Dev. Chem. Eng. Mineral Process. 13(S/6), pp. 61 7-626, 2005.

Mechanical, Physical and Antimicrobial

Characterization of Edible Films Based on

Alginate and Chitosan Containing Garlic Oil

Yudi Pranoto, Sudip Kumar Rabbit* and Was Mahadeo Salokhe School of Environment, Resources and Development, Asian Institute of Technology, P. 0. Box 4, Klong Luang, Pathumthani 121 20, Thailand

Antimicrobial edible films based on alginate and chitosan were made by incorporating garlic oil (GO) as a natural agent. With incorporation of GO up to 0.4% there was a decrease in tensile strength and elongation at break, and slight increase in water vapor permeability. Alginate and chitosan films containing GO at levels of 0.2% and 0.1%, respectively are shown to have an inhibitory effect on Staphylococcus aureus and Listeria monocytogenes by using agar dzmion assay. However, when pieces of the film were added into liquid culture of indicator organism, it was found that the GO-incorporated alginate film exhibited inhibitory activity against Staphylococcus aureus and Listeria monocytogenes, while chitosan films inhibited all three indicator bacteria. The micrographs show the GO is distributed throughout the matrix and indicate that the change in physical characteristics leads to the changes in mechanical properties and water vapor permeability. These results confirmed that GO has good potential for incorporation into edible films for food applications.

Introduction Microbial contamination of ready-to-eat products is of concern for human health. Meat products are extremely susceptible to pathogenic and spoilage bacteria. Since bacterial growth in food occurs mainly at the surface, attempts have been made to using antibacterial sprays or dips [l]. However, direct surface application of antibacterial substances has considerable limitations, because the active substances could be neutralized, evaporated or diffuse rapidly into the bulk of the food [2, 31. Edible films and coatings have been investigated for their ability to retard the transport of moisture, oxygen, aromas, and solute [4]. The capability of edible films or

* Author for correspondence (rakshit@ait.ac.th).

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Yudi Pranoto, Sudip Kumar Rakshit and V i h Mahadeo Salokhe

edible coatings to contain food additives, such as antioxidants, antimicrobial, colorants, flavors, fortified nutrient and spices, are being explored [5]. In such cases, the functional effect of food additives are localized at, and slowly released to, the food surface [ 11. Studies on the antimicrobial action of films have been reported with acetic acid and lactic acid [2], nisin, lysozyme, EDTA and grape fruit seed extract incorporated in alginate film [6], and on chitosan-based film [l, 7, 81.

The use of natural extracts as antimicrobial agents is gaining popularity as consumer demand for chemical-free foods increases [9]. Ouattara et al. [ l ] investigated the use of spice extract of cinamaldehyde as an antimicrobial agent to be incorporated into chitosan film, in order to reduce S. liquefaciens and L. sakei growth in meat products. Grape fruit seed extract was incorporated into alginate and carrageenan films to inhibit some food contaminant bacteria [6]. Garlic oil is obtained by steam distillation of the crushed fresh garlic bulbs. It is mainly composed of sulfur- containing compound such as allicin, diallyl disulfide and diallyl trisulfide, which possess more antimicrobial activity than the corresponding ground form [lo]. Ross et al. [ 111 investigated the antimicrobial efficacy of garlic oil against a broad range of gram-positive and gram-negative human enteric bacteria, including food borne pathogens. However, there is limited information on the use of spice oil to produce antimicrobial edible films and edible coatings based on alginate and chitosan.

The aim of this study was to investigate antimicrobial edible films based on alginate and chitosan, and incorporating garlic oil. The films formed were characterized for mechanical and physical properties, as well as their microstructure. The antimicrobial activity was tested against the food indicator microorganisms: Staphylococcus aureus, Escherichia coli and Listeria monocytogenes.

Materials Used and Experimental Methods (0 Organisms and cultures The microorganisms used in the study were Staphylococcus aureus, Escherichia coli and Listeria monocytogenes. They were obtained from culture collection at the Bioprocess Laboratory of our institute and the Department of Fishery, Bangkok, Thailand. The bacterial cultures were grown on an agar slant and maintained at 4°C.

(ii) Preparation of antimicrobial edible firm Alginate edible film was prepared by modification of the method used by Pavlath et al. [12] which involved an immersion step. Glycerol (0.4 mL) was added to 100 of 1% alginate mixture to improve its film property. Garlic oil (GO) was previously diluted into 10% v/v with ethanol and then incorporated into solution, in order to obtain final GO concentrations up to 0.4% v/v of the edible film solution. It was then cast on a 12 x 16 cm polyacrylic plate, followed by oven drying at 4OoC for 24 hours. Unpeeled film was dipped into 45 mL, of calcium chloride solution containing 1% Ca ion, and re-dried for 4 hours. The dry films were peeled off and stored in a chamber at 50% RH and 25°C until evaluation. Chitosan edible film was prepared by dissolving 1 g of shrimp chitosan (degree of deacetylation approximately 95%) in 100 mL of 1% acetic acid solution. The solution was then filtered through a silk screen to remove undissolved material. Incorporation of GO and the casting processes were done as in the case of alginate-based films.

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Edible Films Based on Alginate and Chitosan-Containing Garlic Oil

(iii) Tensile strength and elongation at break Tensile strength (TS) and elongation at break (EB) of films were tested according to the ASTM standard method [13] by using Lloyd Instrument Testing Machine type LRX 5K (Lloyd Instrument, Ltd., Fareham, UK). Edible films were cut into strips of 1.5 x 10 cm. The films were held parallel with an initial grip separation of 5 cm, and then pulled apart at a head speed of 25 d m i n . TS was calculated by dividing the maximum force at break by cross sectional area of film (N/m2 = Pa). Percent EB was calculated based on the extended and original lengths of the films.

(iv) Water vapor permeabi1iQ Water vapor permeability (WVP) was determined using a gravimetrical method. A cup containing silica gel as a desiccant was covered with films being tested, and then placed in a desiccator filled with distilled water. The cups for test were 5 cm diameter and 3 cm depth, with an exposed film area of 0.001963 m2. Temperature was maintained at 25"C, and RH inside the desiccator was checked using a hygrometer. The cup was weighed at 4 hour intervals for 24 hours. The constant rate of increased weight was obtained by linear regression. WVP was expressed in g.mm/m2.day.kPa, and calculated as follows:

where Aw = the weight of water absorbed in the cup (g); At = time for weight change (days); A = area of the exposed film (m2); x = film thickness (mm); p2 - pI = vapor pressure differential across the film (kPa) calculated based on relative humidity and temperature inside and outside the cup.

(v) Antimicrobial activily Antimicrobial activity of edible films was carried out using agar diffusion and liquid culture tests. In the agar diffision assay, film samples were cut into a disc shape, 17 mm diameter, using a circular knife. Film cuts were placed on Mueller Hinton agar plates (Merch, Darmstadt, Germany) which had been previously seeded with 0.1 mL of inoculum containing 10' - lo6 CFU/mL of indicator bacteria. The plates were incubated at 37°C for 24 hours. The diameter of inhibitory zone surrounding the film discs was then measured.

The liquid culture test was taken by cutting antimicrobial edible films into a rectangular form of 2 x 5 cm. Three sample cuts (30 cm2 total surface area) were immersed in 20 mL nutrient broth (Merck, Darmstadt, Germany) in a 50 mL. flask. A control was prepared for each bacterial culture without edible film strips. The flask was then inoculated with 200 pL cultures containing lo5 - lo6 CFU/mL of bacteria tested, and then transferred to a shalung incubator (Edmund Biihler TH 25) at 37°C and 150 rprn Samples (1 mL) were periodically withdrawn and its optical density measured at 660 nrn using a spectrophotometer.

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Yudi Pranoto, Sudip Kumar Rahhit and Vilas Mahadeo Salokhe

(vi) Microstructure offirm The microstructure of film samples was determined by scanning electron microscopy (SEM), JEOL JSM-5410 (Japan Electron Optics Limited, Japan). Samples were observed in cross-sectional, obtained by fracturing the film samples in liquid nitrogen. Film samples were prepared using standard techniques, mounted on aluminum stubs and sputter coated with gold. Cross-sectional edible film analysis was viewed at 2000x magnification in order to fit the entire cross section into the picture.

Results and Discussion (i) Tensile strength and elongation at break Tensile strength (TS) and elongation at break (EB) of alginate and chitosan edible films incorporated with GO are presented in Table 1. TS value of films based on alginate ranged from 38.67 to 66.11 Mpa, and that of the chitosan edible film from 38.96 to 49.63 MPa. The results indicated that the incorporation of GO into edible film foxming solution significantly reduced (p < 0.05) the TS of alginate-based films. This is conceivable as GO is a hydrophobic material [ll]. The presence of this material in the alginate structure reduced existing ionic interactions, and it disturbed the cross-linking reaction of the carboxyl or hydroxyl groups. Therefore, higher addition of GO can increasingly reduce the TS value. Cagri et al. [14] stated that incorporation of additives other than cross-linking agents generally lowers the TS value. On the other hand, the incorporation of GO up to 0.4% did not change significantly (p < 0.05) the TS of chtosan-based edible film,

Table I . Mechanical and water vapor permeability properties of alginate and chitosan films containing garlic oil.

Edible film

A lginate

Chitosan

Garlic oil

Control 0.1 0.2 0.3 0.4

Control 0.1 0.2 0.3 0.4

(?A v/v) Tensile strength

(MPa) 66.11 5 4.29' 64.69 5 9.90'

55.21 2 11.26sb 49.09 _+ 7.98& 38.67 2 4.87' 49.63 2 1.83& 47.54 5 7.Ub' 45.30 2 2.98bC 41.89 5 7.36' 38.96 + 4.08'

Elongation at

-%%% 4.10 5 0.52ab

4.84 5 1.33& 2.73 5 0.03' 6.22 5 2.64' 4.81 5 0.88bC 3.86 5 1.18'b 4.70 5 O S k 3.79 2 0.24'b

4.35 5 1.2lUbb"

Water vapor permeability

(g.mm/m'.day.kPa) 20.32 2 2.37ab 18.73 0.98" 21.84 5 3.95" 23.42 2.42b 30.89 5 2.72d 14.99 5 3.18" 14.86 5 1.94" 17.79 5 1 .80'" 17.61 2 2.31" 19.93 + 1.94ab

Note: mean 2 standard deviation (n=3); means in same column with diferent superscript letters are significantly diferent (p < 0.05).

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Edible Films Based on Alginate and Chitosun-Containing Garlic Oil

In the chitosan film, hydrogen bonds between hydroxyl groups and amino groups occurred. This reaction with the incorporation of GO during mixing in chitosan solution, and thus has less effect on TS as compared to alginate film. Elongation at break value (EB) of alginate film ranged from 2.73 to 4.84%, whereas EB values of chitosan film varied from 3.79 to 6.22%. A significant reduction of EB value is obviously seen with 0.4% GO incorporation in both edible films.

(ii) Water vapor permeability Water vapor permeability (WVP) is a measure of ease of moisture to penetrate and pass through a material. WVP of alginate and chitosan edible films incorporated with GO are shown in Table 1 . WVP value of the alginate films varied from 18.73 to 30.89 g.mm/m2.day.kPa, whereas WVP value of the chitosan films ranged from 14.86 to 19.93 g.mm/m2.day.kPa. The WVP value of chitosan films is lower than alginate film In general, incorporation of GO into alginate and chitosan edible films leads to an increase in WVP values. The results showed that 0.4% GO increased significantly (p < 0.05) the WVP value in alginate and chitosan-based films. Generally, the presence of hydrophilic groups in the material tends to cause poor moisture barrier [14]. GO is a hydrophobic material and should not increase WVP values of films. However, in this study GO caused a slightly increased WVP value. This might be because the presence of GO in the alginate and chitosan films affected their structural matrix dominantly rather than increasing the moisture barrier. The GO contributed to extend intermolecular interaction, and resulted in loosening the compactness of the structure. It enhanced moisture passage through the edible films, and thereby increased WVP values of the films. This is found to be the case from a study of the Scanning Electron Microscopy (SEM) micrographs discussed later.

(iii) Antimicrobial activity Antimicrobial activity of GO-incorporated edible films evaluated using agar diffusion test against indicator organisms are shown in Table 2. Idubitory activity was measured based on a clear zone surrounding circular film strips. The measurement made included the diameter of film strips. Therefore, the values are always higher than diameter of film strips (17 mm). If no clear zone is present it means that there was no antimicrobial effect, and has been indicated to have zero value. GO- incorporated edible films revealed inhibition against Staphylococcus aureus and Listeria monocytogenes. The clear zone did not occur in Escherichia coli which is gram negative bacteria. Alginate edible film showed antimicrobial effect against the former two microorganisms after 0.2% GO incorporation, while in chtosan film 0.1% GO started to exhibit the effect. Antimicrobial activity increased as hgher levels of GO were incorporated. Gram negative bacteria (Escherichia coli) used in this study is more resistant than gram positive bacteria. Generally, gram positive bacteria are more sensitive to the antimicrobial compounds in spices than gram negative ones [lo]. However, the resistance of gram negative bacteria is not an overall trend, since some strains exhibit reversed phenomenon [IS].

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Yudi Pranoto, Sudip Kumar Rakshit and V i h Mahadeo Salokhe

Table 2. Antimicrobial activity of alginate and chitosan edible films containing garlic oil against some food pathogenic bacteria with agar difision assay.

Edible film

dlginate

Chitosan

Garlic oil

Control 0.1 0.2 0.3 0.4

Control 0.1 0.2 0.3 0.4

(% vh)

Diamc Staphylococcus

aureus 0' 0"

20.13 2 1 .27b 40.67 f. 1 .89def 46.58 2 6.42'"

OP 20.58 f. 0.38b

46.42 f. 7.07'fg 47.00 2 7.40efg

33.79 f. 3.Wd

?r of inhibition z Escherichia

coli OS 0= 0' 0' 0' On 0' On 0' 0'

!e (mm) Listeria

monocytogenes 0' 0'

40.25 f. 3.85de 45.75 2 12.79'"

50.00 2 2.50" 0'

27.75 0.66& 36.03 f. 2.17d

46.53 5 9.47'" 48.50 f. 6.28'"

Note: mean 2 standard deviation (n=3); means with dzflerent superscript letters are significantly different (p < 0.05).

Antimicrobial activity of edible films tested in liquid culture was conducted in GO-incorporated films which previously had exhibited antimicrobial activity (0.2% GO). Figure 1A to 1C represented the cultural growth of Staphylococcus aureus, Escherichia coli and Listeria monocytogenes, respectively. These figures show the bacterial growth pattern of cultures added with strips of alginate film incorporated with 0.2% GO (AIg-GO 0.2) and chitosan film incorporated with 0.2% GO (Chi-GO 0.2) and comparison with a control.

There was a distinct difference in antimicrobial activity of alginate films incorporated with GO as compared to the film without GO on Staphylococcus aureus culture (see Figure 1A). The bacterial growth of culture with unincorporated alginate film was similar to the control (no film), and revealed higher population. However, cultures with chitosan films, GO-incorporated and unincorporated, exhibited noticeable antimicrobial activity.

Antimicrobial effect of edible films based on alginate and chitosan against Escherichia coli is shown in Figure 1B. The growth of Escherichia coli in culture containing unincorporated alginate film was similar to the control, and the population was slightly higher after 12 hours in the stationary stage. GO-incorporated alginate film suppressed bacterial growth by extending the lag phase to double that in the control. Both chitosan films, GO-incorporated and unincorporated, showed noticeable antimicrobial activity by exhibiting no increased growth.

Antimicrobial activity of alginate and chitosan edible films in liquid culture against Listeria monocytogenes is depicted by Figure 1C. The presence of alginate film (without incorporation of garlic oil) in the culture enhanced the growth, causing the population to be twice that of the control. The reason for this is not clear. However, it is possible that in the presence of Listeria monocytogenes growth there is leachmg of the alginate film leading to the increase in optical density.

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Edible Films Based on Alginate and Chitosan-Containing Garlic Oil

A

0 4 a 12 16 20 24

Time (h)

B

0 4 8 12 16 20 24

Time (h)

C

0 4 a 12 16 20 24

Time (h)

Figure 1. Antimicrobial activity of alginate and chitosan edible films containing garlic oil 0.2% against (A) Staphylococcus aureus, (B) Escherichia coli, and (C) Listeria.

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Yudi Pranoto, Sud@ Kumar Rakshit and Was Mahadeo Salokhe

Antimicrobial activity tested with liquid culture c o n f i e d that the antimicrobial efficacy of GO was more pronounced when incorporated into alginate than into chitosan films. Alginate has no antimicrobial activity by itself. Unincorporated chitosan films did not show significant inhibitory effect when examined using agar diffusion test, due to chitosan being incapable of diffusion through the adjacent agar medium [7]. However, in liquid culture, chitosan strips have more chance to contact and interact with bacteria intensively, therefore, the innate antimicrobial characteristic took place [16, 171. Thus chitosan film had greater efficacy as antimicrobial agent, especially in liquid culture, as both chitosan films (GO-incorporated and unincorporated) had an inhibitory effect on the microorganisms studied.

Figure 2. Scanning electron micrographs of the cross-sectional edible films at 2000x magnification: (A) Alginate film, (B) Garlic oil-incorporated alginate film; (C) Chitosan film, and (0) Garlic oil-incorporated chitosan film.

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Edible Films Based on Alginate and Chitosan-Containing Garlic Oil

(iv) Microstructure offirms The micrographs (from SEM) of cross-sections of alginate and edible films are shown in Figures 2A to 2D. These describe the effect of 0.2% GO incorporation in alginate and chitosan edible films as viewed by a cross-sectional part. The microstructure of alginate film (see Figure 2A) is dense and a very rare hole is shown. The cross sectional surface is rough, indicating the difficulty to break or crack during preparation. Incorporation of GO affected the structure dramatically leading to expansion of the film (see Figure 2B). The film has a sponge-like appearance with pores formation in the presence of GO. The GO was found to be distributed throughout the alginate film matrix.

Figure 2C shows the cross-sectional view of chitosan film. It was dense and very compact. No holes and cracks are observed. The cross-sectional surface is smooth, and the film was found to break easily as compared to alginate film. GO-incorporated chitosan film looked thicker and expanded (see Figure 2D). Some pores are also observed, and GO was distributed throughout the matrix. These micrographs suggest that the distribution of GO in an alginate film matrix was more uniform than in chitosan. The micrographs support the mechanical and water vapor permeability data reported earlier.

Conclusions Incorporating 0.4% GO into alginate film leads to a decrease in TS, while in chitosan films the TS remained the same. The EB was also affected by GO incorporation. WVP values of alginate and chitosan films increased after adding 0.4% GO. The incorporation of GO led to these films having antimicrobial characteristics against some food pathogenic bacteria. SEM micrographs show the distribution of the GO entire films matrix. The antimicrobial alginate and chitosan edible films produced have a good potential for food applications, if the consumers do not mind the presence of garlic flavor. Chitosan itself has antimicrobial properties, and added to the antimicrobial effect of GO. The antimicrobial nature of GO, which is a natural extract, would add to the physical barrier provided by the edible films and increase the shelf life of food products.

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