Table of Final Year Project 3

No. Title Sample preparation Sample analysis Result

1. Antilisterial effect of Chitosan-based (CH) and methylcellulose-
citrus essential oils based (MC) films were used to perform the
and their performance antilisterial assay. High molecular weight
in edible film chitosan (1.2 Pa s viscosity at 1% w/w in
formulations 1% w/w glacial acetic acid, acetylation
degree: 4.2% was dispersed at 1% w/w in
an aqueous solution of acetic acid (1% v/w)
and stirred overnight at room temperature.
Methylcellulose (0.3e5.6 Pa s viscosity at
1% w/w in water solution, was dispersed in
distilled water (1% w/w) and heated up to
80 C to promote solubilization.
Antilisterial effect of edible EOs-based films
Once the polymer solutions were obtained, each EO was
added at a concentration of 0.5% (polymer: EO ratio 2:1)
and stirred for 10 min. The mixtures were then sonicated by
the Vibra Cell VCX750 sonicator at 20 kHz and 40% power
for 480 s (1 s on and 1 s off) in order to obtain the film
forming dispersions (FFD). FFDs were casted in plates
(diameter 53 mm), weighted up to 6.7 g, to keep polymer
amount constant in dry films (30 g polymer/m2 ). The films
were dried at room temperature and 60% relative humidity
(RH). The surface of TSA plates (10 g) was seeded with
0.35 mL of cell suspensions (104 CFU/mL) and covered
with CH and MC films. Inoculated coated TSA and
inoculated non-coated TSA dishes were used as controls.
Plates were then sealed with parafilm to avoid dehydration
and incubated at 37 C for 0, 8 and 24 h and at 8 C for 0, 1, 3
and 7 d. The two temperatures were chosen to investigate
the effect of the EOs at the optimal growth temperature for
the test strains (37 C) and simulating the conditions of a
domestic refrigerator (8 C). The agar layer was then
aseptically removed from each Petri dish and placed into a
sterile stomacher bag with 90 mL of Peptone Water and
A stronger antilisterial effect was evidenced for the CH-
based films, alone and in combination with EOs.
Specifically, CH-films were more effective in reducing
the microbial growth at 8 C rather than 37 C. In fact, CH-
films added with EOs led to a reduction up to 3 and 6 log
CFU/cm2 , in the case of LM35 and LM69, respectively,
when incubated at 8 C for 7 days (Fig. 1E and G). The
highest significant antibacterial effect evidenced in case
of the incubation at 8 C may be related to the influence of
the temperature in promoting the permeability of cell
membranes and, thus, dissolving more easily EOs in the
lipid bilayer when low temperatures occur (Sanchez-
Gonz alez et al., 2011 ). Fig. 2 shows the SEM
microstructures of the cross-sections of CH and MC
films. Pure MC and CH films (Fig. 2A, D) exhibited a
homogeneous and continued microstructure in line to that
observed in previous studies (Vargas, Albors, Chiralt, &
Gonzalez-Martínez, 2011). The addition of the lemon
EOs to the film matrix promoted discontinuities (Fig. 2B,
C, E, F), in agreement with the results reported by
Perdones et al. (2012) in CH-based films containing
 homogenized for 60 s in the stomacher Bag Mixer 400 essential oil. The presence of EO droplets is more
(Interscience, Saint Nom, France). Serial dilutions were set noticeable in CHbased films (Fig. 2B, C), and especially
up with Ringer's solution and 0.1 mL of cell suspensions in films containing EO L2 (droplets size 1e8 mm). The
were spread plated onto TSA plates. Colonies were observations pointed to a better incorporation of the EOs
enumerated after 24 h at 37 C. The experiment was carried in CH matrix, where a higher amount of oil droplets was
in duplicate. distinguished. Furthermore, the higher inhibition activity
recorded for EO L2 included into CH matrix can be due
not only to the better incorporation, but also to the
subsequent release of the active compounds. A good
incorporation of EO into the films slows down the
diffusion rate of the antimicrobial compounds, keeping
high concentrations of EOs for extended period of time
and reducing the levels of microorganisms on the surface.
2. Antimicrobial Activity Chitosan solution was prepared with 2 g Microbial analysis Coliform account
of Lemon and chitosan and 1 g acetic acid in 100 g The microbiological counts were specified by placing 10 g Escherichia coli, the fecal indicator organism, were part
Peppermint Essential distilled water. To achieve complete fish specimen in 90 ml of 0.9% Nacl solution and were of the microflora of fresh rainbow trout. The differences
oil in Edible Coating dispersion of chitosan, the solution was homogenized in a stomacher for 1 min. Other decimal between bacterial loads are shown in Figure 1d. Many
Containing Chitosan stirred for 3 h at room temperature. dilutions were prepared from this dilution and plated in the researchers believe that presence of coliform bacteria is a
and Pectin on Chitosan received an addition of Glycerol suitable media total viable counts (TVC) and direct consequence of fecal contamination. The initial
Rainbow Trout as a plasticizer at 0.75 ml/g concentration Psychrotrophic count were determined via surface plate coliform in control samples of rainbow trout fillets was
(Oncorhynchus and was beated for 10 minessential oil, method, using plate count agar. The cultures were incubated 0.97 which is below the 2.3 or 2.4 known as maximum
mykiss) Fillet mingled with Tween 80 was added to the at 37°C for 3 days for TVC, and at 10°C for 7 days for allowable bacterial load for coliform]. Additionally, the
chitosan solution to help distribute and psychrotrophilic counts. growth pattern of coliform showed the same behaviour as
completely incorporate the essential oil. that of TVC and PTC and increased during storage time
 The final coating solution was formed
including chitosan (2%), acetic acid (1%),
glycerol (0.75%), Tween 80 (0.2%), and
each essential oils (0.5, 1%). The final
coating solution was homogenized under
aseptic conditions at 21600 rpm for 1 min.
The control solution was made in the
absence of Lemon and Peppermint oil as
described by others
3. Antimicrobial Essential oil
influence of Essential oils of lemon were removed by
nanoemulsified lemon hydrodistillation using an industrial type of
essential oil and pure Clavenger device during period of 4 h. The
lemon essential oil on Clevenger apparatus composed of a 1000-
food-borne pathogens mL round–bottomed flask, a volatile oil
and fish spoilage determination tube and a reflux condenser.
bacteria All parts are connected via ground glass
joints. The essential oil was kept in
refrigerator at +4 °C until analysis of
chemical composition
in all groups. Control being the highest at day 16 whereas
lower count was detected in treated sample with 1% PEO
(P>0.05).
Screening of antimicrobial activity
Comparison of antimicrobial effects of oil-in-water The antimicrobial activities of two different
nanoemulsion based on lemon essential oil and two concentrations of lemon essential oil and its
different concentrations of lemon essential oil (100% and nanoemulsion against food-borne pathogen bacteria were
10%) on six fish spoilage and four food-borne pathogen presented in Table 2. Lemon nanoemulsion and 10%
bacteria was carried out using paper disc diffusion method essential oil showed higher antimicrobial activity (P <
(Murray, 1995) with minor modifications. Antibiotics 0.05) against S. aureus with 16.63 and 16.25 mm
(tetracycline (30 μg), streptomycin (10 μg) and neomycin (5 inhibition zone diameters than that of 100% lemon
μg) were used on fish spoilage and food-borne bacteria as essential oil (11.63 mm).
positive control. Tween 80 was also used as negative
control. Nutrient agar was carried out as the standard test
medium for bacteria. Fifty microliters of nanoemulsions
(10% lemon essential oil, 1% surfactant and 89% water) and
lemon essential oil (100% and 10%) were pipetted on sterile
filter paper disc (diameter 6 mm), which were permitted to
 dry in an open sterile petri dish in a biological cabinet
vertical laminar flow. Bacteria at the concentration of 108
CFU/mL was spread onto the surface of agar media in petri
dishes. Afterwards, four-paper disc with nanoemulsion,
essential oil (100% and 10%), antibiotics and tween 80 were
set on the inoculated agar surface separately. The diameter
of an inhibition zone around the disc was measured after
incubation of the bacterial plates at 37 ± 1 °C for 18–24 h.
The values were recorded by average (mm) of four-disc
diameter measurements.
4. Citrus lemon essential Antimicrobial activity Agar diffusion method
oil: chemical Microorganisms and growth conditions Antibacterial and antifungal tests were performed by agar The antibacterial activity of C. limon essential oil was
composition, Authentic pure cultures of bacteria and well diffusion method as described by Tagg and McGiven evaluated against Gram + positive (B. cereus, E. faecalis,
antioxidant and fungi were obtained from international and broth microdilution assay using sterile Mueller–Hinton S. aureus, S. epidermis, B. subtilis, L. monocytogenes
antimicrobial activities culture collections: American type culture media (Bio-Rad, France) for bacterial strains and potato and M. luteus) and Gram-negative (P. aeruginosa, E.coli,
with its preservative collection (ATCC) and the local culture dextrose agar (Bio-Rad,France) for antifungal tests. Fifteen S. enteritidis and K. pneumoniae) bacteria. The
effect against Listeria collection of the Center of Biotechnology of milliliters of the molten agar (45 °C) were poured into antibacterial activity was assessed by evaluating the
monocytogenes Sfax, Tunisia. They included Gram-positive sterile petri dishes (Ø 90 mm). Working cell suspensions inhibition zone (IZ) and the determination of MIC values.
inoculated in minced bacteria: Bacillus subtilis ATCC 6633, were prepared and 100 μl were evenly spread onto the As can be seen in Table 2, ClEO showed varying degrees
beef meat Bacillus cereus ATCC 14579, surface of the agar plates of MuellerHinton agar for of antibacterial activity against all strains tested. The
Staphylococcus aureus ATCC 25923, bacteria, or potatoes dextrose agar medium for fungi. Once inhibition zones were in the range of 13–26 mm. Among
Staphylococcus epidermidis ATCC 12228, the plates had been aseptically dried, 06 mm wells were Gram positive bacteria, highest inhibitory zone was
Enterococcus faecalis ATCC 29212 and punched into the agar with a sterile Pasteur pipette. The observed against L. monocytogenes (26 mm) followed by
Listeria monocytogenes ATCC 19117 and ClEO were dissolved in dimethylsulfoxide (DMSO)/water B. cereus (24 mm) and S. aureus (22 mm). Among Gram
 Gram-negative bacteria: Salmonella (1/1) and sterile water to a final concentration of 50 mg/ ml. negative, highest inhibitory zone was observed against S.
enterica ATCC 43972, Escherichia coli Thus, 50 μl were placed into the wells and the plates were enteritidis (18 mm). The inhibition zone for Gentamicin
ATCC 25922 and Pseudomonas aeruginosa incubated at 37 °C for 24 h for bacterial strains and 72 h for (10 μg/well), which was used as positive controls for
ATCC 9027. The following fungal strains fungi at 28 °C. Gentamicin (10 μg/wells), Amphotericin B bacteria, ranged from 12 to 25 mm. Negative control did
were also tested: Aspergillus niger CTM at 20 μg/wells and DMSO served as positive and negative not show any inhibitory effect against the tested bacteria.
10099, Aspergillus flavus (food isolate), control. Antimicrobial activity was evaluated by measuring
Aspergillus nidulans (food isolate), the diameter of circular inhibition zones around the well.
Aspergillus fumigatus (food isolate), Tests were performed in triplicate
Fusarium graminearum (ISPAVE 271),
Fusarium oxysporum (CTM10402),
Fusarium culmorum (ISPAVE 21w) and
Alternaria alternata (CTM 10230). The
bacterial strains were grown on Mueller
Hinton broth (Bio-Rad, France) at 37 °C for
12–14 h while potato dextrose agar (PDA)
(1.5% agar) at 28 °C for 4 days were used
for fungi. Inocula were prepared from an
overnight broth culture by their dilution in
saline solution to approximately 107
colony-forming units (CFU)/ ml for bacteria
and 105 spores/ml for fungus
5. Effect of chitosan– Preparation and characterization of the Fungal decay
lemon essential oil film-forming dispersions To enable the comparison with the results obtained in the in Fig. 6 shows the development of fungal decay of
coatings on storage- vitro studies, 50 strawberries per coating formulation were inoculated strawberries during cold storage, expressed as
keeping quality of Chitosan (1%, w/w) was dispersed in an
strawberry aqueous solution of glacial acetic acid
(0.5%, v/w). This FFD was designated CH.
To prepare chitosan-essential oil FFD,
chitosan was dissolved as described above
and lemon essential oil was added to reach
a final concentration of 3% (w/w). The
mixture was emulsified using a rotor–stator
homogenizer at 21,500 rpm for 4 min. This
film-forming dispersion was designated
CH–LO. This coarse emulsion was
submitted to a second homogenization at
165 MPa in a single pass by using
microfluidizaion to obtain CH–LO-M FFD.
An aqueous solution of glacial acetic acid
(0.5%, v/v), was used for the immersion of
a control sample (control). The particle size
of CH-OA FFD was measured by using a
laser diffractometer.. The FFD were diluted
in a sodium acetate buffer solution under
the appropriate solvent conditions at 2000
rpm until an obscuration rate of 10% was
obtained. The Mie theory was applied by
considering the following optical properties
inoculated with B. cinerea (105 spores/mL) by sample
immersion in a spore suspension before applying the
coating. 50 non-coated inoculated strawberries were used as
control. The strawberries that showed any sign of surface
mycelia development were considered decayed. Results
were expressed as percentage of infected strawberries. Two
replicates were performed
the percentage of visibly infected samples out of the total
amount of stored samples. Chitosan coatings reduced the
percentage of infected strawberries as compared to non-
coated ones (control) after three storage days. Pure
chitosan coatings have previously been reported to have
antifungal effect when applied to cold stored strawberries.
Chitosan antifungal activity was enhanced by the addition
of lemon essential oil, and no significant differences were
shown due to microfluidization of the FFD, despite what
was observed in the in vitro study, where chitosan
coatings with lemon essential oil prepared by means of
microfluidization (CH–LO-M) exhibited a high anti-
Botrytis effect. No clear explanation for this fact was
found, although interactions between the coating
components and the fruit surface can lead to divergent
behaviour with respect to that observed in the in vitro
studies. In this sense, a higher antifungal effect of
limonene than peppermint essential oil, encapsulated in
coatings of modified chitosan, when applied to cold-
stored strawberries, although the in vitro antifungal effect
of the free (non-encapsulated) oils showed the opposite
trend.
for dispersed droplets: a refractive index of
1.52 and absorption of 0.00. Three samples
of each FFD were measured in triplicate.
The rheological behaviour of FFD was
analysed in triplicate after one day of
storage at 25 ◦C by means of a rotational
rheometer with a type Z34DIN Ti sensor
system of coaxial cylinders. Rheological
curves were obtained after a stabilization
time of 5 min at 25 ◦C. The shear stress was
measured as a function of shear rate from 0
to 512 s−1. The power law model was
applied to determine the consistency (K)
and the flow behaviour (n) indexes of the
FFD. Apparent viscosity values were
calculated at 100 s−1, which could be a
typical gradient generated during the
sample coating application process with the
FF.