Food Research International 38 (2005) 267–272

www.elsevier.com/locate/foodres

Physical and antibacterial properties of alginate-based edible

film incorporated with garlic oil
Yudi Pranoto, Vilas M. Salokhe, Sudip K. Rakshit *

Food Engineering and Bioprocess Technology Program, School of Environment, Resources and Development,
Asian Institute of Technology P.O. Box 4, Klong Luang, Pathumthani 12120, Thailand

Received 14 January 2004; accepted 26 April 2004

Abstract

Antibacterial alginate-based edible film has been studied by incorporation of garlic oil as a natural antibacterial agent. Initially,
0.1% v/v garlic oil was tested in in vitro experiments against some food pathogenic bacteria. The presence of 0.1% v/v garlic oil in the
nutrient broth decreased viable cell counts for Escherichia coli, Salmonella typhimurium, Staphylococcus aureus and Bacillus cereus
by 2.28, 1.24, 4.31 and 5.61 log cycles, respectively after 24 h incubation. Meanwhile, an increased cell population occurred on all
accompanying controls. Antimicrobial alginate films were prepared by incorporating garlic oil up to 0.4% v/v. They were charac-
terized for antibacterial activity, mechanical and physical properties. The edible film exhibited antibacterial activity against Staph-
ylococcus aureus and B. cereus among bacteria tested by using agar diffusion assay. Tensile strength and elongation at break were
significantly (p < 0.05) changed by incorporation of garlic oil at 0.3% and 0.4% v/v, respectively. Water vapor permeability decreased
significantly (p < 0.05) with 0.4% v/v garlic oil incorporation, whereas total color difference remained same until 0.4% v/v. These
results revealed that garlic oil has a good potential to be incorporated into alginate to make antimicrobial edible film or coating
for various food applications.

2004 Published by Elsevier Ltd.

Keywords: Edible film; Antibacterial activity; Garlic oil

1. Introduction the active substances are neutralized on contact or dif-

Microbial growth on food surfaces is a major cause
of food spoilage. In particular, bacterial contamination
of ready-to-eat products is of serious concern to human
health. Meat products, for example are extremely sus-
ceptible to pathogenic and spoilage bacteria. Since bac-
terial growth in foods occurs mainly at the surface,
attempts have been made to solve this by using antibac-
terial sprays or dips (Ouattara, Simard, Piette, Begin, &
Holley, 2000). However, direct surface application of
antibacterial substances has limited benefits, because
*
Corresponding author. Tel.: +66 2 524 6115; fax: +66 2 524 6200/
6111.
E-mail address: rakshit@ait.ac.th (S.K. Rakshit).
fused rapidly into the bulk of food (Siragusa & Dickson,
1992; Torres, Motoki, & Karel, 1985).
The possibility of edible film or edible coating to car-
ry some food additives such as antioxidants, antimicro-
bials, colorants, flavors, fortified nutrients and spices are
being studied (Han, 2001; Pena & Torres, 1991). The
method is different from direct application, as the incor-
poration of antimicrobial agents into edible film or edi-
ble coating localizes the functional effect at the food
surface. The antimicrobial agents are slowly released
to the food surface, and therefore, they remain at high
concentrations for extended periods of time (Coma,
Sebti, Pardon, Deschamps, & Pichavant, 2001; Ouattara
et al., 2000). Antimicrobial agents used in food applica-
tion include organic acids, bacteriocins, enzymes,

0963-9969/$ - see front matter
2004 Published by Elsevier Ltd.

doi:10.1016/j.foodres.2004.04.009
268 Y. Pranoto et al. / Food Research International 38 (2005) 267–272
alcohols and fatty acids (Han, 2000; Ouattara, Simard,
Holley, Piette, & Begin, 1997). In addition, spice extracts
have been introduced for their ability to control meat
spoilage (Ouattara et al., 2000). The beneficial effects ob-
tained by using edible film and coating in terms of phys-
ical, mechanical, and biochemical benefits have been
reported in many publications (Han, 2001; Krochta &
Johnston, 1997). Gennadios and Weller (1990) reported
the ability of edible film in retarding moisture, oxygen,
aromas, and solute transport.
Spices such as garlic, onion, cinnamon, cloves, thyme
and sage have been investigated for their antimicrobial
activity. The antimicrobial compounds in plant materi-
als are commonly present in the essential oil fraction
and it has more inhibitory effect than the corresponding
ground form (Frazier & Westhoff, 1978; Nychas, 1995).
Garlic oil is an essential oil product extracted from gar-
lic bulbs by using steam distillation. The compounds of
garlic oil mainly are diallyl disulfide (60%), diallyl trisul-
fide (20%), allyl propyl disulfide (16%), a small quantity
of disulfide and probably diallyl polysulfide (Warade &
Shinde, 1998). However, there is limited information in
the utilization of such natural antimicrobial agents to be
incorporated into edible film or coating. Therefore, it is
very important to investigate the possibility of produc-
ing antimicrobial edible film by incorporation of garlic
oil. The objective of this research was to assess antibac-
terial activity of garlic against the food pathogenic bac-
teria Escherichia coli, Salmonella typhimurium,
Staphylococcus aureus and Bacillus cereus. The study in-
cluded forming antibacterial alginate edible film by the
incorporation of garlic oil. Physical and mechanical
property changes of the alginate film due to garlic oil
incorporation were also investigated.
2. Materials and methods
2.1. Organisms and cultures
Typical meat product bacterial contaminants used in
this study were E. coli, Salmonella typhimurium, Staphy-
lococcus aureus and B. cereus. They were obtained from
the culture collection of Bioprocess laboratory (Biopro-
cess Technology, AIT, Thailand). The bacterial cultures
were grown on nutrient agar slants (Difco Laboratories,
Detroit, MI, USA) and kept at 4 C. Subculturing was
carried out every month to maintain bacterial viability.
2.2. Inhibitory activity of garlic oil in nutrient broth
The experiments were carried out with 50 mL of
nutrient broth (Difco Laboratories, Detroit, MI, USA)
in a 125 mL flask. Garlic oil (ABBRA Co. Ltd., Bang-
kok, Thailand) was initially diluted with ethanol into
10% v/v concentration. In each flask, 0.5 mL of dilute
garlic oil, 49 mL of nutrient broth and 0.5 mL of a
24 h grown bacterial culture were added. Ethanol was
used instead of garlic oil as a control. Three replicate
flasks were prepared for each treatment. Media growths
in flasks were incubated in an incubator shaker (Ed-
mund Bühler TH 25) at 125 rpm, 37 C for 24 h and
0.5 mL of culture was withdrawn as periodical sam-
plings. The samples were serially diluted in sterile dis-
tilled water and 0.1 mL of each dilution was spread
onto Tryptic soy agar (Merch, Darmstadt, Germany)
plates. The plates were then incubated at 37 C for
24 h and viable bacteria were counted.
2.3. Preparation of antibacterial edible film
Alginate-based edible films were prepared by modifi-
cation of the method used by Pavlath, Gossett, Cami-
rand, and Robertson (1999). Sodium alginate (1 g) was
dissolved into 100 mL of distilled water and rotary shak-
ing was done concurrently. As the alginate film was brit-
tle, 0.4 mL of glycerol was added into the edible film
solution. Garlic oil was initially diluted into 10% con-
centration using ethanol and then incorporated into
the edible film solution at various final concentrations
of 0 (control), 0.1%, 0.2%, 0.3% and 0.4% v/v of edible
film forming solution. The solutions were cast onto
12 · 16 cm of polyacrylic plates followed by oven drying
at 40 C for 20–24 h. The unpeeled film was dipped in
45 mL of calcium chloride solution containing 1% Ca
ion and re-dried again in oven for 4–6 h. The dry films
obtained were peeled off and stored for evaluation.
2.4. Antibacterial activity
Antibacterial activity testing of the edible films was
carried out using the agar diffusion method according
to Chen, Yeh, and Chiang (1996). The edible films were
cut into 17 mm diameter discs and then placed on Muel-
ler Hinton agar (Merch, Darmstadt, Germany) plates,
which had been previously seeded with 0.1 mL of inoc-
ulum containing approximately 105–106 CFU/mL of
tested bacteria. The plates were then incubated at
37 C for 24 h. Observations on the diameter of the
inhibitory zone surrounding film discs and contact area
of edible film with agar surface were made. Experiments
were done in triplicate.
2.5. Tensile strength and elongation at break
Tensile strength and elongation at break of films were
tested using a Lloyd Instrument Testing Machine type
LRX 5K (Lloyd Instrument, Ltd., Fareham, UK). The
four films were cut into 1.5 · 10 cm strips. Films were
held parallel with an initial grip separation of 5 cm,
and pulled apart at a head speed of 25 mm/min. Tensile
strength was calculated by dividing the maximum force
 Y. Pranoto et al. / Food Research International 38 (2005) 267–272 269

at break (read from machine or chart) by the cross-sec- yellow (+). Measurements were taken as the average of
tional area of film (Newton/m2 = Pascal). Percent elon- at least three points of each sample. Total color differ-
gation at break was calculated on the basis of length ence (DE) was calculated as follows:
extended as compared to the original length of the film. qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
DE ¼ ðL
LÞ2 þ ða
aÞ2 þ ðb
bÞ2 ,
2.6. Water vapor permeability
where, L*, a* and b* are the standard values of white
plate, L, a and b are the values of samples measured.
Water vapor permeability was determined gravimet-
rically similar to those reported by Gontard, Duchez,
2.8. Statistical analysis
Cuq, and Guilbert (1994). A cup containing silica gel
as a desiccant was covered with the film to be tested
Experimental data was analyzed using Excel (Micro-
and placed in a controlled desiccator. The temperature
soft Inc.) and SPSS software (SPSS Inc.). The one way
and relative humidity inside the desiccator chamber
ANOVA procedure followed by LSD test was used to
were periodically checked. The weight gained by the
determine the significant difference (p < 0.05) between
cup was measured at 4 h intervals within 24 h to deter-
treatment means.
mine water vapor transmission rate and thereafter was
used to calculate the water vapor permeability value.
The water vapor permeability value was expressed in
g mm/m2 day kPa. 3. Results and discussion

2.7. Color measurement 3.1. Inhibitory activity of garlic oil in the nutrient broth

Samples were monitored for their surface colors by
using a Color and Color Differential Meter model TC-
PIIIA (Tokyo Denshoku Co. Ltd, Japan). Instrumental
color readings are L, a and b. These values are L black
(
) to white (+), a green (
) to red (+), and b blue (
) to
The inhibitory effect of garlic oil against the four
selected bacteria is shown in Fig. 1. Garlic oil at approx-
imately 0.1% v/v was able to reduce the growth of all
bacteria tested. A greater inhibitory effect was observed
on B. cereus followed by Staphylococcus aureus, which

10
10
9
9
8
8
7
(Log CFU/mL)

7
(Log CFU/mL)

Viable cells

6
Viable cells

6
5 5
4 4
3 3
2 2
Garlic oil Garlic oil
1 Control 1 Control
0 0
0 4 8 12 16 20 24 28 0 4 8 12 16 20 24 28
(a) Time (h) (b) Time (h)

10 10
9 9
8 8
7 7
(Log CFU/mL)

(Log CFU/mL)
Viable cells

Viable cells

6 6
5 5
4 4
3 3
2 2
Garlic oil Garlic oil
1 Control 1 Control
0 0
0 4 8 12 16 20 24 28 0 4 8 12 16 20 24 28
(c) Time (h) (d) Time (h)

Fig. 1. Bacterial viability of (a) E. coli, (b) Salmonella typhimurium, (c) Staphylococcus aureus and (d) B. cereus with 0.1% v/v garlic oil. Values are
means of three replications and bars represent standard errors.
270 Y. Pranoto et al. / Food Research International 38 (2005) 267–272

are Gram-positive bacteria. The reduction of B. cereus
and Staphylococcus aureus growth were 5.61 and 4.30
log cycles, whereas the controls increased 0.76 and
1.72 log cycles, respectively, after 24 h incubation. Less
inhibition was observed on E. coli and Salmonella
typhimurium by decreases of 2.28 and 1.24 log cycles,
respectively. Both are Gram-negative bacteria. In addi-
tion, viable cells in the controls increased by 2.8 log
and 2.59 log cycles respectively. It confirmed that the
presence of 0.1% v/v garlic oil inhibited growth on all
bacteria tested and was dependent on the Gram charac-
ter of the microorganisms. Generally, Gram-positive are
more sensitive than Gram-negative bacteria to the anti-
microbial compounds in spices (Nychas, 1995). How-
ever, the greater resistance of Gram-negative bacteria
against spice oils is not an overall trend, since even some
Gram-positive bacteria show such resistance (Ouattara
et al., 1997).
3.2. Antibacterial activity
The results of the antibacterial assessment of edible
film incorporated with garlic oil against four selected
bacteria is presented in Table 1. The bacteria selected
here are commonly associated with meat products.
Staphylococcus aureus and B. cereus were observed to
be more sensitive to garlic oil-incorporated film as com-
pared to E. coli and Salmonella typhimurium. Fig. 2
shows the inhibitory effect of alginate film incorporated
with 0.3% garlic oil against Staphylococcus aureus and
B. cereus in comparison with the control. Incorporation
of garlic oil at higher than 0.2% v/v started to exhibit a
clear inhibitory zone indicated by the absence of bacte-
rial growth around the film strips. At a garlic oil concen-
tration up to 0.4%, the clear zone of inhibition was not
observed with E. coli and Salmonella typhimurium.
However, incorporation of garlic oil at higher than
0.1% v/v revealed a weak inhibitory effect, indicated by
minimal growth underneath film discs. In addition, the
growth was obviously observed in all bacteria tested
with edible film without garlic oil incorporation (con-
trol). This result was consistent with the previous in vi-
tro test in nutrient broth, in which E. coli and
Salmonella typhimurium were more resistant than the
two other Gram-positive bacteria Staphylococcus aureus
and B. cereus. These results prove that the active com-
pound of garlic oil could be immobilized in the alginate
film and subsequently released, thereby inhibiting target
microorganisms.

3.3. Tensile strength and elongation at break

Table 1
Antibacterial activity of garlic oil-incorporated edible film against
some bacteria Tensile strength is a measure of film strength, whereas
Bacteria Garlic oil % Observation at 24 h
elongation at break is a measure of film stretch ability
(v/v) prior to breakage. Both properties are important char-
Inhibitory Contact
zoneA areaB
acteristics for packaging material (Krochta & Johnston,
1997). The tensile strength and elongation at break
Escherichia coli 0 (Control) 0a

0.1 0a +
changes of the edible film incorporated with garlic oil
0.2 0a + are summarized in Table 2. The tensile strength varied
0.3 0a + from 38.67 to 66.12 MPa. Incorporation of garlic oil
0.4 0a + markedly affected film tensile strength, as seen in the re-
Salmonella typhimurium 0 (Control) 0a
duced tensile strength value at increased amounts of gar-
0.1 0a + lic oil. A significant difference (p < 0.05) in tensile
0.2 0a + strength was markedly shown after 0.3% of garlic oil
0.3 0a +
incorporation. It is reasonable due to the presence garlic
0.4 0a +
oil as an additive material. The presence of garlic oil in
Staphylococcus aureus 0 (Control) 0a
the alginate probably interferes with ionic interactions
0.1 0a +
0.2 20.13b +
facilitated by Ca ions, which help in forming a network.
0.3 40.67e + Because in making this film, the garlic oil was incorpo-
0.4 46.58f + rated before providing Ca ions. Therefore, the higher
Bacillus cereus 0 (Control) 0a
amounts of garlic oil incorporated caused a greater
0.1 25.67c + reduction of tensile strength. These values were similar
0.2 26.67c + to those reported by Pavlath et al. (1999) in forming
0.3 33.17d + alginate film by immersion into 5% of calcium ion solu-
0.4 51.42g +
tion. The elongation at break value of alginate film var-
+: represents an inhibitory effect;
: represents no inhibitory effect. ied from 2.73% to 4.84%. On the other hand,
A
Values are measurements of diameter of inhibitory zone and
incorporation of garlic oil at certain levels increased
expressed in mm. Values (n = 3) with different superscript letters are
significantly different (p < 0.05). elongation at break. However, addition of garlic oil
B
Contact area is the part of agar on Petri dish directly underneath higher than 0.3% v/v reduced the elongation at break va-
film pieces. lue. Incorporation of garlic oil at less than 0.4% revealed
 Y. Pranoto et al. / Food Research International 38 (2005) 267–272 271

Fig. 2. Inhibitory zone of alginate edible film incorporated with 3% v/v garlic oil (right strips) compared to control (left strips) against (a)
Staphylococcus aureus and (b) B. cereus.

Table 2
hydrophobic property of garlic oil. In this system, gar-
Tensile strength and elongation at break of garlic-incorporated edible
film lic oil might contribute to extend intermolecular inter-
actions of the structural matrix in alginate film,
Garlic oil (% v/v) Tensile strength (MPa) Elongation at break (%)
a
therefore, it enhanced moisture passing through the
0 (Control) 66.12 4.05ab
edible film. The water vapor permeability value of film
0.1 64.70a 4.10ab
0.2 55.21ab 4.35a or coating material should be taken into account when
0.3 49.09bc 4.84a applying onto a moist product such as precooked beef
0.4 38.67c 2.73b patties. The films ability to retard moisture loss from
a–c
Means (n = 3) in same column with different superscript are sig- the product (Wu, Weller, Hamouz, Cuppett, &
nificantly different (p < 0.05). Schnepf, 2001) is an important characteristic that af-
fects product quality.

elongation at break values slightly higher than the algi- 3.5. Color measurement
nate film reported by Pavlath et al. (1999), who did not
use any such additive. The values of color measurement taken into account
were L, a, b, and DE. The color performances of garlic
3.4. Water vapor permeability oil-incorporated edible film can be seen in Table 4. Algi-
nate edible film without garlic oil incorporation ap-
The water vapor permeability value varied from peared clear and transparent. Addition of garlic oil
18.73 to 30.89 g mm/m2 day kPa as presented in Table affected the appearance of edible film in both color
3. Incorporation of garlic oil affected the water vapor and transparency. The color tended to yellowish as indi-
permeability of the alginate edible films. The water va- cated by the increase of b value. The b value produced
por permeability value tended to increase as higher by the incorporation of garlic oil below 0.3% were lower
amounts of garlic oil were incorporated. A significant than the b value of alginate film investigated by Pavlath
difference (p < 0.05) was shown after incorporation of et al. (1999), who made alginate film by immersing in
0.4% v/v garlic oil. It probably occurred due to the copper solution to provide multivalent ions. Opposite
results were revealed when garlic oil at 0.3% and higher
Table 3 were incorporated, in which L values decreased as the
Water vapor permeability of garlic oil-incorporated edible film amount of garlic oil incorporated increased. It indicates
Garlic oil (% v/v) Water vapor permeability (g mm/m2 day kPa) that the color of the edible film tends to darken. Total
color change was observed by reading DE values. Exper-
0 (Control) 20.32a
0.1 18.73a iments showed that there was no significant change
0.2 21.84a (p < 0.05) of DE value, which indicated no color change
0.3 23.42a due to the incorporation of garlic oil. Therefore, incor-
0.4 30.89b poration of garlic oil in alginate films or coatings will
a,b
Means (n = 3) with different superscript are significantly different not affect the appearance of the food product when in
(p < 0.05). use.
272 Y. Pranoto et al. / Food Research International 38 (2005) 267–272

Table 4
Color measurement of garlic oil-incorporated edible film
Garlic oil L (black–white) a (green–red) b (blue–yellow) DE (color difference)
(% v/v)
0 (control) 83.22a 2.26a
3.35a 11.64ab
0.1 81.02ab 1.56a
0.94b 12.45ab
0.2 82.19ab 1.44a 0.66b 10.62ab
0.3 82.25a 1.15a 3.38c 10.27a
0.4 78.94b 2.51a 4.65c 13.84b
a–c
Means (n = 3) in same column with different superscript are significantly different (p < 0.05).

4. Conclusions ca.us/~han/CyberFoodsci/volume2001.htm. Downloaded in May

The results showed that garlic oil had antibacterial
activity on the four bacteria used in this study. Incorpora-
tion of garlic oil into alginate edible film at levels more
than 0.2% led to a significant inhibitory effect on Staphy-
lococcus aureus and B. cereus. At this level, there was no
effect on the physical and mechanical properties of the edi-
ble film formed as observed. Therefore, an antibacterial
alginate edible film incorporated with garlic oil is promis-
ing and has good potential in many food applications.
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