapsulated and rest is in free form. However, the nanoliposomes
jacket. To recover the liposomes containing nisin, emulsions were obtained from emulsion by size exclusion chromatography have
passed through Sephadex G-50 (fractionation range for protein nisin in encapsulated form only. For active and nano-active films,
1500e30,000 M.W.) column and nisin-encapsulating liposomes nisin was added to obtain a final concentration of 1 mg mL 1 in the
were eluted by size exclusion chromatography using Eppendorf film forming solution. As a homogenous solution was achieved
centrifuge (Eppendorf, Hambourg, Germany). after mixing, it was degassed at 50e60  C under vacuum
(YamatoÒ). Films were made by pouring approximately 7 g FFS in
2.3. Particle size characterization the lids of Petri-dishes (Optilux e NunclonÔ Fisher, DK-4000
Roskilde, Denmark) and left to dry them at room temperature
The mean diameter and particle size distribution of liposomes (20  C) and relative humidity (w50%) for 24e48 h. Films were
were determined using dynamic light scattering (DLS) technique either stored under similar conditions of drying or at wzero relative
employing a Zetasizer Nano-ZS (Malvern instruments, UK). The humidity using phosphorus pentoxide (P2O5) depending upon film
apparatus is equipped with a 4 mW He/Ne laser emitting at 633 nm, characterization experiment.
a measurement cell, a photo multiplier and a correlator. Prior to size
measurement, the samples were diluted (1:400) with ultra pure 2.5. Film characterization
water. The samples were taken in vertical cylindrical cuvettes
(10 mm diameter). The scattering intensity was measured at 2.5.1. Optical microscope
a scattering angle of 173 relative to the source using an avalanche To study the surface properties of nanoliposomes embedded
of photodiode detector, at 25  C. Results are presented as an average films, the pre-conditioned films were stained with toluidine blue.
diameter of the liposome suspension (z-average mean) with the Afterward, film topography was examined by using optical micro-
polydispersity index (PDI) which evaluates the size distribution scope (Olympus, Ax 70) with 100 magnification. Random areas on
width. In the dispersion, particles are in a constant random Brow- each film were observed and the representative images were
nian motion that causes the fluctuation of the scattered light captured and digitized by an Olympus DP 70 (U-CMAD-2, Japan)
intensity with time. Therefore, droplets sizes were obtained from using the software DP manager version 2.1 (Olympus corporation).
the correlation function calculated using algorithm of the disper-
sion technology software (DTS). All measurements were carried out 2.5.2. Scanning electron microscope (SEM)
at 25  C, with a medium refractive index 1.335. The measurements Microstructural analyses of cross-sections of dry films (previ-
were performed in five replicates. ously conditioned in desiccators with P2O5 for at least 15 days) were

Table 1
Mechanical characteristics (tensile strength TS, Young’s modulus Y, ultimate elongation UE) and thickness of non-active, active (nisin) and nano-active (liposome-encapsulated
nisin) biodegradable HPMC coatings.

Formulations Thickness (mm) Tensile strength Young’s modulus Ultimate elongation

TS (MPa) Y (MPa) UE (mm)
F1 HPMC 43.5  1.3 59.0  6.8 2727  361 6.0  3.3
F2 HPMC þ N 47.7  5.6 45.1  6.6** 2133  578 6.2  1.2
F3 HPMC þ G 50.8  2.7* 32.0  3.0*** 1229  383** 26.5  2.3***
F4 HPMC þ GþN 59.7  2.0*** 30.6  7.3*** 1074  066** 28.3  1.1***
F5 HPMC þ lecithin 75.7  3.4*** 22.5  2.8*** 1759  326 1.9  0.5*
F6 HPMC þ lecithin þ N 77.9  4.1*** 21.1  1.6*** 1538  601* 1.8  0.1*
F7 HPMC þ emulsion 62.8  0.9*** 25.7  3.0*** 1887  146 0.9  0.1**
F8 HPMC þ N (emulsion) 63.2  0.8*** 21.0  4.0*** 1985  389 0.8  0.2**
F9 HPMC þ N (liposome) 50.4  1.6 37.0  2.5*** 2228  657 2.6  0.7

Non-active, F1, F3, F5, F7; active, F2, F4, F6; nano-active, F8, F9.
HPMC, hydroxypropyl methylcellulose; N, nisin 1 mg mL 1, i.e. w104 IU; G, glycerol (30% w/w D.M.); lecithin, 2.5% w/v; N (emulsion), nisin 50% encapsulated and 50% free
form; N (liposome), nisin 100% encapsulated.
Dunnett test, *p < 0.05; **p < 0.01; ***p < 0.001.
410 M. Imran et al. / Food Hydrocolloids 29 (2012) 407e419

carried out by cryo-fracturing of films. Film cross-sections were placed in a controlled temperature and RH chamber. The water
prepared by dropping a film into liquid nitrogen followed by frac- vapor transport was determined from the weight variation of the
turing with a pre-chilled razor. Freshly fractured film pieces were cell with time. Three replicates were made from each film compo-
retrieved from the liquid nitrogen bath and placed as quickly as sition. Water Vapor Transmission Rate (WVTR) was determined
possible into a Petri dish containing a piece of filter paper and from the slope of weight gain versus time, once the steady state was
placed in a desiccators to warm and dry to room temperature. reached.
Fractured film pieces were then mounted up on a SEM stub. All

1 2
samples were then viewed and photographed in a Hitachi S-4800 WVTR ¼ dm=ðA$dtÞ gh m (1)
scanning electron microscope (Hitachi, Japan) at 0.5e2 kV. Topo-
graphic analyses of upper and lower surfaces, which faced air and

1 1 1
Petri plate during dehydration, respectively, were analyzed by WVP ¼ ½WVTR$X=ðDp$3600ފ gs m Pa (2)
using pre-conditioned films stuck onto a cylindrical aluminum stub
by a double-sided tape to observe the morphology of the surfaces as where dm is the weight gain of the cup over time (dt), A is the area
explained above. of exposed film, Dp is the vapor pressure differential across the film,
and X is the film thickness.
2.5.3. Transmission electron microscope (TEM)
The microstructure of nano-active films was analyzed to study 2.9. Water sorption isotherms
the morphological character of the liposomes in the biodegradable
coating matrices. The pre-conditioned films (using P2O5, at 20  C, Dynamic vapor sorption system (DVS, SMS Ltd, UK) was used to
for 15 days) were dehydrated with ethanol for 1 h. After ethanol obtain the sorption isotherms of films. The sample was equilibrated
treatment, a pre-impregnation in blend of resin Embed 812 and at a constant temperature for different relative humidity values.
propylene oxide (1:1) was performed. Progressive impregnation Film were cut into small pieces (5 mm  5 mm) and dried in
steps with increasing amounts of resin blend (Embed 812 and vacuum desiccators at 20  C over Phosphorus pentoxide (P2O5) for
propylene oxide 0.3:0.7, 0.5:0.5, 0.7:0.3) were tested. The samples 2 weeks. The programmed relative humidities were from 0 to 95%,
were finally placed in the latex molds covered with the resin and divided in 10% increments (10 points). The temperature was set at
Reynolds dye and polymerized at 56  C overnight. The ultra-thin 25  C. The samples were considered to be at equilibrium when the
sections of <100 nm were achieved by an ultra-microtome, these value dm/dt (slope of the change in mass with time) was set to be
ultra-thin film pieces were contrasted with uranyl acetate to <0.002 Dmass%/min. Where Dmass% is the percent of mass varia-
observe under TEM. The mesh was examined using a Transmission tion of the sample at given time.
Electron Microscope (Philips CM-20) at an operating voltage of The modelisation of the sorption isotherms was done using GAB
200 kV. (Guggenheim-Anderson-de Boer) model. The procedure used for
estimating the parameters was non-linear regression (curve
2.6. Film thickness measurement fitting), using Origin 6.1 software (Origin Lab corporation, USA).

X ¼ Xm $CGAB $K$aw =½ð1 K$ aw Þ$ð1 K$ aw þ C$ K$ aw ފ (3)

The thickness of films was measured using the standard NF Q
03-016 with a manual micrometer (Messmer, London, England) X is the mass of water adsorbed at a given aw, Xm is the monolayer
equipped with a head of 1 cm in diameter and with a sensitivity of value (% db). Constant CGAB is related to the energy associated with
2 mm. The thickness of film was measured at ten randomly selected the binding between the water molecules and the matrix primary
points on each film to calculate the average value. interactions sites or monolayer. K is a temperature-dependent
coefficient related to the heat of sorption of the multilayer.
2.7. Tensile properties
2.10. Oxygen permeability
The tensile characteristics of films consist of tensile strength (TS,
MPa), ultimate elongation (UE, percent at break point), and Young’s The basis of this dynamic method is the oxygen transfer through
modulus (Y, MPa). Maximum tensile strength is the largest stress the film. To measure oxygen permeability, the film is placed
that a film is able to sustain. Ultimate elongation is the maximum between two Teflon rings, in a permeation cell. O2 permeability of
percentage change in the length of a film before breaking. Young’s pre-conditioned films (20  1  C and 50  2% RH) was determined
modulus, calculated from the slope of the initial linear region of the using a gas chromatographic system (Shimadzu GC-4A; Shimadzu
stressestrain curves, reflects the film stiffness. The tests were Corp., Kyoto, Japan). Gas concentrations were measured by inject-
performed using the universal testing machine Lloyd instrument ing samples with a gas sampling syringe (Dynatec Pressure Lok,
(Hants, United Kingdom) on 6 specimens previously stored for 7 Baton Rouge, LA, USA) into a gas chromatograph equipped with
days at 20  1  C and 50  2% RH. Sample films of approximately a thermal conductivity detector and two columns (Hayesep for N2
5 cm  2 cm (analyzed area ¼ 3 cm  2 cm) uniaxially stretched and molecular sieve for O2). The flow rate of helium carrier gas was
(sensor of force of 5-KN and constant speed of 20 mm min 1). The 25 mL min 1 and column temperature was 50  C. This method was
stressestrain curves were computer-recorded and exploited with based on measurement of the amount of oxygen diffusing through
Nexygen software. the film over time. The film was first sealed into a test cell, which
was filled with oxygen. At suitable time intervals, gas samples were
2.8. Water vapor permeability (WVP) withdrawn from the lower compartment of the cell via a sampling
port and analyzed by gas chromatography (Khwaldia, Banon,
WVP of composite films was investigated with the gravimetric Desobry, & Hardy, 2004). Oxygen content is reported as percent
method described in the AFNOR NFH00-030 standard (1974) as of detected peaks (O2 and N2). The oxygen permeability was
reported earlier (Imran, El-Fahmy, et al., 2010). The film was sealed determined by using Eq. (4).
in a permeation cell containing a desiccant (silica gel). The glass
permeation cells were 5.8 cm (i.d.)  7.8 cm (o.d.)  3.6 cm deep a x V
PO2 ¼ (4)
with an exposed area of 26.42 cm2. The permeation cells were S  60  0:65
 M. Imran et al. / Food Hydrocolloids 29 (2012) 407e419 411

where a is the coefficient of the curve slope representing the 2.13. Statistical analysis
percentage of oxygen as a function of time, x is the thickness of film
expressed in m, V is the volume of the permeation cell which Statistical analyses were carried out by using the software KyPlot
corresponds to 0.0001 m3 and S is the film surface area exposed i.e. version 2.0 (Koichi Yoshioka, Department of Biochemistry and
0.0025 m2. Biophysics, Graduate school of Allied health Sciences, Tokyo, Japan).
For comparison between HPMC film and films containing plasticizer
2.11. Optical properties or active agent, a parametric multiple test (Dunnett test with HPMC
film as control) was performed. Furthermore different composite
2.11.1. Transparency/light transmission films containing nisin were compared with their respective
Film transparency against ultraviolet (UV) and visible light was formulations without nisin using Tukey parametric multiple test.
measured for a wavelength spectrum between 200 and 900 nm,
using a UVeVisible recording spectrophotometer (Ultrospec 4000 3. Results and discussion
UV/visible, Pharmacia Biotech, UK) according to the procedure
given by Fang and others (Fang, Tung, Britt, Yada, & Dalgleish, 3.1. Particle size characterization of liposome encapsulating nisin
2002). The transparency of the films was calculated by the equa-
tion (Han & Floros, 1997): In most cases, reactive or sensitive material, such as poly-
nucleotides and polypeptides (e.g. nisin), can be turned into stable
ingredients through encapsulation or entrapment by nano-carrier
Transparency ¼ logT600 =x (5)
systems (e.g. liposome) (Mozafari, 2006). The study of liposome
where T600 is the transmittance at 600 nm and x is the film thick- average size, as well as their distribution, is of interest because of its
ness. Three replicates of each treatment were tested. impact on the properties of the films, such as water vapor perme-
ability, mechanical properties and general barrier properties (Fabra,
2.11.2. Color measurement Jiménez, Atarés, Talens, & Chiralt, 2009). At 2000 bar pressure, five
Edible films and coating’s color is one of the sensory charac- passes in CCDS resulted in the average particle size of 151  4 nm
teristics, which must be as neutral as possible, so that they are not for nisin-encapsulating liposomes (Fig. 2). Particle size distribution
detected during consumption. To evaluate the color of films, was uni-modal with a narrow polydispersity index (PDI) of
a Minolta CM, CR-210 colorimeter (Minolta, Colombes, France) 0.23  0.02, which had reflected the efficiency of CCDS technique
employing the Hunter and CIE scale was used. The parameters L* for liposome preparation. Size distribution considering vesicles
for lightness and chromatic coordinates a* and b* are measured. number percentage was found highest between 80 and 130 nm. In
The parameter L* is the luminosity of the sample between the contrast to mm sized carriers, nano-carriers provide more surface
black and the white, the parameter a* is the value of variation of area and have the potential to increase solubility, enhance
the color of the treatment between the green and blue whereas bioavailability, improve time-controlled release (Mozafari et al.,
the parameter b* is the variation of color of the treatment 2008). Thus, nano-sizer results indicated that nanoliposomes con-
between the yellow and the red. The whiteness index (WI) of the taining nisin formulated using soybean-lecithin are sufficiently
composite films was also calculated by the following equation smaller to carryout above-mentioned advantages.
(Sanchez-Gonzalez, Vargas, Gonzalez-Martanez, Chiralt, & Chafer,
2009). 3.2. Characterization of the films

qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi Different film formulations characterized in this study are non-

2 
100 L* þ a* 2 þ b* 2 active (F1, F3, F5, F7), active (F2, F4, F6) and nano-active (F8, F9)
 
WI ¼ 100 (6)
(Table 1). The formulations (F3, F4, F5, and F6) described the effects
of two plasticizers glycerol and lecithin, whereas formulations (F7,
2.12. In-vitro antimicrobial tests F8, F9) illustrated the effect of nano-restructuring of soy-lecithin in
the form of liposomes. The term nisin emulsion is used to express
The minimal inhibitory concentration (MIC) of nisin was both forms of nisin (free in solution and encapsulated in liposome).
determined by the critical dilution method in 96-well plates (Nunc,
Roskilde, Denmark). The target strain L. monocytogenes CIP 82110T 3.3. Microstructural properties
was prepared in TSB-YE medium to a final OD 660 nm of 0.2. Equal
volume of inoculated medium and nisin dilutions were added to 3.3.1. Surface study
the 96-well plate. The plates were shacked and the initial OD Taking into consideration the lack of miscibility of film forming
660 nm was determined with a Titertek Multiscan MCC/340 solution (FFS) components, film microstructure is assumed as
(Huntsville, AL). The plates were incubated for 24 h at 37  C. The a continuous matrix of hydrocolloids, embedding lipid aggregates
MIC value (7.8 mg L 1) is the inverse of the highest dilution where of different sizes, depending on the film composition and the
no growth is detected. Afterward, the corresponding MIC dilutions component interactions developed during both emulsification and
of film forming solutions were incubated with the target strain and film drying steps (Debeaufort & Voilley, 1995). The surface images
its growth kinetics was recorded at 660 nm absorbance for more obtained for control and nano-active films by optical microscope
than 3 days. are shown in Fig. 3. The films were stained with toluidine blue (TB).
To find out the antilisterial activity of films, 1 cm diameter disks It is a quick stain for light microscopic “orientation sections”, which
were cut from different composite bioactive films and placed on are used afterward for electron microscopy. The HPMC film
inoculated TSA-YE nutrient medium (TSB with 12% agar) by pour demonstrated clearly a continuous hydrocolloid matrix. However,
plate method. The method was previously standardized by the captured images for nano-active film (F9) reflected the inner
adjusting the microbial inoculation (0.1% v/v) and the volume of the complexity generated in the film with homogenously dispersed
agar medium layer (12 mL). Dishes of 9 cm diameter were refrig- liposomes on the surface of the dried biopolymer film. The size of
erated at 4  C for 4 h to allow the process of bacteriocin diffusion the liposomes ranged from nano- to micrometer, which could be
without microbial growth and then incubated at 37  C. due to the aggregation/complexation between the liposomes
412 M. Imran et al. / Food Hydrocolloids 29 (2012) 407e419

Fig. 2. Size distribution of nisin encapsulated nanoliposomes using soybean-lecithin (5% w/v) prepared by constant cell disruption system at 2000 bar pressure with five cycles. Size
distribution of liposomes at logarithmic scale presented as vesicles intensity percentage (A); and vesicles number percentage (B).

during film drying. The complexity observed is associated with the encapsulating nisin appear lighter than the HPMC continuous
discontinuities in the refractive index through the material in the matrix and, hence, are easily identifiable in the films. The HPMC
different liposomeehydrocolloid interfaces. The size of the nisin- cross-section displays a relatively smooth inner-structure (Fig. 4a),
encapsulating liposome alone or conjugated in the dried film is the which is even more smooth with glycerol plasticized nisin con-
main factor involved in the optical heterogeneity degree of the film taining HPMC film (Fig. 4b). Whereas the matrix in the nano-
matrix. Nevertheless, the representative image of nano-active film composite films is rougher (Fig. 4e, f). The nanoliposomes are
surface structure revealed homogenous dispersion of liposomes evenly distributed within the polymeric matrix; however, hardly
embedded in biopolymer HPMC network (Fig. 3b). any of the nanoparticles are aggregated which shows good repul-
sion between lecithin liposomes resulting in homogenous dis-
3.3.2. Topographic & morphologic study (scanning electron persibility inside the biopolymeric films.
microscopy) Considering topographic micrographs, the nanoliposome
The permeability of films can be influenced by the composition, embedded films showed non-uniform surface with dome-shaped
morphology and homogeneity of the coating matrix (Bilbao-Sainz, zones and holes (Fig. 4c, d; upper and lower surfaces respec-
Avena-Bustillos, Wood, Williams, & McHugh, 2010). Scanning tively). These semi-spherical structures represented mainly the
electron microscope (SEM) was used to characterize the topog- liposome structures half-implanted in HPMC matrix, in conse-
raphy and morphology of the composite biodegradable films. quence to the drying process of films. Phospholipid-based lipo-
Fig. 4a, e represents the cross-sections of the unfilled and liposomes somes are well distributed through the film on both upper and
reinforced HPMC films, respectively. The nanoliposomes lower sides. These physical surfaces may support lower WVP of

Fig. 3. Ultra semi-fine micrographs (osmium staining) of (a) HPMC film and (b) nano-active HPMC film containing liposome-encapsulated nisin. *Represents the HPMC film while
the arrow sign corresponds to film border.
 M. Imran et al. / Food Hydrocolloids 29 (2012) 407e419 413

Fig. 4. Micrographs of scanning electron microscope (SEM); cross-section view, (a) control HPMC film; (b) plasticized glycerol and nisin containing HPMC film; (e, f) embedded
liposomes encapsulating nisin in HPMC film; (g, h) zoom micrographs to observe liposome degradation/lamella opening/pore formation by nisin; surface-view, (c) and (d) upper
and lower surface of nano-active films containing encapsulated nisin. *Represent the film area as compared to support.
414 M. Imran et al. / Food Hydrocolloids 29 (2012) 407e419

HPMC-liposome-encapsulated nisin (F9) films compared to those for nisin-encapsulating liposomes before and after embedding/
with lecithin incorporation directly without nano-structurization drying in HPMC films. TEM permits us to analyze the liposome
(F6). Liposome hindered the transfer of water molecules inside distribution in emulsified films and to characterize the destabili-
the film; therefore, a homogeneous distribution of active liposome/ zation phenomena such as aggregation and/or coalescence due to
emulsion would give the film a lower WVP. Similar results were the fabrication process (spread and dry) and due to the addition of
previously mentioned for fat or essential oil incorporation in the nisin. The micrograph of liposome embedded films (ultra-thin
biodegradable films (Hambleton, Debeaufort, Bonnotte, & Voilley, slice of <100 nm) had indicated a homogenous dispersion without
2009; Sanchez-Gonzalez et al., 2009). It had been suggested by conjugation occurrence between liposomes inside the film (Fig. 5a).
previous studies that the fat matter is more concentrated at the air HPMC matrix and difference of focused zone had shown different
side, because during drying there is a film retraction that changes liposomes more or less identifiable but their homogenous distri-
its structure, becoming denser thus the lipid migrates toward the bution is quite obvious. In parallel, the nisin emulsion before drying
air side (Hambleton et al., 2009). Indeed fat density is lower than process of FFS (swollen state) had revealed comparable results of
the FFS aqueous phase, and thus fat tends to migrate toward the dispersibility proving sufficient electrophoretic repulsion between
evaporated surface. However, as amphiphilic soya-phospholipids soya phospholipid nanoparticles (Fig. 5b). However, the represen-
have density (1.04) more or less equivalent to the aqueous phase, tative micrograph revealed heterogeneity concerning the lamel-
it had resulted in similar appearance of liposome at both upper larity of liposomes. Generally, the liposomes measuring above
(Fig. 4c) and lower (Fig. 4d) surfaces. This reveals that very little 100 nm diameter belong to multilamellar group in contrast to
creaming occurred during the film drying, possibly owing to the unilamellar liposomes (<100 nm) (Imran, Revol-Junelles et al.,
highly viscous effect of HPMC, which, furthermore, increased when 2010). These multilayer liposomes may improve the controlled
drying progressed. Most probably the density, viscosity, gravimetric release of nisin by providing numerous hindrances as lamellas.
and evaporation pull equilibrated in a way that the dispersion of
nisin-encapsulating liposomes is quite homogenous in both inner 3.4. Film thickness
and outer film structure.
The stability of liposomes and their homogenous distribution in Film thickness relies primarily on its composition (Table 1);
emulsion had fulfilled the prerequisite objective to encapsulate the however the use of pure nisin had not influenced the active film
nisin in liposome, further these nano-active liposomes were thickness as compared to control film (F1), stored at 50% RH and
embedded in HPMC matrix to slow down release of nisin in the food. 20  C. These results had shown improvement by producing
Considering the antimicrobial mechanism of action, nisin is able to homogenous active films as compared to the previous study using
form pores in the liposomal membranes, which had been previously NisaplinÒ (heterogenous mixture of nisin, salt and milk solids)
explained for cell model membranes (liposome) and different (Imran, El-Fahmy, et al., 2010). The observed increased thickness of
bacterial species (Breukink & de Kruijff, 2006; Breukink et al., 2000). films containing glycerol might be there because glycerol retain
Zoom micrographs had revealed interesting micrographs of pore- higher moisture content at the end of film drying due to its hydro-
formation phenomenon in individual liposome embedded in philic nature (Chen & Lai, 2008). The addition of soybean-lecithin
HPMC after 30 days of storage (Fig. 4g, h). Similarly, the represen- alone or in combination with nisin (F5, F6) had significantly
tative micrograph had exposed the lamellar opening/degradation of increased the film thickness to 77.9  4.1 as compared to F1 (Dunnett
the liposomes. Nisin-encapsulating liposomes are slightly enlarged test, p < 0.001). However, the incorporation of soybean-lecithin in
probably due to the deformation forces that act during the polymer the form of nano-emulsion (F7, F8) resulted in significant decrease of
chain aggregation during the solvent evaporation. As nisin is thickness as compared to F5 (Tukey test, p < 0.01). The active-nano
amphiphilic in nature, thus it is encapsulated in both core and film (F9) had eventually decreased the size to 50.4  1.6, which is
lamellar phases of liposomes. The slow degradation of multilamellar non-significantly different than control film (Dunnett test, p > 0.05).
liposomes and pore formation by nisin and storage time may
possibly, as a consequence, play a role in controlled release of nisin 3.5. Tensile properties
both in the film or once transferred into the food system (Fig. 1).
Tensile strength (TS) indicates the maximum tensile stress that
3.3.3. Microstructure (transmission electron microscopy) the film can sustain, ultimate elongation (%UE) is the maximum
For the confirmation of above-mentioned microscopic obser- change in length of a test specimen before breaking, and Young’s
vations, transmission electron microscopy (TEM) was carried out modulus (Y) is a measure of the stiffness of the film (Table 1). Values

Fig. 5. Transmission electron micrographs of (a) nano-encapsulated nisin embedded in HPMC film matrix and (b) nanoliposomes encapsulating nisin observed after fabrication by CCDS.
 M. Imran et al. / Food Hydrocolloids 29 (2012) 407e419 415

of mechanical properties obtained for non-active (HPMC) and nature and to reduce the water sorption capacity of the films
active (HPMC þ nisin) films agreed with previous study using (Sanchez-Gonzalez, Chafer, Chiralt, & Gonzalez-Martinez, 2010).
similar 6% (w/v) concentrated FFS (Imran, El-Fahmy, et al., 2010), in The behavior of lecithin is different from other lipids (essential oils,
spite of the fact that in the present study FFS was prepared in fatty acids) as it comprises mainly of phospholipids which are
distilled water as compared to 35% alcoholic solution utilized amphiphilic in nature. Slight increase in WVP had been previously
earlier. The incorporation of the lecithin leads to softer, less resis- documented for gelatinelecithin composite films (Andreuccetti
tant to break and less stretchable films. However, glycerol had et al., 2009). On the other hand, the incorporation of glycerol
shown an exception by increasing significantly the elongation as provoked a reorganization of the HPMC network which becomes
compared to control. The nano-active film (F9) had shown signifi- less dense with a larger free volume, facilitating greater mobility
cant improvement for TS as compared to lecithin active film (F6). and as a consequence greater WVP by the film matrix. Glycerol
The treatment which contains the nano-encapsulated nisin had plasticized film had given significantly higher permeability (Dun-
a better tensile strength and a better percentage of elongation than nett test, p < 0.001), which may be caused by the higher number of
the treatment containing nano-emulsion nisin. Thus free nisin in available polar (eOH) groups in HPMCeglycerol composite films
HPMC network decreased the TS as shown also by F2 in comparison (Hambleton et al., 2009; Imran, El-Fahmy, et al., 2010).
to control. But treatment containing free nisin had better tensile However, our study confirmed that structuring lecithin into the
strength and a better percentage of elongation than film containing form of nanoliposomes (F7, F8, F9) decreased the WVP as compared to
liposomes. This could be explained by discontinuities in the poly- direct lecithin incorporation. The nano-sized particles are homoge-
mer matrix introduced by the lecithin [at macro- (as such) and nously distributed which may have reduced the water diffusivity,
nano-scale (liposome)] incorporation and by changes in the poly- due to better interaction of the hydrocolloid HPMC and polar-head
mer chain interactions when lipid components are present, which groups of liposome outer membrane. The liposome/emulsion
lead to a weak mechanical response. Similarly the stiffness of film introduces an increase in the tortuosity factor for water migration in
differed non-significantly from F1, when incorporated in the form of the matrix, thus increasing the distance traveled by H2O molecules to
nanoliposomes/emulsion. Similar trends had been observed earlier permeate through the films. The tortuosity factor was higher in these
for the plasticizers analyzing the mechanical properties of nano-active films, which depends mainly on lipid ratio and particle
biopolymer films blended with glycerol and different fatty acids (liposome) size (Pérez-Gago & Krochta, 2001).
(Andreuccetti, Carvalho, & Grosso, 2009; Imran, El-Fahmy, et al.,
2010; Sebti, Ham-Pichavant, & Coma, 2002). Thus different 3.7. Water sorption isotherms
behavior can be attributed to the type of plasticizer (hydrophobic,
hydrophilic) and particular processing of ingredients resulting in Moisture adsorption isotherms (equilibrium moisture content
dissimilar interactions with the biopolymer network, which is also on a dry basis versus water activity) were constructed for non-
affected by the bio-additive nisin incorporated to add functionality active, active and nano-active films at 25  C (Fig. 6). The water
to biodegradable coatings. adsorption curves of HPMC-based films were sigmoid in shape,
showing a slower increase in equilibrium moisture content until
3.6. Water vapor barrier aw ¼ 0.6, after which the increase in humidity led to a large mass
gain, signifying a swelling phenomenon as water activity increased
The WVP allowed us to estimate the water barrier efficiency of and promoted solubilization. This negligible convexity attained at
HPMC edible films with or without encapsulated nisin. In the food low water activity was associated with type III sorption isotherm, an
industry, the difficulty with composite films is the relatively high attribute of components rich in hydrophilic components and similar
water vapor permeability. Permeability in packaging is controlled behavior was observed by other authors for HPMC-based films
by the diffusivity and solubility of water within the film matrix. (Imran, El-Fahmy, et al., 2010; Villalobos, Hernandez-Munoz, &
Thus implementing food nanotechnology, new organization of Chiralt, 2006).
firmly linked three dimensional networks can be fabricated to Sorption isotherm behavior specified that, at low aw, HPMC-
prevent diffusion of water in foodstuffs (de Moura et al., 2009). In based films adsorb moisture with or without plasticizers, which
general, the plasticizers increased the WVP obtained (Table 2). The also interacts with water molecules independently. As aw increased,
incorporation of lecithin directly (F5) slightly increased the WVP. the water increasingly penetrated the HPMC network, partially
Moreover, nisin addition alongside lecithin significantly increased dissolving them to form a gel, that initiated higher molecular
the WVP. Lipid compounds are recognized to improve the water mobility and component interactions, which had influenced the
barrier properties of biopolymer films due to their hydrophobic sorption pattern. The plasticized films containing glycerol (F3, F4)

Table 2
Oxygen permeability, water permeability and water sorption properties (parameter values estimated from the aw 0e0.95 curves at 25  C fitted by GAB model) of biodegradable
composite films. Experimental DVS values were averaged and fitted by GAB model equation (3).
1 1 1 11
Formulations H2O permeability (g m s Pa ) (10 ) O2 permeability m3 m m 2
s 1
kPa 1
(10 17
) Xm C K
F1 HPMC 0.77  0.03 7.4  1.6 4.99 2.73 0.88
F2 HPMC þ N 0.87  0.01 7.5  3.6 4.73 3.07 0.90
F3 HPMC þ G 1.45  0.10*** 10.3  1.3 8.37 1.49 0.89
F4 HPMC þ GþN 3.2  0.11*** 6.7  2.2 10.2 1.21 0.91
F5 HPMC þ lecithin 0.98  0.10* 9.6  2.0 5.04 1.84 0.81
F6 HPMC þ lecithin þ N 1.78  0.04*** 6.9  0.1 4.69 1.98 0.85
F7 HPMC þ emulsion 0.83  0.03 16.3  4.3** 5.29 1.87 0.83
F8 HPMC þ N (emulsion) 0.93  0.04 4.8  0.9 4.86 2.11 0.83
F9 HPMC þ N (liposome) 0.95  0.10 10.8  0.8 4.98 2.29 0.86

Non-active, F1, F3, F5, F7; active, F2, F4, F6; nano-active, F8, F9.
HPMC, hydroxypropyl methylcellulose; N, nisin 1 mg mL 1, i.e. w104 IU; G, glycerol (30% w/w D.M.); lecithin, 2.5% w/v; N (emulsion), nisin 50% encapsulated and 50% free
form; N (liposome), nisin 100% encapsulated.
Dunnett test, *p < 0.05; **p < 0.01; ***p < 0.001.
416 M. Imran et al. / Food Hydrocolloids 29 (2012) 407e419

70 the HPMC film network (Table 2). When nisin is mixed with
glycerol there is a slight decrease in the OP, which is even lower
60 than control film. These modifications are non-significant (Dun-
nett test, p > 0.05), because the changes in lateral chain gap of
HPMC is not sufficient to alter oxygen molecule diffusion. In fact,
g water/100g dry matter

50
the molecular diameter of oxygen is about 11 A while that of H2O
is 2.4 A (Hambleton et al., 2009). Even if plasticization of HPMC
40
network occurs, most probably it is not sufficient to favor oxygen
diffusion. The film containing the nisin in nano-emulsion form
30
(free and encapsulated simultaneously) indicated the lowest
permeability to oxygen. However, the film containing total nisin
20
encapsulated in nanoliposomes had a little elevated OP. Thus the
presence of nisin may render the HPMC composite film matrix
10 more compact and improve the barrier against the O2, since the
formulation F8 indicated significantly lower OP as compared with
0 F7 (Tukey test, p < 0.05). Considering the barrier potential of
0.0 0.2 0.4 0.6 0.8 1.0 cellulose derivatives against O2 and H2O molecules, similar
Water activity
tendency had been observed earlier when OP was not changed
Fig. 6. Moisture sorption isotherms of non-active, active (nisin) and nano-active significantly for carboxymethylcellulose-based edible films when
(liposome-encapsulated nisin) biodegradable HPMC coatings at 25  C and aw essential oils were added, whereas WVP was modified (Bifani
0e0.95. Experimental DVS values were averaged and fitted by isotherm equation; solid et al., 2007).
lines represent the GAB model fitted to the data. (A) HPMC film; (,) HPMC þ nisin;
HPMC þ glycerol (:); HPMC þ glycerol þ nisin (); HPMC þ lecithin þ nisin (B);
HPMC þ nisin emulsion (6); and HPMC þ liposomal nisin (-). 3.9. Optical properties

had the highest water affinity due to the large amount of hydro- 3.9.1. Transparency attributes
philic groups. The difference in water holding capacity of glycerol The color and transparency are pertinent properties since they
containing films as compared to control films can be clearly have a direct impact on the consumer acceptability and quality of
observed after the initial phase of water molecules fixation at the packaged product. Indeed, a consumer will choose a food
monolayer. The active (F2, F6) and nano-active (F8, F9) composite product with a transparent package through which the food is
films had shown non-significant difference of moisture adsorption visible to judge its visual quality, rather than food that has an
compared to pure HPMC films in the complete range of aw, with opaque package. It is observed that HPMC film has given excellent
least water-binding capacity for nisin encapsulated in nanoparticles transparency of film, while lecithin incorporation significantly
emulsion. The lower water-binding capacity could be due to inter- decreased light transmittance (Fig. 7). The treatment containing
actions between the liposomes and the hydrophilic sites of the glycerol had lower transmission than the control film and it is
HPMC chain, which substitute the HPMCewater interactions that further reduced non-significantly when nisin is added to HPMC
predominate in control films. Water adsorption in the high relative film. Generally plasticizer are incorporated in composite films to
humidity range was identical for nano-emulsion or direct lecithin improve the light transmission, however HPMC native films
incorporation. Thus lecithin incorporation (direct or liposome) in demonstrates such an exceptional transparent feature that
HPMC film slightly decrease the moisture adsorption as compared composite film having plasticizers or active agents are relatively
to control film. However, the above-mentioned increase in WVP for less transparent (Imran, El-Fahmy, et al., 2010) (Table 3). Indirectly,
HPMCelecithinenisin composite film could arise due to non- this supports the idea of using HPMC as biopolymer films for food.
homogenous dispersion of lecithin as compared to nano- In nano-active films, the nanoliposome/emulsion incorporation
emulsions in HPMC native network, this irregularity could have reduced the film transparency; however the transparency value at
weaken the film barrier responsible for WVP increase in plasticized 600 nm had shown that it is significantly higher (Tukey test,
active films.
The GAB model was used to fit the DVS data of the composite 80
films. The value of monolayer water content (Xm) is of particular 70
interest, as it refers to strongly adsorbed water to specific sites and is
60
considered as optimal value at which the film is most stable. Xm
value of 4.99  0.19 g/100 g for HPMC film was observed (Table 2) 50
which is close to the previous findings (Imran, El-Fahmy, et al., 2010;
40
Villalobos et al., 2006). Glycerol increased the Xm values for plasti-
cized films. For active films containing nisin in free or encapsulated 30
form, the values of Xm were quite similar. The constant C, related to
20
the total heat sorption, decreased markedly for glycerol plasticized
films. Its decrease suggested that polyol may perhaps occupy 10
sorption sites of polymer and in consequence reduced the bonding 0
energy. The nano-active films, however, had higher bonding energy 100 200 300 400 500 600 700 800 900 1000
for water molecules as exhibited by control also.
Fig. 7. Transmission of light (%T) in UV, visible and NIR spectra through biodegradable
3.8. Oxygen permeability films. Non-active films, (A) HPMC film, (B) HPMC þ glycerol, (:) HPMC þ lecithin,
() HPMC þ liposome; active films, (>) HPMC þ nisin, (C) HPMC þ glycerol þ nisin,
(6) HPMC þ lecithin þ nisin; nano-active films, (þ) HPMC þ nisin emulsion (50%
The addition of plasticizers increased the oxygen permeability encapsulated), ( ) HPMC þ liposomal nisin (100% encapsulated). Mean of triplicate
(OP), as lecithin and the glycerol can modify the basic structure of analysis.
 M. Imran et al. / Food Hydrocolloids 29 (2012) 407e419 417

Table 3
Whiteness index (WI), a and b color values and light transparency of composite active films as affected by nisin, plasticizer or both at macro and nano-scale.

Formulations Transparency Color whiteness index (WI) Chromatic coordinates a* b*

log (T600  X)
F1 HPMC 2.22  0.02 32.0  0.1 0.18  0.01 0.38  0.03
F2 HPMC þ N 2.13  0.01 32.0  0.1 0.2  0.01 0.42  0.05
F3 HPMC þ G 2.14  0.01 31.7  0.1 0.18  0.01 0.37  0.07
F4 HPMC þ GþN 2.05  0.01*** 32.2  0.2 0.21  0.02 0.39  0.02
F5 HPMC þ lecithin 1.76  0.09*** 32.1  0.5 0.06  0.03*** 1.7  0.23***
F6 HPMC þ lecithin þ N 1.78  0.01*** 30.7  0.3 0.2  0.01 0.39  0.03***
F7 HPMC þ emulsion 1.99  0.01*** 31.6  0.4 0.07  0.05*** 1.33  0.21***
F8 HPMC þ N (emulsion) 1.86  0.03*** 31.4  2.3 0.33  0.04*** 1.3  0.38***
F9 HPMC þ N (liposome) 2.06  0.01*** 27.6  1.1 0.23  0.02 0.8  0.01*

Non-active, F1, F3, F5, F7; active, F2, F4, F6; nano-active, F8, F9.
HPMC, hydroxypropyl methylcellulose; N, nisin 1 mg mL 1, i.e. w104 IU; G, glycerol (30% w/w D.M.); lecithin, 2.5% w/v; N (emulsion), nisin 50% encapsulated and 50% free
form; N (liposome), nisin 100% encapsulated.
Dunnett test, *p < 0.05; ***p < 0.001.

p < 0.001) as compared to the use of lecithin at the macro scale between green and blue, and yellow and red respectively. The L*
(direct incorporation). It can be suggested that the structurization parameter values ranged between 29 and 32 (data not shown),
of lecithin into nano-scale liposomes for nisin encapsulation which is not significant variation. Similarly, the whiteness index
increases the transparency aspect. Comparing the transmittance of (WI) due to the changes in diffuse reflectance provoked by light
UV region with visible region, we observed that the percentage of scattering had not shown significant variations. However, the
transmittance was lower at 210 nm, signifying that composite films parameter b*, which gives the variation of color from yellow to red is
of HPMC have a good preventive ability against middle ultraviolet more critical: as one can observe data values range
MUV radiations (200e300 nm). from 0.37  0.07 to 1.7  0.23 (Table 3). Treatments having
a positive value contained lecithin, and therefore had a yellow color.
3.9.2. Color attributes The restructuring of the lecithin into nanoliposomes encapsulating
Addition of various compounds that structurally bind with film nisin significantly decreased the value of the parameter b (Tukey
forming solution could change the native color of the film (Rhim, test, p < 0.001). Moreover, as nisin gives a white color, it masks the
Gennadios, Handa, Weller, & Hanna, 2000). The parameters L*, a* yellow color of film. Therefore active films containing nisin were less
and b* had been measured corresponding luminosity, variation yellow than non-active composite treatments.

Fig. 8. (A) Growth kinetics of Listeria monocytogenes incubated with different film forming solutions at MIC dilutions for more than 3 days at 37  C. The film forming solutions; ( )
control, ( ) HPMC, ( ) HPMC þ nisin, () HPMC þ liposome-encapsulated nisin, and (B) HPMC þ nisin emulsion (free and encapsulated nisin). (B) The inhibition zone of the
growth of L. monocytogenes CIP 82110T inoculated in TSA-YE by nisin containing active films: from left to right e non-active HPMC film; nisin active HPMC film; liposome-
encapsulated nisin embedded HPMC film; nisin emulsion (free and encapsulated nisin) embedded HPMC film at 37  C with overnight incubation.
418 M. Imran et al. / Food Hydrocolloids 29 (2012) 407e419

3.10. Antimicrobial effectiveness similar to native HPMC films and significantly improved than using
lecithin directly without nano-scale restructuring. However, the
As an initial test for the antilisterial activity, different film tensile strength and percentage elongation were decreased signif-
forming solutions (corresponding MIC) were tested on inoculated icantly compared to the control film of HPMC. The results of sorp-
tryptic soya broth (TSB-YE) medium. As expected, HPMC film tion isotherms of water and GAB modelisation have shown a slight
forming solution alone had not shown antimicrobial action against improvement in resistance to water adsorption when nisin was
L. monocytogenes as compared to control with no FFS added (Fig. 8). embedded as nano-emulsion in HPMC matrix. The observation of
Active HPMC film forming solution with nisin inhibited the listerial the structure and morphology of the film by SEM and TEM estab-
development up to 24 h but afterward there was gradual growth of lished the new concept of biodegradable packaging containing
bacteria due to nisin deactivation and bacterial resistance. The nano-encapsulated nisin may help to improve the slow release of
nano-active FFS with liposome-encapsulated nisin had demon- the active agent. The antimicrobial results suggested that incor-
strated lesser antimicrobial activity as bacterial growth started poration of nisin in nano-emulsion form (encapsulated and free)
before 10 h of incubation. Nevertheless the bacterial growth was can possibly be an overall effective approach to control food
reduced by half (peak values) as compared to control. This reduc- pathogen without compromising the basic physico-chemical
tion in pathogen growth was due to nisin controlled release from attributes of composite HPMC biodegradable films.
liposome and probably as a result of active-nanoliposomes inter-
actions with bacteria. However nano-emulsion film forming solu- Acknowledgments
tion (Fig. 8A) with free and encapsulated nisin had indicated better
control of pathogen as compared to 100% encapsulated nisin and The authors acknowledge the technical assistance of Alain
free nisin. This effect is possibly due to the fact that free nisin Kohler, Jacqueline Chanél, Dr. Jaafar Ghanbaja and Dr. Catherine
controlled the initial bacterial growth burst and eventually fresh Strazielle for their help to accomplish scanning and electron
nisin release from liposome and nanoliposome interactions with microscopy at SCMEM and Faculté de Medecine, Nancy, France.
L. monocytogenes could have improved the antimicrobial potential.
The interaction of liposome with target cells can occur by
References
adsorption onto the cell surface, fusion with the cell membrane,
release of drug by micro-pinocytosis, or due to a specific or Andreuccetti, C., Carvalho, R. A., & Grosso, C. R. F. (2009). Effect of hydrophobic
nonspecific endocytosis (Torchilin, 2005). To exhibit the actual plasticizers on functional properties of gelatin-based films. Food Research
International, 42(8), 1113e1121.
potential of these nano-active films for improving control over
Benech, R. O., Kheadr, E. E., Lacroix, C., & Fliss, I. (2002). Antibacterial activities of
Listeria monocytogenes, different films of 1 cm2 were tested on nisin Z encapsulated in liposomes or produced in situ by mixed culture during
inoculated tryptic soya agar (TSA) medium. As expected, non- Cheddar cheese ripening. Applied and Environmental Microbiology, 68(11),
active HPMC had not given any inhibition zone (Fig. 8B) against 5607e5619.
Bifani, V., Ramirez, C., Ihl, M., Rubilar, M., Garcia, A., & Zaritzky, N. (2007). Effects of
L. monocytogenes. Active HPMC film with nisin produced inhibition murta (Ugni molinae Turcz) extract on gas and water vapor permeability of
zone slightly larger than actual covered size by film due to release carboxymethylcellulose-based edible films. LWT e Food Science and Technology,
and diffusion of nisin in the nutrient medium. The nano-active 40(8), 1473e1481.
Bilbao-Sainz, C., Avena-Bustillos, R. J., Wood, D. F., Williams, T. G., & McHugh, T. H.
films had demonstrated different activity, as nano-emulsion film (2010). Composite edible films based on hydroxypropyl methylcellulose
with free and encapsulated nisin had indicated better control of reinforced with microcrystalline cellulose nanoparticles. Journal of Agricultural
pathogen as compared to 100% encapsulated nisin (Fig. 8B). Thus, and Food Chemistry, 58(6), 3753e3760.
Breukink, E., & De Kruijff, B. (1999). The lantibiotic nisin, a special case or not?
both the antibacterial tests in broth and solid medium had shown Biochimica et Biophysica Acta e Biomembranes, 1462(1e2), 223e234.
that the strategy of incorporating encapsulated and free nisin Breukink, E., & de Kruijff, B. (2006). Lipid II as a target for antibiotics. Nature Reviews
either directly in growth medium or embedded in bioactive films Drug Discovery, 5(4), 321e323.
Breukink, E., Ganz, P., De Kruijff, B., & Seelig, J. (2000). Binding of nisin Z to bilayer
in-contact with nutrient medium could control efficiently the
vesicles as determined with isothermal titration calorimetry. Biochemistry,
outburst of food pathogen. 39(33), 10247e10254.
Cao-Hoang, L., Chaine, A., Grégoire, L., & Waché, Y. (2010). Potential of
nisin-incorporated sodium caseinate films to control Listeria in artificially
4. Conclusion
contaminated cheese. Food Microbiology, 1e5.
Chen, C. H., & Lai, L. S. (2008). Mechanical and water vapor barrier properties of
Due to novel, formerly unknown, properties attributed to tapioca starch/decolorized hsian-tsao leaf gum films in the presence of
engineered nanoparticles many new products are introduced in the plasticizer. Food Hydrocolloids, 22(8), 1584e1595.
Davies, E. A., Milne, C. F., Bevis, H. E., Potter, R. W., Harris, J. M., Williams, G. C., et al.
agro-food area. Liposomes are recognized as an excellent carrier for (1999). Effective use of nisin to control lactic acid bacterial spoilage in
controlled release and target delivery of numerous active agents vacuum-packed bologna-type sausage. Journal of Food Protection, 62(9),1004e1010.
(Mozafari, 2006; Mozafari et al., 2008). However, one major limi- de Moura, M. R., Aouada, F. A., Avena-Bustillos, R. J., McHugh, T. H., Krochta, J. M.,
et al. (2009). Improved barrier and mechanical properties of novel
tation is that they have a tendency to leak and lose encapsulated hydroxypropyl methylcellulose edible films with chitosan/tripolyphosphate
components over time, moreover, gradual coalescence and desta- nanoparticles. Journal of Food Engineering, 92(4), 448e453.
bility may occur in food system (Laye, McClements, & Weiss, 2008). Debeaufort, F., & Voilley, A. (1995). Methyl cellulose-based edible films and coatings
I. Effect of plasticizer content on water and 1-octen-3-ol sorption and transport.
Thus innovative approach of fusion of two concepts for improved Cellulose, 2(3), 205e213.
bioavailability i.e. AMP nanoencapsulation and biopolymer immo- Degnan, A. J., Buyong, N., & Luchansky, J. B. (1993). Antilisterial activity of pediocin
bilizing to formulate the next generation biodegradable films AcH in model food systems in the presence of an emulsifier or encapsulated
within liposomes. International Journal of Food Microbiology, 18(2), 127e138.
embedded with either active agent, nano-encapsulated active Ercolini, D., Ferrocino, I., La Storia, A., Mauriello, G., Gigli, S., Masi, P., et al. (2010).
agent or both of them had resolved liposome delivery limitations. Development of spoilage microbiota in beef stored in nisin activated packaging.
For active films, nisin had demonstrated no negative impact on Food Microbiology, 27(1), 137e143.
Fabra, M. J., Jiménez, A., Atarés, L., Talens, P., & Chiralt, A. (2009). Effect of fatty acids
transparency, thickness and water sorption behavior. However, the
and beeswax addition on properties of sodium caseinate dispersions and films.
presence of plasticizer glycerol significantly improved the film Biomacromolecules, 10(6), 1500e1507.
elastic capacity, but negatively altered the permeability and tensile Fang, Y., Tung, M. A., Britt, I. J., Yada, S., & Dalgleish, D. G. (2002). Tensile and barrier
strength. For nano-active films, the results clearly showed that properties of edible films made from whey proteins. Journal of Food Science,
67(1), 188e193.
different physico-chemical properties including barrier (oxygen Hambleton, A., Debeaufort, F., Bonnotte, A., & Voilley, A. (2009). Influence of
and water vapor permeability), color and transparency remained alginate emulsion-based films structure on its barrier properties and on the
 M. Imran et al. / Food Hydrocolloids 29 (2012) 407e419 419

protection of microencapsulated aroma compound. Food Hydrocolloids, 23(8), Pérez-Gago, M. B., & Krochta, J. M. (2001). Lipid particle size effect on water vapor
2116e2124. permeability and mechanical properties of whey protein/beeswax emulsion
Han, J. H., & Floros, J. D. (1997). Casting antimicrobial packaging films and films. Journal of Agricultural and Food Chemistry, 49(2), 996e1002.
measuring their physical properties and antimicrobial activity. Journal of Plastic Rhim, J. W., Gennadios, A., Handa, A., Weller, C. L., & Hanna, M. A. (2000). Solubility,
Film and Sheeting, 13(4), 287e298. tensile, and color properties of modified soy protein isolate films. Journal of
Imran, M., El-Fahmy, S., Revol-Junelles, A. M., & Desobry, S. (2010). Cellulose Agricultural and Food Chemistry, 48(10), 4937e4941.
derivative based active coatings: effects of nisin and plasticizer on physico- Roberts, R. F., & Zottola, E. A. (1993). Shelf-life of pasteurized process cheese spreads
chemical and antimicrobial properties of hydroxypropyl methylcellulose made from cheddar cheese manufactured with a nisin-producing starter
films. Carbohydrate Polymers, 81(2), 219e225. culture. Journal of Dairy Science, 76(7), 1829e1836.
Imran, M., Revol-Junelles, A. M., Martyn, A., Tehrany, E. A., Jacquot, M., & Desobry, S. Sanchez-Gonzalez, L., Chafer, M., Chiralt, A., & Gonzalez-Martinez, C. (2010). Phys-
(2010). Active food packaging evolution: transformation from micro- to nano- ical properties of edible chitosan films containing bergamot essential oil and
technology. Critical Reviews in Food Science and Nutrition, 50(9), 799e821. their inhibitory action on Penicillium italicum. Carbohydrate Polymers, 82(2),
Joerger, R. D. (2007). Antimicrobial films for food applications: a quantitative analysis 277e283.
of their effectiveness. Packaging Technology and Science, 20(4), 231e273. Sanchez-Gonzalez, L., Vargas, M., Gonzalez-Martanez, C., Chiralt, A., & Chafer, M.
Khosravi-Darani, K., Pardakhty, A., Honarpisheh, H., Rao, V. S. N. M., & (2009). Characterization of edible films based on hydroxypropylmethylcellulose
Mozafari, M. R. (2007). The role of high-resolution imaging in the evaluation of and tea tree essential oil. Food Hydrocolloids, 23(8), 2102e2109.
nanosystems for bioactive encapsulation and targeted nanotherapy. Micron, Sanjurjo, K., Flores, S., Gerschenson, L., & Jagus, R. (2006). Study of the perfor-
38(8), 804e818. mance of nisin supported in edible films. Food Research International, 39(6),
Khwaldia, K., Banon, S., Desobry, S., & Hardy, J. (2004). Mechanical and barrier 749e754.
properties of sodium caseinateeanhydrous milk fat edible films. International Sebti, I., Ham-Pichavant, F., & Coma, V. (2002). Edible bioactive fatty acid-cellulosic
Journal of Food Science and Technology, 39(4), 403e411. derivative composites used in food-packaging applications. Journal of Agricul-
Laridi, R., Kheadr, E. E., Benech, R. O., Vuillemard, J. C., Lacroix, C., & Fliss, I. (2003). tural and Food Chemistry, 50(15), 4290e4294.
Liposome encapsulated nisin Z: optimization, stability and release during milk Taylor, T. M., Bruce, B. D., Weiss, J., & Davidson, P. M. (2008). Listeria monocytogenes
fermentation. International Dairy Journal, 13(4), 325e336. and Escherichia coli O157:H7 inhibition in vitro by liposome-encapsulated nisin
Laye, C., McClements, D. J., & Weiss, J. (2008). Formation of biopolymer-coated and ethylene diaminetetraacetic acid. Journal of Food Safety, 28(2), 183e197.
liposomes by electrostatic deposition of chitosan. Journal of Food Science, 73(5). Taylor, T. M., Gaysinsky, S., Davidson, P. M., Bruce, B. D., & Weiss, J. (2007). Char-
Marcos, J. F., & Gandia, M. (2009). Antimicrobial peptides: to membranes and acterization of antimicrobial-bearing liposomes by zeta potential, vesicle size,
beyond. Expert Opinion on Drug Discovery, 4(6), 659e671. and encapsulation efficiency. Food Biophysics, 2(1), 1e9.
Mastromatteo, M., Conte, A., & Del Nobile, M. A. (2010). Combined use of modified Torchilin, V. P. (2005). Recent advances with liposomes as pharmaceutical carriers.
atmosphere packaging and natural compounds for food preservation. Food Nature Reviews Drug Discovery, 4(2), 145e160.
Engineering Reviews, 2(1), 28e38. Villalobos, R., Chanona, J., Hernandez, P., Gutierrez, G., & Chiralt, A. (2005). Gloss
Mozafari, M. R. (2006). Bioactive entrapment and targeting using nanocarrier and transparency of hydroxypropyl methylcellulose films containing
technologies: an introduction. In M. R. Mozafari (Ed.), Nanocarrier technologies: surfactants as affected by their microstructure. Food Hydrocolloids, 19(1),
Frontiers of nanotherapy (pp. 1e16). The Netherlands: Springer. 53e61.
Mozafari, M. R., Johnson, C., Hatziantoniou, S., & Demetzos, C. (2008). Nano- Villalobos, R., Hernandez-Munoz, P., & Chiralt, A. (2006). Effect of surfactants on
liposomes and their applications in food nanotechnology. Journal of Liposome water sorption and barrier properties of hydroxypropyl methylcellulose films.
Research, 18(4), 309e327. Food Hydrocolloids, 20(4), 502e509.
O’Sullivan, L., Ross, R. P., & Hill, C. (2002). Potential of bacteriocin-producing lactic Wu, Y., & Wang, T. (2003). Phospholipid class and FA compositions of modified
acid bacteria for improvements in food safety and quality. Biochimie, 84(5e6), soybeans processed with two extraction methods. JAOCS, Journal of the Amer-
593e604. ican Oil Chemists’ Society, 80(2), 127e132.