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The Isolation and molecular identification of antimicrobial-producing Lactic

Acid Bacteria from chao, South Sulawesi (Indonesia) fermented fish product

Article in Biodiversitas Journal of Biological Diversity · March 2019

DOI: 10.13057/biodiv/d200418

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BIODIVERSITAS ISSN: 1412-033X
Volume 20, Number 4, April 2019 E-ISSN: 2085-4722
Pages: 1063-1068 DOI: 10.13057/biodiv/d200418

Isolation and molecular identification of antimicrobial-producing Lactic

Acid Bacteria from chao, South Sulawesi (Indonesia) fermented fish
product

RISKY NURHIKMAYANI1, BUDI SETIADI DARYONO2, ENDAH RETNANINGRUM1,

1
Microbiology Laboratory, Faculty of Biology, Universitas Gadjah Mada. Jl. Teknika Selatan Sekip Utara, Sleman 55281, Yogyakarta, Indonesia.
Tel.: +62-274-580839, Fax.: +62-274-6492355,  email: [email protected]
2
Genetic and Breeding Laboratory, Faculty of Biology, Universitas Gadjah Mada. Jl. Teknika Selatan Sekip Utara, Sleman 55281, Yogyakarta, Indonesia

Manuscript received: 23 May 2018. Revision accepted: 23 March 2019.

Abstract. Nurhikmayani R, Daryono BS, Retnaningrum E. 2019. Isolation and molecular identification of antimicrobial-producing
Lactic Acid Bacteria from chao, South Sulawesi (Indonesia) fermented fish product. Biodiversitas 20: 1063-1068. Chao is traditional
fermented fish made of anchovy (Stolephorus sp.), rice, salt, and fermented naturally on controlled environment. The product comes
from Pangkep Regency, South Sulawesi. Chao has the potential for the source of high-grade lactic acid bacteria isolates, especially for
antimicrobial compound. Antimicrobial compound produced by lactic acid bacteria is a biopreservative compound with many
advantages. The purposes of this investigation were to isolate antimicrobial-producing lactic acid bacteria from chao and to identify
their isolates based on the molecular properties. Lactic acid bacteria were isolated from chao using Man Ragosa Sharpe Agar (MRSA)
medium added with 0.1 % CaCO3, followed by the ability test to produce antimicrobial compound against Staphylococcus aureus FNCC
0047 as an indicator. The spectrum of crude antimicrobial as cell-free supernatant (CFS) was determined by antagonism against S.
aureus FNCC 0047 and Escherichia coli FNCC 0049 on agar well diffusion test. Selected isolates of antimicrobial-producing lactic acid
bacteria molecularly identified from 16S rRNA marker using primer 27F and 1492R. We obtained four isolates of lactic acid bacteria
that showed the antimicrobial activity. This crude antimicrobial compound could inhibit both gram-positive Staphylococcus aureus
FNCC0047 and gram-negative bacteria Escherichia coli FNCC0049. The highest crude antimicrobial activity of isolates was observed
on IB1C strain. The results of molecular characterization suggested that the IB1C and IB3B strain were identified as Lactobacillus
plantarum, whereas IB3E and IB3F were identified as Pediococcus pentosaceus.

Keywords: Antimicrobial, chao, lactic acid bacteria, Lactobacillus plantarum, Pediococcus pentosaceus

INTRODUCTION motile and producing antimicrobial components (Axelsson

2004). Antimicrobial substances of LAB such as lactic
Chao is a local fermented fish product of coastal acid, hydrogen peroxide, diacetyl, and bacteriocin can
communities from Pangkep Regency, South Sulawesi, inhibit pathogenic bacteria and spoilage bacteria. The
Indonesia. It is produced by soaking the raw materials, ability of LAB to produce antimicrobial components can be
including anchovy (Stolephorus sp.), rice and salt, then used as a natural preservative (Adams and Moss 2008). The
fermented naturally on controlled environment (Karim et LAB are known as GRAS (Generally Regarded as Safe)
al. 2013). The salt content of chao fermentation ranges because they are not toxic and do not produce a toxin, so
from 15-20% that inhibit the growth of the non-tolerant they are not harmful to health, even these microorganism
microbes. Lactic acid bacteria are tolerant and adaptable to are useful for health (USFDA 2015). The use of LAB as a
saline environment with concentrations of 10-18% natural preservative agent can reduce the use of harmful
(Sanches 2008; Agussalim 2017). This makes lactic acid synthetic preservatives. The ability of LAB as natural
bacteria becomes the dominant microorganism in chao preservative has been widely reported, especially from
fermentation products. Chao is also known to have a sour fermented food. Indonesia has many fermented food but
taste that indicates the main microorganism for only few studies about Indonesian’s fermented fish are
fermentation activity is the lactic acid bacteria (LAB) available in literature such as jambal roti, peda (Indriati et
group (Lakong 2003). The LAB was divided into 12 al. 2006), bekasam (Desniar et al. 2015; Budiarto et al.
genera, i.e. Lactococcus, Leuconostoc, Pediococcus, 2016), and rusip (Koesoemawardani et al. 2018). As far as
Streptococcus, Lactobacillus, Enterococcus, Aerococcus, we know, there has been no research on antimicrobial-
Vagococcus, Tetragenococcus, Carnobacterium, producing lactic acid bacteria from chao. This research
Weissella, and Oenococcus. These bacterial species in the aimed to isolate and identify antimicrobial-producing LAB
LAB group are designated as LAB according to their from chao, South Sulawesi Fermented Fish Product.
ability to metabolize lactic acid (Ray 2005).
Lactic acid bacteria are gram-positive bacteria,
endospores-negatives, catalase-negative, usually non-
1064 B I O D I V E R S I T A S 20 (4): 1063-1068, April 2019

MATERIALS AND METHODS Identification of the Antimicrobial-Producing Strain

Cell from bacterial cultures were collected by
Indicator strains and growth conditions centrifugation at 14.000 x g for 1 min. After discarding the
Staphylococcus aureus FNCC 0047 and Escherichia supernatant, the total genomic DNA was extracted by using
coli FNCC 0049 were obtained from the Food and a PrestoTM Mini gDNA Bacteria Kit (Geneaid, Taipei,
Nutrition Culture Collection of Universitas Gadjah Mada. Taiwan). The extracted DNA samples were amplified by
All strains were maintained in Nutrient Agar at 370C. polymerase chain reaction (PCR) using 16S ribosomal
RNA gene. PCR amplification used My Taq Red Mix
Isolation of Lactic Acid Bacteria (Bioline) and universal primers (27F:
The sample of chao was obtained from the local market AGAGTTTGATCMTGGCTCAG and 1492R:
in Pangkep Regency, South Sulawesi, Indonesia. The TACGGYTACCTTGTTACGACTT) (Chao et al. 2008;
sample was diluted 10-fold in 0.85% NaCl solution and Pringsulaka et al. 2012; Miao et al. 2014; Ahn et al. 2017).
mixed thoroughly. Then, 0.1 mL of each dilution was The DNA extract from isolates was mixed with PCR
poured into MRS (Man Ragosa Sharpe) agar (Oxoid) with cocktail. The DNA templates used were 1 µL for each
0.1 % CaCO3 and incubated for 48 h at 370C (Assohoun- reaction. PCR cocktail contain 12,5 µL MyTaq Red Mix
Djeni et al. 2016; Sirichokchatchawan et al. 2018). 2x, 1 µL both 27F Primer and 1492R Primer with final
Colonies producing clear halo were selected and re- concentrations 20 µmol/µL, and ddH2O 9,5 µL. The PCR
streaked on MRS agar plate to obtain pure cultures. condition contained pre-denaturation at 950C for 1 min,
Selected colonies were preliminary screened for lactic acid followed by 35 cycles consisting of 950C for 15 sec,
bacteria by Gram staining, catalase testing, endospore annealing at 520C for 15 sec and extension at 720C for 45
staining, and motility test. Colonies which were gram- sec, and final extension 720C for 10 min. PCR products
positive, catalase negative, endospore negative and non- were confirmed by gel electrophoresis on a 1.5 % (w/v)
motile were subjected for further test. agarose gels, stained with ethidium bromide. The
amplicons were purified by ZymocleanTM Gel DNA
Detection of antimicrobial activity Recovery Kit (Zymo Research) according to the
Antimicrobial activity was determined by well diffusion manufacturer's protocol and used as sequencing templates.
assay of LAB growing cells culture and cell-free The PCR products were sequenced by bi-directional
supernatant (CFS) against indicator strain Staphylococcus sequencing at 1st BASE (Singapore).
aureus FNCC 0047. The antimicrobial activity of LAB
growing cell was performed using the method described by Phylogenetic analysis
Assohoun-Djeni et al. (2016) and Sirichokchatchawan et al. The sequences of bacterial isolates were analyzed using
(2018). Overnight cultures of each LAB strains were Basic Local Alignment Search Tool (BLAST) at
placed in wells cut in Nutrient soft agar (0.7 % agar) www.ncbi.nlm.nih.gov to the database of 16S ribosomal
inoculated with 100 µL of an overnight active culture of RNA sequences (Bacteria and Archaea). The gene
indicator bacteria. After incubation at 37 0C for 24 h, the sequences were aligned with multiple alignments using
plates were examined for the existent of clear halo around Clustal W and Bioedit was used to remove the gap's region
the well. The presence of distinguishable halo was (Nguyen et al. 2014).
considered as positive inhibition. Positive inhibition LAB A phylogenetic analysis was performed by MEGA 7 to
strains were grown in MRS broth for overnight at 37 0C, determine the closest bacterial species (Ahn et al. 2017).
then centrifuged (12.000 rpm for 10 min at 4 0C, Sigma Model selection was determined using Find Best
Santorius 3-30 K, England). Each supernatant was DNA/Protein Models (ML) in MEGA7. The selection was
collected and filtered (0.2 µpore sizes, Corning, Germany) based on the lowest AICc (Akaike Information Criterion,
to obtain CFS (Cell Free Supernatant). The CFS was placed corrected) score. The lowest AICc score was estimated to
in wells cut in nutrient soft agar seeded (100 µL) with be the best model to be used in Maximum Likelihood
indicator strain. The antimicrobial spectrum of CFS was analysis (Tamura et al. 2011).
analysed by antagonism against Escherichia coli FNCC
0049 on agar well diffusion test. Each experiment was
performed in triplicate. Antimicrobial activity was RESULTS AND DISCUSSION
measured based on an inhibition score (IS). The inhibitory
score was obtained based on the following formula
Isolation of Lactic Acid Bacteria
(Tremonte et al. 2017)
As much as twenty-nine isolates of LAB with clear
zone on MRS agar with CaCO 3 were isolated from chao.
Inhibition Score (IS) = Inhibition zone diameters
They were all screened on the basis of gram stain, catalase,
(mm)/well diameter (mm)
motility and oxidase test. Six isolates (IB1C, IB3B, IB3C,
IB3E, IB3F, IB6C) showed gram-positive properties,
The inhibitory effect of an antimicrobial compound
catalase negative, non-motile and endospore-negative
considered to be low if 1 <IS <3, moderate 3≤ IS <5, strong
(Table 1).
5 ≤ IS <7, and very strong 7 ≤ IS <9.
 AUTHORS et al. – Antimicrobial-producing LAB from chao 1065

Detection of antimicrobial activity This study has also performed an assay that applied
Antimicrobial activity was detected based on the ability CFS to the isolates. The CFS was used in the test may
of growth cells and CFS in inhibiting the growth of provide an overview of the antimicrobial activity which is
indicator strain. Among six LAB isolated from chao, four the extracellular metabolite compound produced by
isolates showed an inhibition zone against S. aureus FNCC bacteria. Bacteriocin, one of the antimicrobial compounds
0047 and E. coli FNCC 0049 (Table 2). is known to be an extracellular substance produced by
The detection of antimicrobial activity by growth cell bacteria (Mesa-Pereira et al. 2017). Antagonistic activity of
assay against S. aureus FNCC 0047 showed that three CFS depends on extracellular bactericidal secretion. In the
isolates (IB1C, IB3B, IB3F) have moderate inhibition antimicrobial test of cell growth, the secretion process is
ability, while IB3E isolate produced a low inhibition zone. controlled by the cell to cell interaction (Goers et al. 2014).
The cell cultures that produce large inhibitory zones However, in the CFS, the pure colony of isolates may have
indicates their potential to produce antimicrobial already secreted extracellular antimicrobial compound
compounds. The inhibition zone diameters were in the (Hettiarachchi et al. 2017). CFS produced by BAL contains
range of 11.33 to 18.57 mm. The widest inhibitory zone organic acids, hydrogen peroxide, and diacetyl (Hor and
diameter was shown by IB1C isolate with 18.67 ± 0.58 Liong 2014).
mm, while the narrowest inhibition zone were IB3C and The results of the CFS activity against S. aureus FNCC
IB6C isolates. They did not show any inhibition zone. The 0047 as a gram-positive bacteria showed that IB1C isolate
result showed that low activity or inactivation of IB3C and has a moderate inhibition, while the other isolates (IB3B,
IB6C isolates do not mean that the two isolates are inactive IB3E, IB3F) have low inhibition. This difference occurs
or unable to produce antimicrobial compounds, but it can due to the amount of antimicrobial involved during the test
be due to the ability of an antimicrobial compound to process. On the testing of antimicrobial cell activity, we
diffuse in agar well medium. According to Valgas et al. used 107 CFU/ml cells for each isolate while the CFS test
(2007) diffusion process is influenced by several factors used a supernatant from isolate culture with a 1.5% initial
such as amount, size, and shape of diffuse substances. concentration of inoculated isolates on MRS broth media
Hence, different bacterial strains have different activities so with 24-hour incubation time. During the incubation
that the antimicrobial compounds produced by each isolate process, growth cell and metabolites produced by each
may also differ both in number, size, and shape. In isolate can also be different because each strain of bacteria
addition, the size of the inhibitory zone depends on the has different activities.
speed of antimicrobial to diffuse with the media, and also CFS inhibition activity depends on the active substance
depends on the growth rate of the indicator bacteria. The released to the supernatant. At low isolates, growth causes
growth of isolate that tends to be slower than the indicator low antimicrobial activity (Mariam et al. 2014). The final
strains can cause the isolate has not enough time to form concentration of metabolite products harvested from
antimicrobial compounds in competition against the cultures can vary depending on strain, which causes the
indicator strains on the test medium (Mariam et al. 2014). results of testing of the antimicrobial activity of IB1C CFS
against CFS other isolates to be different (Abushelaibi et al.
Table 1. Characteristic of lactic acid bacteria from chao 2017). Interestingly, IB1C isolate, produced a low
inhibitory activity against gram-negative E. coli FNCC
0049. The cell wall of E. coli isCharacteristic
Isolated much more complex than cell walls of gram-positive bacteria. The outer membrane contains
Cell
lipopolysaccharides
no. (LPSs).
Gram The main function
Catalase of LPSs
Endospore is creating a permeability barrier that restricts the entry of antibiotic
Motility
shape
andIB1C
other toxicRod
substances + that can- kill or injure
- the bacterium
- (Willey et al. 2008). Moreover, based on antimicrobial from
growth
IB3B cell and CFS
Rod assay,
+ IB1C isolate
- had the
- highest inhibition
- zone. The isolates
Note
IB3C: + = positive,
Cocci - = negative
+ - - - with inhibition zone appeared were selected for further
IB3E Cocci + - - - studies.
IB3F Cocci + - - -
Table
IB6C 2. Inhibition
Cocci zone+ diameters- of LAB isolated
- from -
Chao
Identification of the antimicrobial-producing strain
using S. aureus and E. coli as indicators Identification of four isolates that have antimicrobial
activity was carried out by molecular characterization
Inhibition zone diameters (mm) based on DNA sequence of 16S rDNA. Two isolates
Isolated Well Growth cell CFS
(IB1C, IB3B) were identified as Lactobacillus plantarum
no. (mm) S. aureus S. aureus E. coli
FNCC 0047 FNCC 0047 FNCC 0049 with 100% similarities to accession number NR115605.1.
IB1C 5 18,67±0,58b 16,67±1,04b 14,67±1,15a Two isolates were identified as Pediococcus pentosaceus
IB3B 5 15,67±1,15b 14,00±3,61a 14,00±1,00a with 99% similarity to accession number NR042058.1
IB3C 5 - - - (Table 3). According to Dighe et al., (2004) and
IB3E 5 11,33±0,58a 11,83±3,55a 9,33±2,51a Greenwood et al., (2012) if isolates have similarity of more
IB3F 5 16,50±2,29b 13,60±1,80a 9,33±0,57a than 97%, they can be considered as the same species.
IB6C 5 - - - Based on the evidence, the IB1C and IB3B isolates were
Note: Different letters in the same column means different IS, a =
low, b = moderate
1066 B I O D I V E R S I T A S 20 (4): 1063-1068, April 2019

considered as L. plantarum species, and IB3E and IB3F According to our investigations, this is the first publication
isolates were considered as P. pentosaceus. The similar about isolation on antimicrobial producing L. plantarum
studies about the isolation of lactic acid bacteria from fish and P. pentosaceus from chao.
fermentation products were reported that antimicrobial In conclusion, Four LABs isolated from chao could
compound produced by L. plantarum and P. pentosaceus produce antimicrobial against both of both gram-positive
(Desniar et al. 2015; Budiarto et al. 2016; Wang et al. and negative bacteria. The highest antimicrobial activity
2016). was observed in IB1C isolate. Based on molecular
Phylogenetic analysis can provide an estimation of the identification, IB1C and IB3B isolates were identified as L.
evolutionary relationship between microbial sequences. plantarum, whereas IB3E and IB3F isolate identified as P.
According to Madigan et al. (2010), 16S rRNA gene is pentosaceus. Therefore further research should be followed
widely used for the construction of phylogenetic trees to use these isolates as an inoculum in controlling of
because the sequence of nucleotide bases of rRNA contamination of food product as a natural preservative.
molecules can precisely be compared, makes it easier to
identify the diversity. The 16S rRNA gene in bacteria has a
high level of similarity because it is found in all
prokaryotes. The 16S rRNA gene also has variable and
conserved regions, and it is this region reflects the
phylogenetic relationship between species (Mitreva 2017).
The results of the 16S rDNA sequence analysis showed
that the IB3E and IB3F isolates clustered to the same
branch as Pediococcus pentosaceus while the IB1C and
IB3B isolates were clustered in the same group as
Lactobacillus plantarum and Lactobacillus pentosus.
Several studies on the isolation of the LA from fish
fermentation products also showed similar results. Wang et
al. (2016) in their study succeeded in isolating L.
plantarum 120 from fish fermentation product from China,
Suanyu. L. plantarum JCM-1149 produce antimicrobial
compounds that inhibit the growth of Listeria
monocytogenes (Nursyam et al. 2016). Kusmarwati et al.
(2014) also reported the isolation of P. pentosaceus I from
the Bangka and Kalimantan regions as a producer of
antimicrobial compounds as bacteriocin. Thus, it can be Figure 1. The results of DNA amplification with 27F and 1429R
concluded that the lactic acid bacteria found in fermented primers. (M: Marker 1 Kb DNA Ladder (Geneaid), 1: IB3B, 2:
fish product are strains of L. plantarum and P. pentosaceus. IB3E, 3: IB3F and 4: IB1C)

Table 3. BLAST result of the isolate from chao

Accession
Isolate Species of LAB homolog Identities Isolate sources
number
IB1C Lactobacillus plantarum strain JCM 1149 100% NR_115605.1 Pickled cabbage
Lactobacillus plantarum strain NBRC 15891 99% NR_113338.1 Pickled cabbage
Lactobacillus plantarum strain CIP 103151 99% NR_104573.1 Frozen peas
Lactobacillus plantarum strain NRRL B-14768 99% NR_042394.1 Jojoba meal fermentation
Lactobacillus pentosus strain 124-2 99% NR_029133.1 Corn silage

IB3B Lactobacillus plantarum strain JCM 1149 100% NR_115605.1 Pickled cabbage
Lactobacillus plantarum strain NBRC 15891 99% NR_113338.1 Pickled cabbage
Lactobacillus plantarum strain CIP 103151 99% NR_104573.1 Frozen peas
Lactobacillus plantarum strain NRRL B-14768 99% NR_042394.1 Jojoba meal fermentation
Lactobacillus pentosus strain 124-2 99% NR_029133.1 Corn silage

IB3E Pediococcus pentosaceus strain DSM 20336 99% NR_042058.1 Dried American beer yeast
Pediococcus stilesii strain FAIR-E 180 98% NR_042401.1 White maize grain
Pediococcus acidilactici strain DSM 20284 98% NR_042057.1 Barley
Pediococcus claussenii strain ATCC BAA-344 98% NR_075029.1 Fermented Beverage spoiled beer

IB3F Pediococcus pentosaceus strain DSM 20336 99% NR_042058.1 Dried American beer yeast
Pediococcus stilesii strain FAIR-E 180 98% NR_042401.1 White maize grain
Pediococcus acidilactici strain DSM 20284 98% NR_042057.1 Barley
Pediococcus claussenii strain ATCC BAA-344 98% NR_075029.1 Fermented Beverage spoiled beer
 AUTHORS et al. – Antimicrobial-producing LAB from chao 1067

Figure 2. Phylogenetic tree isolates of lactic acid bacteria from chao using Maximum Likelihood (bootstrap = 1000)

ACKNOWLEDGEMENTS (bekasam) and their antimicrobial activity against pathogenic bacteria.

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