Antibacterial Properties of Tannic Acid Incorporated Rice Starch-

gelatin Film

J. Bakar , R. Ahangari, R. Abdul Rahman and R. Karim

Department of Food Technology, Faculty of Food Science and Technology, Universiti Putra Malaysia,

43400, Serdang, Selangor, Malaysia.

Abstract. Many food products either handled chill or frozen are frequently packed in plastic-based

materials, although these packaging materials are of environmental concerns due to their non-biodegradable

characteristics. However, for a short handling period at chill temperatures, for products such as beef, chicken

or fish sausages, safety of consuming the products is the utmost important issue. Hence, these products are

formulated with preservatives such as nitrates to prolong their shelf-life. This brings in the concept of

employing active packaging material as an additional measure to ensure minimal surface contamination to

the packaged foods for better shelf-life. Therefore, the objective of this paper is to determine the antibacterial

activity of tannic acid when incorporated in rice starch-gelatin biodegradable/edible film at 0 to 0.6%

concentrations. The antibacterial activities of the incorporated film were carried out against Escherichia coli

O157:H7, Listeria monocytogenes, Salmonella typhimurium, and Bacillus cereus for the duration of sixteen

days under ambient temperatures (~ 28 oC). Upon storage, the increasing concentration of tannic acid

incorporation resulted in a significant (p<0.05) increase in inhibition effect as evidence by the zone of

inhibitions obtained. The zones of inhibition were 4.5-7 mm for Escherichia coli, 0-7 mm for Salmonella

typhimurium, 0-6 mm in Listeria monocytogenes 0.5-4.5 mm for Bacillus cereus. The result also showed that

E. coli O157:H7 was most sensitive to the presence of tannic acid than L. monocytogenes, S. typhimurium,

and B. cereus. Film incorporated with 0.45 and 0.6% tannic acid were the films with the highest antibacterial

activities. Higher incorporation with tannic acid is limited by the darkening of the film and stiffer texture.

Tannic acid can be a potential antibacterial ingredient in active biodegradable/edible packaging formulation.

Keywords: biodegradable film, tannic acid, rice starch-gelatin film, antibacterial activity

1. Introduction

Biodegradable/edible films function as a barrier to moisture, gas, aroma, and lipid besides protecting a

food product after the primary package is opened [1]. The degradability of these films is so rapid as such

they will help reduce the environmental pollution issue unlike their counterpart the non-degradable plastic

films. Starch-based films have minimum barrier to moisture [2] low permeability to oxygen [2]-[4].

Therefore, they form weak brittle films and hence limit their application as a food packaging material with

good functional properties. However, physical and/or chemical modifications could improve the overall

performance of the film [3]. Proteins such as soy proteins, whey protein and gluten have been used in various

modifications of the starch-based films to influence their barrier properties. Gelatin could also be used in

modifying the barrier properties of starch-based films. Starch-based films and coatings have been reported to

be used for produce packaging such as strawberries, mushrooms and other vegetables [5].

Tannic acid, a gallic ester of D-glucose is known for its antioxidant capacity because of their multiple

phenolic groups that is able to interact with biological macromolecules [6]. It exists in a range of fruits and

plants and is listed as a ‘generally recognized as safe’ (GRAS) food additive [7] and [8]. Several researchers

have reported that tannic acid has antimicrobial activity against foodborne pathogens such as Escherichia

coli and Listeria monocytogenes [8]-[11]. The application of biodegradable film with tannic acid

incorporation for prevention of wound infection was recently reported [12]. There is no previous report for Corresponding author. Tel.: + 603- 8946 8396; fax: +603-8948 5970.

E-mail address: jamilah@upm.edu.my.

International Proceedings of Chemical, Biological and Environmental Engineering, V0l. 100 (2017)

DOI: 10.7763/IPCBEE. 2017. V100. 2

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the usage of tannic acid in rice starch-fish gelatin edible films to impart additional functional properties to

the film for food application. Therefore, the objective of this paper is to determine the effect of tannic acid

incorporation in the rice starch-gelatin film on the microbial inhibition properties of the film against

Escherichia coli O157:H7, Listeria monocytogenes, Salmonella typhimurium, and Bacillus cereus which

could be reflective of their potential to be used as active packaging for the reduction of surface

contamination in actual food application system. The effectiveness of the tannic acid incorporation in the

film was studied at ambient temperature of approximately 28 oC.

2. Material and Methods

2.1. Film formation and Observation

Aqueous solutions of 5 % (w/w) rice starch and the addition of fish gelatin (1 %) including 40 % sorbitol

(w/w, starch and gelatin blend basis) were prepared with the addition of tannic acid at different

concentrations (0.15, 0.30, 0.45, and 0.60 %). The film mixtures were then adjusted to about pH 8 to 9 by

Na2CO3. The mixture was thoroughly mixed before casting into plastic petri dishes (15 cm×15 cm×1.5 cm)

and was allowed to dry in a ventilated oven at 65 oC around 24 h. Dried films were peeled off and kept in

desiccators (RH 56 % at 28 oC) until use.

2.2. Storage Study of Film

The prepared films were cut into 6 mm diameter discs and stored in the respective desiccators for the

total duration of sixteen days. They were removed at pre-determined intervals and placed on streaked agar of

the different tested microbes prior to standard incubation time as described in the antimicrobial test. The

initial day was designated as day one (D1).

2.3. Antimicrobial Test

The disc-diffusion assay was carried out according to [13] and [14]. Films’ inhibition against

Escherichia coli O157:H7 (ATCC 43895), Listeria monocytogenes (ATCC 19115), Salmonella typhimorium

(ATCC 14028), and Bacillus cereus (ATCC 33019) were conducted. Each microbial culture was incubated

at 37 °C in tryptic soy agar (TSA) (Becton Dickinson, Maryland, USA) for 18 h with shaking at 120 rpm.

Cell numbers were adjusted to 5 logs CFU/ml with peptone water (Becton Dickinson, Maryland, USA)

before their use. Each culture was homogenously streaked on to Mueller-Hinton agar (Becton Dickinson,

Maryland, USA) and a disc of 6 mm in diameter of rice starch-gelatin films of various tannic acid

concentrations were then placed on the agar and the plates were incubated at 37 °C for 18 h. The clear zone

around the film disc on the media was measured. Film without tannic acid incorporation was run separately

as the control. The test was carried out three times for each microbe evaluated. The inhibition zone reported

was the clear zone diameter after the deduction of the diameter of the film discs.

2.4. Statistical Analysis

Data were analyzed by one-way analysis of variance (ANOVA) using Minitab release 16. The one-way

ANOVA was used to analyze the effect of rice starch-fish gelatin edible films incorporated with tannic acid.

The Tukey’s test with 95 % confidence interval was used for mean comparison when a significant variation

was found by the ANOVA test.

3. Results and Discussion

3.1. Film Observation

The appearance of plasticized-rice starch-fish gelatin films was slightly opaque and smooth, but rice

starch-fish gelatin films incorporated with tannic acid were brownish and stiffer than the untreated film. This

is due to the cross-linking property of tannic acid itself (Fig. 1).

9

(a) (b)

Fig. 1: Rice starch-fish gelatin film without tannic acid (a) and incorporated with tannic acid (b)

3.2. Antimicrobial Activity

Control film without tannic acid exhibited no inhibitory influence on the growth of tested microbes.

Film incorporated with tannic acid at concentration from 0.15 to 0.60 % per disc demonstrated antimicrobial

activities against E. coli, L. monocytogenes, S. typhimurium, and B. cereus. Fig. 2-Fig. 5 show the inhibition

zone plates against the tested microbes.

Fig. 2: Agar diffusion inhibition zone of Escherichia coli

Fig. 3: Agar diffusion inhibition zone of Listeria monocytogenes

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Fig. 4: Agar diffusion inhibition zone of Salmonella typhimurium

Fig. 5: Agar diffusion inhibition zone of Bacillus cereus

With increasing tannic acid concentration the zone of inhibition increased significantly (p<0.05).

Previous researches had demonstrated that tannic acid have antimicrobial activity on an extensive range of

microorganisms as well as foodborne pathogens such as E.coli 0157:H7 and L. monocytogenes based on log

reduction of their growth; however, the studies reported were not done with edible films [8], [11] and [15].

The zone of inhibition were 4.5-7 mm for Escherichia coli (Fig. 6), 0-6 mm in Listeria monocytogenes (Fig.

7), 0-7 mm for Salmonella typhimurium (Fig. 8) and 0.5-4.5 mm for Bacillus cereus (Fig. 9). The result also

showed that E. coli O157:H7 was more sensitive to the tannic acid present than L. monocytogenes, S.

typhimurium, and B. cereus. This variation might reflect differences in cell surface structures between Gram-

negative and Gram-positive bacteria. The outer cell membrane of bacteria containing lipopolysaccharide may

delay some water-soluble antimicrobial molecules’ ability to influence cytoplasmic membranes. The cause of

stabilization of lipopolysaccharide layer is divalent cations such as Mg2+ and Ca2+ ions. There is a strong

chelating property of these divalent cations with tannic acid [8]. Tannic acid penetration removed these

cations which would be the lipopolysaccharide cell surface layer of Gram-negative bacteria’s destabilization

but would not have any effect on Gram-positive bacteria. On the membrane, the antimicrobial effect’ mode

of tannic acid might be correlated to its inactivation ability of enzymes, microbial adhesions, mineral uptake,

and cell envelope transport proteins [8], [9], and [12]. In contrast, gram-positive bacteria has preventive

barrier against water-soluble tannic acid due to their thick peptidoglycan layer.

There was no significant (p> 0.05) inhibition against S. typhimurium and B. cereus in lower

concentrations of tannic acid, 0.15 and 0.30 % (Fig. 8 and Fig. 9) respectively. There was significant

inhibition against all studied microbes in higher concentration of tannic acid (0.45 and 0.6 %); however,

there was no significant difference between the inhibitory effects of the two concentrations of tannic acid

against the studied microbes.

On the overall, by day sixteen, moderate to strong inhibition could not be obtained for L. monocytogenes,

S. typhimurium and B. cereus where the tannic acid concentration were less than 0.3, 0.4 and 0.4 percent

incorporation, respectively.

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Fig. 6: Zone of inhibition for E. coli

Fig. 7. Zone of inhibition for L.monocytogenes

0

2

4

6

8

D1 D4 D8 D12 D16

Zon

e o

f in

hib

itio

n (

mm

)

Days of storage

0 0.15 0.3 0.45 0.6

S. typhimurium

Fig. 8: Zone of inhibition for S.typhimurium

Fig. 9: Zone of inhibition of B.cereus

4. Conclusions

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The addition of tannic acid to the base film resulted in a stiffer and darker film biodegradable/edible rice

starch-fish gelatin film with increasing concentration of tannic acid incorporation. Tannic acid incorporation

in the rice starch-gelatin film can render antibacterial property to the film for food application. Hence, the

film studied can be used as an active packaging material. The starch-gelatin film incorporated with tannic

acid at 0.45 and 0.60 % showed better inhibition against E. coli O157:H7, L. monocytogenes, S. typhimurium,

and B. cereus activities. E. coli is most sensitive among the four studied microbes.

5. Acknowledgment

The authors like to thank Universiti Putra Malaysia, Malaysia and the Ministry of Science and

Technology, Malaysia for supporting this work through the grant PRGS/1/2015/SG06/UPM/01/2.

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