Title
Sample preparation
Chitosan films incorporated with
Apricot (Prunus armeniaca)
kernel essential oil as active food
packaging material
Improvement of active chitosan
film properties with rosemary
Abstract
Sample analysis
Apricot kernel essential oil
(AKEO) was extracted from
kernels of bitter apricot which is
considered to be a major
agricultural seed waste. The
chemical composition of AKEO
revealed that oleic acid is the
major fatty acid present in
AKEO. Also, N-methyl-2-
pyrrolidone (NMP), which is a
strong antioxidant and
antimicrobial agent, is present in
a significant quantity. Chitosan
films incorporated with AKEO
in different concentrations were
prepared using solvent casting
method and were used for active
food packaging application for
the very first time. Its successful
incorporation in the film was
confirmed by Fourier
Transform-Infra Red (FT-IR)
spectra and Field Emission
Scanning Electron Microscope
(FE-SEM) images. The films
also show excellent
antimicrobial and antioxidant
properties as compared to neat
chitosan films and successfully
inhibited the fungal growth on
packaged bread slices.
Films containing REO showed
more antibacterial activity and
Method
Preparation of chitosan ⁄rosemary
solutions Aqueous solution of
Antimicrobial activity
The assessment of antimicrobial activity
of the films was carried out on Gram
positive bacteria Bacillus subtilis (B.
subtilis) and Gram negative bacteria
Escherichia coli (E. coli). For each
bacterial strain, inoculums from the stock
were revived in Nutrient Broth and were
incubated at 37 °C for 6 h until mid log
phase. Thereafter, the bacterial broth was
diluted serially till final concentration of
107 –108 CFU/ml (colony forming units
per millilitre) is achieved. The diluted
culture broth was taken in a test tube and
test film of specific dimension was cut
and immersed into the culture broth.
Similar dilutions were prepared for each
film type and were incubated at 37 °C for
4 h to allow the interaction between the
film and the bacteria. Nutrient agar plates
were prepared and after completion of 4
h, 10 μl of the bacterial culture from the
test tubes was spread-plated onto the agar
plates. Bacterial colonies, visible on the
plates after 12 h incubation under similar
conditions, were counted and reported in
terms of CFU/ml.
Antibacterial activity Antibacterial
properties of REO, film-forming solution
Result
The bacterial growth, in both cases, gets
retarded as the concentration of AKEO in
chitosan films is increased. The
antibacterial activity is expressed in terms
of colony forming units per millilitre
(CFU/ml) in Fig. 6 which shows the
bacterial population after an incubation
period of 4 h. It was observed that CS/A0
film interaction resulted in B. subtilis and
E. coli populations of 3.5 × 109 CFU/ ml
and 10.1 × 109 CFU/ml, respectively, that
were reduced to 0.8 × 109 CFU/ml and 1.6
× 109 CFU/ml, respectively, for CS/A0.5
film. The bacterial growth is eventually
inhibited for CS/A1 and no viable bacterial
colony was observed at this concentration
However, antimicrobial activity is also
exhibited by neat chitosan films, which is
attributed to the amine groups present in
the chitosan chain. These free amine
groups get protonated to form NH3 + and
interact with negatively charged phosphate
groups present on bacterial cell membrane
leading to its disruption resulting in
bacterial cell death
Its inhibitory activity on gram-negative
bacteria (especially on E. coli) was also
essential oil for food packaging total phenol content. The films
containing REO showed
potential to be used as active
film in food preservation.
chitosan was prepared by
dissolving 20 g of chitosan
powder in 1000 mL of aqueous
acetic acid solution (1%, v ⁄ v),
using a magnetic stirring plate at
90 C and 80 g for 20 min, and
then cooled to room temperature.
Then, 0.2% (w ⁄ v) of Tween 80,
as an emulsifier, was added to the
mixture and stirred in 40 C for 30
min. Finally, an appropriate
amount of REO was added to the
solution, to reach a final
concentration of 0.5, 1.0 and
1.5% (v ⁄ v), and homogenised
with Ultra Turrax (IKA T25-
Digital Ultra Turrax, Staufen,
Germany) at 4000 g for 2 min.
After cooling the resultant
mixture at room temperature, it
was degassed under vacuum for 5
min to remove all bubbles.
Preparation of films
The chitosan ⁄rosemary solutions
(160 mL) were cast in a simple
cubic mould made from Teflon-
coated steel with dimension of
25.5 · 28.5 cm2 and then dried for
72 h at ambient conditions (25 C)
to prepare the films. Dried films
were then peeled and stored in a
desiccator (containing saturated
and discs were studied using the agar
diffusion method. Five different
pathogenic and spoilage bacteria
including Listeria monocytogenes (PTCC
1163), Pseudomonas putida (PTTC
1694), Streptococcus agalactiae (PTCC
1768), Escherichia coli (PTCC 1533) and
Lactococcus lactis (PTCC 1336) were
used for testing. Bacterial strains were
cultured overnight in Brain Heart
Infusion Broth at 37 C; 30 lL of REO and
different film forming solutions was
poured into Mueller Hinton agar wells (5
mm diameter), after their plates had been
seeded with 0.1 mL of inoculums
containing approximately 106 –107 CFU
mL)1 of the indicated bacteria. In the
same way, films were punched into discs
of 6 mm diameter and then placed on the
plates. Next, the plates were incubated in
chamber at 37 C for 24 h, and afterwards,
the zone of inhibition was measured and
was used to evaluate the antimicrobial
potential of the essential oil and the films
notable and interesting. Bozin et al. (2007)
also reported good inhibitory activity for
REO against E. coli strain. As explained
before, high antimicrobial properties are
mainly related to phenol diterpenes, such
as carnosic acid, carnosol, rosmanol,
isorosmanol and rosmarinic acid.
Incorporation of REO into chitosan
showed antibacterial activity in higher than
1% REO in the film-forming solution and
film discs. Moreover, REO showed less
antibacterial activity in film-forming
solution and film disc, respectively, in
comparison with pure essential oil. The
possible reason for the decrease in activity
of the EO incorporated in the chitosan
films compared with activity of pure EO
may be due to lower amount of the EO in
the film solution and film discs in
comparison with the well test for pure EO.
The other reason may be due to slower⁄
controlled release of active compounds
from the chitosan film than from well.

Shelf Life Assessment of Fresh
Poultry Meat Packaged in Novel
Bionanocomposite of
Chitosan/Montmorillonite
Incorporated with Ginger Essential
Oil
Active packaging incorporated
with natural extracts is a
promising technology to extend
shelf life of perishable food.
Therefore, this study aimed to
produce a bionanocomposite
based on chitosan reinforced
with sodium montmorillonite
(MMT) and incorporated with
ginger essential oil (GEO). In
vitro activity was assessed
through migration assay and
antimicrobial study against
foodborne bacteria. Phenolic
compounds were diffused within
48 h of contact, and retained
some of their antioxidant
activity. Films demonstrated
antimicrobial activity against
both Gram-positive and -
negative bacteria tested. The
effect on the shelf life of fresh
poultry meat was determined on
samples wrapped in the
biopolymers and stored under
refrigeration for 15 days,
through physicochemical and
microbiological analyses.
Compared to unwrapped poultry
meat, samples wrapped in the
bionanocomposites showed a
reduction in microorganisms
count of 1.2–2.6 log CFU/g,
maintained color and pH values
magnesium nitrate solution)
Films Production
Bionanocomposites were
prepared according to Souza et al.
[30] and Dias et al. [31]. Chitosan
(1.5%, w/v) film form dispersion
(FFD) in 1% (v/v) of glacial
acetic acid solution was prepared
with continuous agitation
overnight at room temperature. A
plasticizer, glycerol, was added to
all samples (30% w/w of
polymer). In order to achieve
good miscibility and dispersion of
the 2.5% (w/w of chitosan) of
MMT into chitosan chains an
exfoliation process consisting of
three agitation cycles (5 min
agitation with the ultraturrax
(15,000 rpm) (IKA® T18,
Staufen, Germany) followed by
15 min (360 W) in an ultrasound
bath (Selecta, Barcelona, Spain))
was carried out. Before the last
agitation cycle GEO (0.5%; 1%
and 2% v/v in FFD) and tween 80
(0.2% w/v in GEO) were added.
Biofilms without the
nanoreinforcement were produced
without the two first agitation
cycles. Also films without
incorporation of MMT nor GEO
were produced in the same
conditions. The resulting
In vitro antimicrobial activity of pure
GEO and films was studied by agar
diffusion method [36] against Gram-
positive bacteria (Bacillus cereus
(ATCC11778), Enterococcus faecalis
(ATCC29212), Listeria monocytogenes
(ATCC15313), Staphylococcus aureus
(ATCC6538)); Gram-negative bacteria
(Escherichia coli (ATCC8739),
Pseudomonas aeruginosa (ATCC9027),
Salmonella enterica (ATCC10708)); and
one yeast Candida albicans
(ATCC10231). Firstly, glycerol stock
cultures stored at −80 ◦C in ultra-low
temperature freezer (New Brunswick
Scientific, St. Albans, Hertfordshire, UK)
were inoculated in TSA or MHA and
incubated at 30 ◦C for B. cereus and C.
albicans or at 37 ◦C for the other
microorganisms during 16–24 h for
bacteria and 48 h for the yeast. Petri dish
(8.5 cm of diameter) containing Mueller
Hinton Agar were inoculated with a
suspension containing 1 × 108 CFU/mL
for the bacteria or 1 × 106 CFU/mL for
the yeast (both previously adjusted to
match a 0.5 McFarland turbidity standard
using a Mc-Farland densitometer (Model
Den-1B, Grant Instruments, Cambridge,
UK)). For the assessment of pure GEO,
wells with 6 mm diameter were cut in the
MHA and filled with 50 µL of the pure
essential oil or also diluted in DMSO
(1:1) for C. albicans. Wells filled with
s. Trajano et al. [55] only found
antibacterial activity of GEO against S.
aureus, and no inhibition against L.
monocytogens, E. coli, B. cereus, S.
enterica, P. aeruginosa and Yersinia
enterocolitica, while Singh et al. [54]
observed good antimicrobial activity
against E. coli, S. aureus, P. aeruginosa
and Klebsiella pneumoniae, similar to
what was observed by López et al. [24].
The differences from present results and
the ones cited may be attributed to the
characteristic composition of each GEO
tested, once the type and concentration of
phenolic compounds are directly correlated
to the antimicrobial activity [52]. Also, the
extraction procedure applied may have
interfered in the final composition of the
GEO and consequently on its biological
activity. The films, on the other hand, only
presented activity against two of the
bacteria tested: B. cereus and S. aureus,
and only underneath the disk specimens of
the samples incorporated with 1% and 2%
of GEO, in the case of B. cereus, and with
0.5%, 1% and 2% of GEO against S.
aureus (Figure 2). No activity was
observed against Gram negative bacteria or
the yeast tested
 and thiobarbituric acid reactive
substances (TBARS) index
increased at a lower rate,
extending fresh poultry meat
shelf life. The incorporation of
GEO enhanced the biopolymer
activity, by reducing lipid
oxidation and microbiological
growth of the poultry meat. In
contrast, reinforcement with
MMT imprisoned the active
compounds in the polymeric
chain, hindering its activity. In
conclusion, the
bionanocomposites tested
represent promising substitutes
to commercial and unsustainable
plastic films
Zinc oxide nanorods/clove Bovine skin gelatin (BSG)
essential oil incorporated Type B composite films incorporated
gelatin composite films and its with 2% zinc oxide nanorods
applicability for shrimp (ZnO NRs) and clove essential
packaging oil (CEO) (25 and 50%, w/w of
protein) were prepared and
composite was casted in glass
molds (18 cm × 25 cm) or in glass
Petri Dish (10 cm diameter) and
dried for 72 h at room
temperature (relative humidity
approximately 50%). Dried films
were peeled and stored protected
from light at 25 ◦C until
evaluation
To incorporate the CEO into the
film, the oil already mixed with
Tween 80 at 25% (w/w, based on
essential oil) was added to the
BSG and BSG/ZnO NRs
suspension at levels of 0%, 25%
DMSO were used as negative control.
For the films, disks (6 mm diameter)
were cut and placed onto inoculated plate
surface and incubated during 24 h in the
fridge (5 ± 2 ◦C) to allow the migration of
the active compounds to MHA, prior to
the incubation at 37 ◦C or 30 ◦C
(depending on the microorganism) for 20
h or 48 h (yeast). Inhibition zone below
films disks was also considered as
positive antimicrobial activity.
Antimicrobial activity of
bionanocomposites was also assessed by
viable cell colony count (CFU) method
[36,37] against Gram-positive (B. cereus
(ATCC11778)) and Gram-negative (S.
enterica (ATCC10708)) foodborne
bacteria. An amount of 0.2 g of each film
was immersed in 4 mL of TSB containing
~106 CFU/mL of the tested bacteria.
Subsequently, the tubes were kept
shaking (150 RPM) incubated at 37 ◦C
(S. enterica) or 30 ◦C (B. cereus) during
24 h. Tubes without films were used as
control. One hundred microliters of serial
dilutions were spread onto TSA plates,
incubated for 16–24 h and the number of
viable microorganism colonies was then
manually counted.
Antimicrobial effectiveness of BSG The in vitro antimicrobial effectiveness of
composite films against L. BSG-related films against L.
monocytogenes and S. Typhimurium was monocytogenes and S. Typhimurium are
established using peeled shrimps as a illustrated in Fig. 3. As expected the
model food following the method control BSG film showed no antibacterial
described earlier by Ahmed, Mulla and activity. A marginal inactivation (1–2 log
characterized. Depending on the
different combinations of ZnO
and CEO concentration, tensile
strength (TS) and elongation at
break (EAB) varied. ZnO NRs
produced a film with low
flexibility and high in
mechanical resistance. The
oxygen and UV barrier property
of BSG/CEO films increased by
incorporating ZnO NR. FTIR
spectra revealed that the
molecular organization of the
BSG composite films changed
significantly. The
microstructural study showed
that the incorporation of ZnO
NRs prevented porosity of
BSG/CEO films. Composite
films loaded with 50% CEO,
especially in combination with
ZnO NRs, showed maximum
antibacterial activity against
Listeria monocytogenes and
Salmonella Typhimurium
inoculated in shrimp during
refrigerated storage. The results
indicate that the developed
BSG/ CEO/ZnO NRs film could
be utilized as active packaging
for peeled shrimp.
and 50% (w/w, protein content).
The final volume was made up to
100 mL using distilled water. To
obtain the uniform distribution of
ZnO NRs and CEO, the
suspensions were homogenized
for 2 min at the speed of 5000
rpm.Suspensions were gently
stirred for 30 min at room
temperature and were referred to
as a film-forming suspension
(FFS). FFS samples were
degassed for 10 min using the
sonicating bath (Elmasonic S 30
H, Singen, Germany), and 4 g of
FFS cast onto a rimmed silicone
resin plate (5 × 5 cm2 ); air-blown
for 12 h at 25 °C followed by
drying in an environmental
chamber (Binder GmbH,
Tuttlingen, Germany) at 25 ± 0.5
°C and 50 ± 5% relative humidity
(RH) for another 24 h. Dried film
samples were manually peeled off
and subjected to analyses.
Arfat (2016). Two sets (10 g per set) of
sterilized peeled shrimp samples were
placed in two petri dishes. To each petri
dish, a 5 mL aliquot of a 107 CFU/mL
inoculum (prepared by diluting the
standardized inoculum of 108 CFU/mL in
peptone water) of L. monocytogenes and
S. Typhimurium were added separately,
spread over the peeled shrimps using
sterile spreaders, and kept under the
laminar hood for 30 min to allow the
attachment. Afterwards, peeled shrimps
contaminated with L. monocytogenes or
S. Typhimurium were wrapped
individually with BSG composite films,
heat sealed, and stored in petri dishes at 4
°C. Wrapped peeled shrimp samples were
collected and examined for the existence
of L. monocytogenes or S. Typhimurium
by enumeration at 1, 5, 10, 15 and 20
days during the storage period. Peeled
shrimp samples wrapped with control
BSG films served as a control. For
enumeration, the films were carefully
removed from the peeled shrimp, and the
samples were diluted with 10 mL of
sterile peptone water (0.1% w/v),
homogenized in a vortex for 2 min. From
this homogenate, serial dilutions were
made in peptone water and spreadplated
on PALCAM agar for L. monocytogenes
and XLD agar for S. Typhimurium,
respectively. Plates were incubated at 37
°C for 48 h (Lmonocytogenes) and 24 h
(S. typhimurium) before bacterial
reduction) was observed for BSG/2% ZnO
NRs film. The antimicrobial activity of
BSG films increased with increasing the
CEO content, and the time of incubation
against both test organisms irrespective of
ZnO NRs. The highest antimicrobial
activity against both pathogens was
observed for the BSG film containing 2%
ZnO NRs and 50% CEO. A complete
inactivation was observed after 7 days
incubation, which showed a synergism
between eugenol/carvacrol −the active
compound from clove oil and the ZnO
NRs. The hydrophobic compounds from
CEO and Zn2+ are capable of penetrating
through the bacterial cell membrane to
disrupt the cell structure (Burt, 2004; Tayel
et al., 2011)of the microorganisms, react
with the interior cell compounds, and
hence, affected the viability of the cells.
Furthermore, H2O2 generated from the
surface of ZnO acts as an effective
oxidizing agent for the inhibition of
bacterial growth by damaging the cell
membrane of bacteria (Tayel et al., 2011).
BSG films incorporated with CEO showed
a sharp reduction in the bacterial
concentration on day 5, and it continued
till the day 20th. A complete inhibition of
microbes was achieved on 15th and 20th
days of storage when samples packed in
BSG/2% ZnO NRs/50% CEO and BSG/
50% CEO films, respectively. A reduction
of 2.35 log cycle was reported for L.
monocytogenes with addition of 2% clove

Studies on the potential application
of various blends of essential oils
as antioxidant and antimicrobial
preservatives in emulsion based
chicken sausages
Sausages were incorporated with
optimum level of four different
blends of EOs containing six
different EOs (Clove oil,
Holybasil oil, Thyme oil, Cassia
oil, Ajowan oil and Beetel oil),
namely, Blend-1 (0.25 per cent),
Blend-2 (0.25 per cent), Blend-3
(0.25 per cent) and Blend-4
(0.125 per cent); vacuum
packaged and stored at −18±1°C
for 60 days. Duplicate samples
were taken for each parameter,
and three trials were conducted
for each experiment, total being
six observations (n¼ 6) for
consistency of the results.
Findings – Significant decrease
(po0.05) in pH of control
products was observed at each
interval of storage period;
enumeration and expressed as log CFU/g oil after 15 days ostorage at 4 °C (Miladi
of peeled shrimps. All samples were Chaieb, Ammar, & Bakhrouf, 2010). In the
analyzed in duplicate and repeated two present work, the highest inhibition has
times (n = 4 been achieved through a synergism
between ZnO NRs and cyclic
hydrocarbons or phenolic compounds such
as carvacrol and eugenol in clove essential
oil. These active components in CEO
impair the microbial activity, resulting
from hydrophobic interaction with the
bacterial membrane affecting the
functioning of the membrane and
membrane-embedded proteins (Sikkema
De Bont, & Poolman, 1994).
EOs, chemicals and non-meat PC was not observed up to day 15 in
ingredients Food grade EOs were control and Blend-4 products, up to day 30
purchased from reputed in Blend-3 and up to day 45 of storage in
commercial suppliers. Refined Blend-1 and Blend-2 products. Control had
salt (Tata Salt, Tata Chemicals significantly higher ( po0.05) PC than
Ltd, Mumbai, India), vegetable treatment products from day 45 onwards.
oil (Fortune, Adani Wilmar Ltd, The general decreasing trend followed was
Ahmedabad, India), refined wheat Blend-4WBlend-3WBlend2~Blend-1.
flour and spice mix were During initial storage time, psyhcrophiles
purchased from local market. For might be absent because of nonconducive
the preparation of condiments, environment for the growth of
onion and garlic paste was used in psychrophiles; first, cooking of the
the ratio of 3:1. Other non-meat products to an internal temperature of
ingredients, i.e., sodium nitrite, 72°C and then vacuum packaging and
sodium tripolyphosphate were storing at freezing temperature, thus
procured from Central Drug leading to retardation of log phase as a
House Pvt. Ltd, New Delhi. The result of reduced metabolic rate due to
culture media and their additives sudden change in the physical environment
used in the study were procured (Sharma, Sharma, Talukder and
from Himedia. LDPE-nylon- Ramasamy, 2015). Since most of
LDPE coextruded multilayer psychrophiles belong to group of gram

Preparation, physicochemical and
biological evaluation of quercetin
based chitosan-gelatin film for food
packaging
however, in case of treatment films (150 µ thickness) for negative bacteria and these are reported to
products, significant decrease vacuum packaging were procured be more resistant than gram positive
(po0.05) was noticed from day from M/s Hitkari Industries Ltd, bacteria to the action of EOs (Burt, 2004;
30 onwards. Blend-2 was New Delhi. Jayasena and Jo, 2013; Perricone et al.,
observed with significantly 2015). In the present study, PC always
lower (po0.05) thio-barbituric remained below the threshold level of
acid reacting substances acceptability for cooked meat products that
followed by Blend-1. have been reported to be log10 4.6 cfu/g
Significantly lower (po0.05) (Cremer and Chipley, 1977).
total phenolics content was
observed in Blend-4 products as
compared to other treatments.
Regarding DPPH activity,
control products showed
significant decrease (po0.05);
however, in case of treatment
products, DPPH activity showed
significant (po0.05) decrease
after day 15 of storage.
Microbial count increased with
progressive storage period;
however, the counts were well
below the permissible limit of
frozen meat products. All the
blend incorporated products
received very good sensory
scores in consistent manner
Ecofriendly chitosan-gelatin The Agar well diffusion method Antibacterial activity. The Agar well It was observed that the 1% acetic acid
(Ch-ge) bio-composite films was used for the determination of diffusion method was used for the showed no apparent activity and from the
containing Quercetin-starch (Q) antibacterial activity of chitosan determination of antibacterial activity of figure the (zone of inhibition) ZOI of Ch-
were synthesized using solution based films against gram positive chitosan based films against gram ge-Q film was greater than other samples
casting method. bacteria B. substilis and gram positive bacteria B. substilis and gram (Fig. S1). The results of antibacterial
Physicochemical characteristics negative bacteria E. coli negative bacteria E. coli according to activity of Ch-ge and Chge-Q film showed
and mechanical properties of the according to previously described previously described study (Naz et al., that the better antibacterial activity of Ch-
resulting chitosangelatin study (Naz et al., 2018). We have 2018). We have taken agar and broth, ge-Q film than Ch-ge film (Jaisinghani,
containing Quercetin-starch
films (Ch-ge-Q) were studied
using UV, FTIR, XRD and SEM
techniques. The films were also
investigated for their swelling,
water-vapor permeability
(WVP), water solubility
properties. The FTIR spectra
confirmed the chemical
interactions between the
chitosan-gelatin and Q. Surface
morphology of prepared film
was analyzed by the SEM
imaging while XRD spectra
suggest the expanded
crystallinity of the film with the
addition of Q. The film also
showed enhanced barrier
property against UV rays. The
reduction of watervapor
permeability and increase in
tensile strength while a decrease
in elongation at break has been
observed in the Ch-ge-Q film
compared to Ch-ge film. The
antibacterial activity of Ch-ge-Q
film against both gram positive
(B. substilis) and gram negative
(E. coli) bacteria suggested the
Q loaded Ch-ge films as more
feasible antibacterial candidate
especially against the strain E.
coli. The antioxidant activity of
the Ch-ge-Q film was evaluated
using the DPPH and ABTS as
taken agar and broth, both
dissolved in distilled water
separately. Agar (14 g) dissolved
in 500 mL of distilled water and
nutrient broth (1.3 g) in 100 mL
of distilled water and then all
prepared (nutrient broth, agar and
petri plates) had been sterilized in
an autoclave at 121 °C ( ± 1 °C),
15 psi for 15 min. Sterilized
nutrient broth and agar had been
placed in a laminar chamber.
Afterwards, Nutrient broth was
dispersed onto solidified agar
plate and it was used as a growing
medium for the bacteria
Escherichia coli (E. coli)
andBacillus subtilis (B. subtilis).
In agar plates, holes had been
created by sterilized test tube, and
then the prepared bacteria
medium was deposited on to agar
plate and in the end 100 μL of
every sample solution was placed
in the wells of agar plates which
were created in the center of the
agar plate. This incubation was
completed for 12 h at 37 °C ( ± 1
°C) and lastly the antibacterial
activity of the films was analyzed
by measuring the zone of
inhibition (ZOI).
both dissolved in distilled water 2017; Wang, Virgilio et al., 2018, 2018b).
separately. Agar (14 g) dissolved in 500 The zone of inhibition (mm) of the
mL of distilled water and nutrient broth chitosan based film against B. subtilis
(1.3 g) in 100 mL of distilled water and
then all prepared (nutrient broth, agar and
petri plates) had been sterilized in an
autoclave at 121 °C ( ± 1 °C), 15 psi for
15 min. Sterilized nutrient broth and agar
had been placed in a laminar chamber.
Afterwards, Nutrient broth was dispersed
onto solidified agar plate and it was used
as a growing medium for the bacteria
Escherichia coli (E. coli) andBacillus
subtilis (B. subtilis). In agar plates, holes
had been created by sterilized test tube,
and then the prepared bacteria medium
was deposited on to agar plate and in the
end 100 μL of every sample solution was
placed in the wells of agar plates which
were created in the center of the agar
plate. This incubation was completed for
12 h at 37 °C ( ± 1 °C) and lastly the
antibacterial activity of the films was
analyzed by measuring the zone of
inhibition (ZOI).

Physicochemical, Antimicrobial
and Antioxidant Properties of
Chitosan Films Incorporated with
Carvacrol
Carvacrol (CAR) is a phenolic
compound found primarily in oils
of oregano, thyme, and marjoram,
and recognized as a safe additive
standards and corresponded to
81.45% of DPPH and 72.2% of
ABTS scavenging activities. It
was observed that the film
containing Quercetin-starch
presented superior antioxidant
activity results in comparison to
Ch-ge film promising its
application in food packaging.
Chitosan films (CF) with
carvacrol (CAR) [0.5%, 1.0%
and 1.5% v/v] were prepared by
the emulsion method. The
retained CAR, water solubility,
water vapor permeability
(WVP), optical, mechanical
properties, antibacterial and
antioxidant capacity of films
were analyzed. The results
indicate that the retention of
CAR in the CF was ≈50%. The
incorporation of CAR to CF
decreased the water solubility,
the WVP, the yellowing and
transparency and the tensile
strength, but increased the
stiffness. Microcapsules with
diameters of 2 to 7 µm were
found on the surface CF-CAR.
The CF-CAR with highest CAR
concentrations showed
antibacterial activity against S.
typhimurium and E. coli
O157:H7. The CF-CAR had
higher antioxidant capacity and
Preparation of Chitosan The
chitosan was obtained by thermo-
alkaline deacetylation of chitin.
Chitin (1 g) was homogenized
with 50% w/v NaOH (15 mL) at
95 °C for 2 h [45]. The degree of
acetylation of chitosan used in
this study was 34%, with an
average molecular weight of 128
kDa as previously described
Escherichia coli O157:H7 (ATCC 43890)
and Salmonella typhimurium (ATCC
14028) were utilized. The agar diffusion
method was used to determine the
bacterial sensitivity when exposed to the
films. An inhibition zone assay was
performed adding 100 µL of inoculate
containing 105 cfu/mL of each tested
strain and streaked out over the surface of
Muller-Hinton agar plates (Difco, Edo.
México, México). Different films were
cut into 6 mm diameter discs and then
placed on inoculated agar (incubated at
37 °C for 24–48 h) as described by Coma
et al. [51]. Sensitivity was classified
according to diameter of the halo as: not
sensitive (20 mm) [52]. One control of
CF without CAR was used. Inhibition
zone assays were performed in triplicate.
The antibacterial activity of CF-CAR
against two major food contaminant
pathogenic bacteria (E. coli O157:H7 and
Salmonella) was evaluated. Salmonella
showed sensitivity against CF-CAR at
1.0% and 1.5% (Table 4). In contrast, E.
coli O157:H7 was sensitive to CF-CAR
with 1.5%. Although 0.5% CF-CAR tested
against Salmonella and 0.5% and 1% for
E. coli O157:H7 had no sensitivity based
on the used classification, there was an
inhibitory effect under the film. It has been
previously reported that CAR can inhibit
the development of Salmonella (0.5
mmol/L) and E. coli (1 mmol/L) [31,32].
The antibacterial mechanism of CAR has
been studied already and it is known that it
is due to the interaction with lipophilic
components of the bacterial membrane.
This can cause changes in the permeability
of H+ and K+ , which can damage the
essential functions and cause cell death
[33]. Olasupo et al. [34] reported that the
minimum concentration of CAR needed to
inhibit the growth of E. coli (1.5 mmol/L)
is greater than the concentration needed to

Characterization and preservation
performance of active polyethylene
films containing rosemary and
cinnamon essential oils for Pacific
white shrimp packaging
an increased protective effect
against oxidation of erythrocytes
in different grades. These results
suggest potential applications of
CF-CAR as active packaging to
preserve food products.
A new active packaging film of
bilayer structure based on low-
density polyethylene (LDPE)
incorporated with rosemary
essential oil (REO) and
cinnamon essential oil (CEO) in
the inner layer has been
designed and developed for
preserving shrimp. Six types of
active packaging films [REO
(1% w/w), REO (2% w/w), CEO
(1% w/w), CEO (2% w/w), REO
(1% w/w) þ CEO (1% w/w) and
control (film without EO)] were
designed by blown film
extrusion method. Tensile
strength (TS), water vapor
transmission rate (WVTR),
oxygen transmission rate (OTR),
thermogravimetric analysis
(TGA), and microstructure of
films were investigated to justify
the effect of EOs on the film
Preparation of shrimp samples
Pacific white shrimps were
freshly caught and were
completely free of additives. The
shrimps were kept fresh and
transported to the Institution of
Food Science and Engineering,
Ocean University of China,
Qingdao within 1 h. Upon arrival,
shrimps were washed in distilled
water and head, tail, legs, and
shells were removed. Then the
shrimps (with peeled) were
packaged by a manual vacuum
sealing machine immediately. Six
shrimps were singly
vacuumpackaged in an active bag
and stored at 4 C. For each
packaging system, six samples
were taken after 0, 2, 4, 6, 8, and
10 days of storage. The
microbiological analysis (total
viable counts, Enterobacteriaceae
Microbiological analysis The shrimp
samples of 10 g were stirred by a sterile
blender (Joyoung, JY-D501) and then
placed in a sterile homogeneous bag
containing 90 mL saline water of 0.85%
w/w. After mixing for 2 min by a sterile
homogenizer (SCIENTZ-11), 0.1 mL
homogenate was pipette transferred into a
centrifuge tube and serially diluted with
0.9 mL saline water of 0.85% w/w.
Thereafter, 0.1 mL of each diluted
homogenate was used for microbiological
analysis by pour plate method. Summary
of the culture conditions of different
microorganism is listed in Table 2.
Microbiological counts were expressed as
log of colony forming units per gram
(CFU/g) of samples
inhibit Salmonella (1 mmol/L). This could
explain why we saw no inhibitory effect of
CF-CAR 1.0% against E. coli O157:H7
while Salmonella was sensitive. Another
explanation of the absence of inhibition in
CF-CAR with the lowest CAR
concentrations could be due to the fact that
microcapsules (observed in SEM) are not
releasing the CAR content and the effect
shown at high concentrations may be due
to the presence of residual CAR in the CF
and not to the encapsulated one.
Generally, a gradual increase in
Enterobacteriaceae count was detected in
all the specimens as the storage time
increased (P < 0.05). Similar results were
reported by Nirmal and Benjakul (2011),
who found that the initial value of
Enterobacteriaceae count of Pacific white
shrimps was 3e4 log CFU/ g. Furthermore,
the active films with EOs remained below
the control film without EOs throughout
the storage time (P < 0.05). Slight
differences were observed between the
experimental groups, which presented a
profile similar to that of the total viable
count. Shrimp packed by using the film
incorporated with 1% w/w REO þ1% w/w
CEO, and 2% w/w CEO, respectively,
increased the inhibition of
Enterobacteriaceae during 10 days of
storage. This might generally be more
sensitive to cinnamaldehyde in CEO for
membrane-embedded proteins of Gram-
negative bacteria, causing a change in the
 physical functionality. The counts, Psychrotrophic bacteria cell membrane permeability for Gram-
outcomes of scanning electron counts and H2S-producing negative bacteria. Previous study
microscopy (SEM) analysis bacteria), TVB-N contents and confirmed that strong inhibitory effect of
revealed that the surface of TBARS values of the samples plasticised polylactic acid biocomposite
active films became relatively were analyzed after removal from film incorporated with CEO at different
rougher by the incorporation of the bags loadings against Gram-negative (E. coli)
REO or CEO into the LDPE bacteria (Anuar et al., 2017). Sow,
matrix. Meanwhile, the films Tirtawinata, Yang, Shao, and Wang (2017)
with different amount of EOs also observed a lower inhibition
proved to have a slight decline concentration (0.05%, w/w) of carvacrol
in the TS, whereas improved the that could be used to inhibit the growth of
barrier properties. In addition, E. coli ATCC 25922 to about 3 log
shrimp packaged in active films reduction
containing EOs revealed lower
microbial counts, total volatile
basic nitrogen (TVB-N)
contents, and thiobarbituric acid
reactive substances (TBARS)
values compared to samples
packed in control films during
storage at 4 C for 10 days. The
results indicated that the blended
film (REO þ CEO) showed more
effective in maintaining the
freshness and extending the
shelf life of packaged shrimps
up to 4 days. This new active
packaging film has potential to
be used for enhancing the
quality and storage stability of
aquatic products.
Effectiveness of Zataria multiflora
Boiss essential oil and grape seed
extract impregnated chitosan film
on ready-to-eat mortadella-type
sausages during refrigerated
storage
Effect of chitosan coatings enriched
with cinnamon oil on the quality of
refrigerated rainbow trout
Development and characterization
of biodegradable chitosan films
containing two essential oils
The effects of a chitosan (Ch)
coating enriched with cinnamon
oil (Ch + C) on quality of
rainbow trout (Oncorhynchus
mykiss) during refrigerated
storage (4 ± 1 C) were examined
over a period of 16 days. A
solution of Ch (2%, w/v) and Ch
+ C (2%, w/v Ch + 1.5%, v/v C)
was used for the coating. The
control and the coated fish
samples were analysed
periodically for microbiological
(total viable count,
psychrotrophic count), chemical
(TVB-N, PV, TBA), and
sensory (raw and cooked fish)
characteristics. The results
indicated that the effect of the
Ch + C coating on the fish
samples was to enable the good
quality characteristics to be
retained longer and to extend the
shelf life during the refrigerated
storage
ctive biodegradable films from
chitosan containing 10% to 30%
w/w of citronella essential oil
(CEO) and cedarwood oil
(CWO) were developed by
casting and solvent-evaporation
method, and their physical,
mechanical and thermal
Fish sample preparation Fresh
water rainbow trouts with an
average weight of 550–600 g
were purchased at a public market
alive and were transferred to the
Meat Processing Laboratory in
Food Science Department at
Tehran University, decapitated
and filleted by hand. The fish
were harvested during the period
of January–March 2009. Two
fillets were obtained from each
fish after removing the head and
bone
Preparation of the film-forming
dispersions Achitosan solution
with concentration of 1% (w/v)
was prepared by dissolving the
chitosan powder in a 1% (w/v)
aqueous acetic acid while stirring
at 250 rpm on a magnetic stirrer
set at 45 ◦C, following the method
properties were investigated.
Possible interactions between
the chitosan chains and the
essential oils were confirmed
using Fourier-transform infrared
spectroscopy (FTIR). Various
amounts of CEO or CWO had
significant effects on the films’
mechanical properties, with the
exception of 10% of CEO,
which did not significantly
affect the tensile strength of the
films. The incorporation of the
two tested oils provoked a
remarkable reduction in the
water-vapor permeability
properties, with a decrease of
about 63% when 30% CEO was
added in chitosan films.
Thermogravimetric analysis
showed that degradation
temperatures of the films
containing CEO and CWO
improved only slightly in
comparison to control films
without essential oils. FTIR
spectra analysis provided some
insights on the possible
interactions between chitosan
and the two essential oils used.
This study suggests that active
films can be developed by
including CEO and CWO in a
chitosan matrix. Such films can
provide new formulation options
of Ojagh et al. [31]. The chitosan
solution was stirred overnight at
room temperature to achieve
complete dispersion. The
emulsions were obtained by
adding either CEO or CWO to the
chitosan solution to reach final
concentrations of 10%, 20% and
30% w/w. Tween 80 was added
as an emulsifier in quantities
proportional to the essential oils
(0.1%, 0.2% and 0.3% w/w) to
help distribute and completely
incorporate the oil. Finally,
emulsions were homogenized for
4 min at 2500 rpm with a Dual-
Range Mixer (IKA, Canada) and
then degassed under vacuum to
remove dissolved air bubbles. The
quantities of Tween 80, CEO and
CWO were defined according to
our preliminary tests and based on
literature data,taking into
accountthe maximum levels
oftwo essential oils which could
be incorporated into the matrix
without oil phase separation
during film drying. Composite
film without essential oil and
emulsifier was also produced and
considered as control. The
suspension were then poured in
polystyrene petri dishes
measuring 65 mm diameter,
which was placed on a leveled

Chitosan-gelatin composites and
bi-layer films with potential
antimicrobial activity
for packaging industries in
developing active packaging
with potential food-technology
applications
The aims of this work were to
develop composite (Chi-Ge) and
bi-layer (Chi/Ge) edible and
biodegradable films based on
gelatin and chitosan. physico-
chemical properties such as
water resistance, transparency
and color were analyzed.
Composite and bi-layer systems
were uniform, homogeneous and
thin; they showed a compact
structure indicating a good
surface and allowed to dry for
approximately 48 h at 30% RH
and 22 ◦C. Uniform film
thicknesses were achieved by
casting the same amount of film-
forming solution onto each plate.
After drying, the films were
peeled offthe casting surface and
maintained at 22 ◦C and 53%
relative humidity (produced with
saturated Ca(NO3)2 solution) in a
conditioning desiccator
untilfurther evaluation. For each
test,three different samples were
prepared by taking three portions
fromeach film at different
positions (two at the edges and
one at the center) with the
exception of the water vapor
permeability analysis, where the
whole sample was used, and
replicates of each type of film
were evaluated
All films were obtained by . Antibacterial activity of films forming The results obtained with edible films
casting from their film forming solutions and edible films. Disc-diffusion (Table 4) showed that only E. coli was
solutions (FFS). Chitosan (Chi) assay Antibacterial activity test on films sensitive to both, Chi-Ge and Chi/Ge
solution (2%, w/V) was prepared was assessed using the agar diffusion films. No significant inhibition halos were
by dissolving chitosan flakes in method according to Ponce, Fritz, Del induced by control films for none of the
1% (V/V) acetic acid solution Valle, and Roura (2003). The zone of pathogens analyzed (Table 4). One of the
under continuous stirring for 2 h. inhibition assay on solid media was used reasons argued by several authors that
Glycerol was added as plasticizer for determining the antibacterial effects found similar results is the limited film
at a concentration of 28 wt% of films against two typical food diffusion that can take place in the agar
(based on dry chitosan weight). pathogens including a Gram-negative medium (Coma et al., 2002). Pranoto et al.
Gelatin (Ge) powder was bacteria, Escherichia coli O157:H7 (2005) analyzed the antimicrobial activity
dissolved in buffer phosphate at (32158, American Type Culture of chitosan films against various
compatibility between
components, which could
interact by strong hydrogen
bonding, as was confirmed by
FTIR. Water vapor permeability
(WVP) was determined. Both,
bi-layer and laminated systems
resulted effective alternatives to
reduce WVP of chitosan control
film. The tensile strength of
composite and bi-layer system
did not differ significantly (P >
0.05), but elongation at break of
composite films was 40% higher
(P < 0.05) than that of bi-layer
film. Antimicrobial activity of
the films was analyzed. The
results indicated that both E. coli
and L. monocytogenes showed
sensitivity to all the films
forming solutions. The
inhibition halos of both
pathogens to the solutions of Chi
and Chi-Ge showed to be
extremely sensitive. Results
obtained with edible films
indicated that only E. coli was
sensitive to the combination
Chi-Ge and Chi/Ge. Neat Chi
film did not induce significant
inhibition halos for none of the
pathogens, which was quite
surprising and still under study
pH 7 (>Ip), under stirring for 15 Collection) and a Grampositive bacteria microorganisms (E. coli, S. aureus, S.
min at 35 C, to produce a 2.5%, Listeria monocytogenes innocua provided typhimurium, L. monocytogenes and B.
(w/V) FFS. An appropriate by CERELA (Centro de Referencia de cereus) and observed no inhibitory activity
amount of glycerol was added to Lactobacilos, Tucumán, Argentina). against any of them.
achieve glycerol/gelatin weight Edible films were cut into a disc shape of
ratio of 0.28. The chitosan-gelatin 15 mm diameter using a circular knife
(Chi-Ge) composite film (0.8:1 and then placed on Mueller Hinton
w/w) was obtained by mixing the (Merck, Darmstadt, Germany) agar
same volume of chitosan (2% plates, which had been previously seeded
w/v, 28 wt% Gly) and gelatin with 0.1 ml of inoculums containing
(2.5% w/v, 28 wt% Gly) FFSs, approximately 105 e106 CFU/ ml of
respectively. The acidity of the tested bacteria. In the same way, 30 mL
resulting mixture was adjusted to of the different film forming solutions
pH 6 with NaOH and then, stirred were poured into agar wells (5 mm
for 30 min at 35 C. According to diameter). The plates were then incubated
data reported in the literature, it at 37 C for 24 h and, afterwards,
was shown that the examined for width of inhibition. The
polyelectrolyte complex between total area was used to evaluate the
chitosan and gelatin can only antimicrobial potential of films and FFSs.
occur at a pH above the Ip of The diameter of
gelatin, where all gelatin chains
are negatively charged, and below
pH 6.2, to prevent that chitosan
precipitates out of solution
(Hamman, 2010; Yin et al.,
2005). The film forming solutions
with the same dry matter (for
guarantee a constant thickness),
were poured onto Teflon Petri
dishes (14 cm of diameter). The
solutions were dried at 35 C in a
convection oven to constant
weight. Films were stored at 23 2
C in a chamber at 65 2% relative
humidity (RH)
Chitosan films incorporated with
Apricot (Prunus armeniaca) kernel
essential oil as active food
packaging material
Apricot kernel essential oil
(AKEO) was extracted from
kernels of bitter apricot which is
considered to be a major
agricultural seed waste. The
chemical composition of AKEO
revealed that oleic acid is the
major fatty acid present in
AKEO. Also, N-methyl-2-
pyrrolidone (NMP), which is a
strong antioxidant and
antimicrobial agent, is present in
a significant quantity. Chitosan
films incorporated with AKEO
in different concentrations were
prepared using solvent casting
method and were used for active
food packaging application for
the very first time. Its successful
incorporation in the film was
confirmed by Fourier
Transform-Infra Red (FT-IR)
spectra and Field Emission
Scanning Electron Microscope
(FE-SEM) images. The
modified films show an
improved water resistance and
41% enhanced water vapor
barrier property when CS to
AKEO ratio is 1:1. The
elongation percentage value
increased significantly for the
film with oil ratio 0.125 with
respect to CS, but thereafter a
sharp decrement is observed.
Film fabrication The films were
fabricated via solvent casting
method as described elsewhere
(Priyadarshi & Negi, 2017). For
fabrication of neat chitosan films
(CS/A0) calculated quantity of
chitosan (2% w/v) was dissolved
in acetic acid aqueous solution
(1% v/v). The solution was
magnetically stirred overnight at
70 °C for proper dissolution. The
hence prepared viscous chitosan
solution was filtered to remove
any undissolved impurities. The
filtration was carried out with the
help of an ASTM 100 mesh sieve
having a pore size of 150
microns. Thereafter, the solution
was poured on flat glass plates
and allowed to dry at room
temperature for 72 h.
Subsequently the films were
stored in a desiccator at about 0%
relative humidity and 25 °C
temperature to avoid any moisture
absorption before the studies were
carried out. For the fabrication of
films incorporated with apricot
kernel oil (AKEO), the same
procedure discussed above was
followed. However, after 12 h of
chitosan dissolution, 0.2% v/v
Tween® 80 was added and the
stirring was continued for 15 min.
Thereafter, AKEO was added in
Antimicrobial activity The assessment of
antimicrobial activity of the films was
carried out on Gram positive bacteria
Bacillus subtilis (B. subtilis) and Gram
negative bacteria Escherichia coli (E.
coli). For each bacterial strain, inoculums
from the stock were revived in Nutrient
Broth and were incubated at 37 °C for 6 h
until mid log phase. Thereafter, the
bacterial broth was diluted serially till
final concentration of 107 –108 CFU/ml
(colony forming units per millilitre) is
achieved. The diluted culture broth was
taken in a test tube and test film of
specific dimension was cut and immersed
into the culture broth. Similar dilutions
were prepared for each film type and
were incubated at 37 °C for 4 h to allow
the interaction between the film and the
bacteria. Nutrient agar plates were
prepared and after completion of 4 h, 10
μl of the bacterial culture from the test
tubes was spread-plated onto the agar
plates. Bacterial colonies, visible on the
plates after 12 h incubation under similar
conditions, were counted and reported in
terms of CFU/ml
Antimicrobial activity The antimicrobial
activity of neat and AKEO incorporated
chitosan films was studied on Gram
positive bacteria, such as Bacillus subtilis
(B. subtilis), and on Gram negative
bacteria, such as Escherichia coli (E. coli).
It can be seen from Fig. 5 that the bacterial
growth, in both cases, gets retarded as the
concentration of AKEO in chitosan films
is increased. The antibacterial activity is
expressed in terms of colony forming units
per millilitre (CFU/ml) in Fig. 6 which
shows the bacterial population after an
incubation period of 4 h. It was observed
that CS/A0 film interaction resulted in B.
subtilis and E. coli populations of 3.5 ×
109 CFU/ ml and 10.1 × 109 CFU/ml,
respectively, that were reduced to 0.8 ×
109 CFU/ml and 1.6 × 109 CFU/ml,
respectively, for CS/A0.5 film. The
bacterial growth is eventually inhibited for
CS/A1 and no viable bacterial colony was
observed at this concentration (Fig. 5). The
reduction in the bacterial population upon
interaction with the AKEO incorporated
films is presumed to be due to the presence
of NMP in AKEO which is antibacterial by
nature. NMP interacts with the bacterial
membranes and dissolves the membrane
lipids resulting in membrane disintegration
and leakage of intracellular fluids and,
consequently, bacterial cell death
(Phaechamud, Mahadlek,
Charoenteeraboon, & Choopun, 2012).
However, antimicrobial activity is also
 However, a continuous
increment in tensile strength
value was observed with
increasing AKEO concentration
and a substantial 94%
enhancement was observed for
the film with AKEO ratio equal
to that of CS. The films also
show excellent antimicrobial
and antioxidant properties as
compared to neat chitosan films
and successfully inhibited the
fungal growth on packaged
bread slices.
Activity of chitosan films In this paper, the in vitro
enriched with the essential oil of antimicrobial activity of Alpinia
Alpinia malaccensis rhizome malaccensis rhizome oil was
against S. aureus, E. coli and C. investigated in its pure form and
musae incorporated into chitosan films,
against Colletotrichum musae,
Staphylococcus aureus and
Escherichia coli. Dry rhizome
oil of A. malaccensis was
extracted using steam
distillation. Chitosan films
enriched with the oil were
prepared by casting as a
potential bio-based food
packaging film. The major
constituent of A. malaccensis oil
was identified as 1,8-cineole by
GC-MS. The antifungal activity
of the essential oil (EO) was
tested using the disk diffusion
method. The minimum
appropriate amount so as to
obtain the final concentration of
the film forming solutions as
denoted in Table 1. After the
addition of AKEO, the solution
was homogenized with the help of
IKA® T25 Ultra Turrax®
homogenizer at 10,000 rpm for 5
min and the films were casted and
stored as described above
Preparation of chitosan films
Chitosan films were prepared
using the method described by
Ojagh et al. (2010). Distilled
water was added to the control
films instead of essential oil of A.
malaccensis. Films formed were
air dried for 72 hrs at room
temperature (28 ± 2 °C). The
dried films were peeled off and
stored under ambient conditions.
Chitosan films containing the dry
rhizome oil of A. malaccensis at levels of
0.000 – 0.028 µg mm-2 were placed in
sterilised conical flasks (100.0 mL)
containing nutrient broth. S. aureus and
E. coli cultures (107 CFU/mL, 100.0 µL)
were inoculated into each flask
separately. The samples were incubated
overnight at 28 (± 2) ºC. The OD610 of
the samples were measured at the end of
the incubation period.
exhibited by neat chitosan films, which is
attributed to the amine groups present in
the chitosan chain. These free amine
groups get protonated to form NH3 + and
interact with negatively charged phosphate
groups present on bacterial cell membrane
leading to its disruption resulting in
bacterial cell death (Mural, Kumar,
Madras, & Bose, 2016; Priyadarshi &
Negi, 2017).
Chitosan films also possess antimicrobial
activity. An enhanced inhibition of all
three pathogens was observed when the
films were enriched with oil. The
inhibition was more pronounced for S.
aureus than E. coli. The reason for the low
inhibition of the growth of pathogens by
the oil when incorporated into the film
may be partly due to the evaporation of the
oil during the drying process of the film
and slow release of the oil by the film. The
release of the oil from the film is affected
by the thickness and moisture permeability
of the film.

Antimicrobial Activity of
Chitosan Films Enriched with
Essential Oils
inhibitory concentration (MIC)
and the minimum lethal
concentration (MLC) for C.
musae was 100.0 and 300.0 μg
μL-1 , respectively. A liquid
bioassay method was used to
determine the antifungal
efficacy of the oil enriched
chitosan films against C. musae.
More than 50 % inhibition of the
growth of the fungus was
achieved when the oil
concentrations in EO-chitosan
films was between 0.08 – 0.40
μg mm-2. The antibacterial
activity of the oil and the oil
enriched films was determined
by measuring the optical density
of the cultures at 610 nm. The
optical density (OD610) values
of S. aureus and E. coli cultures
showed a sharp and gradual
reduction, respectively with
increasing oil concentration. The
growth of S. aureus was
negligible at oil concentrations
higher than 5.0 μg μL-1. The oil
enriched films showed a lower
inhibition of the growth of both
bacteria.
Antimicrobial and . To achieve complete dispersion chitosan films The bacteria suspensions Pure chitosan films, with no EO, served as
physicochemical properties of of chitosan, the solution was were prepared by following the same a control to determine potential
chitosan films and chitosan stirred overnight at room procedure as for the paper disc diffusion antimicrobial effects of chitosan films per
films enriched with essential oils temperature, filtrated through test. The inoculum was evenly spread on se, but we did not observe any inhibition of
(EO) were determined in vitro miracloth (Calbiochem- Mueller Hinton II Agar plates resulting in the tested bacteria by the control films
and on processed meat.
Antimicrobial effects of pure
EO of anise, essential oils (EO)
were determined in vitro and on
processed meat. Antimicrobial
effects of pure EO of anise,
basil, coriander, and oregano,
and of chitosan-essential oil
films against , and of chitosan-
essential oil films against
Listeria monocytogenes and
Escherichia coli O157:H7 were
determined by an agar diffusion
test. The antibacterial effects of
the EO were similar when
applied alone or incorporated in
the films. The intensity of
antimicrobial efficacy was in the
following order: oregano > >
coriander > basil > anise. The
chitosan films and chitosan-
oregano EO films were applied
on inoculated bologna samples
and stored 5 d at 10 C. Pure
chitosan films reduced C. Pure
chitosan films reduced L.
monocytogenes by 2 logs, by 2
logs, whereas the films with 1%
and 2% oregano EO decreased
the numbers of whereas the
films with 1% and 2% oregano
EO decreased the numbers of L.
monocytogenes by 3.6 to 4 logs
and E. coli E. coli by 3 logs.
Pure chitosan films were 89 by 3
Novabiochem Corp., San Diego,
Calif., U.S.A.) to remove
impurities, and sterilized at 121 C
for 15 min. The EO were 1st
mixed with Tween 20 (Aldrich
Chemical Co.) to help distribute
and completely incorporate the
oils in chitosan matrix and then
added to the chitosan stock
solution. The final film forming
solution (FFS) consisted of 1%
chitosan, 1% acetic acid, 0.5 %
Tween 20, and 1%, 2%, 3%, or
4% EO. The FFS was
homogenized under aseptic
conditions at 21600 rpm for 1 min
(Polytron Kinamatica Inc. PV,
Cincinnati, Ohio, U.S.A.) and
poured into 50-mm inner dia
sterile petri dishes. All the films
were prepared with 20 g of FFS
per petri dish (1 film), which
ensured 10 mg chitosan/cm2.
Control films were prepared
identically but without addition of
EO. After drying under 5 psi
vacuum at 30 C, films were kept
in sealed petri dishes at 4 C until
analysis.
106 CFU/ plate. Uniform 6.6-mm-dia (Figure 3). There are 2 possible reasons for
discs were cut with a hole-puncher from these results. First, the inoculum in this
the prepared chitosan films, and 1 film experiment was 106 CFU per petri dish
disc was placed in the center of the
inoculated petri dish. Concentrations of
the EO in the film forming solutions of
1%, 2%, 3%, and 4% corresponded to
3.1, 6.2, 9.3, and 12.3 L EO per film disc,
respectively. Chitosan films with no EO
served as control. The plates were
incubated at 35 C, and measurements
were taken after 48 h. During the
incubation, the film discs slightly swelled
due to water absorption and resulted in
enlarged diameter (6.9 mm). Therefore,
both the inhibition zone and the disc
diameter were measured, and the width of
the inhibition ring was recorded. The
tests were performed in duplicates.

Development and structural
characterization of chitosan films
containing Eucalyptus globulus
essential oil: Potential as an
antimicrobial carrier for
packaging of sliced sausage
logs. Pure chitosan films were
89 m thick, whereas addition of
1% and 2% oregano EO
increased thickness to 220 and
318 thickness to 220 and 318 m,
respectively. During application
on bologna discs, the films
absorbed moisture, resulting in
the final thickness of 143, 242,
and 333 esulting in the final
thickness of 143, 242, and 333
m, respectively. Addition of
oregano essential oil into the
chitosan films decreased water
vapor permeability, puncture
and tensile strength, but
increased elasticity of the films.
The films have the potential to
be used as active biodegradable
films with strong antimicrobial
effects. films. The films have the
potential to be used as active
biodegradable films with strong
antimicrobial effects. Keywords:
chitosan, edible films, essential
oils, antimicrobial films
Novel antimicrobial biopolymer ilm preparation Chitosan solution . Evaluation of antimicrobial effect of Antimicrobial efficiency of active chitosan
films based on the incorporation was prepared by dissolving 1.5 g active chitosan film in a real food system films in the real food system The
Eucalyptus globulus essential oil of chitosan in 100 ml acetic acid Chicken sausage was obtained from local antimicrobial efficiency of active chitosan
(EGO) in chitosan were solution containing 0.7% (v/v) market. They were sliced by professional films was tested on sliced sausage, which
successfully developed and under a magnetic stirrer at 40 °C cutter. Chitosan alone and chitosan film previously inoculated with L.
characterized as an active film until chitosan was completely containing EGO was prepared in sterile monocytogenes after 3 days at 23 °C. As
for packaging of sliced sausage. dissolved, then glycerol as a conditions in a laminar flow chamber. can be seen in Fig. 4, the active films
Characterization of the films plasticizer (0.15 g glycerol/g Five grams of sliced sausage was containing 0.5, 1, and 1.5% produced a
revealed that incorporation of chitosan powder to the inoculated with 100 μl of L. reduction of 0.26, 0.7, and 1.01 Log (CFU)
EGO (0.5, 1.5, and 1.5%) in the
biopolymer led to change in
morphology, mechanical and
barrier properties. Antibacterial
activity of EGO was assayed
against Salmonella entertidis
and Escherichia coli as Gram-
negative bacteria; furthermore,
Bacillus cereus and
Staphylococcus aureus as Gram-
positive bacteria. Results
showed that the EGO was able
to generatean excellent
antibacterial effect in liquid and
vapour phases. Chitosan films
with 1.5% EGO in liquid
medium produced 4.22, 3.98,
4.55, and 4.71 log reduction
against S. aureus, E. coli, B.
cereus and S. entertidis,
respectively. However, only
minor population reduction of
bacteria was observed in the
vapour phase. The highest
antibacterial activity in sliced
sausage was obtained by
chitosan films containing 1.5%
EGO (1.01 log reduction). This
work highlighted that EGO
incorporation in chitosan is a
promising approach in order to
control risk of foodborne
contamination in a real food
system.
formulation) was added to the
solution and stirred for 10 min.
Different concentrations (0.5, 1,
and 1.5% (v/v)) of EGO were
incorporated into the
chitosanbased film formulation.
Then Tween 80 at level of 0.2%
(v/v) of EGO was added as an
emulsifier. Afterward, the film
forming solutions were
homogenized with 12,000 rpm for
4 min by using a homogenizer
(Heidolph, Germany) to uniform
oil dispersion. Air bubbles in the
solution were then removed by
ultrasonic treatment for 5 min. 25
ml of the film solution was casted
on a petri dish with 10 cm
diameter. After drying the film in
the oven at 38 °C for 24 h, they
were peeled from the plate
surface (O
monocytogenes on its surface, then each /ml, respectively. The results showed the
sample was individually wrapped with films containing antimicrobial capability to
the films (diameter 10 cm). Control inhibit the growth of the L. monocytogenes
samples was wrapped with chitosan films during the storage.
without EGO. After 48 h incubation at 23
± 2 °C, each sample was transferred into
individual sterile stomacher bags and
then mixed with 90 ml of peptone and
homogenized in a stomacher laboratory
blender for 2 min. Antimicrobial activity
of chitosan-based films was tested by
performing serial dilutions with peptone
and consequently plating in Palcam
Listeria Selective Agar. The bacterial
populations were enumerated by counting
the colony forming units (CFU). All
measurements were conducted on three
separate samples in duplicate