FYP Table ziqah nurin

No. Title Sample preparation Sample analysis Result

1. Antimicrobial and antioxidant Preparation of film-forming solutions
activities of edible coatings enriched Chitosan solution was prepared by dissolving
with natural plant extracts: In vitro 20 g of chitosan in 1 kg of 1% acetic acid and
and in vivo studies 1% glycerol solution. To achieve complete
chitosan dispersion, the solution was stirred
overnight at room temperature and centrifuged
to remove impurities. It was then sterilized at
121 ◦C for 15 min
Studies in vivo
Before film application, squash slices
were washed by immersion in tap water
for 60 s and then drained. Afterwards,
squash slices were immersed in the film-
forming solutions for 180 s at 20 ◦C and
then drained. Two types of controls were
used: (1) fresh raw squash slices with no
treatment (fresh control samples) and
(2) fresh raw squash slices immersed in
distilled water and subjected to the same
drying conditions (dry control samples).
After film application, squash slices were
dried by exposure to flowing air at 30 ◦C
with 40–50% relative humidity for 50
min. For microbiological studies, diced
squash (25 g) was macerated in PO4K3
buffer solution (pH 7.2) with a conferred
homogenizer (Stomacher 400 Circulator
Homogenizer). The enumeration and
differentiation of mesophilic aerobic
bacteria were performed on PCA (plate
count agar) after 48 h at 35 ◦C (ICMSF,
Changes in mesophilic bacterial counts in butternut
slices coated with edible films enriched with rosemary
and olive during refrigerated storage. Reductions in
bacterial counts were measured as log N0 − log Nw,
where N0 represent samples with no oleoresins (in
CFU kg−1), and Nw represent samples with oleoresins
(in CFU kg−1). For dry control samples, initial
reference values were 5910 ± 820; for chitosancoated
samples, they were 5720 ± 980 CFU kg−1; for samples
coated with casein, 5930 ± 810 CFU kg−1; and for
CMC-coated samples, 6170 ± 120 CFU kg−1. These
results reveal that the application of these edible films
would produce no significant initial bactericidal effect
(p < 0.05). An initial reduction in total bacterial counts
was observed in all samples treated with rosemary and
olive oleresins. No combinations of film–oleoresins
significant advantages regarding the
antibacterial properties observed when oleoresins were
applied to dry control samples. A possible explaination
could be that the oleoresins antibacterial compounds
would be dispersed in the coating and, thus, bacterial
cells would be less exposed to them. Antimicrobial
 1983; Mossel and Moreno Garcıa, ´ agents may selectively and gradually migrate from the
1985). Microbial counts were conducted film to the food surface, producing their antimicrobial
in duplicate on six independent lots. effects during storage. Actually, the antimicrobial
Peroxidase and polyphenoloxidase activity of the oleoresins coatings increased during the
activities were determined just as first 2 d of storage in all coating/oleoresin pairings
described in Section 2.5, and were except olive in CMC. After 2 d of storage, the
performed in duplicate on three antimicrobial activity decreased. Chitosan and olive
independent lots and rosemary oleoresins presented, by themselves,
antibacterial activity in in vitro assays. Still, when the
combination of chitosan and these oleoresins was
employed in both in vitro and in vivo assays, the
antibacterial activity did not increase, ruling out any
additive or synergic effects. In fact, the antibacterial
activities of olive and rosemary oleoresins were
reduced when they were applied to the coating
material. These results are probably due to the
dispersion effect of the active compounds and the
interactions among these compounds
2. Chitosan-acorn starch-eugenol edible Synthesis of edible films The antibacterial activity of the films was Antimicrobial activity
film: Physico-chemical, barrier, Chitosan solution was prepared by dispersing measured using the agar diffusion The antimicrobial effects of CH/AS films incorporated
antimicrobial, antioxidant and 1.5% w/v chitosan in 1% v/v acetic acid at 30 method. Spread plates of BHI agar were with Eu at different concentrations, against Escherichia
structural properties oC for 4 to 6 h and 18% w/w glycerol was inoculated with 100 μL (107 -109 coli and Staphylococcus aureus were shown in Fig 5
added as the plasticizer. Acorn starch was CFU/mL) of different microorganisms and Table 2. Neat CH/AS film did not show significant
stirred in distilled water at concentration of (Escherichia coli, and Staphylococcus effective against any of the two tested bacteria. This
1.5% w/v, and then heated to 95 oC for over 20 aureus). Then, samples were cut into 6 effect of CH/AS film may relate to the fact that CH
min. After gelatinization, glycerol was added at mm diameter discs and laid onto the does not diffuse through the adjacent agar media in the
a concentration of 18% w/w and the liquid was
subjected to further stirring for 10min. A series
of CH-AS film-forming solutions were
prepared by mixing 100 mL of CH solution
with different volumes of AS solution, and then
allowed to cool at 45 oC before adding Eu. In
order to achieve better emulsification of Eu,
different contents Eu was firstly dissolved in 10
mL ethyl alcohol and then 10% (g/g total Eu)
Tween 80 was added as the emulsifier. After
cooling, different concentrations of Eu
emulsion was added to the CH-AS film-
forming solutions. Then, the mixtures were
homogenized at 45 oC at 8000 rpm using a
homogenizer for 4 min. After it, the mixtures
were filtered with cheesecloth to remove
insoluble substances and allowed to stand for
1.5 h for air bubbles removal. Finally, the
mixtures were poured into cubic Plexiglas
moulds (110 mm×110 mm×8 mm) and dried at
40 oC for 36 h at a relative humidity (RH) of 60
% to obtain the films. After peeling from the
moulds, the films were stored at a temperature
of 25 oC and 69% RH (saturated potassium
iodide solution) for 72 h before testing.
Sample’s nomenclature was CH-xAS-yEu, the
inoculated plate’s surface. Next, the plate
was incubated at 37℃ for 24 h. The
diameter of the inhibitory zone
surrounding film discs was measured
with a vernier caliper. The whole
inhibitory zone was calculated then
subtracted from the film disk area, and
this difference in area was reported as the
zone of inhibition.
agar diffusion test method; only organisms in direct
contact with the active sites of chitosan were inhibited.
There were some strong hydrogen bond interaction and
good compatibility between CH and starch molecules
in the films. Therefore, CH molecules were presumed
to be immobilized within the film and not diffuse to
generate an inhibition zone. These results are in
concordance with the results of other published data.
No any inhibition zone was observed for the gelatin-
chitosan film against S.aureus and E. coli. However,
significant positive inhibitions were observed when
Eu-containing films were measured. As expected, the
better inhibition of the films was observed as higher Eu
concentrations. It indicated that the antibacterial effect
of CH-AS-Eu films was attributed to Eu. It was also
notable and interesting to fund that the inhibition zone
of CH-AS-Eu films against E. coli (Gram-negative
bacteria) were slightly larger than that of S.aureus
(Gram-positive bacteria). The result coincides with the
result of Hafsa et al.[51] about chitosan films
containing eucalyptus globules essential oil and Altiok
et al.[52] about chitosan films incorporating with
thyme oil. But some authors have reported that
essential oils were more effective against gram-positive
bacteria than the gram-negative bacteria tested. The
principal antibacterial mechanism of Eu was associated
 x represent the mass ratio of AS and CH in the
film, and the y represent the concentration of
Eu in the CH-AS blend film.
3. Effect of different animal fat and Six different types of the film samples were
plant oil additives on produced as follows: Chitosan was (600 mg)
physicochemical, mechanical, was dissolved in 60 mL of acetic acid solution
antimicrobial and antioxidant (%1 v:v) under constant stirring at 100 rpm at
properties of chitosan films 25°C for 24 h. After filtration of each solution,
100 µL of glycerol (as a plasticizer) and 10 µL
of Tween 40 (as an emulsifier agent) were
added into each of chitosan solution. Chitosan
solution without oil or fat content was used as a
control. For the blend films, 0.15 mL of olive
oil, corn oil, sunflower oil, melted butter and
melted animal fat were put into respective
chitosan solution. The mixtures were
homogenized using a homogenizer at 25.000
rpm for 5 min. Following the homogenization
process, all the samples were poured into the
plastic Petri dishes (with 15 cm diameter) and
dried at 25°C for 48 h.
Microorganisms used in the study
The food-borne and human pathogen
microorganisms (Escherichia coli (E.
coli) ATCC 25922, Staphylococcus
aureus (S. aureus) ATCC 25923, Proteus
microbilis (P. microbilis) ATCC 14153,
Proteus vulgaris (P. vulgaris) ATCC
13315, Pseudomonas aeruginosa (P.
aeruginosa) ATCC 27853, Enterobacter
aerogenes (E. aerogenes) ATCC 13048,
Bacillus thuringiensis (B. thuringiensis),
Salmonella enterica serotype typhmurium
(S typhmurium) SL 1344 and
Streptococcus mutans (S. mutans) ATCC
25175) were used to evaluate the
antibacterial properties of chitosan-oil/fat
blend films. Bacteria were maintained on
Luria Bertoni (LB) agar culture medium
at 4°C. All bacteria strains were sub-
cultured on LB agar culture at 37°C for
24 h
with its increasing non-specfic permeability of
cytoplasmic membrane, thus result in the disruption of
the membrane. These results showed that Eu could be
immobilized in the CH/AS film and subsequently
released, thereby inhibiting target microorganisms.
Antimicrobial activity of chitosan-oil/fat blend films
was investigated against food-borne and human
pathogen bacteria
Mean inhibition values for antimicrobial properties
chitosan-oil/fat blend films are presented in Fig. 8. It is
interesting that antimicrobial activity of all tested
chitosan films was greater than negative control (only
solvent). Plant oils and animal fats blended chitosan
films were strongly effective against all of the tested
bacteria strains with mean inhibition zone values range
of 14.560.81 mm (P. microbilis) to 29.840.92 mm
(P. aeruginosa) as their inhibition zone diameter
increased about 45.02.2% against tested pathogens
compared with negative control. This might be
corresponded to the entrapment of oil compounds into
the chitosan film improves the stability of film
mechanical properties which leading to efficient
activity of antimicrobial compounds against pathogens.
In all test, control (only solvent) showed inhibition
zone ranged with 6.320.45 mm 13.810.78 mm.
Interestingly, the common antibiotic gentamicin

4. Improvement of the Quality and
the Shelf Life of Figs (Ficus carica)
Using an Alginate–Chitosan Edible
Film
Chitosan layer was prepared by the
methodology. One gram of chitosan was diluted
in 100 mL of a 1 % (v/v) lactic acid solution,
and afterward, 0.5 g of soy lecithin, 2 g of
glycerol, and 5 mL of olive oil were added. The
mixture was homogenized at 15,000 rpm during
10 min using an Ultraturrax T18. Finally, the
emulsion formed was cooled to 25 °C before its
application.
exhibited lower values of inhibition zone (ranged from
16.560.62 to 20.730.84 mm) against almost of the
tested bacteria than chitosan film blended with
different oil compounds. Sunflower oil and olive oil
incorporated chitosan films displayed higher
antibacterial activity than other film samples (corn oil,
animal fat and animal butter). These results suggest
that chitosan blended with plant oils or animal fats
could be used as a new generation, non-toxic and
hydrophobic antimicrobial agent. In general,
antimicrobial film’s functionality in food packaging
can be evaluated from the extent of microorganism
growth. Chitosan-oil/fat blend films produced in this
study are nontoxic, biodegradable and mechanically
stable. Thus, they could be used as food packaging
material that can protect both the food content and the
environment.
Fruits’ Visual Contamination
The application of the A–Ch BE film also retarded the
fruits’ visual contamination (Fig. 5). Thus, uncoated fig
fruits presented visual fungal infection after just 3 days
of storage. On the contrary, fungal infection was only
observable in coated figs after 15 days of storage. In
fact, at the end of storage, coated figs presented a
visual fungal contamination on, approximately, 5 % of
the fruits, whereas about 80–87 % of the uncoated figs

5. Investigation of the physicochemical, Preparation of films
antimicrobial and antioxidant The edible films were prepared by casting
properties of gelatin-chitosan edible technique. The pigskin gelatin (GEL) and
film mixed with plant ethanolic chitosan (CH) solutions were prepared
extracts separately and then conveniently mixed to
obtain the following formulations: films of pure
biopolymers (GEL100 or CH100) and films
based on blends of gelatin and chitosan with
25% (GEL75:CH25) or 50% (GEL50:CH50) of
CH in blends. The different ethanolic extracts
were incorporated at 1%. Gelatin solution was
prepared, where gelatin (4g GEL/100ml of
distilled water) was hydrated at room
temperature for 30min and solubilized later in a
thermostatic bath at 55ºC (±0.5ºC). After this,
glycerol (0.2%) was added. In parallel, chitosan
solution was prepared. Chitosan (1% w/w) was
dispersed in an aqueous solution of glacial
acetic acid (1.0% v/w) for 12 h using a
were contaminated. The chitosan coatings could reduce
microbial and fungal contamination in strawberries
stored at 20 °C and 95–98 % RH for 4 days. The
coatings of carboxymethyl cellulose, hydroxypropyl
methyl cellulose, and their combination with the
antifungal agent chitosan reduced fungal contamination
in strawberry fruit.
Quantitative analyses Antimicrobial activity
The disk diffusion method was used to The results of antimicrobial activity tests for pure
determine the antimicrobial activity of biopolymers films (GEL100 and CH100) and
the films. E. coli and S. aureus were GEL50:CH50 blend films with different extracts,
selected for this study. The plates against S. aureus and E. coli, are presented. As
containing Müller-Hinton agar were expected, the chloramphenicol, used as antibiotic
inoculated after preparation of the control, presented the highest (sensitive) inhibitory
bacterial suspension, and then a sterile effect in comparison to all tested films. The diameter of
swab was used for dipping the bacterial the inhibition zone of pure chitosan films (CH100)
suspension in all the plate avoiding indicated inhibition on growth against S. aureus and E.
leaving uncovered areas. Filter paper coli strains tested, but, contrarily, the pure gelatin films
disks (18 mm diameter) were soaked with did not show any antimicrobial activity. Different
15 ml of absolute ethanol and authors have demonstrated the antimicrobial activity of
chloramphenicol (2.5%) used as negative chitosan and explained this behavior for its positively
and positive controls, respectively. Then charged amino group which interacts with negatively
films samples (18 mm diameter) and charged microbial cell membranes, leading to the
controls were laid onto the inoculated leakage of proteinaceous and other intracellular
plate’s surface. These plates were constituents of the microorganisms. An increase of
incubated for 24 h at 37ºC. At the end, chitosan proportion and the presence of rosemary,
 magnetic stirrer at 40ºC. The film-forming
solutions (FFS) were obtained by mixing GEL
and CH solutions in different ratios using a
rotor–stator homogenizer at 10,000 rpm for 5
min at room temperature. The FFS were cast in
petri plates (14 cm diameter) and dried at 30ºC
and 60% relative humidity (RH) for about 24 h,
in a conventional oven. The films were peeled
off from the casting plates and conditioned for
7 days at 25ºC-53% RH in chambers containing
saturated solutions of Mg (NO3)2 prior to all
analyses.
6. The antimicrobial, mechanical, Film-making materials
physical and structural properties of Chitosan (95-98% deacetylated, MV ¼ 8.0 -
chitosan gallic acid films 105 Da) (Moreira et al., 2011) and glacial acetic
acid (99%, analytical reagent grade) were
obtained as a plasticizing agent, and gallic acid,
as an antimicrobial agent, were purchased from
Fisher Scientific Inc. (Pittsburgh, PA, USA).
2.2. Film preparation. The edible films were
prepared by dissolving 1 g of chitosan in 100 g
of 1% acetic acid solution and stirred, at room
the halos formed by inhibition zones
surrounding clear areas were considered
as a measurement of the antimicrobial
activity. All analyses were performed in
triplicate.
Antimicrobial activity
Antimicrobial properties of the crafted
films were determined by the log
reduction method with a slight
modification. Briefly, culture medium
broth was inoculated with certain amount
of suspension of bacteria. The bacterial
concentration in the seeding culture was
approximately 6 - 108 CFU/mL. Serial
dilutions of the suspension were
cinnamon, boldo do-chile and guarana extracts in films
(GEL50:CH50) allowed to observe an increased
diameter of the inhibition zone during the period
analyzed. The films showed their antimicrobial activity
with diameters of the inhibition zone ranging from 23
to 26 mm against S. aureus with rosemary and
cinnamon extracts added, respectively, and from 21 to
24 mm against E. coli with boldo-do-chile and guarana
extracts added, respectively. Bodini et al., (2013)
studied gelatin-based films with ethanol-propolis
extract added in different concentrations (0, 5, 40 and
200 g of extract/100 g of gelatin) and concluded that
only the films with concentrations of 40 and 200 g of
extract inhibited the growth of S. aureus, possibly
associated with the increase in polyphenol
concentrations.
Antimicrobial properties
To examine the antimicrobial properties of the studied
edible films, E. coli, S. typhimurium, B. subtilis, and L.
inoculate, which are very significant pathogens in the
food industry, were tested. The results are shown in
Fig. 1. The edible films incorporated with different
concentrations of gallic acid significantly improved the
antimicrobial activities of the chitosan film against all
the tested bacteria (p < 0.05). The log reduction
increases with the increase of gallic acid concentration,
temperature, until chitosan was completely
dissolved. Glycerol at 0.3 g/100 g was added as
a plasticizer. Film without gallic acid was
designated as film 0 (F0) which was used as a
control. Gallic acid was added at varying
concentrations: 0.5 g/100 g in film 1 (F1), 1.0
g/100 g in film 2 (F2) and 1.5 g/100 g in film 3
(F3), respectively. Equal volumes (150 mL) of
the film solutions were spread on glass plates
(200 - 200 mm) and dried for 12 h at 35 - 2 C in
an incubator (New Brunswick Scientific
Excella* E24 ， Fisher Scientific Inc. PA,
USA). The films were removed from the glass
plate with a thin spatula and conditioned at 23
2 C and 50 2% relative humidity (RH) before
running further test
performed, and the optical density values
were tested to achieve a standard curve.
Square film pieces (20 20 mm) were
sterilized and introduced into a test tube
containing 5 mL fresh suspension of
bacteria and incubated at 37 C for 24 h.
Optical density of culture media was
measured at 620 nm using a Perkine
Elmer HTS 7000 Bioassay reader, and
cell concentrations were determined. All
samples/standards were run in triplicates.
which illustrates the antimicrobial activity of gallic
acid. The results show that the log reductions of B.
subtilis, ranged from 1.24 to 5.75, are demonstrated to
be higher than other bacteria. The minimum inhibitory
concentration (MIC) of chitosan against B. subtilis is
0.10 g/L. The log reductions of E. coli ranges from
0.57 to 2.31. The MIC of chitosan against E. coli is
0.75 mg/mL and gallic acid demonstrated significant
antimicrobial activity against E. coli (MIC ¼ 1 g/L).
Combining gallic acid with chitosan shows a potent
antimicrobial effect according to our results. The log
reductions of S. typhimurium ranged from 1.07 to 1.75.
Furthermore, the combination of gallic acid in chitosan
films exhibited obvious reduction in the growth of L.
inoculate, resulting in an approximate 2.5-log
reduction. Listeria growth inhibition was recorded for
gallic acid at 0.45 g/L (A. The diameters of the zone of
inhibition (mm) of chitosan against E. coli and B.
subtilis were 18 mm and 40 mm respectively, which
verified that B. subtilis is more sensitive than E. coli to
chitosan. Furthermore, the film showed a higher
effectiveness against B. subtilis, and L. inoculate
compared to E. coli and S. typhimurium which may be
rationalized by the characteristic difference of the outer
membrane between Gram-positive bacteria and Gram-
negative bacteria.