www.elsevier.com/locate/foodres

Food Research International 38 (2005) 267–272

Physical and antibacterial properties of alginate-based ediblefilm 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

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 humanhealth. 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

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

doi:10.1016/j.foodres.2004.04.009

* Corresponding author. Tel.: +66 2 524 6115; fax: +66 2 524 6200/

6111.

E-mail address: rakshit@ait.ac.th (S.K. Rakshit).

the active substances are neutralized on contact or dif-

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,

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 beenreported 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 fractionand 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 inthe 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 Buhler TH 25) at 125 rpm, 37 �C for 24 h and0.5 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 filmsobtained 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-

tional area of film (Newton/m2 = Pascal). Percent elon-

gation at break was calculated on the basis of length

extended as compared to the original length of the film.

2.6. Water vapor permeability

Water vapor permeability was determined gravimet-

rically similar to those reported by Gontard, Duchez,

Cuq, and Guilbert (1994). A cup containing silica gel

as a desiccant was covered with the film to be tested

and placed in a controlled desiccator. The temperature

and relative humidity inside the desiccator chamber

were periodically checked. The weight gained by thecup was measured at 4 h intervals within 24 h to deter-

mine water vapor transmission rate and thereafter was

used to calculate the water vapor permeability value.

The water vapor permeability value was expressed in

gmm/m2daykPa.

2.7. Color measurement

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

0

1

2

3

4

5

6

7

8

9

10

0 4 8 12 16 20 24 28

Time (h)

Via

ble

cells

(L

og C

FU

/mL)

Garlic oilControl

Via

ble

cells

0

1

2

3

4

5

6

7

8

9

10

0 4 8 12 16 20 24

Time (h)

Via

ble

cells

(Log

CF

U/m

L)

28

Garlic oilControl

Via

ble

cells

(a) (b

(c) (d

Fig. 1. Bacterial viability of (a) E. coli, (b) Salmonella typhimurium, (c) Stap

means of three replications and bars represent standard errors.

yellow (+). Measurements were taken as the average of

at least three points of each sample. Total color differ-

ence (DE) was calculated as follows:

DE ¼ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiðL� � LÞ2 þ ða� � aÞ2 þ ðb� � bÞ2

q,

where, L*, a* and b* are the standard values of white

plate, L, a and b are the values of samples measured.

2.8. Statistical analysis

Experimental data was analyzed using Excel (Micro-

soft Inc.) and SPSS software (SPSS Inc.). The one way

ANOVA procedure followed by LSD test was used to

determine the significant difference (p < 0.05) between

treatment means.

3. Results and discussion

3.1. Inhibitory activity of garlic oil in the nutrient broth

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 allbacteria tested. A greater inhibitory effect was observed

on B. cereus followed by Staphylococcus aureus, which

0

1

2

3

4

5

6

7

8

9

10

0 4 8 12 16 20 24 28

Time (h)

(Log

CF

U/m

L)

Garlic oilControl

0

1

2

3

4

5

6

7

8

9

10

0 4 8 12 16 20 24 28

Time (h)

(Log

CF

U/m

L)

Garlic oilControl

)

)

hylococcus aureus and (d) B. cereus with 0.1% v/v garlic oil. Values are

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 ediblefilm incorporated with garlic oil against four selected

bacteria is presented in Table 1. The bacteria selected

Table 1

Antibacterial activity of garlic oil-incorporated edible film against

some bacteria

Bacteria Garlic oil %

(v/v)

Observation at 24 h

Inhibitory

zoneAContact

areaB

Escherichia coli 0 (Control) 0a �0.1 0a +

0.2 0a +

0.3 0a +

0.4 0a +

Salmonella typhimurium 0 (Control) 0a �0.1 0a +

0.2 0a +

0.3 0a +

0.4 0a +

Staphylococcus aureus 0 (Control) 0a �0.1 0a +

0.2 20.13b +

0.3 40.67e +

0.4 46.58f +

Bacillus cereus 0 (Control) 0a �0.1 25.67c +

0.2 26.67c +

0.3 33.17d +

0.4 51.42g +

+: represents an inhibitory effect; �: represents no inhibitory effect.A Values are measurements of diameter of inhibitory zone and

expressed in mm. Values (n = 3) with different superscript letters are

significantly different (p < 0.05).B Contact area is the part of agar on Petri dish directly underneath

film pieces.

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 andB. 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 byminimal 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

Tensile strength is a measure of film strength, whereas

elongation at break is a measure of film stretch abilityprior to breakage. Both properties are important char-

acteristics for packaging material (Krochta & Johnston,

1997). The tensile strength and elongation at break

changes of the edible film incorporated with garlic oil

are summarized in Table 2. The tensile strength varied

from 38.67 to 66.12 MPa. Incorporation of garlic oil

markedly affected film tensile strength, as seen in the re-

duced tensile strength value at increased amounts of gar-lic oil. A significant difference (p < 0.05) in tensile

strength was markedly shown after 0.3% of garlic oil

incorporation. It is reasonable due to the presence garlic

oil as an additive material. The presence of garlic oil in

the alginate probably interferes with ionic interactions

facilitated by Ca ions, which help in forming a network.

Because in making this film, the garlic oil was incorpo-

rated before providing Ca ions. Therefore, the higheramounts of garlic oil incorporated caused a greater

reduction of tensile strength. These values were similar

to those reported by Pavlath et al. (1999) in forming

alginate film by immersion into 5% of calcium ion solu-

tion. The elongation at break value of alginate film var-

ied from 2.73% to 4.84%. On the other hand,

incorporation of garlic oil at certain levels increased

elongation at break. However, addition of garlic oilhigher than 0.3% v/v reduced the elongation at break va-

lue. Incorporation of garlic oil at less than 0.4% revealed

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

Tensile strength and elongation at break of garlic-incorporated edible

film

Garlic oil (% v/v) Tensile strength (MPa) Elongation at break (%)

0 (Control) 66.12a 4.05ab

0.1 64.70a 4.10ab

0.2 55.21ab 4.35a

0.3 49.09bc 4.84a

0.4 38.67c 2.73b

a–cMeans (n = 3) in same column with different superscript are sig-

nificantly different (p < 0.05).

Y. Pranoto et al. / Food Research International 38 (2005) 267–272 271

elongation at break values slightly higher than the algi-

nate film reported by Pavlath et al. (1999), who did not

use any such additive.

3.4. Water vapor permeability

The water vapor permeability value varied from

18.73 to 30.89 gmm/m2daykPa as presented in Table3. Incorporation of garlic oil affected the water vapor

permeability of the alginate edible films. The water va-

por permeability value tended to increase as higher

amounts of garlic oil were incorporated. A significant

difference (p < 0.05) was shown after incorporation of

0.4% v/v garlic oil. It probably occurred due to the

Table 3

Water vapor permeability of garlic oil-incorporated edible film

Garlic oil (% v/v) Water vapor permeability (gmm/m2daykPa)

0 (Control) 20.32a

0.1 18.73a

0.2 21.84a

0.3 23.42a

0.4 30.89b

a,bMeans (n = 3) with different superscript are significantly different

(p < 0.05).

hydrophobic property of garlic oil. In this system, gar-

lic oil might contribute to extend intermolecular inter-

actions of the structural matrix in alginate film,

therefore, it enhanced moisture passing through the

edible film. The water vapor permeability value of filmor coating material should be taken into account when

applying onto a moist product such as precooked beef

patties. The films ability to retard moisture loss from

the product (Wu, Weller, Hamouz, Cuppett, &

Schnepf, 2001) is an important characteristic that af-

fects product quality.

3.5. Color measurement

The values of color measurement taken into account

were L, a, b, and DE. The color performances of garlic

oil-incorporated edible film can be seen in Table 4. Algi-

nate edible film without garlic oil incorporation ap-

peared clear and transparent. Addition of garlic oil

affected the appearance of edible film in both color

and transparency. The color tended to yellowish as indi-cated by the increase of b value. The b value produced

by the incorporation of garlic oil below 0.3% were lower

than the b value of alginate film investigated by Pavlath

et al. (1999), who made alginate film by immersing in

copper solution to provide multivalent ions. Opposite

results were revealed when garlic oil at 0.3% and higher

were incorporated, in which L values decreased as the

amount of garlic oil incorporated increased. It indicatesthat the color of the edible film tends to darken. Total

color change was observed by reading DE values. Exper-

iments showed that there was no significant change

(p < 0.05) of DE value, which indicated no color change

due to the incorporation of garlic oil. Therefore, incor-

poration of garlic oil in alginate films or coatings will

not affect the appearance of the food product when in

use.

Table 4

Color measurement of garlic oil-incorporated edible film

Garlic oil

(% v/v)

L (black–white) a (green–red) b (blue–yellow) DE (color difference)

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–cMeans (n = 3) in same column with different superscript are significantly different (p < 0.05).

272 Y. Pranoto et al. / Food Research International 38 (2005) 267–272

4. Conclusions

The results showed that garlic oil had antibacterialactivity 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 andmechanical 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.

References

Chen, M. C., Yeh, G. H. C., & Chiang, B. H. (1996). Antimicrobial

and physicochemical properties of methylcellulose and chitosan

films containing a preservative. Journal of Food Processing and

Preservation, 20, 279–390.

Coma, V., Sebti, I., Pardon, P., Deschamps, A., & Pichavant, F. H.

(2001). Antimicrobial edible packaging based on cellulosic ethers,

fatty acids, and nisin incorporation to inhibit Listeria innocua and

Staphylococcus aureus. Journal of Food Protection, 64(4), 470–475.

Frazier, W. C., & Westhoff, D. C. (1978). Food Microbiology. New

York: McGraw Hill.

Gennadios, A., & Weller, C. L. (1990). Edible films and coatings from

wheat and corn proteins. Food Technology, 44(10), 63–69.

Gontard, N., Duchez, C., Cuq, J. L., & Guilbert, S. (1994). Edible

composite films of wheat gluten and lipids: water vapor perme-

ability and other physical properties. International Journal of Food

Science and Technology, 29, 39–50.

Han, J. H. (2000). Antimicrobial food packaging. Food Technology,

54(3), 56–65.

Han, J. H., (2001). Edible and biodegradable films/coatings

carrying bioactive agents. Available from: www.den.davis.

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

2001.

Krochta, J. M., & Johnston, C. D. M. (1997). Edible and biodegrad-

able films: challenges and opportunities. Food Technology, 51(2),

61–74.

Nychas, G. J. E. (1995). Natural antimicrobial from plants. In G. W.

Gould (Ed.), New Methods of Food Preservation (pp. 59–89).

Glasgow: Blackie Academic Professional.

Ouattara, B., Simard, R. E., Holley, R. A., Piette, G. J. P., & Begin, A.

(1997). Antibacterial activity of selected fatty acids and essential

oils against six meat spoilage organisms. International Journal of

Food Microbiology, 37, 155–162.

Ouattara, B., Simard, R. E., Piette, G., Begin, A., & Holley, R. A.

(2000). Diffusion of acetic and propionic acids from chitosan-based

antimicrobial packaging films. Journal of Food Science, 65(5),

768–773.

Pavlath, A. E., Gossett, C., Camirand, W., & Robertson, G. H. (1999).

Ionomeric films of alginic acid. Journal of Food Science, 4(1),

61–63.

Pena, D. C. R., & Torres, J. A. (1991). Sorbic acid and potassium

sorbate permeability of an edible methylcellulose-palmitic acid

films: water activity and pH effects. Journal of Food Science, 56(2),

497–499.

Siragusa, G. R., & Dickson, J. S. (1992). Inhibition of Listeria

monocytogenes on beef tissue by application of organic acids

immobilized in a calcium alginate gel. Journal of Food Science,

57(2), 293–296.

Torres, J. A., Motoki, M., & Karel, M. (1985). Microbial stabilization

of intermediate moisture food surfaces. I. Control of surface

preservative concentration. Journal of Food Processing and Pres-

ervation, 9, 75–92.

Warade, S. D., & Shinde, K. G. (1998). Garlic. In D. K. Salunke & S.

S. Kadam (Eds.), Handbook of Vegetable Science and Technology

(pp. 397–413). USA: Marcel Dekker, Inc.

Wu, Y., Weller, C. L., Hamouz, F., Cuppett, S., & Schnepf, M. (2001).

Moisture loss and lipid oxidation for precooked ground-beef

patties packaged in edible starch-alginate-based composite films.

Journal of Food Science, 66(3), 486–493.