amarila malik12*, iman santoso , andi yehuda2,...

Available online athttp://jurnal.permi.or.id/index.php/mionline

DOI: 10.5454/mi.8.1.3ISSN 1978-3477, eISSN 2087-8575Vol 8, No 1, Maret 2014, p 16-23

*Corresponding author; Phone/Fax: +62-21-7270031/7863433, Email: [email protected]

+62-21-

Recently, we reported on isolates from Tangkuban

Perahu, a volcano in West Java and culture growth in

laboratories in Regensburg, Germany, and Depok,

Indonesia (Handayani 2012). We could

demonstrate growth of (Huber and

Prangishvili 2004) in Regensburg by electron

microscopyand obviously cultured members of the

order (Huber 2002) in

Depok, but the latter could not be characterized

et al.

Sulfolobales

Thermoplasma tales et al.

(Handayani 2012). Now, we focused on the

isolation and culture growth of

et al.

Thermoplamsa

species,e.g. and . These

organisms are cell wall-less thermoacidophilic archaea

with unique tetraetherlipids, which have raised our

interest in biomedical and biotechnical applications

(Freisleben 1999). was

first isolated by Darland . (1970) from sulfuric acid

milieu in self-heated coal refuse piles. Later,

was found in solfataric hot springs

(Segerer 1988). Solfataric environments appear

to be the natural habitat also of (Yasuda

T. acidophilum T. volcanium

Thermoplasma acidophilum

et al

T.

volcanium spp.

et al.

T. acidophilum

Archaea is an organisme with unique feature because of its ability to inhabit an extremophyle conditions. Ourexpeditions to Tangkuban Perahu, West Java aimed to obtain archaealstrains from the solfatara fields located inDomas crater. From the samples, we intended to culture species growing around 55 C below pH2, which until now have not yet been fully characterized. We collected five samples from mud holes withtemperatures from 52 C to 57 C and pH below 2. In serial cultures of up to 8 transfers in Freundt’s medium wegrew tetraetherlipid synthesizing species as documented by phase contrast microscope. Totalmembrane lipid extracts were analysed by thin layer chromatography; the pattern matched total lipid extractsfrom DSM 1728 membranes. For confirmation, 16S rDNA identificationperforming PCR and sequencing were carried-out. Analysis using BLAST showed identities asthe highest similarity of 99%, followed by also with99% similarity (ANKF776908 andANKF776909). This is the first report of culturing cell-wall-less thermoacidophilicarchaea,in particular

species in Indonesian laboratories.

Key words:

Thermoplasma

Thermoplasma

Thermoplasma acidophilumT. acidophilum

T. volcanium,

Thermoplasma

°

° °

archaea, Indonesian volcanoes, Tangkuban Perahu, tetraether lipid,

Archaea merupakan organisme dengan fitur unik karena kemampuannya mendiami kondisi extrem.Ekspedisi ke Tangkuban Perahu, Jawa Barat dilakukan dengan tujuan untuk memperoleh galur archaea dariladang solfatara yang terletak di kawah Domas. Dari sampel yang diperoleh selanjutnya dilakukan pengulturanspesies pada kondisi berkisar 55 C dan di bawah pH 2; pengulturan seperti ini belum dilaporkanada yang melakukan. Sebanyak lima sampel diambil dari lubang lumpur dengan suhu berkisar 52 C sampaidengan 57 C dan pH di bawah 2 telah diperoleh. Dalam kultur seri hingga 8 kali transfer menggunakan mediaFreundt telah berhasil diperoleh kultur spesies pensintesis tetraether lipid yang kemudian didokumentasikan menggunakan mikroskop fase kontras. Ekstrak total lipid membran dianalisis dengankromatografi lapis tipis; pola yang didapat sesuai dengan jumlah pola ekstrak lipid membran

DSM 1728. Untuk konfirmasi, identifikasi menggunakan 16S rDNA juga dilakukan dengan PCRdan kemudian dilanjutkan dengan sequencing DNA. Analisis menggunakan BLAST menunjukkan identitas

dengan kesimilaritasan tertinggi 99 %, dan identitas jugadengan kesimilaritasan tertinggi 99 % (AN KF776908 dan AN KF776909). Penelitian ini merupakanpengulturan archaea termoasidofilik tanpa dinding sel yang pertama di laboratorium di Indonesia, khususnyaspesies .

Kata kunci: archaea, gunung api Indonesia, lipid tetraeter, Tangkuban Perahu,

Thermoplasma

Thermoplasma

Thermoplasma

Thermoplasmaacidophilum

Thermoplasma acidophilum Thermoplasma volcanium

Thermoplasma

Thermoplasma

°

°

°

Characterization of Thermoplasma Species Cultured fromSampling on Tangkuban Perahu, Indonesia

AMARILA MALIK *, IMAN SANTOSO , ANDI YEHUDA , SERUNI K.U. FREISLEBEN ,

SEPTELIA INAWATI WANANDI , HARALD HUBER , ZESSINDA LUTHFA ,

ROSARI SALEH , HANS-JOACHIM FREISLEBEN

1 2 2 2

3 4 2

2 3AND

1 2 3

4

Facultyof Pharmacy, Faculty of Mathematics and Natural Sciences, Faculty of Medicine,Universitas Indonesia, Depok 16424, Indonesia;

Department of Microbiology, Archaea Centre, University of Regensburg, Germany

et al Thermoplasma

et al

. 1995). In Indonesia species,

which have not yet been further characterized, have

been reported from TangkubanPerahu (Huber .

1991), an easily accessible volcano in West Java, South

of Jakarta, near the city of Bandung.Formerly, growth of DSM 1728 was

achieved in fermenters under laboratory conditions at

pH 1.5 to 2.0 and an optimal growth temperature of 59

C (Freisleben . 1994) as a major source of

tetraether lipid, because the organism lacks a cell wall,

and thus the membrane lipids are ‘naked’ and easily

accessible. In nature, members grow

autotrophically metabolizing elemental sulfur, but they

can also grow mixo- and heterotrophically, from

anoxic to oxic conditions (Huber . 1991).

It is intended to optimize growth conditions, to

identify and characterize the archaeal cells and to

extract and purify their tetraether lipids for application

in the biomedical field (e.g. liposomes, archaeosomes

as drug and vaccine delivery systems) and in

nanotechnology (e.g., monomolecular thin film

surface coating) (Freisleben .1995; Bakowsky

. 2000; Patel . 2000; Schiraldi . 2002;

Krishnan and Sprott 2008; Thavasi . 2008). In the

present study, the identification by molecular

techniques performing PCR and sequencing was used.

We applieda pair of primers designed by aligning 16S

rRNA genes specifically targeting the gene encoding

for 16S rRNA, followed by DNA sequence analysis

using BLAST, as well as Clustal W and TreeView X .

On June 11, 2012 (samples KD 1,2) and

January 21, 2013 (samples KD 3,4,5) sampling was

carried out on the Indonesian volcano Tangkuban

Perahulocated in West Java, Bandung. Samples were

taken from solfatara fields in the Domas crater (Kawah

Domas, KD), under similar conditions as described by

Handayani . 2012. In contrast to the former

sampling the temperature range of our new samplings

was quite lower; all samples weretaken from mud holes

between 52 Cand 57 C, because we wanted to

concentrate on and species,

wich are known to grow in this temperature range.

The samples were obtained from acidic mud

holesand warm springs from where a strong smell of

H S was rising. Previous reports regarding archaeal

habitats had shown that the growth temperature of

speciesis around 50 C up to 60 C

(Huber 1991; Yasuda . 1995) which was also

T. acidophilum

et al

Thermoplasma

et al

et al et

al et al et al

et al

et al


Thermoplasma

et al. et al

°

° °

° °

R

MATERIALS AND METHODS

Sampling.

2

confirmed in fermentor growth (Freisleben . 1994).

None of samples from five different mud holes and

et al

warm springs had a pH above 2; KD1 and KD5 were

sampled at 57 C; KD2 at 52 C; KD3 at 56 C; and

KD4 at 54 C. Samples were collected into 140 mL

screw-capped glass bottles, filled to the top and firmly

closed.

Freundt’s medium was prepared according to

Freisleben . (1994). All substances were dissolved

to 1 L in aquabidest; the pH was adjusted to 2 with 10%

H SO (v/v). The medium was autoclaved at 121 C for

20 min.

The culture medium was composed of 1 L

Freundt’s medium, 200 mlof a solution containing

glucose (20 g) and Difco yeast extract (DYE, 1 g) and

50 mlinoculum from KD samples. Aliquots of 300 mL

were cultured micro-aerobically in closed 500 mL-

culture bottles at pH 1-2 in anincubator at 55 C with a

shaker at 110 rpm. The culture bottleswere prepared

with a syringe through the rubber top for limited

(“micro-aerobic”) oxygen supply.

After 5 to 7 d the cultures were examined and

documented with an Olympus Phase Contrast

Microscope Model BX41-32000-2. Photos were

takenusing a Digital Microscope Camera Model Dp20

with its manufacturer-provided Camera Software. Cell

counts were accomplished by means of a Neubauer

Chamber.For serial cultures, aliquots of 30 mL were

transferred to new culture bottles containing 300 mL of

Freundt’s medium pre-heated to 55 C.Additional

purification steps, e.g. either by plating, were not

carried out since we considered enrichment by 8 serial

transfers sufficiently selective.

Subsequently, cells from transfers 5 to 8 were

harvested for further examination, such as genetic

characterization and extraction of total membrane

lipids. Aliquots of cells harvested from these cultures

were kept as frozen stocks ( MBFKD-

W2 and MBFKD-B2) in the repository

of the Laboratory of Microbiology and Biotechnology,

Faculty of Pharmacy, Universitas Indonesia.

One gram of cells

(Freisleben . 1994) was extracted three times with

a total amount of 65 mL chloroform/methanol 1:1

(v/v). The cells were centrifuged after each extraction

step and finally the combined extracts centrifuged at 1

500 g for 30 min.

For removal of hydrophilic-

contaminants the lipid extracts were made biphasic by

° ° °

°

°

°

°

×

et al

Thermoplasma

Thermoplasma

et al

2 4

Culture.

Extraction of Total Membrane Lipids from

Cells (Antonopoulos . 2013).

Removal of Hydrophilic Contaminants by Two

Phase Separation.

et al

Volume 7, 2013 Microbiol Indones 17

the addition of chloroform and 0.1 M aqueous NaCl

solution to achieve a ratio of chloroform/methanol/salt

solution of 2:1:0.8 (v/v/v) (Folch . 1957). The

separated chloroform phase was extracted for a second

time with 1/4 of its volume methanol/aqueous NaCl 1:1 (v/v).

The lower phase was filtered by a phase separation paper

(MN 616wa Macherey-Nagel, Düren, Germany) and

evaporated to drynessin a Rotavapor-R (Büchi, Flawil,

Switzerland) with repeated addition of chloroform/

methanol 3:1 in order to readily remove the water.

Thin layer

chromatography (TLC) was carried out on 0.25 mm

layers of silicagel (Merck, Darmstadt, cut to 10 5 cm)

and developed in chloroform/methanol/water 65:25:4

(v/v/v).

Lipids were detected with sulphuric acid/methanol

1:9 (v/v) and heating at 140 C. If heating was

accomplished slowly the isoprenoid-derived lipids

showed characteristic colours of red, brown or yellow

before turning to black.

For comparison, total lipid extracted from

DSM 1728 (Freisleben

. 1994) was chromatographed under the same

condition. Apart from the total lipid extract of strain

DSM 1728, we had a fraction with mild hydrolization

of the phoshoester of MPL (Antonopoulos . 2013).

In TLC the respective band of MPL disappeared and

instead, the MGL band at the front increased

(Antonopoulos . 2013).

Genomic DNA extraction were performed as

described (Herrera and Cockell 2007; Bergmann et al.

2010) modified by the following procedure: After

harvesting by centrifugation at 1 500 g for 30 min the

cells were homogenized and washed twice with 500

and 750 μL STET buffer, respectively (NaCl 100

mM;Triton X-100 5% v/v;EDTA 1 mM; Tris-HCl 10

mM, pH 8.0) by centrifugation for 3 min at 23 C. The

pellet was re-suspended in 557 μL of the same buffer

and incubated with 10 μL lysozyme solution (10 mg

lysozyme from chicken egg white (Sigma, St. Louis,

MO) in 1 mL Tris-HCl 10 mM, pH 8) and 4 μL

proteinaseK(25mgml )inthewaterbathfor1hat37 C.

Subsequently, 65 μL of 5M NaCl and 80 μL of 4%

(w/v) hexadecyltrimethylammonium bromide, CTAB

(Sigma, St. Louis, MO) were added, vortexed,

incubated in the water bath at 65 C and after addition

of 4 μL proteinase K further incubated in the shaking

water bath at 37 C for 1 h. The samples were taken

from the water bath, cooled to RT, and incubated with

RNase in the water bath at 37 C for 15 min. DNA was

et al

Thermoplasma acidophilum et

al

et al

et al

Thin Layer Chromatography.

Molecular Genetic Identification DNAExtrac-

tion.

×

°

× ,

°

°

°

°

°

-1

precipitated by adding 650 μL chloroform-isoamyl

alcohol (24:1, v/v), vortexing for 10 sec and

centrifugation at 1200 for 20 min

Again, 65 μL of 5M NaCland 80 μL CTAB 4%

were added, vortexed and incubated in the water bath at

65 C. Supernatant was carefully removed into new

Eppendorf cups. The pellet was re-suspended in 650 μl

chloroform-isoamyl alcohol, vortexed for 10 sec and

centrifuged at 1 200 for 20 min. The supernatant

was carefully removed into new Eppendorf cups.

Into 500 μL of supernatant, 400 μL of cold

isopropanol were added and shaken very gently until

white threads became visible. Concentrations

measured in our samples were between 29.55 and

54.55 μg mL . The DNA precipitates were dried

in a desiccator for 10 min, then re-suspended in

20 μL TE buffer (10 mM Tris-HCl, 1 mM disodium

EDTA, pH 8.0) and storedat -20 C until used.

For PCR identification of archaeal

isolates, we applied primer design as reported

(Slobodkina . 2004; Baker . 2001), modified to

our condition. First, six species 16S

rDNA sequences ( 2 sequence data, and

4 sequence data) were downloaded from

NCBI database and aligned using Clustal W2

(http://www.ebi.ac.uk/To ols/msa/clustalw2/). Second,

by using the 16RS rRNA gene sequence

(accession number AJ299215.1) as reference, we

designed the primers performing Clone Manager

Suite . The result was further analyzed using the

program available at http://sg.idtdna.com/analyzer/

Applications/OligoAnalyzer/. The forward primer was

5’-GGAGATGGACTCTGAGACAACAG-3’ and the

reverse primer 5’-CTACGGTACGAGCTGACG

ACG-3’. PCR was run in a reaction mixture (50 mL)

containing approximately 0.20 μg genomic DNA, 8

pmol of each primer, 1 μL 10 buffer, 1.6 mM MgSO ,

2 mM of each deoxyribonucleotide triphosphate

(dNTPs), and 1 U of KOD DNA polymerase on a

thermal cycler. The temperature profile was as follows:

initial DNA denaturation for 4 min at 95 C; then 34

cycles of denaturation at 94 C for 30 s and 58 C

primer annealing for 30 s, extension at 70 C for 45 s,

and final extension at 70 C for 4 min.

Amplification

products (2 μL) were visualized in agarose gel

according to standard protocols. For casting the gels,

agarosewas dissolved in 1% Tris-acetate-EDTA (TAE)

buffer (Cytryn . 2000). Ethidiumbromide was

added prior to gel casting with very careful handling.

×

×

g

g

.

°

°

×

°

° °

°

°

1

R

in

vacuo

et al et al

Thermoplasma

T. acidophilum,

T. volcanicum,

T. volcanicum

et al

Identification Using Polymerase Chain

Reaction (PCR).

Visualization of PCR products.

4

Microbiol Indones18 MALIK ET AL.

After running the gels for 30 min on a horizontal gel

electrophoresis device, DNAbands could be visualized

on UV transilluminator connected to a documentation

system. The PCR product was further analyzed by

DNAsequencing (1 BASE, Singapore).

The DNA sequence

information obtained was analyzed by BLASTserver

maintained at the National Center for Biotechnology

Information, Bethesda, MD (http: //www.ncbi.nlm.nih.

gov), i.e. nucleotide BLAST. Furthermore, the partial 16S

rDNAsequences were analyzed by using Clone Manager

Suite and aligned with known 16S rDNA of

species downloaded from database

GenBank (http://http://www.ncbi.nlm.nih.gov/genbank/).

The search for similarity or homology of DNA

sequenceswas done on-line using the BLAST server

maintained at the National Center for Biotechnology

Information (NCBI), Bethesda, MD (http://www.ncbi.

nlm.nih.gov). To check the relationship of our strain to

other existing species, we performed

neighbor-joining method by Clustal W to create a

phylogenetic tree of closely related 16S rDNA

sequences. The tree was edited by means of

TreeViewX software.

The DNA sequences obtained in this study have

been deposited under GenBankAN KF776908 andAN

KF776909 in NCBI database from where they can be

uploaded by employing BankIt .

st

R

R

R

R

Analysis of DNA Sequence Data and Nucleotide

Sequence Accession Numbers.

Thermoplasma

Thermoplasma

RESULTS

Culture Condition.

Comparison of Total Lipid Extract.

From KD samples up to 8

serial cultures were grown under conditions, which are

preferred by species, i .e . ,

microaerophilically at 55 C in Freundt’s medium at

pH below 2. To follow the culture growth, samples

were taken from the cultures at times indicated and

optical density (OD) was read . Fig 1 shows

KD3 culture development of transfers 1, 3, and 5 over a

period of two weeks. We observed a lag phase of 5 days

in culture 1, reduced to 3 days after five transfers with

OD<0.1, then a log/exponential phase to 7-8 d and

OD=0.35. Highest OD of 0.4 was measured in the

culture after 5 transfers on day 10, which was already

considered as late stationary phase, where the OD

already started to decrease. From growth behaviour, it

was decided that cultures should be harvested not later

than on day seven (between 160 and 170 hours). Fig 2

shows serialculture 3 in the phase contrast microscope.

Diameter determination ranged from 0.9 to 1.7μm with

a mean value of 1.2 μm. Counts in the Neubauer

Chamber resulted in 57 10 cells mL .

Cells were harvested on the 7 day of culture and

used for the extraction of total membrane lipids and the

extraction of DNA.

From two

serial cultures total lipid was extracted according to

Thermoplasma

°

×

at λ 578nm

6 -1

th

350300250200150100500

0.45

0.40

0.35

0.30

0.25

0.20

0.15

0.10

0.05

0.00

hours

OD

Fig 1 Serial cultures from KawahDomas KD3 sample to optimize growth conditions for Thermoplasma species.and : KD3 Gen 1, :

Depicted aregenerations 1, 3, 5 monitored by OD at λ 578 nm. KD3 Gen 3, and : KD3 Gen 5.


Antonoploulos . (2013) and compared to total lipid

extract from DSM 1728. The result is

shown in Fig 3. Mild hydrolysis of the main

tetraetherglycophospholipid (MPL) splits the

phosphoester and yields the main tetraetherglycolipid

(MGL). This reaction demonstrates the position of the

two lipids in the chromatogram. The band of MGL at

et al

T. acidophilum

the top of the chromatogram mixes with the apolar dye

present in the total membrane lipid extract from all

cultures. In general, the pattern of the extracts from TP

isolates matches exactly that of DSM

1728 (Antonopoulos . 2013).

T. acidophilum

et al

Primer for PCR and Molecular Identification.

Alignment of and 16ST. acidophilum T. volcanium

Fig 2 Phase contrast image of the third serial culture of sample KD3. Magnificationx200 (scale bar = 5 μm), characteristic cellsof Thermoplasma.

front, apolar

MGL

apolar dye

MPL

start, polar

other polar lipidsPhospholipids &

glycolipidsapolar lipids,

Fig 3 Total membrane lipid extracts from cultures MBFKD-W2 and MBFKD-B2 (left plate, lanes 1 and 2) of sample KD3; Fig.3(b) (right plate, lanes 3 and 4) show the comparison of the total membrane extract fromstrain DSM 1728. Lane 3; the phosphoesters or the tetraetherglycophospholipids of the latter had been mildlyhydrolyzed; the phospholipid bands in the middle part of the chromatogram are not present, especially the thickest bandof the main phospholipid (MPL) disappeared. Instead, the band of the main glycolipid at the top of the chromatogram ismuch thicker than in the other lanes. Lane 4 is total membrane extract from grown underconditions for maximum MPL yield (Freisleben . 1994; Antonopoulos . 2013). MPL, main polar lipid = main(glyco)phosholipid; MGL, main glycolipid.


Thermoplasma acidophilumet al et al


(oxygen) supply was obtained by a syringe through the

tight rubber stopper of the half-litre culture bottles

(empty half-litre aquabidest bottles filled 2/3 volume

with culture medium). Cultures can certainly still be

optimized for faster growth to higher cell concentration

and higher contents of desired lipids, e.g. MPLor MGL.

Our main intention is to obtain special tetraether

lipids from these cells. Hence, we extracted the

membranes with the method described in

Antonopoulos . (2013) and compared the extracts

from cultured Kawah Domas samples with those from

strain DSM 1728. To denote the

position of the main glycophospholipid (MPL) in TLC,

we applied mild hydrolysis to split the phosphoester in

MPL (the only ester in the compound) to yield the main

glycolipid MGL (Antonopoulos . 2013). Hence,

the thick band of MPL in TLC should disappear and

concomitantly, the band of MGL should become

thicker (see TLC, right plate, lane 3). The total

membrane extract contains a contaminating yellow-

brownish apolar dye almost co-migrating with the

solvent front and merging with the high amounts of

MGL in lane 3. Smaller amounts of the latter separate

from the dye (TLC lanes 1 and 2). Summarizing this

part of our study we can state that the TLC pattern of

total membrane extracts from cultured KD isolates

matches exactly the pattern obtained from

DSM 1728 extracts indicating that we

cultured species from the isolates.

In Indonesian biotopes species have

not been further identified except for Huber .

(1991). Hence, it is essential to compare their results

with ours. Starting from the description of isolate

sampling, the location and the habitat conditions were

identical with ours: solfataric mud holes with strongly

acidic pH and moderately hot temperatures around 50

C (in our case pH 2 and 52-57 C). Huber . (1991)

enriched [citation] “cell wall-less highly irregular

coccoid thermo acidophilic archaea” in Darland’s

medium . 1970); we used Freundt’s

medium as published (Freisleben . 1994).

Huber . (1991) cloned the cultured

microorganisms on starch-solidified medium at 60 C

under an air-reduced atmosphere and obtained small

”fried egg”-shaped colonies after 4 d of incubation.

The authors concluded that the Indonesian

strain differed from strains

isolated from other places in the world, but had

alsosome indication of : Cell extracts of

their isolate KD3 DSM 4300 showed serological cross-

reaction with antibodies preparedagainst the histone-

et al

T. acidophilum

et al

T.

acidophilum

Thermoplasma

Thermoplasma

et al

et al

(Darlandet al

et al

et al

T.

volcanium T. volcanium

T. acidophilum

° °

°

rRNA Genes. Several 16S rRNA gene sequences of

Thermoplasma species were aligned by performing

Clustal W. Using T. volcanium (AJ299215) as

reference for primer design the resulting target region

was between nt255 and nt978. The pair of primers was

designed from the regions at nt 255-277 and nt 956-

978, both for forward and reverse primers.

Two PCR products(W2 and B2) were generated

and chosenfor DNA sequencing after gel visualization

as shown in Fig 4, each was obtained from two genomic

DNAsamples extracted from two different cell cultures

but from the same starter culture.

Alignment ofthe first PCR product exerted 99%

similarity with , followed by

with the same percentage (99%).The tree of

T.acidophilum T.

volcanium

phylogenetic relationship (Fig 5) shows the closest NR

028235 and M38637.1, which are both .

Hence, we conclude that our cultures from KD isolates

contain strains belonging to the

species but also contain

strains.

From our sampleswe grew serial enrichment

cultures with up to 8 transfers. As expected, growth

behavior did not change significantly in the serial

cultures. We used the medium and pH according to the

culture conditions of Freisleben . (1994); however,

growth temperature was slightly lower (55 C) than the

optimum laboratory temperature of 59 C. Limited air

T. acidophilum

Thermoplasma

T. acidophilum, T. volcanium

et al

DISCUSSION

°

°

Fig 4 Agarose gel electrophoresis of 16S rDNA PCR ofgenomic DNAs; lane 1 = MBFKD-W2; lane 2 = andMBFKD-B2; lanes M = DNAladder (250 - 1000 kb).


2012). Hence, the identity of “KD3” with the one of

Huber . (1991) is unintentional.

The authors are thankful to the Department of

Biology and the Department of Physics, Faculty of

Mathematics and Natural Sciences, the Department of

Biochemistry and Molecular Biology, Faculty of

Medicine and to the Laboratory of Microbiology and

Biotechnology, Faculty of Pharmacy,Universitas

Indonesia for providing the laboratory facilities. The

authors declare no conflict of interest.

et al

ACKNOWLEDGMENTS

REFERENCES

Antonopoulos E,Freisleben HJ, Krisnamurti DGB,Estuningtyas A, Mulyanto C, Ridwan R, FreislebenSKU. 2013. Fractionation and purification ofmembrane lipids from the archaeon

DSM 1728/10217. Separ Purific Technol.110(7):119-126. doi:10.1016/j.seppur.2013.03.014.

Baker GC, Gaffar S, Cowan DA, Suharto AR. 2001.Bacterial community analysis of Indonesian hotsprings. FEMS Microbiol Lett. 200(1):103-109.10.1111/j.1574-6968.2001.tb10700.x.

Bakowsky U, Rothe U, Antonopoulos E, Martini T, HenkelL, Freisleben HJ. 2000. Monomolecular organization ofthe main tetraether lipid from

at the water-air interface. Chem PhysLipids. 105(1):31-42. doi:10.1016/S0009-3084(99)00131-0.

Bergmann I, Mundt K, Sontag M, Baumstark I, Nettmann E,Klocke M. 2010. Influence of DANN isolation on Q-



like protein of (DSM 1728).

From our results, we interpret that our samples from

Kawah Domas (TP) and subsequent cultures contain

both, and and we assume

that they can be found together also in other Indonesian

biotopes.This clarifies the question raised by Huber

. (1991) concerning the above cross-reaction.

Furthermore, the authors discuss the differences in GC-

content of 46% in and 38% in

isolated so far from several habitats around

the world,whereas the Indonesian isolate KD3 DSM

4300 exerted a GC-content of 40%. No wonder that

GC-content is in between, since we found both species.

It is our intention to scale-up

cultures sampled from Tangkuban

Perahu in fermenters to obtain sufficient amounts of

tetraether lipid for the production of acid-stable

liposomes or archaeosomes which can be applied to oral

drug and vaccine delivery. Indonesia, a hotspot

ofextremophilicarchaea has been left behind for decades

in this area of research. We hope that our successful

cultivation and identification of will

induce enthusiasm to further investigate extremophilic

forms of life from biotopes in this country.

. It should be mentioned that it was not

intended to have the same isolate ID in our study as

Huber . (1991), i.e., “KD3”. We had various

isolates with varying pH and temperature, numbered

KD1-KD5 in three samplings; our KD3 from the last

sampling on January 21 , 2013 turned out successful in

culturing a species. From other samples,

we managed to culture (Handayani .

T. acidophilum


et

al

T. acidophilum T.

volcanium

Thermoplasma

Thermoplasma

et al

Thermoplasm

Sulfolobus et al

Outlook.

Note

st

B2 KD3/Thermoplasma

W2 KD3/Thermoplasma

T. acidophilum M38637.1

T. acidophilum NR 028235.1

T. volcanium AF339746.1

T. volcanium AJ299215.1

T. volcanium NR 028185.1

T. volcanium NR 074223.1

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*Corresponding author; Phone/Fax: +67-721-781498, Email: [email protected]

Sweet potato flour is generally produced

conventionally by drying of sliced peeled tubers

followed by grinding and sieving to pass different

mesh size. This process has disadvantages such as

limited use in application, primarily in food system,

and low whiteness index. Sweet potato flour has

viscosity profile, rehydration, and solubility that are

less favorable than those of wheat flour. In addition

native flour did not show any thermo pasting behavior

when subjecting to amylograph brabender (Yuliana

and Nurdjanah 2013). The native sweet potatoes flour

has low viscosity that makes it just useful for the food

that require lower viscosity (Aprianita et al. 2009)

In this research we tried to improve or modify

sweet potato flour properties trough spontaneous

fermentation of the fresh tuber, so that the flour is

whiter and has broader application in various food

products. Previous studies showed lactic fermentation

has been implemented in some flour and starch

production, such as in the production of sweet potato

starch (Deng et al. 2013), sour cassava flour (Putri

2011), cocoyam flour (Oke and Bolarinwa 2012) and

rice flour (Lu et al. 2005). Mutwiri (2007) and Kasim

(2012) worked on naturally fermented sweet potato

flour that was prepared by drying a fresh sweet potato

pulp before fermenta sausages filling (Mutwiri 2007).

In African countries, lactic acid fermentation has been

practiced to improve nutritional value and taste. For

examples, amylolytic LAB are mainly distributed in

directly Nigerian fermented meals (Sanni et al. 2002),

and traditional African fermented sorghum (Yousif et

al. 2010).

Lactic acid bacteria as dominant organisms in food

fermentations will convert free sugars to lactic acid,

produce amylolytic enzymes that degrade starch

granules and hydrolyze the short chain amilose and

amylopectin in amorphous region of starch granules.

The degradation of starch granule by lactic acid during

fermentation could change the porosity and surface

et al.

Native sweet potato flour is usually has low whiteness index and limited application to food systems due to its inherent functional properties. Therefore, it needs modification process to improve this property. In this study, sweet potatoes cubes were lactic spontaneously fermented for 120 h before being processed to flour to modify its properties. Selected physico-chemical properties of flour were then determined and compared with the control (without fermentation). The results showed that lactic acid fermentation significantly caused more changes on flour properties. The lactic acid fermentation caused an alteration in the starch granules as evident by Scanning Electron Microscopy. When compared to the control flour, spontaneous fermented flour had lower solubility, higher swelling power, and paste viscosity. The results suggested lactic spontaneous fermentation within 120 h period of time could provide a greater extent of flour modification.

Key words: modified sweet potato flour, spontaneous lactic acid fermentation

Tepung ubi jalar biasanya mempunyai indeks putih yang rendah dan sifat fungsional alami yang menyebabkan keterbatasan aplikasi dalam sistem pangan. Dengan demikian tepung ubi jalar perlu dimodifikasi untuk memperbaiki sifat-sifat fungsionalnya. Pada penelitian ini, potongan ubi jalar difermentasi laktat secara spontan selama 120 hari sebelum diproses menjadi tepung untuk memperbaiki sifat fungsionalnya. Sifat fisiko kimia terpilih kemudian diukur dan dibandingkan dengan tepung hasil perlakuan yang tidak difermentasi (kontrol). Hasil penelitian menunjukkan bahwa fermentasi asam laktat secara nyata menyebabkan perubahan sifat-sifat tepung. Fermentasi menyebabkan perubahan granula pati seperti yang ditunjukkan pada gambar hasil Scanning Electron Microscopy. Jika dibandingkan dengan kontrol, tepung hasil fermentasi mempunyai kelarutan yang lebih rendah, kemampuan pembengkakan granula, dan viskositas pasta yang lebih tinggi. Hasil ini mengindikasikan bahwa fermentasi secara spontan selama 120 jam dapat menghasilkan proses yang berdampak besar terhadap modifikasi tepung.

Kata-kata kunci: fermentasi asam laktat spontan, tepung ubi jalar termodifikasi

Effect of Spontaneous Lactic Acid Fermentation on Physico-Chemical Properties of Sweet Potato Flour

NETI YULIANA*, SITI NURDJANAH, RIBUT SUGIHARTO, AND DEARY AMETHY

Department of Agricultural Product Technology (THP), Agriculture Faculty, Universitas Lampung, Jalan Sumantri Brojonegoro No 1, Bandar Lampung 35145, Indonesia

area of the granule and would modify the properties of

flour. Information about these physicochemical

properties is important to determine the usability of

fermented sweet potatoes flour on various food

products.

Beside amylolitic enzymes, lactic acid bacteria

also produce proteolitic enzymes as well as organic

acids which degrade protein and inactivate polyphenol

oxidase in sweet potatoes. Lower protein content,

inactivation of polyphenol oxidase together with low

free sugar content as a result of the conversion of free

sugar to lactic acid in fermented sweet potatoes flour

may reduce the non enzymatic browning during flour

drying. Thus, it will result in increasing whiteness of

sweet potatoes flour.


Sweet Potato Fermentation. White-fleshed sweet

potato samples were obtained from Pasar Gintung, a

local market at Bandar Lampung, Province of

Lampung-Indonesia. The sweet potatoes were washed, 3peeled, and cut into cubes (1x1x1 cm ). The potato

cubes (40 g) were put into a 150 mL-fermenting

container, then it was made up to 150 mL volume with

the 3% brine solution. The fermenting containers

containing the samples were pasteurized using a

microwave oven (Sharp) at high level setting for 10 min

to reach temperature around 72-73 °C, and then they

were left at room temperature to cool. The fermentation

was conducted at room temperature (30 °C) for 120 h to

let them reach completely fermentation. This was

indicated by drop in pH to 4.0 and unique acid aroma of

fermented sweet potatoes cube as described in

previously author (Yuliana et al. 2010). Microbial

growth during fermentation was evaluated by

enumerating total of lactic acid bacteria (LAB) using

total plate count method. Appropriate dilutions were

placed on duplicate plates of MRS medium (Oxoid)

with 0.1% (w/v) CaCO . The cultures were incubated at 3

30 °C for 48 h. LAB was identified by the presence of

clear zones around the colonies; The experiment was

designed in three replicates. The control (non

fermented flour) was prepared by drying fresh cubes of

sweet potatoes followed by milling and sieving.

Sweet Potatoes Flour Production. After fermenta-

tion has been completed, the cubes were washed with

running tap water until the water runs clear and then

drained. The fermented cubes of sweet potatoes were

dried in an oven at 65 °C overnight until the moisture

content reaches approximately 12%. These dry

2 YULIANA ET AL. Microbiol Indones

fermented cubes were then ground into flour to pass

through an 80 μm mesh sieve size. The same procedure

was applied to dry cubes from fresh sweet potatoes

without fermentation to make control sweet potatoes

flour. The flour was packed in polythene bags and

stored at ambient temperature of around 25±2 °C for

further analysis.

pH and TA. Sweet potatoes flour samples (5 g)

were weighed in triplicate into a beaker, mixed with 20

ml of distilled water. The resulting suspension stirred

for 5 min and left to settle for 10 min. The pH of the

water phase was measured using a calibrated pH meter

(AACC, 2000). The TA was measured using titration

method with phenolpthalein as indicator of end

titration.

Morphology of Sweet Potatoes Starch Granules.

The morphology of starch granules was evaluated by

scanning electron microscope (JSM-6510LV SEM).

Flour samples were suspended in 95% ethanol and

mounted on circular aluminium stubs with double-

sided sticky tape. The flour granules were evenly

distributed on the surface of the tape, and the ethanol

was allowed to evaporate. The samples were then

coated with 12 nm gold, examined and photographed at

an accelerating voltage of 15 kv with a magnification of

2000×.

Swelling power and solubility. Swelling power

and water solubility index (WSI) determinations were

carried out in the temperature range 60-80 °C at 10 °C

intervals. Briefly, 0.5% flour suspensions were

prepared in 15 mL tubes and heated in a water bath at

60, 70, and 80 °C for 30 min with constant agitation to

avoid sedimentation. This was followed by

centrifugation at 1000× g for 15 min at 20 °C. The

sediment fraction was weighed and its mass related to

the mass of dry starch was expressed as swelling

power (w/w). The solubility was reported as the ratio of

the weight of total soluble starch to the weight of dried

sample.

Pasting properties of flour. The pasting

properties of the flour were evaluated by using a Micro

Visco Amylo Graph (Brabender). Flour suspensions

(10%, w/w) were equilibrated at 30 °C for 1 min, -1heated at 95 °C for 5.5 min, at a rate of 6 °C min , held

at 95 °C for 20 min, cooled down to 50°C at a rate of 6 -1°C min and finally held at 50 °C for 20 min. It was a

programmed heating and cooling cycle. Parameters

recorded were pasting temperature (PT), peak

viscosity (PV), minimum viscosity (MV), or trough

viscosity (TV), final viscosity (FV), and peak time

(PTime). Breakdown viscosity (BV) was calculated as

the difference between PV minus MV, while total

setback viscosity (TSV) was determined as the FV

minus MV. All determinations were performed in

duplicate.

Flour Whiteness. Sweet potatoes flour whiteness

was determined using a Powder Whiteness Tester

Model C 100, Kett Electric Laboratory. A 25 g sample

of flour was weighted and put in the sample container.

The top of container was then closed and container was

inserted into the sample holder in the system. The

values of whiteness were the average from three

measurements.

RESULTS

pH and TA of Flour. Changes in pH and titratable

acidity (TA) of flour are shown in Table 1 and Fig 1.

Fermentation was found to cause a gradual reduction in

a pH from 6.49 to 4.5. Concomitant with the drop in

pH, there was a rise in TA of SP flours throughout the

fermentation process from 0.02 % to 1.68 %.

Flour Whiteness. Fermentation improved the

whiteness of SP flour (Fig 2). Fermented flour was

observed to have higher whiteness index compared to

that in control.

Solubility. Sweet potato flour is difference in

solubilitty. In general, fermented flour has lower

solubility in spite of the temperature over 60 to 80 °C.

Swelling Power. Any significant change in

swelling power of SP flour, as shown in Fig 4. At 60 °C

the swelling power of both treatments was very low,

and this value increased when the heating reached 70

°C. The granules of control sweet potato flour swelled

at a lower temperature (~73 °C) in comparison to those

of spontaneous sweet potato flour, which swelled at

~75 °C. The swelling power of control sweet potatoes

flours increased steadily with a temperature rise from

60 to 80 °C, as opposed to fermented sweet potato flour

with a rapid change of swelling power in this

temperature region. At 80 °C, the swelling power of the

fermented sweet potato flour was greater than that of

control flours.

Pasting Properties. Table 2 demonstrates the

viscosity profile of different flour. The fermented flour

samples had the higher maximum viscosity, break

down and set back than those of control. However, the

control had the higher peak time (10 min), and pasting

temperature (93.87 °C) which may indicate different

structural rigidity in comparison to fermented sweet

potato flour.

Morphology of Granule. Fig 5 shows mild

superficial corrosions on some starch granules by


Control Spontaneous

8

7

6

5

4

3

2

1

0

pH

2

1.8

1.6

1.4

1.2

1

0.8

0.6

0.4

0.2

0

TA

(%

)

Control Spontaneous

Fig 1 pH and titratable acidity (TA) of control (fresh SP) and spontaneous fermented SP flour.

Table 1 pH and titratable acidity (TA) at control (fresh SP) and spontaneously fermented SP flour

Treatment TA (%) pH

Control 0.02±0.005 6.49±0.63

Spontaneous 1.68±0.16 4.5±0.61

Microbiol Indones4 YULIANA ET AL.

Whi

tene

ss (

%)

0

10

20

30

40

50

60

70

80

90

Control Spontaneous

Fig 2 Whiteness (%) of control and spontaneous fermented SP flour.

60 °C 70 °C 80 °C

Temperature

Sol

ubil

ity

(g/g

)

25.00

20.00

15.00

10.00

5.00

0.00

60 °C 70 °C 80 °C

Temperature

14.00

12.00

10.00

8.00

6.00

4.00

2.00

0.0

Sw

elli

ng p

ower

(g/

g)

Fig 3 Solubility of control (fresh) and spontaneous fermented SP flour. : control and : fermented.

Fig 4 Swelling power of control (fresh) and spontaneous fermented SP flour. : control and : fermented.


during fermentation in this research was around 0.619

at day 1 to 7.803 (log 10 CFU) at day 5 (120 h). This

could explain the apparent increase in lactic acid

towards the end of fermentation accompanied by

decrease in pH.

Fermentation was observed to improve the

whiteness index of SP flour (Fig 2). This can be ascribed

to flour purification by spontaneous fermentation and

the decrease of ash, protein and sugar content. It is

generally accepted that the ash content, protein and free

sugar are factor affecting the whiteness of flours such as

those from cassava and rice (Sobowale 2007; Lu et al.

2005). During fermentation, bacteria would produce

proteolityc which degraded protein in sweet potatoes,

convert free sugar to lactic acid thus the content of

protein and free sugar in fermented flour was lower than

those at control. A higher protein and free sugar content

in control flour may cause the non enzymatic browning

during flour drying which result in darker color, thus

fermentation. The spontaneous fermented samples

were much smoother surface angle, etched and had

very shallow pits but the control samples had no pits

when examined using scanning electron microscopy.

DISCUSSION

Fermentation was found to cause a gradual

reduction in a pH. This result is in agreement with

Yuliana et al. (2013) and Adebayo-Oyetoro

(2012) who reported that lactic acid fermentation

causes a rapid drop in pH as in sweet potatoes cube and

cassava tubes fermentation. Spontaneous fermentation

of carbohydrate-rich biomass such as SP, cassava,

sorghum, and caper berries, is mainly lactic acid

fermentation (Yuliana et al. 2013; Kakou et al. 2010;

Yousif et al. 2010; Pulido et al. 2005). The pH of the

fermented SP flour is lowered due to the production of

organic acids by lactic acid bacteria. Total LAB grew

et al.

Table 2 Pasting properties of control and fermented SP flour

A B

Fig 5 Scanning electron micrographs of control and fermented SP flours (15 kV, 2000 ). The arrows show etches on the starch granules. A: control flour and B: spontaneous fermented flour .

Beginning of gelatinization 73.78 ±0.65 75.38±0.83

Maximum viscosity (BU) 215.00±4.16 473.50±89.41

Peak time 10 min 7.5 min

Temperature at max viscosity 93.87±0.69 84.30±2.63

Breakdown (BU) 21.50±10.47 216.00±6.98

Setback (BU) 62.25±5.50 156.50±20.14

Pasting properties Control Fermented

amorphous areas in the starch granule are relatively

susceptible to hydrolytic agents such as various

enzyme and acids . Beside producing enzyme, LAB

also produced organic acids, mainly lactic acid during

fermentation.

The pasting behavior of the control and

spontaneous SP flour was studied by observing

changes in the viscosity of a flour system based on the

rheological principals. During heating in water, starch

of flour began to gelatinize as the granules became

swollen and partially solubilized, contributing to a

viscous starch paste. The control flour samples, had the

higher peak time (Table 2), which may indicate a

greater structural rigidity in comparison to fermented

sweet potato flour. This structural rigidity was also

observed from the lower swelling power as discussed

previously.

The control sweet potato flour had higher (93.87

°C) pasting temperature than the fermented flour

having the lower (84.30 °C). Also control sweet potato

flour had lower peak viscosity as opposed to the

fermented SP flour (Table 2). This observation might

have been influenced by lower rigidity of starch

granules in fermented sweet potato, which in turn

caused instability and consequently disruption upon

the heating and stirring treatment (Leon et al. 2006).

On the other hand, the higher peak viscosity of the

spontaneous fermented flour compared to control flour

samples could be due to the increase of granule size

(Fig 1A), which also led to higher swelling power (Fig

2A) and subsequently higher viscosity. The high

viscosity of fermented SP would make them very

useful in food applications where high thickening

power is required. However, the viscosity of this flour

decreased substantially afterwards. This phenomenon

is probably likely due to lower protein content and free

leaching of amylose and amylopectin from the

granules (Leon et al. 2006).

Fermented flour also showed a retrogradation

tendency, indicated by the rise of viscosity during

cooling period as shown in its setback value. Setback is

a measure of recrystallization of gelatinizatied starch

during cooling or measurement of retrogradation. The

spontaneous fermented SP flour had setback value

higher than control flour. A higher retrogradation

tendency of fermented SP makes it suitable for use in

jelly foods and noodle. A good quality of noodle is

thought to result from the pasted starch that exhibit a

high set back value on cooling (Lii and Chang 1981).

Breakdown viscosity measure of the vulnerability

or susceptibility of the cooked starch to disintegration.

reducing whiteness of flour.

Fermentation was found to make difference in

solubility of sweet potato flour as shown in Fig 3. This

could be caused by starch structural differences, such

as chain length distribution and granular size. Bello-

Perez et al. (2000) reported that the distributions of

chain length in the starches cause differences in

solubility, and Tian et al. (1991) stated that granular

size also affects solubility of the starches where the

smaller the granule size, the higher the starch

solubility. In this study it was revealed that based on

SEM analysis, the granular size of the fermented flour

was bigger than that of control.

Fermentation caused any significant change in

swelling power of SP flour. There was difference start

in rapid swell beetween the granules of control sweet

potato flour and those of spontaneous sweet potato

flour. The starch granules start to swell rapidly only

after the temperature reached the onset of the

gelatinization temperature (Jacguier et al. 2006). The

onset gelatinization temperature of control sweet

potatoes flour was 73.78±0.65 °C, and those of

spontaneous sweet potatoes flour was 75.38±0.83 °C

as determined by micro visco amylograph, correspon-

ded to the start of the rapid increase of swelling power

of these flours.

There was also difference pattern in rapid change

of swelling power between control and fermented SP

flour. The swelling power of flour samples is often

related to their protein and starch contents (Woolfe

1992). During fermentation, bacteria would produce

proteolytic enzymes which degrade protein in sweet

potatoes, thus the content of protein in fermented flour

was lower than those at control. A higher protein

content in control flour may cause the starch granules

to be embedded within a stiff protein matrix, which

subsequently limits the access of the starch to water

and restricts the swelling power. Furthermore, during

fermentation, lactic acid bacteria would produce

amylase that hydrolyzes amylose thus reduce amylose

content. Lower amylose content would increase the

swelling factor of starch (Tester and Morisson 1990).

According to Leach et al. (1959), the major factor that

controls the swelling behavior of a starch is the strength

and character of the micellar network within the

granule. The control flour samples may have a greater

structural rigidity in comparison to fermented sweet

potato flour. LAB is known to produce amylases, and

when starch were treated with amylases there was an

initial attack on the amorphous regions of the starch

granule. French (1984) observed that intercrystalline



Bello-Pérez LA, Contreras-Ramos SM, Jimenez-Aparicio A, Paredes-Lopez O. 2000. Acetylation and characterization of banana (Musa paradisiaca) starch. Acta Cientifica Venezuela 51:143-149.

Deng F-M, Mu T-M,Zhang M, Abegeunde OK. 2013. Composition, structure and physicochemical properties of sweet potato starches isolated by sour liquid processing and centrifugation. Starch/Starke 65(12):162-171.

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Lii C-Y, Chang S-M. 1981. Characterization of red bean starch and its noodle quality. J Food Sci. 46(1):78-81. doi:10.1111/j.1365-2621.1981.tb14535.x.

Leon AE, Barrera GN, Perez GT, Ribotta PD, Rosell CM. 2006. Effect of damaged starch levels on flour-thermal behaviour and bread staling. Eur Food Res Technol. 224(2):187-192. doi:10.1007/s00217-006-0297-x.

Lu Z-H, Li L-T, Min W-H, Wang F, Tatsumi E. 2005.The effects of natural fermentation on the physical properties of rice flour and the rheological characteristics of rice noodles. Int J Food Sci Technol. 40(9):985-992. doi:10.1111/j.1365-2621.2005.01032.x.

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Kakou CA, Guehi ST, Olo K, Kouame FA, Nevry RK Koussemon CM. 2010. Biochemical and microbial changes during traditional spontaneous lactic acid fermentation process using two varieties of cassava for production of a “Alladjan” starter. Int Food Res J. 17:563-573.

Kassim, SM. July, 2012. Effect of fermentation period on functional and pasting properties of sweet potato flour “Elubo”. Food Sci and Technol, Federal University of Agriculture, Abeokuta.

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Oke MO, Bolarinwa IF. 2012. Effect of Fermentation on physicochemical properties and oxalate content of cocoyam (Colocasia esculenta) flour, ISRN Agronomy Volume 2012:1-4.

Pulido RP, Omar NB, Abriouel H, Lo pez RL,Canamero MM, Galvez A. 2005. Microbiological study of lactic acid fermentation of caper berries by molecular and culture-dependent methods. Applied and Environ Microbiol. 71(12):7872-7879 doi:10.1128/AEM.71.12.7872-7879.2005.

Putri WDR, Haryadi DW, Marseno, Cahyanto MN. 2011.

The higher the breakdown in viscosity, the lower the

ability of the starch sample, to withstand heating and

shear stress during cooking (Adebowale et al. 2005).

Therefore fermented SP flour might be less able to

withstand more heating and shear stress compared to

control flour because of their higher breakdown value.

Other modified technique fermentation was needed to

reduce the breakdown value of this flour. The stability

of fermented flour against heat and mechanical

treatment would also be useful in many other food

applications.

In regard to morphology of granule, there was

presence of the pits and smoother surface angle in the

starch granules seems to indicate some breakdown of

the starch. We presume that they might be caused by the

digestion of some starch by lactic acid bacteria. In

addition, cell wall material on control flour still

attached to the starch granules in control flour, while

those has been released in fermented flour. The size of

starch granules in fermented flour become bigger than

those in control. Fermentation may thus change the

amorphous region of the starch granule, size of the

granule as well as the chemical components and

thereby modify both physical properties of SP flour

and its rheological characteristics.

Based on the systematic analysis of the physical

properties of SP flour and its rheological characteristics,

it can be concluded that lactic spontaneous fermentation

had great effect on starch crystalline and the

amorphous region. The results revealed that

fermentation may change the amorphous region of the

starch granule, size of granules, as well as the chemical

components and it may modify the physical and pasting

properties of SP flour. Therefore, fermented SP flour is

easily swollen and soluble. Further research work is

needed to apply this fermented flour for making product

such as noodle that is suitable from these change

properties.

REFERENCES

AACC, American Association of Cereal Chemists. 2000. Approved methods of the AACC. Methods 02-52

Adebayo-Oyetoro AO, Olatidoye OP, Ogundipe OO, Balogun IO, Apara TO. 2012. Effect of local cassava fermentation methods on functional pasting and sensory properties of lafun. Continental J Agricl Sci. 6(2):1-8.

Aprianita A, Purwandari U, Watson B, Vasiljevic T. 2009. Physico-chemical properties of flours and starches from selected commercial tubers available in Australia. Int Food Res J. 16:507-520

Yousif NMK, Huch M, Schuster T, Cho G- S , Dirar HA, Holzapfel WH, Franz MAP. 2010. Diversity of lactic acid bacteria from Hussuwa, a traditional African fermented sorghum food. Food Microbiol. 27(6):757-768. doi:10.1016/j.fm.2010.03.012.

Yuliana N, Nurdjanah S. 2013. Development of sweet potatoes pickle as modified flour: Effort to balance the need of wheat. Research Report. Universitas Lampung.

Yuliana N, Nurdjanah S. 2009. Sensory properties of spontaneously fermented purple sweet potato pickle at various salt concentration. Jurnal Teknologi dan Industri Hasil Pertanian 14:120-128.

Yuliana N, Nurdjanah S, Ocatrini ZH. 2010. Some biochemical and total lactic acid bacteria changes during natural fermentatioan of the purple sweet potatoes. Proceeding International Seminar on Holticulture to support Food Security: B209-B214.

Yuliana N, Nurdjanah S, Margareta M. 2013.The Effect of a mixed-starter culture of lactic acid bacteria on the characteristics of pickled orange-fleshed sweet potato (Ipomoea batatas L.). Microbiol Indones. 7(1):1-8. doi:10.5454/mi.7.1.1.

Effect of biodegradation by lactic acid bacteria on physical properties of cassava starch. Int Food Res J. 18(3):1149-1154

Sanni A, Morlon-Guyot J, Guyot JP. 2002. New efficient amylase-producing strains of Lactobacillus plantarum and L. fermentum isolated from different Nigerian traditional fermented foods. Int J Food Microbiol. 72(1-2):53-62. doi:10.1016/S0168-1605(01)00607-9.

ShimelisE, Meaza M, Rakishit S. 2006. Physicochemical properties, pasting behaviour and functional characteristics of flours and starches from improved bean (Phaseolus vulgaris L) varieties grown in East African. CIGR E- Journals 8:1-18.

Sobowale AO, Olurin TO, Oyewole OB. 2007. Effect of lactic acid bacteria starter culture fermentation of cassava on chemical and sensory characteristics of fufu flour. Afr J Biotechnol. l 6(16):1954-1958.

Tester RF, Morrison WR. 1990. Swelling and gelatinization of cereal starches. I. Effect of amylopectin, amylose and lipids. Cereal Chem. 67:551-559.

Tian SJ, Rickard JE, Blanshard JM. 1991. Physicochemical properties of sweet potato starch. J Sci Food Agri. 57(4):451-491. doi:10.1002/jsfa.2740570402.




*Corresponding author; Phone/Fax: +62-21-5703306/5727615 ext. 450/+62-21-5719060, Email: [email protected]

Tempeh is an indigenous Indonesian fermented

food and has become an important part of the

Indonesian diet for hundreds years. Tempeh is

consumed in relatively large portion and can be found

in a variety of types of cooking and processing

methods. Excellent protein quality has made tempeh

become a meat substitute and becomes popular among

vegetarians (Liem et al.1977).

The process of tempeh making in Indonesia is still

using conventional methods with uncontrolled

condition (Barus et al. 2008). During the fermentation

process, not only fungi involved but also bacteria play

important roles in the formation of flavor and nutrition.

Bacterial growth during tempeh production begins in

the process of soybean soaking. During the

fermentation process, the producers inadvertently

adding bacteria so that the bacteria eventually become

an inseparable part of tempeh, and even has an

important role in determining the quality of tempeh

(Barus et al. 2008; Seumahu et al. 2013).

Several studies have reported the presence of

bacteria in tempeh as Klebsiella pneumoniae and

Citrobacter freundii (Keuth and Bisping 1994), and

also bacteria of the phylum Proteobacteria and

Firmicutes (Seumahu et al. 2012) which are reported as

bacteria producing vitamin B in tempeh. Other 12

contaminants such as Brevibacterium epidermidis and

Micrococcus luteus known to play a role in the

formation of antioxidants in tempeh (Klus and Barz

1995). Furthermore, bacteria in tempeh is also known

as one of the factors that play a role in the formation of a

bitter taste in tempeh (Barus et al. 2008). A diverse

array of lactid acid bacteria and yeasts also play

important role in Indonesian tempeh production

(Efriwati et al. 2013).

K. pneumoniae which is included in family

Enterobacteriaceae is also known both of causing

Tempeh is important traditional Indonesian fermented food made from soybeans employing Rhizopus oligosporus or R. microsporus. During the process of tempeh production, some bacteria from the environment and tempeh starter become an integral part of tempeh, and even have important roles in determining the final quality of tempeh it self. Several studies reported the presence of Klebsiella pneumoniae in tempeh as one of vitamin B12 producing bacteria in tempeh. However, K. pneumoniae also known as opportunistic pathogens causing pneumonia and liver abscess in human. In this study, Enterobacterial Repetitive Intergenic Consensus-Polymerase Chain Reaction (ERIC-PCR) was employed to determine genetic diversity of K. pneumoniae isolated from tempeh and compared them with medical isolates. The result indicated that isolates from tempeh were genetically distinct from those of medical isolates.

Key words: ERIC-PCR, tempeh

Tempe merupakan salah satu makanan utama tradisional Indonesia yang terbuat dari kacang kedelai dengan fermentasi menggunakan cendawan Rhizopus oligosporus atau R. microsporus. Selama proses pengolahan, bakteri yang berasal dari lingkungan dan inokulum awal menjadi bagian yang tidak terpisahkan dari tempe, bahkan memiliki peranan yang penting dalam menentukan kualitas akhir pada tempe. Beberapa penelitian telah melaporkan keberadaan Klebsiella pneumoniae pada tempe sebagai salah satu bakteri penghasil vitamin B12 pada tempe. Akan tetapi, K. pneumoniae juga dikenal sebagai patogen oportunis penyebab penyakit pneumonia dan abses hati pada manusia. Pada penelitian ini, metode Enterobacterial Repetitive Intergenic Consensus-Polymerase Chain Reaction (ERIC-PCR) digunakan untuk membandingkan keragaman genetik K. pneumoniae pada tempe dibandingkan dengan isolat medis. Hasil penelitian ini menunjukkan bahwa secara genetik pneumoniae pada tempe berbeda dengan isolat medis.

Kata kunci: ERIC-PCR, tempe

Klebsiella pneumoniae,

Klebsiella pneumoniae,

Klebsiella pneumoniae from Indonesian Tempeh were Genetically Different from that of Pathogenic Isolates

1 1,2 1EVELINE AYU , ANTONIUS SUWANTO *, AND TATI BARUS

1Department of Biology, Faculty of Biotechnology, Universitas Katolik Atma Jaya,Jalan Jenderal Sudirman 51, Jakarta 12930, Indonesia;

2Department of Biology, Faculty of Mathematics and Natural Sciences, Institut Pertanian Bogor, Darmaga Campus, Bogor 16680, Indonesia

pneumonia disease, an acute infection that attacks the

alveoli (Gori et al. 1996) and liver abscess in human

(Wang et al. 1998). Therefore, the study of genetic

diversity is important for the identification and

characterization of bacterial pathogenicity (Rademaker

and de Bruijn 1997). One of the many molecular

techniques for the study of genetic diversity is

Enterobacterial Repetitive Intergenic Consensus

(ERIC)-PCR. ERIC sequences are short sequences,

which is 126 bp long, with sequences that are conserved

as internal repeat and as non-coding sequences (Lupski

et al. 1992). This technique is often used because it is

simple, rapid, reproducible, and discriminative (Olive

et al. 1999) and has been successfully analyzed the

diversity of different types of bacteria, such as

Mycobacterium tuberculosis (Sechi et al. 1998) and

Vibrio parahaemolyticus (Khan et al. 2002). ERIC-

PCR has also been used to analyzed genetic diversity of

Klebsiella spp. isolated from tempeh (Barus et al.

2013), as well as to study genetic heterogeneity of

many types of Vibrio cholerae (Waturangi et al. 2012).


This research was conducted in Research

Laboratory, Faculty of Biotechnology, Atma Jaya

Catholic University of Indonesia from May to

December 2013.

Medical isolates of K. pneumoniae. Four medical

isolates ware used for comparison with K. pneumoniae

from tempeh, i.e. K. pneumoniae ATCC BAA-2146, K.

pneumoniae subsp. pneumoniae ATCC 10031, K.

pneumoniae ATCC 35657, and one K. pneumoniae

isolate originated from pneumonia patient, named as

FK isolate, collection of Department of Microbiology,

Faculty of Medicine, Atma Jaya Catholic University.

K. pneumoniae isolates from Tempeh. Tempeh

EMP and WJB was produced in Bogor, West Java,

Indonesia (Barus et al. 2008). A 10 g of fresh tempeh

was placed into 90 mL of sterile physiological salt

0.85% (w/v) NaCl and homogenized in orbitar shaker

(Yih Der) at a speed of 24 x g for one min. Dilution was -1 -6made from 10 until 10 and a 100 µL from dilution of

-4 -5 -610 , 10 , and 10 was spread on Eosin Methylene Blue

(EMB) Agar (Oxoid) and incubated overnight at 37 °C.

A single purple mucoid colony was typical character of

K. pneumoniae. These colonies were further analyzed

by cultivating them on Simmons’ Citrate Agar (SCA)

(Difco), and incubated at 37 °C for 24 h. Tempeh

sampling were conducted twice, i.e. August and

October 2013. Klebsiella sp. 135 isolated from tempeh

10 AYU ET AL. Microbiol Indones

was used for control (Maysella 2010).

Analysis of 16S rRNA Genes. Suspected blue

colonies on Simmons’ Citrate Agar were further

verified using sequencing of genes encoding 16S

rRNA. The whole cells from single colonies on plates

were used directly in PCR reaction as described by

Rademaker and de Bruijn in 1997. The 16S rRNA

gene was amplified employing a PCR machine

(Applied Biosystems, 2720 Thermal Cycler) using

primer 63f (5’- CAGGCCTAACACATGCAAGTC-

3’) and 1387r (5’- GGGCGGWT GTACAAGGC -3’)

(Marchesi et al. 1998). Total PCR volume was 50 µl

containing 2 µL DNA template, 25 µL GoTaq Green®

Master Mix (Promega), 2 µL primer forward dan -1reverse (25 pmol µL ) dan 19 µL nuclease free water.

The PCR protocol was as follows: initial denaturation

at 94 °C for 5 min, denaturation at 92 °C for 30 s,

annealing at 62 °C for 30 s, elongation at 72 °C for 30 s,

and post extention at 72 °C for 7 min. The cycle was

repeated for 30 times. A 5 µL of PCR amplification

products were further verified by electrophoresis in

1% agarose (Bioline) in 1x TAE buffer for 60 min, 80

V. Sequencing of PCR products were performed in

Macrogen Inc., Korea, and were analyzed employing a

program SeqTrace. Sequencing results were compared

to the database with the Basic Local Alignment Search

Tool (BLAST) program which is provided by National

Centre of Biotechnology Information (NCBI).

Genetic Profiling of K. pneumoniae Isolates. 13

bacterial isolates were identified as K. pneumoniae

from EMP tempeh and 10 isolates from WJB tempeh.

A total PCR volume used was 25 µL containing 12.5

µL GoTaq Green® Master Mix (Promega), 1 µL of 25

pmol ERIC1R (5’-ATGTAAGCTCCTGGGGATTCA

C-3’), 1 µL of 25 pmol ERIC2F (5’-AAGTAAGTGAC

TGGGGTGAGCG-3’)Give references for the primers

(Versalovic et al. 1991), 9.5 µL nuclease free water, and

1 µL DNA template which was obtained directly from

the isolates using a sterile toothpick. The PCR protocol

was as follows: initial denaturation at 95 °C for 7 min,

denaturation at 95 °C for 30 s, annealing at 49 °C for 1

min, elongation at 65 °C for 8 min, and post extention at

65 °C for 16 min (Applied Biosystems, 2720 Thermal

Cycler). The PCR cycle was used 30 times. A 5 µL of

PCR products was verified by electrophoresis for 90

min and 70 V, on 1% agarose in 1x TAE buffer. Formed

band profiles were observed under the UV

transilluminator. Formed band profiles were then

compared as biner number and analyzed using FreeTree

(Hampl et al. 2001) and TreeView to construct a

phylogenetics tree (http://taxonomy.zoology.gla.ac.uk/

rod/treeview.html.

RESULTS

Isolation of K. pneumoniae. A total of 58 bacterial

isolates (Table 1) were isolated from EMP and WJB

tempeh. The bacterial colonies were purple in the

center of colony, mucoid, and rounded shape on EMB

medium. The colonies of these isolates changed from

green to blue on Simmons’Citrate medium, which is

specific character of Klebsiella sp.

Fig 1A and 1B showed hypermucoviscocity of K.

pneumoniae ATCC35657 or FK colonies when

touched with inoculating loop. However, colonies of

K. pneumoniae from EMP (Fig 1C) and WJB tempeh

(Fig 1D) did not show hypermucoviscosity. This

character showed significant phenotypic difference

between tempeh and those of medical isolates.

Analysis of 16S rRNA Genes. A total of 18 isolates

from the EMP tempeh and 25 isolates from WJB

tempeh were selected for further amplification of

genes encoding 16S rRNA. Based on 16S rRNA gene

sequence alignments with the NCBI database, K.

pneumoniae were identified in 13 isolates obtained

from EMP tempeh and 10 isolates from WJB tempeh

(Table 1). The other microorganisms were found from

EMP tempeh based on 16S alignment analysis were:

Klebsiella sp., Rhizobium sp., and Enterobacter sp.

while Bacterium sp., Klebsiella sp., and Cronobacter

sakazakii were isolated from WJB tempeh.

Genetic Profiling of K. pneumoniae Isolates. A

total 23 isolates which were identified as K.

pneumoniae were further subjected to ERIC-PCR

analysis in order to compare their genetic diversity

from those of medical isolates. ERIC-PCR of 13

isolates of K. pneumoniae from EMP tempeh showed a

similar pattern (Fig 2). The ERIC-PCR profiles of

medical isolates showed a diverse patterns but

distinctively different from tempeh isolates. This result

indicated that K. pneumoniae presence in tempeh was

not the same as pathogenic K. pneumoniae. Similar

result were also found when we analysed isolates from

WJB tempeh (Fig 3) which showed different ERIC-

PCR profiles from the medical isolates. Although

genetic profiles of K. pneumoniae isolates in WJB

tempeh were more varied than EMP tempeh, we found

noi dentical profiles when compared to those of

medical isolates.

The resulting electrophoresis band profiles were

further converted into binary data matrix. The results

were used to construct a phylogenetic tree using

FreeTree program and TreeView. Fig 4 showed the

genetic relationship of K. pneumoniae isolated from

EMP tempeh. Based on this analysis, the medical

isolates clustered into a separate and distict group.

Tempeh isolates formed two groups, the first one

contains isolates which have similar ERIC profiles,

i.e., I EMP16, I EMP13, I EMP9, I EMP8, I EMP5, I

EMP4, and I EMP. The other group contains of I EMP1,

II EMP5, II EMP2, II EMP4, II EMP1, and II EMP3.

Isolates from WJB tempeh formed a separate group

under one branch except one isolate, I WJB2 (Fig 5).

Again, in this analysis, the medical isolates formed a

separate group outside the branches which contained

isolates from WJB.

DISCUSSION

K. pneumoniae presence in both of tempeh samples

(EMP and WJB) and consistantly exist from the first


Tempeh sample Number of sampling

Number of IsolatesSequencing of

16S rRNA EMB agar SCA

DNA Sequence Alignment

(K. pneumoniae)

EMP I 18 18 10 8

II 8 8 8 5

Total of EMP 26 26 18 13

WJB I 17 10 10 7

II 15 15 15 3

Total of WJB 32 25 25 10

Grand Total

58

51

43

23

Table 1 Klebsiela pneumoniae from fresh tempeh

Microbiol Indones12 AYU ET AL.

and second sampling of each of these tempeh samples.

The presence of K. pneumoniae in tempeh have been

reported before (Keuth et al. 1994, Barus et al. 2008).

Growth of K. pneumoniae on EMB medium showed

distinctive colony morphology, which were dark

purple in the center, mucoid, and rounded shape.

C

D

A B

Fig 1 The phenotype of K. pneumoniae ATCC 35657 (A), FK isolate (B), K. pneumoniae from EMP (C), and WJB tempeh (D).

700 bp

Fig 2 ERIC-PCR from K. pneumoniae from EMP tempeh, ie. (M) Molecular markers, (A) ATCC 10031, (B) ATCC 2014, (C) ATCC 35657, (D) FK, (E) Klebsiella sp. 135, (F) I EMP1, (G) I EMP3, (H) I EMP4, (I) I EMP5, (J) I EMP8, (K) I EMP9, (L) I EMP13, (N) I EMP16, (O) II EMP1, (P) II EMP2, (Q) II EMP3, (R) II EMP4, and (S) II EMP5.


700 bp

Fig 3 ERIC-PCR profiles of K. pneumoniae from WJB tempeh, ie. (M) Molecular markers, (A) ATCC 10031, (B) ATCC 2014, (C) ATCC 35657, (D) FK, (E) Klebsiella sp. 135, (F) I WJB1, (G) I WJB2, (H) I WJB3, (I) I WJB4, (J) I WJB5, (K) I WJB6, (L) I WJB16, (N) II WJB3, (O) II WJB5, and (P) II WJB8.

Medical isolateMedical isolates

Group 2

Group 3

Medical isolates

Group 2

Group 3

Medical isolate

Fig 5 Philogenetic tree generated from ERIC-PCR profiles of K. pneumoniae isolated from WJB tempeh.

Fig 4 Philogenetic tree generated from ERIC-PCR profiles of K. pneumoniae isolated from EMP tempeh.

disease associated with K. pneumoniae in tempeh. On

the other hand, their presence in tempeh might be

beneficial due to their ability to synthesize vitamin B12

and their immunomodulatory effects in human.

To conlude tempeh produced in Indonesia naturally

harbors K. pneumoniae with unique genomic profiles.

K. pneumoniae from tempeh samples in this study

showed that they were genetically different from

isolates known to be pathogenic to human.

ACKNOWLEDGMENT

This work was supported by DANA DIPA IPB

(code : 2013.089.521219). We also acknowlegde the

Department of Microbiology, Faculty of Medicine,

Universitas Katolik Atma Jaya for the FK isolate.

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Fig1showed the phenotype difference tha the

colonies of medical isolates showed more sticky on solid

media than those isolated from tempeh. Mutation in

magA is one of the factors that mutants lost the

hypermucoviscosity phenotype and became susceptible

tophagocytes and a virulent to mice (Fang et al. 2004).

16S rRNA gene sequences are the most common

house keeping genetic marker used for identifying

bacteria in the laboratory (Janda et al. 2007). Although

16S rRNA gene (approximately 1 500 bp ) is large

enough for bioinformatics purposes, but usually it is

not reliable for intra-strain differentiation. For

example, Escherichia coli O104: H4 that is known

pathogenic as well as isolate in recent out breaking

Germany (Mellmann et al. 2011) were confirmed only

as E. coli based on 16S DNA sequence analysis.

We confirmed colonies obtained from tempeh

samples as K. penumoniae based on 16S DNA

sequence analysis before subjected them for ERIC-

PCR analysis to reveal intra-species genetic diversity

(Barus et al. 2013).

In this study, ERIC-PCR method has been

successfully employed to differentiate genetic profiles

of K. pneumoniae isolated from tempeh and those of

medical isolates. The ERIC-PCR profiles of K.

pneumoniae from EMP tempeh (Fig2) and WJB

tempeh (Fig3) were different from those of medical

isolates. Keuth and Bispingin 1994 reported that K.

pneumoniae isolated from Indonesian tempeh were

negative for three known enterotoxins, i.e. Shiga-like

toxin SLTIIA, heat-labil enterotoxin LTIh, and heat-

stable enterotoxin STIh. Therefore, intraspecies

genetic diversity within K. pneumoniae might reflect

different phenotypes which could make the pathogenic

or non-pathogenic.

The phylogenetic analysis showed two separate

clusters representing K. pneumoniae from tempeh and

the medical isolates. The phylogenetic trees generated

from EMP tempeh showed unambiguously that tempeh

isolates were genetically different from pathogenic K.

pneumoniae (Fig4). That figure showed three main

groups were formed from tempeh isolates, while the

medical isolates were clearly separated in a different

group. Similar result was also obtained from isolates

derived fromWJB tempeh (Fig 5)

Our results suggested that K. pneumoniae isolates

inIndonesian tempeh could be a distinctive non-

pathogenic group of this species. This is also in line

with the facts that tempeh production and consumption

have been practiced in Indonesia for centuries and, to

our knowledge, there is no single report on infectious

Microbiol Indones14 AYU ET AL.


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Meacham KJ, Zhang L, Foxman B, Bauer RJ, Marrs CF. 2003. Evaluation of genotyping large numbers of Escherichia coli isolates by enterobacterial repetitive intergenic consensus-PCR. J Clin Microbiol. 41(11):5224-5226. doi:10.1128/JCM.41.11.5224-5226.2003.

Mellmann A, Harmsen D, Cummings CA, Zentz EB, Leopold SR, Rico A, Prior K, Szczepanowski R, Ji Y,

Indonesia has marginal land, such as dryland,

swamp, swampy, tides and peat. However, they are not

utilized optimally (RENSTRA the Ministry of

Agriculture 2009). Based on wide area, ecological

Composition and abundance of culturable bacteria of four soil samples (Ktr50II, D50II, G50II, and A50II)were analyzed. The soil samples were collected from maize rhizosphere that planted in dryland Lombok Island.Each soil sample give different growth performance of maize in greenhouse experiment. This study was toinvestigate the relation of maize growth performance with culturable bacterial community of their rhizosphereand the effect of culture media on number of bacterial isolates recovery. The rhizosphere bacteria were culturedand isolated on commercial media (SEA) and non commercial modification media (NA n, NA n-SE and NA n-RE). rhizosphere bacteria were obtained from four maize rhizophere soil samples. D50II isthe soil sample that caused the better growth performance to the maize, contrary to Ktr50II. D50II hassignificantly highest number of culturable bacterial types, while significantly lowest on Ktr50II. In D50II, atleast 17 bacterial isolates contributed to better growth performance in maize and have relative abundance ofdominant isolate not more than 35 34%. In comparing the rhizosphere bacteria recovered using different culturemedia, bacteria cultivated on SEA have different growth characteristic compared with bacteria cultivated onNA n, NA n SE and NA n-RE. Six bacterial isolates showed antagonistic ability when grew on SEAbut not in allof three media. Compared with commercial media, non commercial modification media can increase totalisolates recovery about 70 6%

culture media, dryland, maize, rhizosphere bacteria

Telah dilakukan kajian komposisi dan kelimpahan bakteri terkultur dari empat sampel tanah (Ktr50II, D50II,G50II, dan A50II). Sampel tanah dikoleksi dari rizosfer tanaman jagung yang tumbuh di lahan kering PulauLombok. Pada penelitian skala rumah kaca diketahui bahwa setiap sampel tanah memberikan performapertumbuhan berbeda pada tanaman jagung. Penelitian ini bertujuan untuk mengetahui hubungan antaraperforma pertumbuhan tanaman jagung dengan komunitas bakteri terkultur penghuni daerah perakarannya sertapengaruh media kultur terhadap perolehan isolat bakteri. Bakteri rizosfer di kultur dan diisolasi menggunakanmedia komersil (SEA) dan media modifikasi non komersil (Naln, NAln-SE and NAln-RE). Sebanyak 34 isolatbakteri diperoleh dari keempat sampel tanah. D50II diketahui memberikan performa pertumbuhan yang lebihbaik pada tanaman jagung, sebaliknya Ktr50II memberikan performa pertumbuhan yang buruk. D50IImempunyai jumlah jenis bakteri terkultur paling banyak dan sebaliknya pada Ktr50II. Pada D50II, sedikitnya 17isolat bakteri terlibat dalam menghasilkan pertumbuhan yang lebih baik pada tanaman jagung, serta denganjumlah kelimpahan relatif isolat dominan tidak lebih dari 35,34%. Bila membandingkan bakteri rizosfer yangdiperoleh dengan media kultur berbeda, terlihat bahwa bakteri yang terkultivasi pada SEA mempunyai karakterpertumbuhan yang berbeda dibandingkan bakteri yang terkultivasi pada NAln, Naln-SE and NAln-RE. Enamisolat bakteri menunjukkan kemampuan antagonis saat tumbuh pada SEA, namun kemampuan tersebut tidakmuncul saat tumbuh pada tiga media lainnya. Dengan media modifikasi non komersil dapat meningkatkanperolehan isolat sekitar 70,6% dibandingkan hanya menggunakan media komersial

bakteri rizosfer, lahan kering, media kultur, tanaman jagung

l l lThirty four strains

.

l l l

. .

Key words:

.

Kata kunci:

Abundance of Culturable Bacteria Isolated from Maize Rhizosphere SoilUsing Four Different Culture Media

ERNIN HIDAYATI , ARIS TRI WAHYUDI *, ANTONIUS SUWANTO ,

RAHAYU WIDYASTUTI

1,3 1 1

2AND

1

2

3

Department of Biology, Faculty of Mathematics and Natural Sciences,Institut Pertanian Bogor, Bogor 16680, Indonesia;

Department of Soil Science and Land Resources, Faculty of Agriculture,Institut Pertanian Bogor, Bogor 16680, Indonesia;

Department of Biology, Faculty of Mathematics and Natural Science,Universitas Mataram, West Lombok 83125. Indonesia

potency, social and economic potency, dryland is

appropriated to optimize. West Nusa Tenggara (NTB)

is one of the provinces in Indonesia with a relatively

wide dryland. From the total area (about 1 673 476.307

ha in Lombok and Sumbawa Islands), only about

626 034.60 ha that can be developed (BAPPEDA NTB

. .

.

ISSN 1978-3477, eISSN 2087-8587Vol 8, No 1, Maret 2014, p 33-40


DOI: 10.5454/mi.8.1.5

*Corresponding author; Phone/Fax: , Email:[email protected]

2003). Now, the dryland is being actively used

primarily for maize plantation

Soil microbes have a significant role in the soil

ecosystem. So that they can be used as a selective

indicator of soil and also as the main index to evaluate

the soil quality and global diversity (Zhang 2013).

Therefore, soil microbe are feasible for fundamental

components that could be considered as one of the

management strategies for sustainable agriculture in

dryland. Soil microbial communities can be influenced

by many factors. Several studies showed that

rhizosphere bacterial communities of maize influenced

by complex interaction, such as soil type (Castellanos

. 2009), plant growth stage (Di Cello . 1997),

cultivar (Dohrmann . 2013) and genotype

(Schmalenberger and Tebbe 2003). Although growing

on the same soil type, same species and age of plant,

sometimes individually of maize has a different growth

performance. This case has not been ever studied yet,

especially in dryland Lombok Island. According to Li

. (2014), rhizosphere bacteria have significant

contribution to crop health, productivity and carbon

sequestration. Thus it is predicted that the difference of

plant growth performances have relation to

rhizosphere bacterial community of that plant.

Rhizosphere bacterial communities have been

analyzed using cultivation technique and metagenome.

Although many molecular techniques used to study

many aspects of environmenal microbe, but this

technique has disadvantage, such as inavailability of

culture isolates. As a result, it is impossible to study

some aspects related to the life and function of the

isolates. Cultivation technique have significantly

contributed to our understanding of living microbes

(Pham and Kim 2012). Until now, cultivation

technique is fundamental tool to provide access to

diverse characteristics and physiology of microbe

(Reid and Buckley 2011). Culture isolates obtained

can be stored for further analysis and application.

Cultivation technique is also not expensive relatively.This

method can be applied by many laboratories, including

laboratories in developing countries. Because of this

technique has been done since long time ago, so that the

results of this technique has been well documented that

could be useful for comparison of metagenome.

The study of microbial communities based on

cultivation techniques highly depend on culture

media. The problem is that soil as complex media for

microbial growth in which they are interacting each

other. Many progress have been obtained from

.

et al.

et al et al

et al

et al

cultivation-based methods, especially related to

modification of nutrient culture media, such as by

reducing nutrient component and concentration of

culture media (Aagot 2001; Connon and

Giovanni 2002; Schoenborn . 2004), adding

growth stimulant (Nichols 2008; D'Onofrio

2010), soil extract amended (Hamaki 2005;

O'Neill 2009; George 2011) plant juice

supplemented (Stamer 1970; Nour 2010)

and adding exudates of artificial root and root exudates

(Baudoin 2003; Kozdroj; Louvel 2011 and

van Elsas 2000). To date, no single media that can bring

satisfaction for microbial cultivation.

Because of the facts that Lombok Island dryland

will be developed for plantation of agricultural crops,

especially maize, it is important to explore many

aspects of culturable bacterial community of the

dryland related to maize plantation. To cover greater

bacterial isolate, this study improved modification of

culture media by supplementing soil extract and root

extract of maize on low nutrient concentration media.

The goal of this study was to explain the contribution of

dryland culturable bacterial community to give good

growth performance of maize. Accordingly, this

investigation examines the relation of maize growth

performance with culturable bacterial community of

their rhizosphere and the effect of culture media on

number of bacterial isolates recovery

Four types of rhizosphere soil

samples used in this study, namely Ktr50II, D50II,

G50II, and A50II. The soil samples were collected

from rhizosphere of two months age of maize (

var BISI 2) that grew in dryland field located at

Lombok Island, West Nusa Tenggara, Indonesia (S

08º13'42.4'', E 116º21'24.4''). Study of soil samples

continued in greenhouse experiment on the growth of

maize for 30 d.As a result, each soil sample application

showed different vegetative growth performance of

maize in greenhouse. D50II gave better growth

performance of maize, A50II and Ktr50II gave worse

growth performance, and G50II gave medium growth

performance. Maize root mass were taken from the

field and placed in plastic bags labeled. The

rhizosphere soil samples were collected as described

by Phillips and Fahey (2006). All soil samples were

placed into sterile plastic bags labeled and stored in the

refrigerator at 6-7 C.

et al.

et al

et al. et al.

et al.

et al. et al.

et al. et al.

et al. et al.

.

Zea

mays

.


Soil Sampling

0

34 HIDAYATI ET AL. Microbiol Indones

Preparation of Culture Media

Cultivation and Growth Ability of Rhizosphere

Soil Bacteria

. Rhizosphere

bacteria were cultivated using commercial culture

media (Soil Extract Agar) and non commercial

modification culture media (NAln, NAln-SE and

NAln-RE). Soil Extract Agar (SEA, HIMEDIA,

Mumbai, India) consisted of 1 g L glucose,

dipotassium phosphate 0.50 g L , 17.75 g L soil

extract, and 15 g L agar. Nutrient Agar low nutrient

(NAln) consisted of 1% (0.08 g L ) Nutrient Broth

(Criterion, Santa Maria, CA) and 18 g L agar powder.

Nutrient Agar low nutrient supplemented with soil

extract (Naln-SE) consisted of NAln and 50% soil

extract. Nutrient Agar low nutrient supplemented with

root extract of maize (Naln-RE) consisted of NAln

and 25% soil extract. On each medium, of

antifungal nystatin was added.

Firstly, soil extract and root extract prepared. Soil

extract was taken from soil in Lombok Island dryland.

One part of soil sample was mixed with two parts of

sterile water ( ). Soil slurry was sterilized for 1 h

(O'Neill 2009) and then allowed 24 h at room

temperature. The supernatant was filtered with thick

layer sterile cotton. The liquid of soil extract was stored

at 6-7 C. The maize root extract was prepared from

maize root mass ( var BISI 2). One part of the

root mixed with two parts of sterile water ( ). Root

slurry was sterilized for 20 min and then allowed 24 h

at room temperature. The supernatant was filtered with

thick layer sterile cotton. The liquid of soil extract was

stored at 6-7 C.

Ten grams of soil samples were

suspended in 90 mLof sterile saline (0.85% NaCl). The

soil suspension was shaken on a rotary shaker for 15

min. From dilutions 10 , 10 , 10 , 10 , and 10

at 28 C for 7

days. During incubation, total colony forming unit

(CFU), number of bacterial isolates, and CFU of each

isolate were determined. Cultural morphology, colony

texture, colony pigmentation, and cell characteristics

were recorded. The colonies were also streaked on each

medium to verify their characteristic to avoid double

identification. Interesting isolates were identificated

based on size of Terminal Restriction Fragment (TRFs)

of 16S rDNA were digested with the MSpI restriction

enzyme. Purified bacterial isolates were stored in 20%

glycerol at -20 C for use throughout the study.

-1

-1 -1

-1

-1

-1

-1

0

0

-3 -4 -5 -6 -7

0

0

50 μg mL

,100 μL

aliquots of suspensionswerespreadedon the surface of

all of four culture media and incubated

w/v

et al.

Zea mays

w/v

.

Antagonistic Assay

StatisticalAnalysis.

bundance of Bacteria Isolated from Maize

Rhizosphere Soil

. Some previous studies

suggested that bacteria in mix culture could inhibit the

growth of other bacteria by producing antibacterial

substance. In this study, the bacterial isolates were also

examined for their antagonistic ability to inhibit other

isolates. The suspected of antagonist isolates were

streaked on the edge of agar plate of all culture media

and incubated at 28 C. Three days after incubation,

tested isolates were streaked horizontally 5 mm in

opposite to antagonist and further prolonged

incubating for 2 weeks. Inhibition activity of

antagonists were determined as the positive ability

when the colony of testing isolates was retarded.

Each treatment was made in 3

replicate. All result enumerations of isolates and

colony were analyzed using Annova 5% and then

followed by using Turkey Method of minitab 16.

. Thirty-four bacteria isolates were

cultivated from four maize rhizosphere soil (Ktr50II,

D50II, G50II, and A50II ) using four culture media

(SEA, NAln, NAln-SE, and NAln-RE). The number

and isolate types in each rhizosphere soil sample are

showed in Fig 1 A. Number of isolate types were

significantly higher in D50II and significantly lowest

in Ktr50II. At least 17 of the 34 isolates (50%) were

found in D50II. A further 12 of 34 isolates (35.29%)

were found in A50II, nine of the 34 isolates (26.47%)

were found in G50II, and five of the 34 isolates

(14.70%) were found in Ktr50II. Based on bacterial

isolates distribution of each soil sample, 18 of 34

isolates (52.9%) were found in certain soil samples and

the other found in more than one soil samples. Based on

colony forming unit (CFU), the number of CFU was

significantly higher in G50II (13.05x10 ) and then

decreased from D50II (5.07x10 ), A50II (1.87x10 ),

and Ktr50II (1.25x10 ) (Fig 1 B).

Comparison of isolate abundance showed that each

rhizosphere soil sample has different relative

abundance pattern. D50II has the most abundance

isolates and then decreasing from A50II, G50II, and

Ktr50II (Fig 2). Each soil sample was inhabited by

different dominat isolate type. For examples, CDL30 is

the most abundant isolate found in Ktr50II, CDL 38

found abundance in D50II, CDL 6 found abundance in

G50II, and CDL33 found abundance inA50II. Relative

0

7

7 7

7

RESULTS

A


abundance of dominant isolate found in D50II showed

not more than 35.34%, while in the other soil samples

more than 37.60%. CDL 6 was detected as a

predominant isolate was found in all soil samples. CDL

6 is known to having 123 TRF size digested with the

MspI restriction enzyme.

. Fourteen of the 34 isolates

were cultivated on NAln-RE, 12 of 34 isolates were

Ability of Each Culture Media to Cultivate the

Rhizosphere Bacteria

cultivated on NAln, 11 of the 34 isolates were

cultivated on Naln-SE, and 10 of the 34 isolates were

cultivated on SEA (Fig 3). At least 5 isolate types

shared cultivated on NAln, NAl-SE and NAln-RE, 6

isolate types shared on NAln-SE, and NAln-RE, while

7 isolate types shared on NAln, NAln-SE and NAln-

RE.At least 29.4% of bacterial isolates were cultivated

on SEA only while the remaining (70.6%) were

cultivated on NAln, NAln-SE, and NAln-RE.

A B

Fig 1 Comparison of number and isolate type (A) and colony forming unit (B) of maize rhizosphere bacteria cultivated frommaize rhizosphere soil samples cultivated using four culture media (SEA, NAln, NAln-SE, and NAln-RE).

Fig 2 Estimation of relative abundance of maize rizosphere bacteria from Lombok Island dryland related to different growthperformance of maize made by cultivation technique using four different culture media.


Growth Differences of Bacterial Isolates on

Each Culture Medium. Nearly all bacterial colonies

developed on SEA have different morphological

appearances compared with NAln, NAln-SE and

NAln-RE (Fig 4). The bacterial colonies were

cultivated on SEA developed during the first day of

incubation. The characteristic of these colonies were as

follows: fast growing, colony diameter average of 2.0

- 2.5 mm with clear differences among each colony

morphology (Fig 4 A). When the dilution higher than

10 , only dominated with growing of certain isolates.5

The growth of these isolates usually followed by

appearance the yellowish color surrounding the

colonies. Bacterial colonies were cultivated on NAln,

NAln-RE, and NAln-SE developed after 3 days of

incubation. The characteristic of the colonies were as

follows: slow growing, colony diameter average of

0.5-2.0 mm, and many of these are very thin and

transparent (Fig 4 B, C, D). Cultivated bacteria from

dilution higher than 10 not showed phenomena such

as on SEA. Physiological differences were also found

only in six isolates such as CDL 12, CDL 19 , CDL 25,

5

Fig 3 Comparison of number and isolate type isolated using four culture media (A) and number of isolate shared amongculture media (B).

5

5

6

7

10

79

6

10

14

11

12

SEA

NAln

NAln-RE

NAln-SE

A B

SEA

NAln

NAln-SE

NAln-RE

C D

A B

Fig 4 Appearance of colony development of maize rhizosphere bacteria on SEA (A), NAln (B), NAln-SE (C), and NAln-RE

(D) at 10 dilution after 6 days of incubation.-3


CDL 26 , CDL 30, and CDL 32, where those isolates

produced yellowish color around the colonies when

grew on SEA. This evidence did not happen when they

grew on NAln, NAln-SE, and NAln-RE.

.

Antagonistic assay was done to six isolates (CDL 12,

CDL 19, CDL 25, CDL 26, CDL 30, and CDL 32) that

produced yellowish color when grew on SEA. This

assay showed that ability to produce yellowish color

is related to antagonistic ability of the isolates in

inhibiting the growth of other isolates (Fig 5). The

antagonistic ability only showed when the antagonist

grew on SEA (Fig 5 A, B, C) and not on NAln, NAln-

SE and NAln-RE (Fig 5 D, E, F). The antagonist

inhibited the growth of certain isolates but not in other

isolates.

The CDL 30 and CDL 32 were detected as the

higher relative abundance in Ktr50II. CDL 30 known

to having 486 TRF size and showed 98% score identity

similar with (NR_114751),

while CDL 32 known to having 485 TRF size and

showed 97% score identity similar with

(NR_037000). In the other soil

samples, relative abundance of the antagonist less than

15%.

his study is the first recorded of the maize

rhizosphere bacterial states in Lombok Island dryland.

Antagonistic Ability in Some Bacterial Isolates

Pseudomonas stutzeri

Pseudomonas

pseudoalcaligenes

DISCUSSION

T

Fig 5 Bacterial antagonist (in vertical streak) isolated from maize rhizosphere soil bacteria. Antagonistic ability developedwhen grew on SEA(A, B, C) but not on NAln (D), NAln-SE (E), and NAln-RE (F).

The results of this experiment showed that based on

total number isolates and colony forming unit (CFU),

D50II has higher number of bacterial isolates

compared with other soil samples. Despite D50II has

lower number of CFU compared with G50II, the

number of CFU about 5.07x10 is more likely

produced better growth.As for the G50II, although that

soil sample has higher of CFU compared with D50II,

less number of isolate likely it was not enough to

produce better growth as D50II. Despite of A50II has

higher number of isolates but less number of CFU may

caused poor growth compared with G50II. In Ktr50II,

lowest number of isolates and CFU may caused the

worse growth. This result explained that the number of

bacterial isolates more important to maize growth than

total CFU. In addition, each kind of bacteria should be

present in a sufficient number to be able to affect the

growth of maize. The results indicated that the maize

growth performance influenced by number and isolate

type on the soil samples.

Although the number of isolate types in A50II

significantly higher than G50II, but the community

seemed to be dominated by only one type of isolate is

CDL33, which the relative abundance about 81.99%

(Fig 2). In addition to number of isolate type and CFU,

the relative abundance pattern that composed each soil

type may contribute to differentiation of the growth

performance of maize. It predicted that more abundant

baceria with balance composition should give better

effect to maize growth.

7


More previous study showed many aspects that

known to influence the microbial community (Di Cello

1997; Schmalenberger and Tebbe 2003;

Dohrmann 2013). In this study, when the

influence of the abiotic factors and plant are reduced, it

appeared that the rhizosphere bacterial community

contributed to influence the growth of maize. It is

assumed that abundance of the origin microbial status

in dryland is the initial factor that influence the growth

of maize.

The results also showed that 52.9% of bacterial

isolates were found in certain soil sample and the other

found on more than one soil samples. The similarity of

isolate type was found in the soil samples may it is

because of the soil samples taken from the same soil

type from the same location. While the variation of

isolate type was found in the soil samples may it is

because of each soil sample has different origin

microbial diversity since they are in the land.

Furthermore, the differences of diversity which

contributed to the differences of maize growth

performance.

By doing variation in composition of media such

as low concentration of nutrients (NAln), low

concentration of nutrients supplemented by soil

extract (NAln-SE) and supplemented by maize root

extract (NAln-RE) increased the number of culturable

bacteria obtained from the soil sample compared with

commercial media only. It was suggested that the

different nutrient composition is likely affected the

number and type of bacterial cultivated on each media.

In this experiment, rich nutrient composition of the

SEA may provide conditions that allow for certain

groups of bacteria to grow rapidly and dominate each

other, while the slow growing bacteria will eliminated

with no space to grow. In contrast to the NAln, NAln-

SE and NAln-RE, poor nutrient composition provide

conditions that allow the slow growing and dormant

bacteria to grow. The similar results also found by

other researcher (Brozel and Cloete 1992; Joseph

2003).

The soil extract and root extract were

supplemented to NAln-SE and NAln-RE respectively,

may also contribute as a selection factor of the types of

rhizosphere soil bacteria. According to Hamaki

(2005), soil bacteria and actinomycetes can grow on

soil extract agar media but not on conventional media.

Some experiments also showed that medium of soil

extract amended was suitable for growth of fastidious

soil bacteria (Davis . 2005) and also reduce

et al.

et al.

et al.

et al.

et al

diversity and dominance of fast growing bacteria

(Kozdroj and van Elsas 2000).

According to Tamaki (2005), bacteria in mix

culture can produced antibacterial substances to inhibit

the growth of other bacteria. Dominance also

contributed to the ability of organisms to produce

extracellular antibacterial component (Rao

2005). Refer to Tamaki (2005) and Rao

(2005), it is likely that the antagonistic ability of six

isolates (CDL 12, CDL 19, CDL 25, CDL 26, CDL 30,

and CDL 32) may caused by antibacterial substances

that produced by the isolates. The antagonistic ability

of these rhizosphere soil bacteria not only when they

are in the form of mixed cultures but also explained the

relationship between type of culture media and

antagonistic ability of the bacteria.This antagonistic

mechanism may one of the factor causing fewer

number of cultivated bacteria and lower total CFU of

bacteria on SEA.

et al.

et al.

et al. et al.

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Pseudoalteromonas tunicate.

.


Currently the use of harmful chemicals as a

preservative of food like tofu, noodles, meatballs,

chicken meat, and fish is prohibited. We must search an

alternatives to the particular safe food and fish

preservatives and for consumption. Bacteriocins from

lactic acid bacteria (LAB), especially their

antibacterial activities, have attracted much attention

and have been the subject of intensive investigation

(Mataragas . 2002). The limited existence of data

regarding bacteriocins from spp. makes this

genus an interesting object to investigate, since it

produces diverse array of antimicrobial peptides

representing several different basic chemical

structures (Adetunji and Olaoye 2011)

The production of bacteriocins or bacteriocin-like

substances has already been described for some

spp., such as , ,

et al

Bacillus

Bacillus Bacillus substilis B. cereus B.

.

Bacillus cereusEscherichia coli

Staphylococcus aureus Salmonella thypi Bacillus subtilis Listeria monocytogenesBacillus cereus

Staphylococcus aureus

Bacillus cereus


SS28 isolated from budu, a fermented fish product from West Sumatra, producedantimicrobial compound that had broad spectrum of inhibition against five microorganisms ( ,

, , , and ). The aims of thisresearch are characterization of SS28 antimicrobial activity and observation of its effect to thecellular morphology of with electron microscope. Antimicrobial compound produced by

SS28 was stable at pH range between 2 and 11 and to heating at 121 C for 15 min. Maximumantimicrobial activity was expressed at pH 2-3 and 70°C for 45 min. The activity remained after 15 min exposureto UV light. The main changes observed under SEM and TEM were the alteration ofstructural cell membrane 48 h after exposure to the antimicrobial compound from Bacillus cereus SS28

o

.

Key words:

Kata kunci:

antimicrobial bacteriocin, SS28, budu, characterization, West Sumatera

SS28 diisolasi dari ikan budu, produk fermentasi ikan yang berasal dari Sumatera Baratyang dapat menghasilkan komponen antimikroba bakteriosin dengan spektrum yang luas dan dapatmenghambat lima bakteri ( dan

). Tujuan dari penelitian ini adalah melakukan karakterisasi komponen antimikroba daribakteri SS28 dan mempelajari pengaruhnya terhadap morfologi sel dari bakteri

. Komponen antimikroba yang dihasilkan oleh bakteri SS28 stabil pada perlakuan pH 2-

11 dan pemanasan suhu 121 C selama 15 menit. Aktivitas antimikrobial yang paling tinggi terdapat pada pH 2-3,

suhu 70 C selama 45 menit.Bakteriosin masih stabil setelah terpapar di bawah sinar UV selama15menit.Denganmenggunakan SEM dan TEM terlihat perubahan struktur membran sel bakteri setelahterpapar selama 48 jam oleh komponen antimikroba dari SS28

antimikroba bakteriosin, SS28, budu, karakterisasi, Sumatera Barat

Bacillus cereus

Bacillus cereus

Escherichia coli, Staphylococcus aureus, Salmonella thypi, Bacillus subtilisListeria monocytogenes

Bacillus cereus Staphylococcusaureus Bacillus cereus

Staphylococcus aureusBacillus cereus

Bacillus cereus

o

o

.

Characterization of Antimicrobial Bacteriocin Produced by SS28Isolates from Budu, a Traditionally Fermented Fish Product of West Sumater

Bacillus cereus

a

YUSRA *, FAUZAN AZIMA , NOVELINA , PERIADNADI1 1 1 2

AND

1

2

Departement of Agricultural Processing Technology, Faculty of Agricultural Technology

Departement of Biology, Faculty of Matematics and Natural Sciences,Universitas Andalas, Padang, 25163, Indonesia

stearothemophilus Bacillus

et al et al

Listeria

monocytogenes Streptococcus pyogenes et

al

B. megaterium

B. amyloliquefaciens et al

Bacillus .

et al

et al

Bacillus

Bacillus cereus

and other spp. (Zheng

1999; Cherif . 2001; Stein . 2002). Some

strains produce bacteriocin with broad spectrum of

activity including important pathogens such as

and (Cherif

. 2001). Some produced well characterized

bacteriocins, such as lichenin and megacin produced

by . Bacteriocin had also been isolated

from (Lisboa . 2006).

A number of general physicochemical properties

has been studied to provide information about the

composition and structure of bacteriocins. Various

studies stated that bacteriocins produced by sp

showed resistance to heat treatment and tolerance to

pH, as described by Sharma . (2009), and Khalil

. (2009) about the effects of pH, heating and

exposure to UV light towards sp MTCC 43

bacteriocins.

SS28 isolated from budu showed

very high antimicrobial activity against all tested

ISSN 1978-3477, eISSN 2087-8575Vol 8, No 1, Maret 2014, p 24-32


DOI: 10.5454/mi.8.1.4

*Corresponding author; Phone/Fax:, Email:

+62-751-7051678/+62-751-55475 [email protected]

strains ( , ,

, and

), with range of inhibition zone 14-35

mm (Yusra 2013). Budu is a fermented fish

product from West Sumatera, mainly originated from

the coastal areas, such a Pariaman, Tiku and Pasaman.

normally made from bigger size marine fish such

as Spanish mackerel ( sp.) and

leatherskin ( sp.), locally, knowns as ikan

tenggiri and ikan talang (Yusra 2012). However,

studies related to the antibacterial characteristics of

these organisms have been limited and not fully

exploited. Therefore, the purpose of this research were

to characterize the antimicrobial compounds isolated

from SS28 and to observe its effect to

cell morphology with electron

microscopy (SEM and TEM)

. Materials used in this study

were isolated from SS28. The indicator

strains used in this work were provided by the

Laboratory of Clinical Microbiology Research,

Faculty of Medicine and Microbiology, Universitas

Indonesia, and Laboratory Microbiology, Department

of Food Science and Technology, Faculty of

Agricultural Technology, Institut Pertanian Bogor.

They include both gram negative and gram positive

strains ( , , , , and

)

. The strain SS28

provided by Yusra . (2013) was maintained

at -4 C and as frozen stock cultures in equal volumes

of 10% glycerol. SS28 was grown in MRS

broth, , , , and

were grown in nutrient broth (NB).

The cultures were grown at 37 C for 24 h in MRS

broth or NB medium.

SS28 was

grown in the MRS broth media as much as 200 ml,

incubated at 37 C for 30 h. One mL culture sample was

taken hourly and put in a test tube. Changes in the

optical density of the cultures were recorded at 600 nm

wavelength (Olivera 2004).

.

SS28 was cultivated in 250 ml erlemeyer flask

Escherichia coli Staphylococcus aureus

Salmonella thypi Bacillus subtilis Listeria

monocytogenes

et al.

Budu

Scomberomorus

Chorinemus

Bacillus cereus


Bacillus cereus

E. coli S. aureus S.thypi B. subtilis L.

monocytogenes .

B.cereus

et al

B. cereus

E. coli S. aureus S. thypi B. subtilis L.

monocytogenes

B. cereus

et al.

Bacillus

cereus

.


Bacterial strains

Bacterial cultures

Growth and production of bacteriocin by

SS28 in a MRSB at 37 C.

Production of Crude Bacteriocin

o

o

o

B.

cereuso

containing 100 mL of MRS broth and incubated for 48 h

at 37 C. Supernatants were harvested by centrifugationo

at 6000 g for 10 min at 4 C. The pH of the cell free

supernatant was adjusted to 6.5 using 1 M NaOH

solution to prevent the inhibitory effect of organic

acids. The supernatants were then filtered using 0.22

μm membrane filter (Millipore). The filtrates were

used for the characterization of bacteriocin.

o

Antimicrobial Act iv i ty of Extracted

Bacteriocin

Characterization of Bacteriocin

Effect of pH on antimicrobial activity

Effect of Temperature on Antimicrobial

Activity

Effect of UV Light on Antimicrobial Activity

Scanning Electron Microscopy.

. Agar well diffusion and paper disc

methods were used to study antimicrobial activity of

the extracted bacteriocin. In the agar well diffusion

assay 0.1 mL culture of the tested microorganisms

(

and ) were spread

on sterile nutrient agar. Twenty L

placed in each well and the

plates were aerobically incubated at 37 °C for 24 hrs.

.

Supernatant from culture was diluted

with deionized water. The diluted supernatant was then

divided into several parts, each of which was adjusted

to different pH levels between 2 to 11 using sterile

10 mM/l NaOH or 10 mM/l HCI solution. The

solutions were then heated at 100 °C for 30 min, before

the pH was adjusted to 6.5 with sterile dH O and

assayed for its activity (Nofisulastri . 2006). The

antimicrobial activity were determined by paper disc

assay.

. Supernatant of SS28 was exposed

to various heat treatments: 40, 55, 70, 85, 100, and

121 ºC. Aliquot volumes of each fraction were then

removed after 0, 30, 60, or 90 min and assayed for

bacteriocin (Ogunbarwo . 2003).

.

Ten ml supernatant of SS28 was placed in a

sterile petri dish and exposed to short - wave UV light

(wavelength 340 nm, 220-240 V, 50 Hz) situated at a

distance of 30 cm from petri dishes. Time of exposure

to UV light is 30 minutes after which the bacteriocin

activity was estimated by the papper disc method

(Ogunbarwo . 2003).

culture

that has been exposed to bacteriocin from

SS28 at 37 C for 48 h were examined by SEM to

visualize any morphological change occuring in the

cell following exposure to bacteriocins and pressure.

The cell suspensions were fixed with 3%

gluteraldehyde in Na-cacodylate buffer (100 mM, pH

7.1). Then the cells were pelleted and washed to

E. coli, Staphylococcus aureus, Salmonella thypi,

B. subtilis, Listeria monocytogenes

B. cereus SS28

et al

B. cereus

et al

B. cereus

et al

S. aureus

B. cereus

μ extracted bacteriocin

preparation (CBP) was

2

o


remove gluteraldehyde before resuspended in the

same buffer. A drop of each suspension was transferred

to a poly-L-lysine-treated silicon wafer chips that

were kept for 30 min in a hydrated chamber to let the

cells adhere. The attached cells were post fixed by

immersing the chips in 1% osmium tetroxide (OsO ) in

cacodylate buffer for 30 min, then rinsed in the same

buffer and dehydrated in ethanol in ascending

concentrations (%): 50, 70, 95 (2x) and 100 (2x), for 10

min each. The chips were mounted on aluminum

stubs and coated with gold-palladium in a sputter

coater (Emitech K550, Ashford, Kent, England). The

chips were viewed at 3 kV accelerating voltage in a

Hitachi S-4000 field emission scanning electron

microscope (JEM-JEOL JSM-5310LV type) and

secondary electron image of cells for topography

contrast were collected at several magnifications

(Bolshakova . 2004).

The

cell suspensions that has been exposed to bacteriocin

from SS28 at 37 C for 48 h were harvested

by centrifugation and washed twice with 0.1 M

phosphate buffer (pH 7.3). The cells were fixed with

2.5% (v/v) glutaraldehyde, 2.0% (v/v) formaldehyde

in 0.12 M phosphate buffer for 10 days and then

postfixed in 2% (w/v) osmium tetroxide in the same

buffer for 45 min. The samples were dehydrated in a

graded acetone series (30-100%) and embedded in

Araldite-Durcupan for 72 h at 60 °C. Thin sections

4

et al

S. aureus

B.cereus

Transmission electron microscopy.

o

(microtome UPC-20, Leica) were mounted on grids,

covered with collodion film and poststained with 2%

uranyl acetate in Reynold's lead citrate. Its preparation

were observed with transmission electron microscope

tipe JEOL-1010 (Bozzola and Russel 1999, with

modification).

The growth and

bacteriocin production of is

slight increase of cell dry weight was observed for 28 h

of fermentation. During log phase (6 - 22 hours

fermentation), medium pH decreased rapidly. It

occured concurrently with the increase of the cell dry

weigt. The data indicated that the alteration of the

medium pH was inversely proportional with growth of

Ss28.

The effect

of pH on bacteriocin activity was studied. It was

observed that bacteriocin produced by SS28

was stable between pH 2-11 (Fig 2).

The inhibitory activity towards the test isolates was

heat stable (Fig 3). The antimicrobial activity remained

constant after heating at 121 ºC for 15 minutes. The

activity was highest when being heated at 70 ºC for

45 min.

RESULTS

Growth of SS28 and the production of

bacteriocin in MRSB at 37 C.

Effect of pH onAntimicrobialActivity.

Effect of Temperature onAntimicrobialActivity.

B. cereuso

B. cereus Ss28

B. cereus

B. cereus

Fig 1 The growth curve of SS28 isolate on MRS broth mediumBacillus cereus

26 ET AL.YUSRA Microbiol Indones

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0 5 10 15 20 25 30

Ab

sorb

an(n

m)

Time (h)

Fig 2 Effect of pH on the activitiy of antimictobial compound from SS28 determined based on the size of theinhibition zone (mm)

Bacillus cereus.

Fig 3 Effect of temperature on activity of antimictobial compound from SS28, determined based on the sizeof the inhibition zone (mm)

Bacillus cereus.

Effect of UV Light on antimicrobial activity.

Scanning Electron Microscopy.

Bacteriocin produced by the test isolates was tested

for their sensitivity (loss of activity) to UV light

exposure. The antimicrobial activity was lost or

unstable after exposure to UV-light for 15 and 30 min

(Fig 4).

SEM has been

widely used in microbiology to study the surface

structure of biomaterials and to measure cell

attachment and changes in morphology of bacteria.

The SEM-generated photomicrograph of pathogen

after treatment with antimicrobial

compound from SS28 is presented in

Figure 5.

S. aureus

B. cereus

pH

0

5

10

15

20

25

30

2 3 4 5 6 7 8 9 10 11

Inhib

itio

nZ

one

Siz

e(m

m)

Escherichia coli

Staphylococcus

aureus

Salmonella thypi

Bacillus subtilis

Listeria

monocytogenes

0

5

10

15

20

25

30

35

40 50 70 85 100 121

Inhib

itio

nzo

ne

size

(mm

)

Temperature (°C)

E. coli

S. aureus

S. thypi

B. subtilis

L. monocytogenes


Fig 5 Scanning electron microscopy of cells control (a) (Bajpal . 2009) and after treatment withantimicrobial compound of SS28 (b).

Staphylococcus aureus et alBacillus cereus

Bacteria

0

5

10

15

20In

hib

itio

nzo

ne

size

(mm

)

UV, 15 menit

UV, 30 menit

Fig 4 Effect of uv light on activity of antimictobial compound from SS28, determined based on the size of theinhibition zone (mm)

Bacillus cereus.

A B

Transmission Electron Microscopy. The effect of

antimicrobial compound from SS28 on

bacterial cells was studied using as a

representative of Gram-positive cells. Morphological

investigations were performed using 48-h

culture treated with antimicrobial compound from

SS28 l). Control has exhibited typical

coccus morphology of (Fig 6). Untreated

B. cereus

S. aureus

S. aureus

B.

cereus

S. aureus

(20 μg/m

S.aureus

S. aureus

cells) shows a typically structured nucleus of

and a perfect cell wall (Fig 6A). After 48

hours of exposure to the antimicrobial compound, a

slight alteration can be observed in the cell cytoplasm

(Fig 6B), the cells exhibited notable alteration in

cell cytoplasm. Bacterial cells completely collapsed

48 h after treatment with the antimicrobial compound

(Fig 6).


DISCUSSION

Bacteriocin activity remained stable up to 24 h

fermentation, then the activity started to drop after 28 h

fermentation. Koroleva (1991) stated that most of the

metabolism products resulted in the log phase were in

the form of lactic acids, which causes the decrase in pH

of the medium. This acidic condition will eventually

inhibit growth of the respective bacteria (negative feed

back effect).

Bacteriocin is extracellular secondary metabolite.

The increase in the amount of biomass produced in the

bacterial culture caused the increase in the amount of

the bacteriocin produced. After reaching the stationary

phase, the amount started to decrese (Boe 1996).

Synthesis of bacteriocin by LAB occurred during the

exponential growth phase, usually following the

protein synthesis (Schnell 1998). Torkar and

Matijasic (2003) who did research on the

characterization of bacteriocins produced by

from milk and other dairy products, found that the

production of bacteriocins entered the stationary phase

after 10-16 hours of incubation. The research by

Naclerio . (1993) on the production and activity of

bacteriocins cerein present in the

stationary phase also demonstrated similar result.

The highest antibacterial activity was exhibited at

pH range 2 to 3, while inactivation occurred between

pH 9 to 11. Khalil . (2009) showed that

bacteriocins produced by 22 has

activity antimicrobial against at pH

range 2-8. Naclerio . (1993) who studied the

antimicrobial activity of bacteriocins cerein from

found that the compound's activity was

et al.,

B. cereus

et al

from B. cereus

et al

B. megaterium

S. thypimurium

et al

B. cereus,

Fig 6 Transmission electron microscopy of cells control (A) (Santhana ., (2007) and after treatmentwith antimicrobial compound of SS28 (B).

Staphylococcus aureus et alBacillus cereus

stable between pH 3-12. Growth temperature plays an

important role and is often correlated with bacteriocin

production (Todorov and Dicks 2006).

Similar to the results of Alam . (2011), who

stated that bacteriocin of BS15 retained

activity up to 80 C for 30 min, other bacteriocin

produced by ssp. diacetilactis was reported to

maintain its activity even after boiling for up to 60 min.

On the other hand, Lactacin F was reported to

completely lose the activity when treated at 50 C for 30

min (Kojic . 1991; Kim . 2005). Cleveland

. (2001) suggested several potential advantages of

bacteriocins to serve as biopreservatives, namely: a)

the material is not toxic and susceptible to degradation

by proteolytic enzymes because it is a protein

compound, b) the material does not harm the intestinal

microflora because it is easily digested by

gastrointestinal enzymes, c) the material can reduce the

chemicals as a food preservative, d) flexibility of use,

and e) stability towards sufficiently broad range of pH

and temperature that it is resistant to treatment

processes involving acids and bases, as well as hot and

cold conditions.

Antimicrobial activity of SS28 was the

highest against with inhibition zone diameter

20 mm, after 15 minutes exposure, which decreased to

10 mm after 30 minutes. species and other gram

negative bacteria were sensitive to nisin and other

bacteriocins after exposure to treatments that change

the permeability barrier properties of the outer

membrane (Stevens . 1991). Khalil . (2009)

19 bacteriocin was stable after 15 min

exposure to UV light and was completely destroyed

after 90 min.

et al

B. subtilis

L. lactis

et al et al et

al

B. cereus

S. thypi,

S.

et al et al B.

megaterium

o

o

A B


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Jack RW, Wan J, Gordon J, Harmark K, Davidson BE,HillierAJ, Wettenhall REH, Hickey MW, Coventry MJ.1996. Characterisation of the chemical andantimicrobial properties of piscicolin 126, a bacteriocinproduced by JG 126. J ApplEnviron Microbiol. 62(8):2897-2903.

Metasequioaglyptostroboides

Pediococcus acidilactici

Bacillus

Lactobacillus

lectronmicroscopy: principles and techniques for biologists

Bacillus thuringenesis

Carnobacterium piscicola

The effect of antimicrobial compound from

supernatant SS28 from wall and cell

membrane was investigated. It could be associated

with the damage in the cell wall and cell membrane and

subsequent lysis and reduction. Immediately after

treatment, 80% of the cell's surface appeared

rough, which is quite different from the normal cells. In

a previous study with ,

which has an inducible autolytic enzyme, bacteriocin

treatment, pressurization or their combination did not

only produce cell death and cell lysis, but also triggered

the autolytic enzyme, which, by hydrolyzing the wall,

disintegrated the cells (Bhunia . 1987;

Kalchayanand . 2002).

Electron microscopy showed cell lysis after

treatment with antimicrobial compound of

SS28. The cell damage caused by antimicrobial

compound resembles that observed with a crude

bacteriocin treatment (Ocana . 1999). Bizani .

(2005) tried to truestigate the effect of cerein 8A

against spore. An approximately 4-5

log reduction was observed when spores were plated

in PCA containing 800 AU ml . As cerein 8A

concentration increased to 1600 AU ml , complete

inhibition of colony development was observed. When

spores were treated with cerein 8A in BHI broth before

plating, similar results were observed. The bactericidal

effect of the antimicrobial compound from

SS28 apparently works by disrupting the

membrane function of target organisms.

To conclude antimicrobial bacteriocin from

was stable over a broad range of pH

(between pH 2 to 11) and to heat-treatment at 121 C for

15 min. The antimicrobial activity was the highest at

being heated at 70 C for 45 min and for 15 min of

exposure to UV light. The main changes observed

under SEM and TEM analyses were structural

disorganization of the cellular membrane

48 h after exposure to the antimicrobial compound of

SS28.

B. cereus

S. aureus

Layconostoc mesenteroides

et al

et al

B. cereus

et al et al

Bacillus cereus

Bacillus

cereus

B.cereus SS28

S. aureus

B. cereus

10

-1

-1

o

o

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budu


amarila malik12*, iman santoso , andi yehuda2,...

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