Leaf Extract

Antibacterial Active Packaging Edible Film

Formulation with Addition Teak (Tectona

grandis) Leaf Extract

Joni Kusnadi and Ponco Budyanto Agriculture Product Technology, Faculty of Agricultural Technology, Brawijaya University, Jl. Veteran, Malang,

Indonesia 65145

Email: [email protected]; [email protected], [email protected]

Abstract—The objective of this research is to investigate the

effect of teak leaf extract as antibacterial to the quality and

antibacterial activity of active packaging edible film. This

research is arranged used Randomized Block Design

consists of 2 factor combinations. First factor is carrageenan

concentration consist of 3, 5 and 7% of tapioca’s weight.

Second factor is teak leaf extract’s concentrations consist of

10, 15 and 20% of edible film’s solution’s volume and

repeated 3 times. The data were analyzed with ANOVA and

continued with BNT or DMRT α=5%. The best treatment

was determined using Multiple Atribute Method. The result

showed that the addition of 7% carrageenan and 20% teak

leaf extract was the best treatment. The edible film

produced had 0,183 mm thickness, 11,635 gram/m2, 24hour

water vapor transmission, 54,334 % elongation, 1,034 N/cm2

tensile strength and 0,600 cm and 0,650 cm antibacterial

activity toward E. coli and S. aureus, respectively.

Index Terms—antibacterial, caragenan, edible film, tectona

grandis extract

I. INTRODUCTION

Seventy percent of food poisoning cases in Indonesia,

caused by the bacterium Staphylococcus aureus,

Eschericia coli and Coliform because of unhygiene

packaging. So it requires packaging of food products that

capable of avoiding the risk of product contamination by

pathogenic and spoilage bacteria. Todate some techniques

have been developed in order to produce active

packaging which is environmentally friendly and

naturally active. Packaging usually contain antibacterial,

absorbent material O2, CO2 absorbent or enhancer [1].

Widely used packaging which can be applied as an

active packaging is edible films. Edible film is made of a

thin layer of edible material, formed on top of the food

component as a barrier to mass transfer, as a carrier of

food and improves food handling [2]. The material for

edible film can use materials such as tapioca, carrageenan

[3], [4] and glycerol. Antibacteria are compounds that

inhibit the activity of bacteria patogen. Antibacteria have

been widely used as bioactive compounds for edible film.

One of Antibacterial compound is phenol. Phenol is one

of compound found in the leaves of teak [5]. Average

Manuscript received January 9, 2015; revised March 9, 2015.

production of PT. Perhutani of 800 thousand m3 per year,

however the utilization of teak leaves is still limited [6].

Addition of teak leaf extract as antibacterial material to

edible made from carrageenan and starch should capable

of extending the shelf life of the edible film and minimize

bacterial contamination of pathogenic and spoilage in

foods such as S. aureus and E. coli and also increase the

utilization of teak leafs.

Starch is a natural polymer that can readily be formed

into films. It consists of (1-4) linked α-D-glucopyranosyl

units. Natural starch, amylopectin and amylose, consist of

this two kinds of chain. The linear polymer, amylose,

makes up about 20 wt% of the granule, and the remain 80

wt% consist of branched polymer, amylopectin. Amylose

is crystalline which has an average molecular weight of

500,000, while amylopectin is highly branched and have

higher molecular-weight [7]. Despite their ease of

preparation, starch films have poor physical properties.

These can be improved by blending with either synthetic

polymers to produce biodegradable materials [8] or other

natural polymers in edible packaging.

Carrageenan is a water soluble linear biopolymer,

increasingly used as natural thickener, formulation

stabilizer, or gelling agent in applications ranging from

food industry (mainly dairy products) to pharmaceutics

[9]. The dairy sector accounts for a large part of the

carrageenan applications in food products, such as frozen

desserts, chocolate milk, cottage cheese and whipped

cream. In addition to this, carrageenans are used in

various non-dairy food products, such as instant products,

jellies, pet foods, sauces, and non-food products, such as

pharmaceutical formulations, cosmetics and oil well

drilling fluid [10], [11]. Except starch, carrageenan is,

with pectin, the main natural gelling polysaccharide

extracted from plants or seaweeds and used as high-value

functional ingredient in foods, cosmetics and

pharmaceuticals [12].

Carrageenan isomers which already known were κ-, λ-,

and ι-carrageenan (κ-kappa, λ-lambda and ι-iota), which

has differences in the number and position of the ester

sulfate groups belongs to the repeating galactose units. κ-

carrageenan has one negative charge per disaccharide

with a tendency to form a strong but rigid gel. κ-

carrageenan has two and λ-carrageenan has an average of

2.7 charges per disaccharide unit. The gelling power of κ-

International Journal of Life Sciences Biotechnology and Pharma Research Vol. 4, No. 2, April 2015

©2015 Int. J. Life Sci. Biotech. Pharm. Res. 79

carrageenan makes it capable of forming good film [13],

[14]. The formation of edible coating with good film

forming and mechanical properties can be done by

blending starch with κ-carrageenan. Starch must be

gelatinized first before blending with carrageenan

The purpose of this research is to investigate the effect

of teak leaf extract as antibacterial to the quality and

antibacterial activity of active packaging edible film. So,

using this edible film which inhibit the growth and kill

pathogens and spoilage bacteria in food will extend the

shelf life and maintain the quality of foods.

II. METHODES

A. Materials and Test Isolates

Materials used for the extraction of teak leaf

antibacterial are fresh Netherlands teak leaf. Materials

used for testing the antibacterial property of the edible

film produced in this researhc were E. coli ATCC 25 922,

S. aureus ATCC 29 213, NA and NB which obtained

from the Laboratory of Food Microbiology Department

of Agricultural Technology UB’s Faculty of Agricultural

Technology.

B. Antibacterial Extraction

In this study, the antibacterial compound was extracted

from fresh teak leaves using a modified method of

Effendi [15], solvent extraction using distilled water and

and 80 Watt microwave for 1 minute.

Edible film was made by mixing 5 grams tapioca, 1 ml

glycerol, carrageenan with various concentration and 100

ml of distilled water. All mixed and heated (temperature

65-70 °C) until the dough gelatinized. After the mixture

is cooled to a temperature of 40 °C, teak leaf extract was

added. The mixture then spread on a mold, and dried.

After the edible film dried, it can be taken from the mold.

C. Parameters Used

The parameters used in this research were the total

phenol [16], thickness, tensile strength, film elongation

[17], water vapor transmission [18], the antibacterial

activity [19], the best treatment [20].

D. Data Analysis

The data were analyzed using Analysis of Varian

(ANOVA) followed by Least Significant Difference

(LSD) using 5% confidence interval or DMRT (Duncan

Multiple Range Test) with a confidence interval of 5%.

The best treatment will be taken by the method of

multiple attributes [20].

III. RESULT AND DISCUSSION

A. The Thickness of Edible Film

The lowest thickness produced by 3% carrageenan and

10% leaf extract and the highest produced by carrageenan

treatment of 7% and 20% leaf extract (Fig. 1). The use of

carrageenan increased soluble solids, causing higher

thickness [21]. More carrageenan added will increase the

edible polymer films, increasing the viscosity of the

suspension together with tapioca starch edible film during

heating and gelatinization [18]. Edible film thickness is

due because of the higher concentration of carrageenan

used. The increase of carrageenan will increase in total

dissolved solids present in the film-forming solution, so

after the drying process will produce thicker films. In

general, the thickness effect on tensile strength,

elongation, and water vapor transmission rate [4].

Figure 1. Average elongation of edible film formed from varies carrageenan concentration with addition of varies teakleaf extract

concentration.

B. Elongation

Elongation is the maximum elongation when the edible

film start to tear of when it streched [2]. The use of

carrageenan in the production of edible film resulted in

the ability to bind water into a well that produced

elongation edible film [21]. The results showed 53.67%-

59.33% elongation (Fig. 2). Elongation chategorized

good when elongation more than 50% and less when the

elongation less than 10% [21].

Figure 2. Average thickness of edible film produced from varies carrageenan concentration with addition of varies teakleaf extract

concentration.

The highest elongation obtained from carrageenan

concentration of 3% and teak leaf extract by 10%. In

carrageenan concentration 5 and 7% decrease in

elongation. This is presumably due to the increase in the

percentage of solids to water volume [21]. More

carrageenan, carrageenan molecules produce a stronger

matrix which caused the non-elastic so that elongation

decreased [4]. The addition of carvacrol can lower

elongation of edible film [22]. The addition of

carrageenan affect the reduced elongation [4]. Elongation

of edible film is due the higher the concentration of

carrageenan used. The carrageenan molecules will form a

stronger film matrix so the films lost its elasticity or

easily broken (brittle), and consequently the percentage



of extension decreases. Other research showed that the

increase of breaking strength of the film will be followed

distantly by decrease in the percentage of elongation.

Edible films with the smallest elongation usually have a

stronger film formed.

C. Water Vapor Transmission

The more the concentration of carrageenan and teak

leaf extract the lower water vapor transmission (Fig. 3).

From the results obtained showed that water vapor

transmission ranges from 10.832 g/m2.24 hour - 27.683 g

/ m2.24 hours.

Glycerol is a substance which can improve the

properties of edible film as a barrier to water vapor [21].

The lowest water vapor transmission is obtained from the

treatment of carrageenan concentration of 7% and 20%

leaf extract. The combination of carrageenan molecules,

tapioca starch and glycerol which binds both to the edible

films caused low water vapor transmission [21]. The

water vapor transmission of edible films depends on

many factors, such as the integrity of the film, the

hydrophilicehydrophobic ratio, the ratio between

crystalline and amorphous zones, and the polymeric chain

mobility [22]. One of the major purposes of edible films

is to block moisture transfer between the food and the

surrounding atmosphere, therefore the WVP should be as

low as possible. Permeability is the contribution of

diffusivity and solubility of the permeant through the

solid matrix, being this parameters changed by the

structure of the matrix when using the same permeant

Figure 3. Average water vapor transmission rate of edible film produced from varies carrageenan concentration with addition of varies

teakleaf extract concentration.

D. Tensile Strength

Mechanical strength and extensibility are generally

required for a film to resist external stress and maintain

its integrity when applied to food products.Tensile

strength is the maximum tensile stress is acceptable

edible film to break up [21]. The results of research show

the tensile strength of edible films ranged 0.434 N / cm2-

1,434 N / cm2.

Lowest tensile strength obtained from carrageenan

concentration of 3% and 10% identity leaf extract and the

highest tensile strength obtained from carrageenan

concentration of 7% and 20% leaf extract (Fig. 4). In

general, the addition of carrageenan will add to the strong

bond so the gel formed becomes stronger and caused

higher tensile strength. Some research showed that the

higher the concentration of carrageenan will increase the

value of tensile strength [18]-[23]. The tensile strength of

the material arising from the reaction between the

polymer bonding between atoms or secondary polymer

chains against external forces is given [23]. An increase

of tensile strength has been also reported when guar gum

(galactomannan) was added to chitosan films, although

further increase of concentration of galactomannan in the

mixtures led to reduction of TS values [24]. The addition

of agar into cassava starch induced an improvement in

EB and TS of cassava starch-based films [25]. Different

polysaccharide ratios can be used to improve mechanical

properties depending on the degree of interaction between

the molecules of the components.

Figure 4. Average tensile strenght of edible film formed from varies carrageenan concentration with addition of varies teakleaf extract

concentration.

E. Antibacterial Activity of Edible Film

The addition of teak leaf extract showed antibacterial

activity on Escherichia coli ATCC 25922 and

Staphylococcus aureus ATCC29213 proven by the

formation of clear zone diameter 0.12cm-1.26cm and

0.14cm-1.37cm. other research showed that teak leaf

extract added on edible film caused inhibition of

Eschericia coli and Staphylococcus aureus; Psedomonas

aeruginosa, Proteus mirabilis, Salmonella typhimurium,

Klebsiella pneumoniae, Escherichia coli and Serratia

marcescens [27].

Teak leaf extract has antibacterial activity against

Escherichia coli [26]. Antibacterial contained by edible

films containing teak leaf extract more effect on

Staphylococcus aureus ATCC 29 213 compared against

E.coli ATCC 25922. This is caused by the structure of the

cell wall of Gram-positive bacteria such as

Staphylococcus aureus is simpler than the cells of Gram-

negative bacteria, Escherichia coli, so cause antibacterial

easier entry into cells of gram-positive bacteria. Gram-

positive bacteria have a cell wall of different sensitivity to

antibiotics, physical and enzyme treatment compared

with gram-negative bacteria which Gram-positive

bacteria more sensitive than Gram-negative antibacterial

[28]. In order antibacterial effect on Gram-negative

bacteria, antibacterial must penetrate the outer membrane

of bacterial sheath advance. After that, the antibacterial

entry through the cell wall and reaches past the

periplasmic enzyme serine protease. The enzyme

responsible for the biosynthesis of the cell wall [29].

Carrageenan with the concentration of 7% and 20%

leaf extract resulted in the largest inhibition zone (Fig. 5).

The higher the concentration of leaf extract the higher

the antibacterial antibacterial activity, meaning that the



bacteria will be killed faster when higher concentrations

of antibacterial given [26]. Phenol is a major component

in teak leaf extract as antibakteri. Fenol can be damaged

by oxidation. The efectiveness of oxidation depending on

the amount of O2 in the air and the material properties of

carrageenan [6] transmission of O2, as the small molecule

of carrageenan fill a void in polymer matrix so as to

minimize O2 that affected the oxidized phenols. The

hydrophobic nature of O2 and hydrophilic structure

carrageenan resulted in O2 difficult to penetrate the

polymer carrageenan [30], so phenol in the network are

better protected if the edible film [31].

Figure 5. Average inhibition zone diameter of edible film formed from varies carrageenan concentration with addition of varies teakleaf extract

concentration.

Teak bark effective against Listeria

monocytogenes and methicillin resistant

Staphylococcus aureus. The antibacterial compound

of teak leaf is 5-hydroxy-1, 4- naphthalenedione

(Juglone) [32]. This compound has been found to be

inhibitory to oral pathogens, notably Streptococcus

mutans, Streptococcus sanguis, Porphyromonas

gingivalis and Prevotella intermedia [33], [34]

Another study showed the synergistic in-vitro

antibacterial activity to formulate new cost effective

antimicrobial agent for multi-drug resistant

organisms, based on the synergistic activity of

Tetracycline with methanol extract of teak. Other

research shows maximum synergistic activity

against different bacteria both Gram-positive and

Gram-negative species. The higher synergistic rate

was achieved against Salmonella typhimurium

(MTCC 98), Klebsiella pneumonia (MTCC 432),

and lowest synergistic shows against Pichia pastoris

(MTCC 34), Escherichia coli (MTCC 729). No

synergistic activity was observed in Citrobacter

freondii (MTCC 1658) [26]. The antibacterial

activity was also tested for leaf, bark and wood

extracts of teak against Staphylococcus aureus

(ATCC 25923), Klebsiella pneumoniae (ATCC

700603), hospital strains of Salmonella paratyphi

and Proteus mirabilis [35]. The use of carrageenan as edible films and coatings

already covers various fields of the food industry such as

application on fresh and frozen meat, poultry and fish to

prevent superficial dehydration [36], ham or sausage-

casings [37], granulation-coated powders, dry solid foods,

oily foods [34], etc., but also manufacturing soft capsules

[39], [40], and especially non-gelatin capsules [41].

Indeed, this protective barrier can also be used in food

domain in order to prevent the transfer of moisture, gases,

flavours, or lipids and thus to maintain or improve food

quality and to increase food product shelf life [42].

Another promising emerging technology that has been

applied to various biopolymers, including, carrageenan-

based coating, is their use as antimicrobial agent carriers

in active packaging systems [14]-[43].

F. Selection of Best Treatment

The best treatment is obtained by using the method

according to the Multiple Attribute [44]. Based on test

results, it is obtained with the use of the best treatment

karagenan 7% concentration and teak leaf extract 20%

(K3E20). It can be shown from the value of each

parameter which can be seen Table I.

TABLE I. THE BEST TREATMENT OF EDIBLE FILM WITH ADDITION OF

CARRAGEENAN AND TEAK LEAF EXTRAC

Parameter Perlakuan Terbaik

Ketebalan 0,180 (mm)

Transmisi Uap Air 11,635 (gram/m2.24jam)

Elongasi 54,330 (%)

Tensile Strenght 1,030 (N/cm2)

Aktivitas Antibakteri

terhadap Eschericia coli

ATCC 25922

6,000 (mm)

Aktivitas Antibakteri

terhadap Staphylococcus aureus ATCC 29213

6,500 (mm)

IV. CONCLUSION

The addition of carrageenan has a significant influence

on the thickness, water vapor transmission and tensile

strength edible film. Addition of teak leaf extract has

significant impact on the thickness, water vapor

transmission of edible film.

The best treatment is obtained on addition of

carrageenan concentration as much as 7% and teak leaf

extract as much as 20% which results in a thickness of

0.183 mm, 11.635 grams of water vapor transmission per

m2.24 hour, 54.334% elongation, 1,034N/cm2 tensile

strength, 0,600 cm inhibition zone against E. coli

ATCC25 922 and 0.650 cm inhibition zone S. aureus

ATCC29213.

ACKNOWLEDGMENT

The author would like to thanks Dr. Estri Laras

Arumingtyas for useful discussions.

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Joni Kusnadi was born in Tanjungkarang, Lampung, Indonesia dated June 12, 1962. He

completed the S1 in the Department of

Agricultural Product Technology, Faculty of Agricultural Technology, Gadjah Mada

University in 1986 and graduated S2 in the Department of Biothecnology, Gadjah Mada

University in 1999. S3 education was

completed in 2008, on the program of agricultural science, Post Graduate UB. In

1988 until now he is a lecturer of Department Completed the S1 in the Department of Agricultural Product Technology, Faculty of Agricultural

technology, Brawijaya University, Malang, Indonesia in the field of

Food Microbiology study. In the year 2009 until now, he is also the head of the post graduate study program of agricultural product

technology at Brawijaya Univegrsity, Malang, Indonesia.



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Leaf Extract