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Food Packaging and Shelf Life 11 (2017) 98–105

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Food Packaging and Shelf Life


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

Edible nano-bio-composite film cargo device for food packaging


applications
Aalok Basua , Sonia Kundua , Santanu Sanaa , Asim Haldera , Md Farooque Abdullaha,b ,
Sriparna Dattaa , Arup Mukherjeea,b,*
a
Division of Pharmaceutical & Fine Chemical Technology, Department of Chemical Technology, University of Calcutta, 92 A.P.C. Road, Kolkata 700 009, India
b
Centre for Research in Nanoscience & Nanotechnology, University of Calcutta, JD-2 Salt Lake, Kolkata 700098, India

A R T I C L E I N F O A B S T R A C T

Article history:
Received 17 August 2016 This work is meant to design newer nano-bio-composite films for food packaging applications.
Received in revised form 28 December 2016 Polylactide nanoparticles non-covalent interaction in chitosan matrix was carefully studied and used
Accepted 18 January 2017 further to develop uniform nano-bio-composite films. Environmentally benign materials and processes
Available online 26 January 2017 were applied throughout for an intended application in packaging of edibles. Nanoparticles array laden in
polysaccharide films served as cargo loaders for active molecules. Quercetin, a bioflavonoid, ubiquitous in
Keywords: many plant species was used as model cargo molecule. Sustained quercetin release imparted a
Nano-bio-composite film synchronized anti-microbial and antioxidant properties in finished packaging films. Microstructures
Polylactide nanoparticles
when examined in FESEM and AFM, suggested nanoparticles homogenous dispersion throughout the
Chitosan
film matrix. Film surface RMS roughness in tapping mode AFM experiment was recorded at
Quercetin
1.63  0.23 nm which typically represented conditions for microbial deterrence. Control chitosan film
radical scavenging capacity was 5.8% in DPPH assay while that was recorded at 23.5% in new active
packaging films.
© 2017 Elsevier Ltd. All rights reserved.

1. Introduction 2007). Most sustainable materials however failed to serve stand


alone due to constrains in filming and barrier properties. Water
Primary packaging materials for food, water and health swell-able starch films and water insoluble guar gum films are
products mostly originate from the fossil derived polymers. known, but both hinders significantly in the moisture and oxygen
Packaging plastics such as polyethylenes, polypropylene, or the passage (Das, Ara, Dutta, & Mukherjee, 2011). This work is meant to
polystyrenes are convenient, but pose imminent health hazards develop newer nano-bio-composite films using poly-lactides
due to migration of toxic additives into the consumables. Recycling (PLAs) interactions in chitosan for safe packaging design.
of plastics increases leaching of additives and results in adverse Polymer nanotechnology is a vibrant area which attempts to
biological interactions. Potential carcinogens like bisphenol A, develop materials loaded with specialized particulates in nano-
diffusing from polymer wastes into the water and environment, are scale (Rhim, Park, & Ha, 2013). Biopolymer nano-bio-hybrids were
quite well known (Fasano, Bono-Blay, Cirillo, Montuori, & Lacorte, studied extensively earlier for food packaging applications
2012). Sustainable biopolymers such as chitosan, guar gum and (Kochumalayil et al., 2013). Chitosan films laden with cellulose
starch are experimented as compatible and safe alternatives for nanocrystals improved film permeation properties. Nano-bio-
packaging of edibles (Danga and Yoksan, 2015; Mikkonen et al., composites are newer class of biopolymer matrices which apply
polymer nanoparticle physico-chemical interactions for superior
materials design. High surface area of incorporated particulates
helps in improvement of biopolymer thermal and barrier
* Corresponding author at: Division of Pharmaceutical & Fine Chemical properties (Konwar, Gogoi, Majumdar, & Chowdhurya, 2015).
Technology, Department of Chemical Technology, University of Calcutta, 92 A.P.C.
Non-covalent polymer functional interactions in nanoscale are
Road, Kolkata 700 009, India.
E-mail addresses: arupm1234@gmail.com, amchemtech@caluniv.ac.in some of the predictable techniques for remarkable property
(A. Mukherjee). enhancement.

http://dx.doi.org/10.1016/j.fpsl.2017.01.011
2214-2894/© 2017 Elsevier Ltd. All rights reserved.
A. Basu et al. / Food Packaging and Shelf Life 11 (2017) 98–105 99

Chitosan exhibits good film present-ability, safety, edibility and stirring and 0.03 g of glycerol for each 10 g of biopolymer was
inherent antimicrobial properties. The cationic polysaccharide is added as plasticizer. Nano-bio-composite films were simulta-
obtained from the exoskeletons of crustaceans like crabs and neously prepared after homogeneous dispersion of Plns. Solutions
shrimps. The compound is however hydrophilic and a poor barrier were degassed under reduced pressure and poured onto polypro-
for moistures. Somewhat dormant antimicrobial property of pylene Petri-dishes for drying at 60  2  C for 3 h in air circulating
chitosan is reported, but the means and mechanisms are not well oven. Final films were removed easily from the Petri-dishes. The
understood. Chitosan therefore expresses properties akin to a food films were conditioned for 48 h at 23  2  C, 70  2% relative
packaging materials if only some of its fundamental characteristics humidity (RH) and used for further studies.
can be enhanced. PLAs are safe biopolymers originating from
catalyzed polymerization of lactic acid monomers. Unlike chitosan,
2.4. Quercetin quantification
PLAs are more hydrophobic at the surface and carry anionic
charges. Quercetin (Qr), is a bio-flavonoid available abundantly in
All quantifications for Qr were carried out in reverse phase
foods and vegetables such as onion, broccoli and grapes. Qr posses
HPLC system, Waters dual pump 515 (Waters, USA) using
strong DNA gyrase inhibitory property and is a proven limiting
acetonitrile: water (40:60, v/v) at a flow rate of 1 mL min1. The
agent against microbial growth. Furthermore, Qr finds use as a
analysis was carried out in C18 column (Phenomenex, USA)
powerful anti-oxidant in dairy products, packaged cereals, and
250  4.6 mm, and PDA detector (2996, Waters, USA) set at 255 nm.
processed foods (Harwood et al., 2007). The bioflavonoid is,
A peak area (y) vs. concentration (x) graph, y = 40367 x  38914,
however, unstable when exposed to pH variations, chemical
R2 = 0.999, was first developed from standard injections and used
environment and light (Scalia & Mezzena, 2009). We postulated
for Qr analysis throughout.
that Qr when entrapped in PLA nanoparticles may express
enhanced bioactivity and stability. Furthermore, Qr cargo nano-
2.5. Characterization of Pln nanoparticles
particles interactions in the chitosan film matrix can be applied
favourably for active films design.
The particle size, polydisperisty index (PDI) and zeta potential
This work is aimed at Qr cargo PLA nanoparticles synthesis and
of Plns were determined in a Zetasizer1 Nano ZS (Malvern
homogeneous embedding in chitosan matrix. Biocompatible
Instruments, UK). Helium–neon laser beam, 4 mW, 633 nm, was
surfactant Pluronic F127 was used for nanoparticles stabilizer in
applied against back scattering angle of 173 . Batch measurements
synthesis and filming. Qr served as a molecular cargo loader. Low
were performed in triplicate and the results were averaged for
ebb Qr release over a prolonged period was considered to
comparison. For determination of zeta potentials, Pln suspension
contribute in antimicrobial and antioxidant film active surface
was filled in disposable dip cells and electrophoretic mobility was
properties. The nano-bio-composite film is proposed as a safe
recorded against an applied electrical field. Cargo mass loading was
alternative for packaging of edibles.
estimated by Qr analysis before and after nanoparticulation in the
supernatant. Percent encapsulation efficiency (% EE) and Qr mass
2. Experimental
loading efficiency (% LE) were calculated from an average of six
batch experiments.
2.1. Materials
Qr Mas s entrapped in Plns
%EE ¼  100 ð1Þ
Poly-Lactide (PLA) (Resomer1 L210S) was received as a gift Qr Mas s initially taken
from Evonik Industries, Germany) and the medium molecular
weight, 75–85% deacetylated chitosan was purchased from Sigma-
Aldrich, (Bangalore, India). Pluronic F127 surfactant, bioactive Qr Mas s entrapped in Plns
%LE ¼  100 ð2Þ
compound quercetin and glycerol, were purchased from Sigma- Total weight of Plns
Aldrich (Bangalore, India). Glacial acetic acid, fused calcium
Qr release from Plns was estimated using dialysis bags, having
chloride, solvents and HPLC grade water were procured from
molecular weight cut off 12,400 (Sigma, USA). Typically, 0.0107 g of
Spectrochem Pvt. Ltd., (Mumbai, India). The microbial media
Plns were weighed accurately, (Sartorius MSE3.6P-000-DM,
constituents such as agar-agar, beef extract and peptone were from
Sartorius AG, Germany) dispersed in 1 mL of 100 mM, pH 7.4
Himedia Laboratories (Mumbai, India).
phosphate buffer and transferred into end-tied dialysis bags.
Individual bags were placed in glass vials containing 35 mL of
2.2. Synthesis of poly-lactide nanoparticles
phosphate buffer added with 10% v/v ethanol (Licciardello,
Wittenauer, Saengerlaub, Reinelt, & Stramm, 2015). Glass vials
Poly-lactide nanoparticles were prepared following oil-in-
were put to shaking at 50 rpm in shaker incubators maintained at
water emulsion evaporation technique (Das et al., 2014). Briefly,
25  2  C. Dissolution samples were withdrawn from the vials at
PLA (10 mg) and Qr (2 mg) were dissolved together in 1 mL of
predetermined time intervals and were replaced with same 5 mL
methylene chloride and was added into 10 mL of 1% w/v aqueous
portion of fresh medium to maintain sink conditions. Qr release at
Pluronic F-127 solution. The mixture was sonicated under probe
each time point was estimated from 20 mL aliquot injections into
sonicator (Sonic, USA) for an emulsion. The emulsion was further
HPLC set up (2.4). Cumulative percentage release over time was
diluted to 20 mL in water and kept stirring for 3 h over magnetic
calculated from six batch experiments and the data was averaged
stirrer at 25  C. The final Qr loaded nanoparticles (Pln), were
for x-y plots.
separated by centrifugation at 16,000 rpm for 45 min at 4  C,
washed twice with water, re-centrifuged and preserved in
2.6. Nano-bio-composite film characterization
desiccators.

Film thicknesses were estimated using a 0–25 mm dial


2.3. Chitosan nano-bio-composite films
thickness gauge with 0.01 mm accuracy. Thickness at six
positions for each of six film samples was recorded and the
Chitosan films with or without the nanoparticles were prepared
average value was used for calculation of opacity and water vapour
following solvent casting technique. Chitosan (1.5% w/v) was
permeability. The film opacity was estimated from the absorbance
dissolved in 0.5% (v/v) aqueous acetic acid solution under magnetic
100 A. Basu et al. / Food Packaging and Shelf Life 11 (2017) 98–105

of six samples at 660 nm (UV 2550, Shimadzu, Japan). Total soluble 2.10. Film anti-oxidant capacity
matter (TSM) was measured by immersion of the film (2 cm x 2 cm)
for 24 h in 50 mL of distilled water placed over an orbital shaker at Film antioxidant capacity at different time points were

120 rpm at 25  2  C. TSM was calculated after drying at 110 C and estimated following DPPH (2, 2-diphenylpicryl hydrazyl) free
expressed as percentage of the original dry weight. Moisture radical scavenging assay (Dinis, Madeira, & Almeida, 1994). The
content was estimated by measuring the weight loss of films upon nano-bio-composite and bare chitosan film were cut in 75 mm
drying in an oven at 110  5  C for 24 h. diameters, weighed and placed separately in glass containing
Water vapour permeability (WVP) for the bare chitosan film 35 mL phosphate buffer added with 25% v/v ethanol. The glass vials
and Pln laden ones were determined following a method described were put to shaking at 50 rpm in shaker incubator at 25  2  C.
earlier (Das & Mukherjee, 2012). Briefly, 3 g fused calcium chloride Analysis samples were prepared by mixing 1 mL solution of DPPH
was placed in each of the two matched diffusion cups. Uniform and 50 mL of the solution aliquot withdrawn at predetermined
circular film of surface area 0.000962 m2 was placed to cover the time intervals. The reaction mixtures were shaken and kept in dark
analysis cups. The cup joints were sealed using vacuum grease and for 15 min and UV absorbance were recorded at 516 nm. The free
weighed. The cups were placed in humidity chambers maintained radical scavenging activity in percentage was derived and plotted
at 25  2  C, 85% RH and were weighed every 24 h until constant for comparative evaluations.
weight. The cup weight increase over time was plotted and WVP
was calculated from the linear portion slope. 2.11. Film anti-microbial property
The thermal properties of the film types were analysed at a
heating rate of 10  C/min in a DT-TGA analyzer (DTG 60 Shimadzu, Antimicrobial growth retardant property was analysed quali-
Japan) under nitrogen flow. X-Ray Diffraction (XRD) patterns were tatively on agar diffusion plates and the viable cell count
recorded in a Philips Model PW 3040/60 XPERT PRO (Philips, experiments were performed for quantitative estimations
Netherlands) operated at 40 kV, scanning rate 8 /min in 0.03 steps (Rojas-Grau et al., 2006). Staphylococcus aureus MTCC 3160 was
per second. Film tensile strength was recorded using a universal used as the standard organism for antimicrobial experiments. For
testing machine 5500R (Instron, USA). Film samples were cut into qualitative evaluations, bacterial culture was first prepared by
60 mm long and 10 mm wide pieces and placed between the flooding 500 mL of 106 cfu mL1 inoculums on Petri dishes
pneumatic jaws of the tester. The initial grip separation was set to containing sterile nutrient agar medium and the cells were
22 mm and crosshead speed 50 mm/min, with 150 kgf load cell. allowed to grow for 6 h. Both film types were cut in strips
Samples were analysed six times to confirm repeatability. (2 cm  4 cm) and placed aseptically on to the plates and incubated
Qr diffusive dissolution from the nano-bio-composite films in inverted position for 24 h at 37  2  C. Growth or no growth
were recorded in a procedure similar to that under Section 2.5. A regions were read and marked under a plate reader for
circular portion of the film 75 mm diameter was cut weighed and comparative purposes.
placed in individual dialysis bags. The dialysis bags were placed in For quantitative estimations, uniformly cut test films (2 cm  4
glass vials containing 35 mL of phosphate buffer. The glass vials cm) were placed in screw capped bottles containing 9 mL of sterile
were put to gentle shaking at 50 rpm in a shaker incubator. saline. Test organism, 1 mL, 1 106 cfu mL1 from broth was
Dissolution samples were withdrawn at predetermined time transferred aseptically in each bottle placed over a shaker
intervals and the medium was immediately replaced each time. incubator maintained at 50 rpm, 37  2  C. Aliquots solutions of
Qr release was estimated from injections in HPLC set up (2.4) and 0.1 mL were withdrawn at regular intervals from each bottle and
x-y plots developed. plated in duplicate for count of survival colonies (Roy, Knapp,
Guthrie, & Perrier, 2008). Bacterial positive and negative controls
2.7. Fourier transform infrared (FT IR) spectroscopy were also run parallel in sterile saline. Three experiment sets were
conducted for each film types and the results were plotted for
FT-IR spectra were recorded in a Jasco-670Plus FT-IR Instrument observations.
(Jasco, Japan). Spectral acquisitions were carried out at a resolution
of 4 cm1 and an average of 128 scans were recorded over the mid 2.12. Statistical analysis
IR ranges of 4000–400 cm1. Pln embedded or bare chitosan films
were placed directly in the transmission path and the gathered Results were presented as mean standard deviation (SD).
data were stacked in Bio-Rad software (Bio-Rad, KnowItAll, USA) Sigma Plot 6.0 and Origin 6.0 Professional were used for the
for comparison of overlap regions and functional group studies. FT- analysis of the experimental data. Student t-test was conducted for
IR spectra for individual components alone or in combinations comparative studies and the difference was considered significant
were also recorded in KBr pallets. when p < 0.01.

2.8. Electron microscopy


3. Results and discussion
Films were analysed in a field emission scanning electron
3.1. Quercetin loaded poly-lactide nanoparticles
microscope (FESEM, JSM-7600F, JEOL, Japan). Samples were
mounted on an aluminium stub using double sided carbon tape,
Qr cargo PLA nanoparticles were synthesized following facile
sputter coated with platinum at 10 Pa for 2 min and images
emulsion evaporation technique. PLA and Qr were both soluble in
captured at 2 kV exposures.
methylene chloride. Tri-block stabilizer, Pluronic F-127, was
preferred for experimental ease and resultant particle parameters.
2.9. Atomic forced microscopy (AFM) analysis
Hydrodynamic diameter of Plns was recorded at 225  16.23 nm
from six batch experiments. Blank nanoparticles without Qr
Molecular visualization of both the film types was carried out
loading were observed to have an average size of 140  11.39 nm.
using a VEECO Multimode Nanoscope, AFM (VEECO Instrument
This difference was considered due to molecular Qr loading in Plns.
Inc., USA). All measurements were completed in tapping mode
PDI was recorded as 0.359  0.02 and the particle zeta potential
under ambient conditions. The images were further analysed using
was 25.5  1.28 mV. The negative z charge was understandably
Nanoscope Analysis ver. 1.40 software (Bruker Corporation, USA).
A. Basu et al. / Food Packaging and Shelf Life 11 (2017) 98–105 101

Fig. 1. Quercetin release from the Pln nanoparticles and nano-bio-composite films.

due to the pluronics stabilization and polylactide surface water inside the polymeric matrix, followed by disruption of van
carboxylic groups. der Waal’s forces between the polymer chains (Turhan & Sahbaz,
2004). The water solubility value of the films dropped significantly
3.2. Quercetin quantification and nanoparticle efficiency when embedded with Plns. This phenomenon was attributed to the
hydrophobic nature of polylactide nanoparticles and low water
Qr was estimated all through in a reverse phase HPLC set up. Qr penetration due to non-covalent interactions of Plns within the
elution time was 6.8 min and a standard equation developed from chitosan matrix. A reasonable moisture content of primary food
different concentration injections were used for estimations. packaging film is important for quality preservation and film
Supernatants before and after nanoparticle harvesting, were properties (Appelqvist, Cooke, Gidley, & Lane, 1993). In case of the
sampled from each preparation batch and were injected in HPLC nano-bio-composite film the moisture content was lowered to that
for analysis. The entrapment efficiency is a determinant of reasonable level of 22.505  0.431%. It was likely due to favourable
preparation process economics. Pln entrapment efficiency (EE) interactions between the chitosan amine functions and the
recorded was 97.4  0.41% indicating no much loss of cargo negatively charged Plns. This has contributed in reduced availabil-
molecules in process. Nanoparticle loading efficiency (LE) indicate ity of functional groups in the nano-bio-composite for water
the cargo mass entrapped in final particles and that for Pln was at binding. Interestingly, incorporation of Plns has increased water
18.66  0.12%. vapour permeability to a degree. This was considered due to
Qr release from Pln was studied at room temperature (Fig. 1). enhanced water permeation through the chitosan and Pln bonded
The release rate was generally sluggish and biphasic in nature. Qr interfaces. Very similar results were reported earlier in case of
cumulative release from the Pln was recorded as 42% over a period functionalized clay laden chitosan films (Casariego et al., 2009).
of 20 days. In case of the Pln the release phase change appeared at Polylactide nanoparticles being more amorphous, the water
24 h and that was 8 h in case of the nano-bio-composite film. Qr vapour permeability in the new nano-bio-composite was incre-
faced a double diffusion layer in the nano-bio-composite film and mental. Tensile strength of nano-bio-composite film was signifi-
that was considered as one reason for slower and sustained cantly higher than the bare chitosan film (Table 1). This increase in
migration. The Qr bioflavonoid release followed fickian mass tensile properties may be attributed to hydrogen bonds formation
diffusion mechanism. between chitosan amino groups and the Plns surface carboxyl
functions.
3.3. Nano-bio-composite film characterization XRD indicate crystallinity parameters and material purity. The
bare chitosan film expressed characteristics 2u reflections at 20.5 ,
Thin and flexible films were obtained from water dispersions. (Fig. 2), due to the uniform crystal lattice of the biopolymer
Cast nano-bio-composite films were easily removed from the (Rubilar et al., 2013). In case of the nano-bio-composite film the

polypropylene Petri dishes upon drying. The average thickness of embedded poly-lactide nanoparticles were evidenced at 16.5
bare chitosan film was 0.046  0.005 mm while that in nano-bio- (Arrieta et al., 2014). The chitosan peak in the nano-bio-composite

composite film was 0.054  005 mm. The nano-bio-composite but experienced a shift to higher angles at 21.0 for possible
films were uniformly opaque (Table 1). This was likely due to Pln bonding interactions. Cellulose nanoparticles when incorporated
nanoparticles embedding in chitosan matrix. The bare chitosan earlier in chitosan recorded very similar observations (Dehnad,
film expressed notably high water solubility than the nano-bio- Mirzaei, Emam-Djomeh, Seid-Mahdi, & Dadashi, 2014). Cellulose
composite. The biopolymer solubilization involves penetration of nanoparticles contributed in film strength only but cannot be used

Table 1
Film physical properties.

Film type Thickness (mm) Opacity WVP  1011 (g cm1 s1 Pa1) TSM (%) MC (%) Tensile Strength (MPa)
Bare chitosan 0.046  0.005 1.793  0.015 0.7137  0.045 55.245  2.001 31.87  1.074 34.32  0.006
Nano-bio-composite 0.054a  0.005 3.175a  0.195 0.7868a  0.056 28.185a  1.269 22.505a  0.431 36.20a  0.008

[*a, P < 0.01].


102 A. Basu et al. / Food Packaging and Shelf Life 11 (2017) 98–105

decomposition was slower and distinct between 253 and 456  C.


Nanoparticles therefore induced reinforcement and heat resistant
stability due to restrictions in polymer chain mobility (Djidi,
Mignarda, & Tahaa, 2015). The DTA studies mostly corroborated
with the TGA observations. The first loss peaks for both films were
recorded at 147  C while the film intrinsic molecular escapade
observed a shift from 256  C in chitosan to 259  C in the nano-bio-
composite film. This was likely due to nanoparticle reinforced lag
in depolymerization and polymer chain breaks. The idiosyncratic
peak endotherm for the nano-bio-composite film at 419  C
confirmed exactly with the TGA observations for Plns bond
reinforcement within the chitosan film matrix. Similar shifts in
nanostructure incorporated biopolymers were reported amply
elsewhere (Konwar et al., 2015). It was also observed often, that the
inorganic nanostructures like iron oxide or clay increased heat flow
and induced early matrix decomposition while carbon based
nanostructures non-covalent interactions induced matrix heat
stability. In our case Pln nanostructures interaction has assisted in
film thermal stability.
Qr mass release from the nano-bio-composite films was very
Fig. 2. XRD study for bare chitosan and the nano-bio-composite film.
precise and at much lower levels than in Plns (Fig. 1). Qr therefore
experienced additional barriers while diffusing into the release
as cargo loaders. Thermal analysis was performed for both the bare
medium under sink conditions. The releasing profile of Qr
chitosan and the nano-bio-composite film. Chitosan carry water
exhibited satisfactory fit in pseudo-first order kinetic models.
molecules and the first stage characteristic degradation appeared
These observations suggesting that Qr release was predominantly
between 35 and 121  C due to loss of surface bound moistures
controlled by the molecular diffusion from the composite nano-
(Fig. 3A). The second stage of mass loss represented disintegration
bio-polymer matrix. The release data was further fitted in
of the chitosan side chains with loss of intrinsic water molecules
Korsmeyer-Peppas dissolution model (Mt/M1 = ktn) (Ramamoor-
(Konwar et al., 2015). Chitosan final decomposition appeared
thy & Rajiv, 2015). The ‘n’ value recorded for the nano-bio-
between 253 and 370  C which corresponded with depolymeriza-
composite film was 0.16 while that in case of the Plns was 0.21. The
tion, breakdown of the polysaccharide structure and organic
fickian mass diffusion appeared the guiding mechanism for a
degradations. In case of nano-bio-composite films minor change
passive concentration dependent Qr molecular diffusion.
was recorded in the first two TGA stages. The final phase

Fig. 3. Thermal analysis of bare chitosan and the nano-bio-composite film.

Fig. 4. FTIR spectra of A) bare chitosan film, Qr, and nano-bio-composite film, B) PLA, Qr, and Qr loaded Pln, C) PLA, chitosan and mixture.
A. Basu et al. / Food Packaging and Shelf Life 11 (2017) 98–105 103

3.4. FT IR studies Plns indicated loading with no physico-chemical interactions


(Fig. 4B). Additionally, the benzopyran response for Qr appeared at
FT-IR spectra over the mid IR ranges were studied in order to 1513 cm1 alongside polylactide CH2 stretching at 2996 cm1
understand individual components interactions and the final and typical  OH stretching at 3563 cm1 indicating no molecular
nano-bio-composite film characteristic. Bare chitosan films interactions.
exhibited amide peaks at 1656 and 1596 cm1 which corroborated In order to understand chitosan interactions in PLA, we further
well with literature reports (Kosaraju, Weerakkody, & Augustin, compared individual components and different composition
2010) (Fig. 4A). A broad overlap region for  N H and  O H mixtures extensively in middle range FT-IR. Chitosan powder
stretching was recorded at 3200–3500 cm1. In case of the nano- recorded the amide II appeared at 1596 cm1. The broad linear
bio-composite films the peak intensity for the overlap region was O H vibrations were recorded as usual at 3436 cm1. PLA when
much narrowed down and recorded a shift at 3320–3729 cm1. incorporated in chitosan pressed KBr pellets shifts were consis-
This was due to non-covalent conjugations of chitosan functional tently recorded in different vibrations (Fig. 4C). The chitosan amide
vibrations. The amide peaks for both the  C¼O and  N H II was reduced in intensity and observed a shift to 1593 cm1. The
vibration regions of chitosan also observed a shift in case of the PLA ester carbonyl vibrations at 1752 cm1 were also almost
nano-bio-composite films and the corresponding peaks were disappeared when impressed in chitosan film matrix. Interestingly,
recorded at 1663 and 1553 cm1. Under favourable conditions similar observations were recorded during synthesis of different
chitosan can extend hydrogen bonding and charge transfer blended membranes (Wan, Wu, Yu, & Wen, 2006). It is thus
interactions due to polymer–polymer interactions (Yang, Tu, Li, obvious that a favourable casting condition assisted in non-
Shang, & Tao, 2010). Similar recordings for the amide ketone shift covalent interactions for Plns embedded in chitosan film.
to the upfield and the  NH vibrations towards the downfield
were recorded earlier for molecular interactions (BenBettaïeb, 3.5. Film microscopy
Karbowiak, Bornaz, & Debeaufort, 2015). In our case, uniformly
dispersed nanostructured Plns in film matrix has provided for an Surface morphology for the bare chitosan and the nano-bio-
intense force binding throughout, which could be easily recorded composite films were studied in FESEM and the selections were
in the mid range FT-IR studies. The peaks due to Qr however presented in Fig. 5A (i) and (ii). Both film surfaces appeared smooth
appeared at 1667, 1513, and 1317 and 1167 cm1 in the Qr cargo suggesting nano-bio-composite films were uniformly dispersed.
nano-bio-composite film indicating no interactions. AFM studies on the exposed surface in tapping mode were carried
Qr alone in KBr pellets exhibited a broad band at 3368 cm1 due out in order to understand the topographic formations in
to hydroxyl groups and a band for ketone stretching appeared at nanoscale. The nano-bio-composite film appeared comparatively
1667 cm1. The atypical aromatic ring vibrations appeared at contoured at the surface (Fig. 5B (i) & (ii)). The root-mean-square
1513 cm1 while the bands at 1317 and 1167 cm1 were assigned to roughness (Rrms) was determined using a formula:-
Qr, CO C vibrations (Fig. 4A). In case of PLA the  CH2  P
Rrms = [ (Zi  Zave)2/N]1/2
stretching vibrations were recorded at 2996 cm1 and the
characteristic peak due to polylactide carbonyl group appeared Where, Zave is the average of the Z height values within an area, Zi is
at 1757 cm1. Qr is most vulnerable at the redox C3O H position. the point height data, and N representing the number of study
Bioflavonoid Qr peak at 3368 cm1 remained unaffected in loaded points in each square observation. Six film samples were applied to

Fig. 5. Film surface study A) FESEM; B) AFM; (i) bare chitosan film, (ii) nano-bio-composite film.
104 A. Basu et al. / Food Packaging and Shelf Life 11 (2017) 98–105

Fig. 6. DPPH scavenging activity of bare chitosan and the nano-bio-composite film.

determine the film surface roughness parameters. The average attained a plateau near the end of 3 h which corroborated exactly
Rrms value for the chitosan film was recorded at 1.02  0.19 nm with the cargo molecular release observations (Fig. 1). A slow
while that for the nano-bio-composite film was 1.63  0.23 nm. sustained diffusion of molecular cargo from Plns in chitosan matrix
Film topographic fine structures were known to affect bacterial film has ideally contributed in film bio-activity.
population drastically in the vicinity (Thiréa, Simãoa, & Andrade, Qr [2-(3,4-Dihydroxyphenyl)-3,5,7-trihydroxy-4H-1-benzo-
2003). The Rrms above which a statistically significant reduction in pyran- 4-one] is known as one of the most powerful natural
cellular proliferation occurs is nearly 1.1 nm (Qian, Xiao, Zhao, & antioxidants (Zhang et al., 2011). The bioflavonoid expresses
He, 2011). This is due to non-adherence and filming retardation. In vitamin C type concentration dependent antioxidant properties.
our case, the pellet contour structures observed for the nano-bio- Autoxidation of Qr is due to transfer of one electron from the
composite film was due to non-covalent interactions of Plns quercetin anions HOMO levels to the half occupied p* orbitals of
embedded in cast films. Furthermore, Pln hydrophobic formations the dioxygen and subsequent formation of radical anions (Castle &
in the hydrophilic chitosan film structure have contributed in Perkins, 1986). Autoxidation of the next most powerful flavonoid
apposite topographic features culminating in a major deterrent for rutin however, requires much more alkaline conditions. The nano-
microbial growth. bio-composite cargo device thus provided a precise release of Qr
which contributed in film anti-oxidant capacity appropriate in
3.6. Nano-bio-composite film anti-oxidant capacity packaging applications.

Chitosan anti-oxidant capacity depends much upon the surface 3.7. Anti-microbial properties of nano-bio-composite film
available free amino and hydroxyl groups but that occur only in
higher pH ranges. The biopolymer water insolubility at pH > 6.5 is Chitosan expresses a very low ebb anti-microbial effect due to
another major limitation in practicable applications. A significant polycationic properties. At pH 7, chitosan no longer exhibits
and sustained antioxidant capacity was observed in new nano-bio- antimicrobial property due to large number of uncharged surface
composite cargo films (Fig. 6). This was due to low critical amines. The biopolymer develops excellent films and coatings but
concentration of Qr on film surface. The film antioxidant capacity the antimicrobial property disappears when in contact with food

Fig. 7. Quantitative antimicrobial activity of bare chitosan and the nano-bio-composite film.
A. Basu et al. / Food Packaging and Shelf Life 11 (2017) 98–105 105

materials like starch or proteins (Vermeulen, Debevere, & Das, D., & Mukherjee, A. (2012). Biomaterial film for soluble organic sorption and
Devlieghere, 2004). Furthermore, earlier works have clearly anti-microbial activity in water environment. Bioresource Technology, 110, 412–
416.
demonstrated that though the gram negative bacteria are Das, D., Ara, T., Dutta, S., & Mukherjee, A. (2011). New water resistant biomaterial
susceptible in presence of chitosan, the gram positive ones behave biocide film based on guar gum. Bioresource Technology, 102, 5878–5883.
quite differentially (Sun, Wang, Kadouh, & Zhou, 2014). Qr, Das, S., Roy, P., Pal, R., Auddy, R. G., Chakraborti, A. S., & Mukherjee, A. (2014).
Engineered silybin nanoparticles educe efficient control in experimental
however is a powerful antibacterial due to inhibition of bacterial diabetes. PLoS One, 9(7), e101818. http://dx.doi.org/10.1371/journal.
Na+/K+ ATPase and DNA gyrases enzymes. Kinetic inhibition of pone.0101818.
Staphylococcus aureus strains was remarkable in nano-bio-com- Dehnad, D., Mirzaei, H., Emam-Djomeh, Z., Seid-Mahdi, J., & Dadashi, S. (2014).
Thermal and antimicrobial properties of chitosan?nanocellulose films for
posite films (Fig. 7). Bare chitosan film exposure though reduced extending shelf life of ground meat. Carbohydrate Polymers, 109, 148–154.
the initial colony forming count in two hours but the effect was Dinis, T. C. P., Madeira, V. M. C., & Almeida, L. M. (1994). Action of phenolic
constant thereafter. Bacterial killing was but observed in case of derivatives (acetaminophen, salicylate, and 5-aminosalicylate) as inhibitors of
membrane lipid peroxidation and as peroxyl radical scavengers. Archieves of
the Qr cargo nano-bio-composite film and the cfu count came to
Biochemistry and Biophysics, 315, 161–169.
zero levels in four hours of exposure. As observed in the dissolution Djidi, D., Mignarda, N., & Tahaa, M. (2015). Thermosensitive polylactic-acid-based
studies Qr mass release from the nano-bio-composite film was networks. Industrial Crops and Products, 72, 220–230.
quite sustained, and a level of Qr concentration in the vicinity of Fasano, E., Bono-Blay, F., Cirillo, T., Montuori, P., & Lacorte, S. (2012). Migration of
phthalates, alklyphenols, bisphenol A and di(2-ethylhexyl)adipate from food
the film surface appeared important for gram positive bacterial packaging. Food Control, 27, 132–138.
elimination. Similar results were observed in agar diffusion Harwood, M., Danielewska-Nikiel, B., Borzelleca, J. F., Flamm, G. W., Williams, G. M.,
experiments. No growth region on bacterial lawn was recorded & Lines, T. C. (2007). A critical review of the data related to the safety of
quercetin and lack of evidence of in vivo toxicity, including lack of genotoxic/
and the zone of inhibition was larger in case of nano-bio-composite carcinogenic properties. Food and Chemical Toxicology, 45, 2179–2205.
film than that in case of bare chitosan. Kochumalayil, J. J., Bergenstrahle-Wohlert, M., Utsel, S., Wagberg, L., Zhou, Q., &
Berglund, L. A. (2013). Bioinspired and highly oriented clay nanocomposites
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4. Conclusions barrier properties. Biomacromolecules, 14, 84–91.
Konwar, A., Gogoi, N., Majumdar, G., & Chowdhurya, D. (2015). Green chitosan–
New nano-bio-composite active film was proposed for safe carbon dots nanocomposite hydrogel film with superior properties.
Carbohydrate Polymers, 115, 238–245.
packaging of edibles. Pluronics stabilized PLA nanoparticles non-
Kosaraju, S. L., Weerakkody, R., & Augustin, M. A. (2010). Chitosan-glucose
covalent interactions in chitosan cast films provided improved film conjugates: Influence of extent of millard reaction on antioxidant properties.
properties and microbial deterrent contoured surface. Bioflavonoid Journal of Agriculture and Food Chemistry, 58, 12455–12499.
Licciardello, F., Wittenauer, J., Saengerlaub, S., Reinelt, M., & Stramm, C. (2015). Rapid
Qr as nanoparticle cargo has further enhanced active film
assessment of the effectiveness of antioxidant active packaging— Study with
properties. Multilayer molecular diffusion of Qr provided consis- grape pomace and olive leaf extracts. Food Packaging and Shelf Life, 6, 1–6.
tent antibacterial and antioxidant film surface properties. Designer Mikkonen, K. S., Rita, H., Hele’n, H., Talja, R. A., Hyvönen, L., & Tenkanen, M. (2007).
nanoparticle laden biomaterial films can find applications in Effect of polysaccharide structure on mechanical and thermal properties of
galactomannan-based films. Biomacromolecules, 8, 3198–3205.
packaging of processed food and other edibles. Qian, L., Xiao, H., Zhao, G., & He, B. (2011). Synthesis of modified guanidine-based
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Acknowledgements Applied Material Interfaces, 3, 1895–1901.
Ramamoorthy, M., & Rajiv, S. (2015). In-vitro release of fragrant l-carvone from
electrospun poly(encaprolactone)/wheat cellulose scaffold. Carbohydrate
Institutional grants from the Centre for Research in Nano- Polymers, 133, 328–336.
science & Nanotechnology, University of Calcutta for this work is Rhim, J. W., Park, H. M., & Ha, C. S. (2013). Bio-nanocomposites for food packaging
applications. Progress in Polymer Science, 38, 1629–1652.
greatly acknowledged. Authors would also like to acknowledge Rojas-Grau, M. A., Avena-Bustillos, R. J., Friedman, M., Henika, P. R., Martin-Belloso,
that there is no conflict of interest in publication of this article. One O., & McHugh, T. H. (2006). Mechanical, barrier, and antimicrobial properties of
of the authors Asim Halder, would like to thank the TEQIP program, apple puree edible films containing plant essential oils. Journal of Agricultural
and Food Chemistry, 54, 9262–9267.
University of Calcutta for a fellowship grant. Santanu Sana and Md Roy, D., Knapp, J. S., Guthrie, J. T., & Perrier, S. (2008). Antibacterial cellulose fiber via
Farooque Abdullah would like to thank the University Grants RAFT surface graft polymerization. Biomacromolecules, 9, 91–99.
Commission, Govt of India for their individual senior research Rubilar, J. F., Cruz, R. M. S., Silva, H. D., Vicente, A. A., Khmelinskii, I., & Vieira, M. C.
(2013). Physico-mechanical properties of chitosan films with carvacrol and
fellowship grant.
grape seed extract. Journal of Food Engineering, 115, 466–474.
Scalia, S., & Mezzena, M. (2009). Incorporation of quercetin in lipid microparticles:
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