2. 540 AntiMICROBIAL (aDOLF).cdr

Antimicrobial Activity of Melinjo Seed and Peel Extract
( ) Against Selected Pathogenic BacteriaGnetum gnemon

ADOLF JAN NEXSON PARHUSIP AZIS BOING SITANGGANG1* 2,3AND

Department of Food Technology, Universitas Pelita Harapan, MH. Thamrin Boulevard, 1100 Lippo Village,
Tangerang, Indonesia;

Department of Food Science and Technology, Institut Pertanian Bogor, Darmaga , Bogor 16680, Indonesia;

Southeast Asian Food and Agricultural Science and Technology (SEAFAST) Center, Institut Pertanian Bogor,

Darmaga, Bogor 16680, Indonesia

Gnetum gnemon

emping

Aspergillus flavus Bacillus cereus , Staphylococcus aureus

Enterobacter aerogenes

B. cereus S. aureus

Gnetum gnemon

Aspergillus flavus

Bacillus cereus

, Staphylococcus aureus Enterobacter aerogenes minimal inhibitory

concentration minimal bactericidal concentration

B. cereus S. aureus

Gnetum gnemon

Melinjo ( ) is an Indonesian native plant which has not been widely accepted due to its limited utilization.

Mainly, melinjo is consumed as an ingredient to make a vegetable dish or as raw material of ' '. The purpose of this

research was to study the antimicrobial activity of the melinjo seed extract and melinjo peel extract. In this study, extraction

from melinjo seed and peel was conducted by maceration using three kinds of solvent: ethanol, ethyl acetate and hexane for 24

h at room temperature. The results showed that none of the melinjo extracts (concentration from 5% - 25% w/v) could inhibit

the growth of IPBCC 88.030; whereas for ATCC 10876 ATCC

25953, and ATCC 13048 there was efficient inhibition by 5% (w/v) of melinjo seed-ethanol extract.

The minimal inhibitory concentration (MIC) value of melinjo extract was ranged from 0.26 g mL to 1.46 g mL , whilst the

minimal bactericidal concentration (MBC) value was ranged from 1.02 g mL to 6.04 g mL . The inhibitory capacity of

extract had a similar level as compared to 10 ppm penicillin G on ATCC 10876 as well as on ATCC 25953.

Furthermore, as compared to 10 ppm streptomycin, the inhibitory capacity of the extract was equal for the all tested bacteria.

Cell wall deformation was observed using SEM, and confirmed by the presence of ions (Ca and K ) outside of the cells,

detected by means of AAS.

Key words: Melinjo, , extraction, antimicrobial, inhibition

Melinjo ( ) adalah tanaman lokal Indonesia yang belum dimanfaatkan secara luas. Umumnya melinjo

dikonsumsi sebagai komponen dalam pembuatan sayur ataupun dalam pembuatan kue kering yang dikenal dengan emping.

Tujuan penelitian ini adalah mengkaji aktivitas antibakteri ekstrak melinjo yang berasal dari biji dan kulit melinjo. Proses

ekstraksi dilakukan dengan metode maserasi menggunakan tiga jenis pelarut, yaitu etanol, etil asetat, dan heksana selama

24 jam pada suhu ruang. Hasil ekstraksi biji melinjo (5% w/v) dengan menggunakan pelarut etanol sampai dengan

konsentrasi 25% (w/v), ternyata tidak menunjukkan aktivitas antifungal khususnya terhadap pertumbuhan

IPBCC 88.030; namun demikian ekstrak melinjo tersebut memiliki aktivitas antibakteri, khususnya terhadap

ATCC 10876 ATCC 25953, dan ATCC 13048. Nilai

(MIC) dan (MBC) dari ekstrak melinjo untuk berbagai bakteri yang

diujikan berturut-turut berada pada selang 0.26 g mL hingga 1.46 g mL , dan 1.02 g mL hingga 6.04 g mL .

Penghambatan ekstrak melinjo menunjukkan kemampuan yang hampir sama dengan 10 ppm penisilin G terhadap bakteri

contoh ATCC 10876 dan ATCC 25953, sedangkan jika dibandingkan dengan streptomisin, kemampuan

penghambatan ekstrak melinjo hampir mirip dalam beberapa variasi konsentrasi. Uji konfirmasi berupa pengamatan struktur

sel bakteri menggunakan SEM menunjukkan terjadinya perubahan pada struktur dinding sel bakteri dan hal ini diperkuat

dengan adanya pengeluaran sejumlah ion yang menyusun dinding sel (Ca and K ) yang dikuantifikasi menggunakan AAS.

Kata kunci: Melinjo, , ekstraksi, antimikrob, penghambatan

μ μ

μ μ μ μ

-1 -1

2+ +

-1 -1 -1 -1

2+ +

Melinjo ( ) is native to Indo-

Malaya, belonging to the Gnetacea family (Kato .

2009; Kato . 2011). The size of the melinjo tree is

about 50 ft in height and it is often found in dry and

humid forests of the region. Particularly in Indonesia,

the distribution areas of this plant cover in Andaman,

Sumatra and Java Island (Manner and Elevitch 2006).

For Sumatra itself, the productivity of melinjo is more

than 20,000 granules per year (Manner and Elevitch,

2006). It is a spontaneous re-growth species in fallow

forests and commonly planted as a cultivated species in

both backyard gardens and orchards or as hedges. In

Gnetum gnemon

et al

addition to those, melinjo has been utilized as a

conservation farming component at the upland

watershed in semi-arid climate (Hafif . 1995).

For local food products, melinjo seeds are

commonly used as raw material for making '

(common name of the traditional cracker in Indonesia)

and as supplementary soup material. The high purine

content of melinjo seeds may cause an increase in uric

acid production which can lead to chronic arthritis due

to bone erosion (Terkeltaub 2010). Therefore many

people are reluctant to consume it. Melinjo peel, as a

waste from the process of making emping are seldom

used as vegetables, most likely being thrown out

(Siswoyo . 2011).

et al

emping'

et al*Corresponding author, Phone: (+62)-21-5460901, ext 1249,
Fax: (+62)-21-5460910, E-mail: adolf.parhusip@uph.edu

ISSN 1978-3477, eISSN 2087-8575
Vol 5, No 3, September 2011, p 103-112

I N D O N E S I A

Available online at:
http://www.permi.or.id/journal/index.php/mionline

DOI: 10.5454/mi.5.3.2

Several bioactive compounds are found in melinjo,

such as saponins, tannins, and flavonoids (Kato .

2011; Santoso . 2010; Siswoyo . 2011). These

compounds have been studied to have potential

utilization as drugs or antibody, antimicrobial,

pigments or even anti-inflammatory agents (Khan

2003; Rauha 2000; Uboh 2010).

Furthermore, a recent study has reported the isolation

of stilbenoids isolated from the seeds of melinjo (Kato

2009) which stated these stilbenoids show

moderate antimicrobial activity via a diphenyl-picril-

hydrazil-hydrate (DPPH) radical scavenging effect,

including lipase and .

However, this study focused itself on isolation process

of stilbenoids with complex steps and studied

biological activities of the extracts generally. On the

other hand, a detail investigation specifically on the

antimicrobial activity of melinjo extracts has not been

reported. In accordance to those findings, where the

melinjo contains bioactive compounds, such as

saponins, tannins, and flavonoids, and stilbenoids;

there is therefore a need to investigate the antibacterial

activity of melinjo seed and peel extract which can be

applied further in food products or even in drugs.

Initially, we hypothesized the mechanism of

antibacterial activity of this extract would be by

interfering with the synthesis of the bacteria cell wall.

Therefore within this study we carried out an anti-

bacterial comparison study between the melinjo extract

and common antibiotics used as antibacterial agents

(penicillin G and streptomycin). In addition, to see the

cell wall deformation, we carried out SEM analysis to

depict clear images of cell wall changes due to the

addition of extract into the growth medium. Selected

microorganisms used within this study were

ATCC 25953,

ATCC 10876, ATCC 13048,

ATCC 07853 and

IPBCC 88.030, which were

considered to represent Gram-negative, Gram-positive

bacteria and also mold. We used bacteria and mold

within this study since their cell wall assemblies are

different. Consequently, this results in different

responses for the cell wall deformation as anti-

microorganism added to inhibit the growth of those

microorganisms.

Several pathogenic bacteria used

for this study, such as ATCC

25953, ATCC 10876,

ATCC 13048, and

ATCC 07853 were purchased from

et al

et al et al

et al.

et al. et al.

et al.

Staphylococcus aureus Bacillus cereus

Enterobacter aerogenes

Pseudomonas aeruginosa

Aspergillus flavus

Staphylococcus aureus

Bacillus cereus Enterobacter

aerogenes Pseudomonas

aeruginosa

α-amylase inhibition activity

MATERIALS AND METHODS

Microorganisms.

American Type Culture Collection (ATCC) -

Biological research center (BRC), Manassas, VA

20108, USA, whilst IPBCC 88.030

was purchased from Bogor Agricultural University,

Bogor, Indonesia. At first glance, all bacteria were

activated on each physical growth medium. For

bacteria, media used were nutrient agar (NA) and

nutrient broth (NB), whereas for fungi (

IPBCC 88.030) media used were potato dextrose

agar (PDA) and potato dextrose broth (PDB). After the

activation, those microorganisms were then preserved

in micro-tubes (containing 0.5 mL activated culture)

with addition of glycerol (50% v/v) and finally stored at

-40 °C until used again.

Several pro-analytical

organic solvents, such as ethanol, ethyl acetate, and

hexane were purchased from Mayky Lovic Tangguh

Perkasa, Indonesia. Such media, nutrient agar (NA),

potato dextrose agar (PDA), nutrient broth (NB), and

potato dextrose broth (PDB) were purchased from

Sigma-Aldrich. Especially for NB, the MDL number

was MFCD01867738 and we confirmed that it does not

have any sodium chloride (NaCl) in its composition.

This was very important, since within this study such

cell leakage due to the addition of the extract would be

carried out in terms of Ca and K ion determinations.

Moreover, two antibiotics, penicillin G and

streptomycin were obtained from Meiji Seiyaku

(Tokyo, Japan).

The ripe melinjo seed and

melinjo peel (red color) used in this study were

collected in September 2009 from the village of

Balekambang Condet, East Jakarta, Indonesia. The

plant was identified by the Research Center of Biology,

Indonesian Institute of Sciences (LIPI), where a

voucher specimen was deposited in Pelita Harapan

University (1283/IPH.1.02/If.8/XII/2009).

. Removal of water from

melinjo seeds and melinjo peels was done using a

cabinet dryer. Thus, the dried materials were crushed to

reduce the size into powder form and sieved using an

abrasive screener (Ø: 35 Mesh). Extraction was carried

out using several pro-analytical organic solvents

(ethanol, ethyl acetate, and hexane) with a ratio

between melinjo materials (seed and peel) to solvent

being 1:4. The extraction was carried out at 30 °C,

shaken at 150 rpm using an orbital shaker incubator

(Type Hotech, Taiwan) for 24 h. The mixture was then

filtered using Whatman filter paper no. 4 by assistance

of a vacuum pump. The extraction process did not

follow the study reported by Sunaryanto . (2010),

since the sources of bio-actives that act as anti-

microorganisms were totally different. Further crude

Aspergillus flavus

Aspergillus

flavus

et al

Reagents and Chemicals.

Seed and Peel Samples.

Extraction of Samples

2+ +

104 P SARHUSIP AND ITANGGANG Microbiol Indones

extract isolation was done by amplification using a

rotary evaporator (Buchi) at 40 °C and shaken at 75

rpm in round-bottomed flask until crystal or paste

appeared. Eventually, the crude extracts were put in a

dark bottle and stored at temperature of 4 °C.

The antimicrobial assay was

carried out using the well-diffusion method according

to Bloomfield (1991) with minor modification

according to Yasni . (2009) for five kinds of

microorganism as pre-mentioned above. Each extract

resulting using several solvents (ethanol, ethyl acetate,

hexane) from different parts of melinjo (seed, peel) was

diluted to give several concentrations of the extract

(b/v), such as 0; 5; 10; 15; 20; 25%. The MIC (Minimal

Inhibitory Concentration) and MBC (Minimal

Bactericidal Concentration, bactericidal term is used

for bacteria) or MFC (Minimal Fungicidal

Concentration, fungicidal term is used for fungus) were

determined according to Bloomfield (1991). By

plotting natural logarithm (ln) of concentration (Mo) in

x-axis and inhibition zone square value (Z ) in y-axis,

the value that crosses x = 0 will be Mt. Furthermore,

MIC was then calculated as 0.25 x Mt and MBC or

MFC as 4 x MIC. The determination of the preferred

extract was based on the inhibition zone value as

reported by Elgayyar . (2001) and Suliantari .

(2008). Following this method, statistical software

SPSS 16.0 was used to run ANOVA in giving a

conclusion as to which extract would be preferred at

low concentration gives higher inhibition zone.

Generally an anti-spore is

defined as a compound that has the ability to inhibit

spore activity (Jenson and Moir 2003). Determination

of anti-sporal activity from a preferred extract onto

spore-forming bacteria ATCC 10876 was

conducted using the well-diffusion method. The

inhibition zone for spores (culture was

incubated for 48 h) was compared with the inhibition

zone for vegetative cells of ATCC 10876

(culture was incubated for 8 h). The statistical analysis

was carried out employing the (p < 0.05). Each

experiment was run in duplicate, and mean values were

calculated. A statistical package (SPSS version 16.0)

was used for the data analysis.

This test was

carried out to compare the performance between

preferred extract of melinjo and several antibiotics that

have antibacterial capacity. The procedure was by

varying the concentration of antiotics and the

inhibition diameter was then evaluated using the well-

diffusion method.

. To determine the presence of leakage of

Antimicrobial Assay.

Anti-Sporal Activity.

Antibiotics Testing Comparison.

Atomic Absorption Spectrophotometer (AAS)

Analysis

et al

et al et al

one way

B. cereus

B. Cereus

t-test

bacterial cells, AAS analysis was conducted within this

study which focused on Calcium ions (Ca ) and

Potassium ions (K ) quantification. The bacterial

strains were activated using NB for 8 h. Freshly

activated bacteria were collected by centrifuging the

broth at 4 032 g force (rotating at 6 000 rpm; radius

of rotor 100 mm) for 15 min. Cells were then washed

for several times using de-mineral water and finally re-

suspended again in 10 ml de-mineral water in a reaction

tube for each bacterial strain. Into the tube, a preferred

extract concentration was added and then incubated for

24 h. The quantification method of those cations

followed the procedure of Prashar . (2003), where

the suspension was then analyzed to determined Ca at

422.7 nm, whilst K at 766.5 nm using AAS instrument

(AAS type Shimadzu AA-680).

To determine the efficacy of extract and the

morphological changes, SEM studies were performed

on tested bacteria treated with preferred melinjo

extract. Controls were prepared without melinjo

extract. The bacterial samples were washed gently and

gradually with 2% glutaraldehyde solution, 2% tannic

acid solution, buffer solution (0.1 M sodium cacodylate

containing 10 mM MgSO , pH 6.7), and 1% osmium

tetraoxide solution. Each specimen was dehydrated

using sequential exposure per ethanol concentrations

ranging from 50 - 100%. The ethanol was replaced

by tertiary butyl alcohol. After dehydration, the

specimen was put onto a stub. Finally, the specimen

was sputter-coated with gold (Aurum) in an ion coater

for 5 min, followed by microscopic examinations

(SEM type JSM-5310 LV) with a magnification of

10,000-15,000 times.

The data was analyzed using

one-way analysis of variance (ANOVA) for repeated

measurements using statistical software SPSS 16.0 for

windows. The Duncan's multiple range tests was used

to determine differences at each point. Differences at

each point were considered significant at .

Crude extracts obtained from melinjo

seed tended to be colored as brownish yellow in color,

while the extract obtained from the melinjo peel was

dark red. Within this study, both melinjo seed and peel

could be extracted with ethanol, 10.25%, and 13.33%,

respectively (Table 1). Conclusively, through the yield

percentage of this investigation, the dominant

components contained in melinjo seed or melinjo peel

were polar, as reflected by higher yield using polar

solvent.

et al

P ≤ 0.05

Scanning Electron Microscope (SEM) Analysis.

Statistical Analysis.

RESULTS

Extraction.

Volume 5, 2011 Microbiol Indones 105

Antimicrobial Activity of Seed-based and Peel-

based Melinjo Extract. The results showed that

IPBCC 88.030 could not be

inhibited by either melinjo seed and peel extract since

even the highest concentration applied (25% w/v) into

the wells, the growth of this fungus could not be

inhibited. In addition, the extract inhibition towards

Gram-positive bacteria ( ATCC 10876 and

ATCC 25953) was positively higher than for

Gram-negative bacteria ( ATCC 13048

and ATCC 0785 (Fig 1, 2, 3, and 4).

Aspergillus flavus

B. cereus S.

aureus

E. aerogenes

P. aeruginosa )

106 P SARHUSIP AND ITANGGANG Microbiol Indones

Table 1 Yield of melinjo seed and peel extracts

Plant sample Solvent Yield (%)

Melinjo seeds
Ethanol 10.25
Ethyl acetate 2.31

Hexane 0.00

Melinjo peels
Ethanol 13.33
Ethyl acetate 2.43

Hexane 1.51

Fig 1 Inhibition diameter of melinjo extracts ( ) againstsource of extract - solvent used Bacillus cereus.

Fig 2 Inhibition diameter of melinjo extracts ( ) againstsource of extract - solvent used Staphylococcus aureus.

Minimum Inhibitory Concentration and

Minimum Bactericidal Concentration.

Anti-sporal Activity

From the

MIC and MBC values (shown in Table 2), melinjo seed

- ethanol extract has the lowest MIC and MBC values.

Therefore, conclusively, melinjo seeds - ethanol

extract was the most effective extract that could inhibit

bacterial growth for both Gram-negative and Gram-

positive bacteria, as compared to other melinjo extracts

through the exception of inhibition to mold growth

( IPBCC 88.030). There were not any MIC

and MFC values for . Representative images

to depict inhibition zone, could be seen in Fig 5

which ethanol was used as the solvent and source of

extract was melinjo peel. In addition, the preferred

extract was chosen according to Suliantari . (2008),

summarized in Table 3.

. Anti-sporal activity of

preferred melinjo extract can be observed by

contacting the extract with spores. The number of

A. flavus

et al

Fig 3 Inhibition diameter of melinjo extracts ( ) againstsource of extract - solvent used Enterobacter aerogenes.

Fig 4 Inhibition diameter of melinjo extracts ( ) againstsource of extract - solvent used Pseudomonas aeruginosa.

are used as a bridge for phospholipid components on

cell wall. The ions Ca and K can be detected after the

bacteria have been in contact with the extract. This

indicates that the destruction or collapse was happened

on the bacteria cell membrane, which might cause the

extraction of ions of Ca and K from the body of a

detected cell by AAS. As seen on the Table 4, the

amount of Ca that has been detected on Gram-

negative bacteria (73.17 mg L and 62.41 mg L ) was

larger as compared to Gram-positive bacteria (58.27

mg L and 67.17 mg L ).

The morphological changes on the bacteria cell surface

can be detected using SEM. There were some

morphological changes in , , and

which treated with 5% melinjo seeds -

ethanol extract and which was treated

with 15% melinjo peels - ethyl acetate extract

(Fig 9). Morphological changes that occurred include

2+ +

-1 -1

Scanning Electron Microscope (SEM) Imaging.

B. cereus S. aureus E.

aerogenes

P. aeruginosa

spores is large when the bacterial culture is incubated

for 48 hours (Fig 6). According to the results of statistic

analysis (p < 0.05), there was a significant difference

between anti-sporal activity of extract onto spores of

(10.18 mm) and anti-bacterial activity of extract

towards vegetative cells of (12.83 mm).

The results

from comparison testing of antibiotics and preferred

extracts were shown (Fig 7 and 8). According to the

overall results, it was known the inhibition capability

of preferred extracts was nearly comparable with

10 ppm of tested antibiotics (Fig 7 for comparison

with penicillin G and Fig 8 for comparison with

streptomycin).

Potassium ions (K ) exist on a bacterial

cytoplasm membranes which have functionality on

membrane transportation process. Calcium ions (Ca )

and magnesium ions (Mg ) on Gram-negative bacteria

cereus

B. cereus

Antibiotics Comparison Testing.

Atomic Absorption Spectroscopy (AAS)

Analysis.
+

Volume 5, 2011 Microbiol Indones 107

Bacterium Melinjo extracts MIC (μg mL
-1

) MBC (μg mL
-1

)

Bacillus cereus

Melinjo seed – ethanol extract 0.76 3.03

Melinjo seed – ethyl acetate extract 0.86 3.45

Melinjo peel – ethanol extract 1.40 5.58

Melinjo peel – ethyl acetate extract 0.26 1.02

Melinjo peel – hexane extract 0.26 1.04

Staphylococcus aureus

Melinjo seed – ethanol extract 0.15 0.58

Melinjo seed – ethyl acetate extract 1.19 4.76

Melinjo peel – ethanol extract 0.90 3.58

Melinjo peel – ethyl acetate extract 1.46 5.82

Melinjo peel – hexane extract 0.00 0.00

Enterobacter aerogenes

Melinjo seed – ethanol extract 0.40 1.58

Melinjo seed – ethyl acetate extract 0.97 3.86

Melinjo peel – ethanol extract 0.00 0.00

Melinjo peel – ethyl acetate extract 1.35 5.39

Melinjo peel – hexane extract 0.00 0.00

Pseudomonas aeruginosa

Melinjo seed – ethanol extract 1.04 4.15

Melinjo seed – ethyl acetate extract 1.51 6.04

Melinjo peel – ethanol extract 0.00 0.00

Melinjo peel – ethyl acetate extract 1.36 5.43

Melinjo peel – hexane extract 0.00 0.00

Tabel 2 Minimal Inhibitory Concentration (MIC) and Minimal Bactericidal Concentration (MBC) values of melinjo extract using different sources (seed,
peel) and different solvents (ethanol, ethyl acetate, hexane)

Fig 5 Representative images of inhibition zone of ethanol extract from melinjo seed.

Bacillus cereus 5% of melinjo seed – ethanol extract

Staphylococcus aureus 5% of melinjo seed – ethanol extract

Enterobacter aerogenes 5% of melinjo seed – ethanol extract

Pseudomonas aeruginosa
15% of melinjo peel – ethyl acetate

extract

Table 3 Preferred extract for each tested bacteria leakage (Fig 9f), pore formation (Fig. 9h) and lysis of

the membranes integrity (Fig 9b, 9h), and bumps

(Fig 9b, 9d, 9f, 9h). Cell shapes became abnormal

caused by the penetration of extract into cells which

caused swelling in some cells (Fig 9d, 9h) and rough

surfaces as compare to the smooth ones of untreated

cells.

108 P SARHUSIP AND ITANGGANG Microbiol Indones

Bacillus cereus

Vegetative cells Spores

D
ia

m
e
t
e
r

o
f

in
h

ib
it

io
n

(
m

m
)

12.83

10.18

Fig 6 Diameter of inhibition resulting from 5% melinjo seed-ethanol
extract against vegetative cells and spores of Bacillus cereus.

Fig 7 Diameter of inhibition of preferred melinjo extracts and penicillin G against selected bacteria.

Fig 8 Diameter of inhibition of preferred melinjo extracts and streptomycin against selected bacteria.

DISCUSSIONS

Melinjo seed and melinjo peel extract showed

antibacterial activity against , ,

, and , but did not show

antifungal activity because it could not inhibit

. None of the extracts could

inhibit the growth of could be explained as

fungus is eukaryotic organism that has cell wall which

contains very stiff chitin (Yokoi . 1998). This chitin

fundamentally contributes to fungal cell wall strength

and stability and prevents outer material from

penetrating. Furthermore, it may be expected that an

extract could not interfere with the permeability of

cytoplasmic membrane because extracts

did not have any reaction with sterol inside a mold's

cytoplasmic membrane (Farkas 1979).

B. cereus S. aureus E.

aerogenes P. aeruginosa

Aspergillus flavus growth

A. flavus

et al

aeruginosa

Volume 5, 2011 Microbiol Indones 109

However, melinjo seed and melinjo peel extract

have antibacterial activity, proven by the facts that the

extract could inhibit the growth of tested bacteria as

seen in the inhibition area, and moreover was

supported by (i) the MIC and MBC values

(0.26~1.46%; 1.02~6.04% respectively), (ii) anti spore

results (5% w/v of extract), (iii) having the equivalent

inhibition capacity as compared to antibiotics

(penicillin G and streptomycin 10 ppm).

The extract has higher inhibitory effect towards

Gram-positive bacteria as compared to Gram-negative

bacteria because Gram-negative bacteria have several

complex layers on their cell walls. The structure of the

cell wall layer on Gram-negative bacteria consists of

p e p t i d o g l y c a n a n d a n o u t e r m e m b r a n e

(lipopolysacharide and lipoproteins) (Lesage and

Bussey 2006). The existence of an outer membrane cell

from Gram-negative bacteria causing the inhibitional

diffusion of antimicrobial extract inside both

peptidoglycan membranes and bacteria cells and leads

to inappropriate conditions for penetrating interacting

with inner cell wall components (Fan . 1975). In

addition, approximately 90% of a Gram-positive

bacteriums cell wall is composed of peptidoglycan,

whereas the peptidoglycan layer in the Gram-negative

bacterium cell wall is only 50-10%. The outer

membrane of Gram-negative bacteria is impermeable

to penicillin G, so that bacteria growth can not be

inhibited (Tompsett . 1947).

For antisporal activity, there was a significant

difference between anti-sporal activity of extract onto

spores of (10.18 mm) and anti-bacterial

activity of extract towards vegetative cells of

(12.83 mm). The layer difference at vegetative cell and

endospore level will cause diameter dissimilarity

which was produced by the extract. Generally layers of

endospore are exosporium layers, spore coats, cortex

(Atrih . 1998). An enormous layer on it along

with its stiff nature, will cause the extract to be harder

to diffuse into the cell and inhibit cell growth.

Therefore, the inhibition diameter of extract toward

spores of was smaller as compared to its

vegetative cells.

For the preferred

extracts were able to inhibit both Gram-positive and

Gram-negative bacteria (Fig 7). However, penicillin G

was also found to effectively inhibit Gram-positive

bacteria (Fig 7). According to Baldwin . (1997),

antibacterial activity of penicillin G was due to its

interference on the synthesis process of the bacteria cell

wall. Though this conclusion needs further

clarification, due to this finding, we hypothesize the

preferred melinjo extracts should follow the

mechanism of penicillin G inhibition in that they were

et al

B. cereus

et al

B. cereus

antibiotics comparison testing,

et al

Bacterium
Total Ca

(mg L
-1

)
Total K

+
(mg L

-1
)

Bacillus cereus 58.27 196.39

Staphylococcus aureus 67.17 190.03

Enterobacter aerogenes 73.17 130.06

Pseudomonas aeruginosa 62.41 111.26

Table 4 Total calcium and potassium ions detected by AAS

Fig 9 SEM photomicrographs showing the morphological changes of tested

bacteria after exposure to preferred melinjo extract ( )

for each bacteria. a, control; b, treated

with 5% melinjo seed ethanol extract; c, S. aureus control; d,

treated with 5% melinjo seed ethanol extract ;

e, control; f, treated

with 5% melinjo seed ethanol extract; g,

control; h, treated with 15% melinjo peel -

ethyl acetate extract.

Gnetum gnemon

Bacillus cereus Bacillus cereus

Staphylococcus aureus

Enterobacter aerogenes Enterobacter aerogenes

Pseudomonas aeruginosa

able to influence the synthesis process of bacteria cell

wall. Streptomycin could inhibit Gram-positive and

Gram-negative bacteria. According to Lin (2000),

the inhibition mechanism of streptomycin was by

attacking the ribosome of microbes which leads to

error reading of the mRNA sequence taking place,

resulting in the formation of polypeptide which

becomes irregular and nonfunctional, and therefore

inhibited bacterial growth. The anti-bactericidal

activity of the each preferred extract for each bacterium

was comparable with the action of streptomycin in

inhibiting bacterial growth (Fig 8). Furthermore, we

et al.

110 P SARHUSIP AND ITANGGANG Microbiol Indones

suggest the overall inhibition mechanisms of melinjo

extracts against bacteria were by interfering with protein

synthesis in bacteria, degrading the existing present

cell wall or interfering which bacterial cell wall synthesis,

and damaging cell membrane of bacteria (as reported by

Yasni . (2009)) and proven by the analysis of mineral

content and SEM imaging system (Fig 9).

According to Parhusip (2006) and Rogers (1970),

destruction of bacterial cell membrane may also cause

the extraction of minerals the from cell inner side such

as, calcium (Ca ) and potassium (K ). Therefore,

within this study we have found the K leakage was

higher for both Gram-negative and positive bacteria as

compared to Ca leakage. Moreover, in the Gram-

positive bacteria the K leakage was higher than Gram-

negative ones. However, this condition was contrary to

Ca leakage, since the Gram-negative leakage was

found to be higher than the Gram-positive ones. This

could be explained since the outer membrane of Gram-

negative bacteria consists of a large number of Ca

ions, whereas Gram-positive bacteria consist of Ca

which is located on its cytoplasmic membrane (Farkas

1979). This was also confirmed by the results

mentioned by Beveridge (1999).

Lastly, those findings mentioned above were totally

confirmed morphologically through SEM imaging

(Fig 9). The same imaging phenomenon was also

reported by Al-Reza (2010) who mentioned that

the essential oil extract of causes the

formation of pores or holes in the cell membrane and

cell lysis in , which indicates the occurrence of

disturbances on the structure of cell membranes.

Within this study the same perspective was also found.

Moreover, changes in morphology in this study were

also similar to results of Shalamanov (2005), including

changes in the morphology on Gram-negative bacteria

( , , and

) that had been contacted with

chlorhexidine gluconate. Changes that occurred were

elongation, variations in shape and size of cells, the

formation of bumps, grooves and wrinkles on the cell

walls. These SEM images were performed to provide

basic supportive data on the occurrence of

morphological changes in bacteria cells so that the

mechanism of melinjo extract action could be deduced.

Finally, from these comprehensive data we could

conclude that melinjo extract has real potential to be

applied in food preservation against selected

pathogenic bacteria.

We would like to thank Universitas Pelita Harapan

(UPH) for the funding, and Elisa FR and Jennifer

Octavia for the assistance.

et al

et al.

Zizyphus jujuba

S. aureus

Enterobacter cloacae Pseudomonas aeruginosa

Serratia marcescens

2+ +

ACKNOWLEDGEMENTS

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