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ASEAN-FEN INTERNATIONAL FISHERIES SYMPOSIUM – 2017 IOP Publishing
IOP Conf. Series: Earth and Environmental Science 137 (2018)
1234567890 ‘’“” 012060 doi:10.1088/1755-1315/137/1/012060

The antagonistic activity of lactic acid bacteria isolated from

peda, an Indonesian traditional fermented fish

T F Putra1, H Suprapto2, W Tjahjaningsih3, and H Pramono3 4

1
Study Program of Aquaculture, Faculty of Fisheries and Marine, Airlangga University,
Surabaya 60115, Indonesia
2
Departement of Fish Health Management and Aquaculture, Faculty of Fisheries and Marine,
Airlangga University, Surabaya 60115, Indonesia
3
Department of Marine, Faculty of Fisheries and Marine, Airlangga University, Surabaya
60115, Indonesia

E-mail: [email protected]

Abstract. Peda is an Indonesian traditional fermented whole fish prepared by addition of salt
prior to fermentation and drying process. Salt used to control the growth of the lactic acid
bacteria for the fermentation process. The objectives of this study were isolating and
characterize the potential lactic acid bacteria (LAB) from peda as culture starter candidate,
particularly its activity against pathogenic bacteria. A total of five samples from five regions of
East Java Province was collected and subjected to LAB isolation. Fifty-seven of 108 colonies
that show clear zone in de Man, Rogosa and Sharpe (MRS) agar supplemented with 0.5%
CaCO3 were identified as LAB. Twenty-seven of the LAB isolates were exhibit inhibition
against Staphylococcus aureus ATCC 6538 and Pseudomonas aeruginosa ATCC 27853.
Isolate Aerococcus NJ-20 was exhibited strong inhibition against S. aureus ATCC 6538 (7.6 ±
1.35 mm inhibition zone) but was not produce bacteriocin. This finding suggests that the
isolate Aerococcus NJ-20 can be applied as biopreservative culture starter on peda production.
Further analysis on technological properties of isolates will be needed prior to application.

1. Introduction
Peda is an Indonesian traditional fermented fish product that contains 20-30 % (w/w) salt [1]. Peda is
often produced from mackerel (Rastrelliger sp.) [2] with the addition of salt and put through a drying
process to prolong the shelf life of the fish. Salt in fermented fish inhibits the growth of spoilage and
pathogenic bacteria, as well as maintaining lactic acid bacteria for the fermentation process [3].
Lactic acid bacteria (LAB) is Generally Recognized as Safe (GRAS) by WHO and plays an
important role in the process of fermentation of food such as inhibiting spoilage bacteria and
producing the flavour, aroma, and texture of fermented food [4,5]. LAB may be isolated from
fermented fish such as plasom, bekasam, fish sauce, and peda [6,7,8,9].
The antagonistic activity of LAB is due to antimicrobial compounds produced by the bacteria such
as lactic acid, hydrogen peroxide (H2O2), diacetyl, reuterin, and bacteriocin [10]. Lactic acid can
disrupt the outer membrane of bacteria that causes lysis, whereas bacteriocins are antimicrobial
proteins or peptides synthesized by ribosomes [11] that initiate pore formation on the bacterial

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ASEAN-FEN INTERNATIONAL FISHERIES SYMPOSIUM – 2017 IOP Publishing
IOP Conf. Series: Earth and Environmental Science 137 (2018)
1234567890 ‘’“” 012060 doi:10.1088/1755-1315/137/1/012060

membrane. Bacteriocin has bactericidal or bacteriostatic properties against spoilage and pathogens
bacteria and is used as a biopreservative in food products [12,13].
The antagonistic activity of LAB was isolated from various fermented fish such as bekasam [14].
The different regions of production and methods performed during the production of peda may lead to
differences in LAB isolation as well as their antagonistic activity against foodborne pathogens. The
objective of this study was to isolate and analyze the antagonistic activity of LAB from peda as a
culture starter of peda production.

2. Methodology
2.1 Samples collection
Sample collection was conducted by the method of Cho [15]. Mackerel Peda (Rastrelliger sp.)
samples were collected from five regions of East Java (Surabaya, Sidoarjo, Gresik, Kediri, and
Nganjuk). All samples were wrapped in sterile plastic bags, stored in an insulated cool box (4oC) and
transported to the laboratory within 5-8 hours.

2.2 Isolation of lactic acid bacteria (LAB)

The isolation of lactic acid bacteria was done according to Angmo et al [16] and Pranomo et al [17]. A
total of 10 g sample of mackerel peda was homogenized with 90 ml of normal saline (0.85 % w/v) and
diluted. One mL of the diluted sample was spread on de Man, Rogosa and Sharpe (MRS, Merck,
Germany) Agar supplemented with 0.5 % CaCO 3, and then incubated at 30oC for 48 hours. Individual
colonies were picked and streaked on MRS supplemented with CaCO3 and incubated at 30oC for 24
hours. Purified cultures were tested for catalase and Gram staining. All catalase negative and Gram-
positive strains were stored in MRS broth with the addition of sterile glycerol (1:1 v/v) at -20oC.

2.3 Antagonistic activity of LAB

The isolates of LAB were cultured on de Man, Rogosa and Sharpe (MRS, Merck, Germany) broth and
incubated at 30°C for 24 hours twice prior to the antagonistic test. The indicator bacteria
(Staphylococcus aureus ATCC 6538 and Pseudomonas aeruginosa ATCC 27853), which represented
Gram-positive and Gram-negative bacteria respectively, were cultured on Trypticase Soya Broth
(TSB, Oxoid, UK) and incubated at 30°C for 24 hours. Each indicator bacteria (107 CFU/ml) was
swabbed on a Mueler Hinton Agar (MHA, Oxoid, UK) medium using a sterile cotton-swab. LAB
culture was applied at 20 μl into a sterilized paper disk. The paper disk was then placed on the surface
of the MHA medium that had been swabbed with indicator bacteria and incubated at 30 oC for 24
hours. LAB isolates that formed the inhibited zone (≥ 6 mm) were selected for the bacteriocin
production test [18].

2.4 Bacteriocin-producing test

The LAB isolates which inhibited indicator bacteria were cultured on de Man, Rogosa and Sharpe
(MRS, Merck, Germany) broth medium and incubated at 30oC for 24 hours. The culture was then
centrifuged (Hettich Rotanta 420 R, Germany) at 4600 rpm at 4°C for 10 minutes, then the pH
adjusted to 7.0 with 1 N NaOH. The neutralized-cell free supernatant is heated at 80°C for 10 minutes
to eliminate hydrogen peroxide and kill the cells. The neutralized-cell free supernatant was filtered
with 0.22 μm millipore filter. The antimicrobial activity of the supernatant was confirmed by the Agar
Well Diffusion Assay (Pringsulaka et al. 2012) against the indicator bacteria S. aureus ATCC 6538.
MHA (Merck, Germany) soft agar (0.75 % agar w/v) which was inoculated with 1 ml of indicator
bacteria 107 CFU/ml. A 50 μL cell free neutralized supernatant was loaded to each well and incubated
at 30 °C for 24 hours. The isolate that exhibited antimicrobial activity in the AWD test was then
characterized by the nature of its activity by adding proteinase K. The neutralized-cell free supernatant
that lost activity by the addition of proteinase K indicates bacteriocin production [30]. Briefly, the
proteinase K of 200 μg (w/v) was dissolved in a 1 ml phosphate buffer solution and then added to the
neutralized-cell free supernatant. The neutralized-cell free supernatant was incubated at 30 °C for 2

2
ASEAN-FEN INTERNATIONAL FISHERIES SYMPOSIUM – 2017 IOP Publishing
IOP Conf. Series: Earth and Environmental Science 137 (2018)
1234567890 ‘’“” 012060 doi:10.1088/1755-1315/137/1/012060

hours. The activity of the proteinase K enzyme was stopped by heating it at the temperature of 100 oC
for 30 minutes. The neutralized-cell free supernatant was tested against S. aureus ATCC 6538 with
Agar Well Diffusion Assay.

2.5 Identifitication of LAB

Identification was performed to find the genus of LAB that showed high antagonistic activity. The
characterization included performing tests on gas production (fermentation type), pH effect,
temperature influence, and salinity and comparing them to the manual identification of LAB.

2.6 Data analysis

All measurements of antagonistic activity were performed in triplicate. The data was expressed in the
mean and standard deviation of the three experiments.

3. Results and Discussion

3.1 Isolation of LAB
A total of 108 colonies that exhibited a clear zone on de Man, Rogosa and Sharpe agar supplemented
with CaCO3 0.5% was purified in MRS. Among 108 isolates, only 57 isolates (52.7 %) were included
as lactic acid bacteria due to it being Gram-positive, rods and cocci-shaped, and negative catalase.
LAB shows a clear zone on MRS supplemented with CaCO3 due to the production of organic acid
(such as lactic acid, propionic acid, and/or acetic acid) [5] that dilutes the CaCO3 in the medium, then
the organic acids can react with CaCO3 forming Ca-lactate and forms a clear zone [6,19]. LAB is a
Gram-positive, non-spore forming, rod or cocci-shaped, catalase negative, and non-motile bacteria. In
addition, from the data of Gram staining (data not shown), 12 and 45 isolates out of 57 isolates LAB
were rod and cocci-shaped respectively.
The isolating rate of lactic acid bacteria was correlated with various factors such as the location,
method, and source of LAB. Angmo et al [16] was an isolated LAB from a fermented beverage of
Ladakh and found 46 LAB isolates from dried cottage cheese and beverage. Pringsulaka et al [20]
isolated 152 LAB from 93 samples of Thai fermented meat and fish product. Furthermore, 160 LAB
strains were isolated from the digestive tracts of marine fish and evaluated of potential use as
probiotics by Buntin et al [21]. Additionally, there are 51 strains of LAB isolated from the intestinal
tract of Kuruma shrimp (Marsupenaeus japonicus) and the selected strain for probiotic [22].
The total bacterial count on the samples is well known to be correlated with the source of the raw
materials. In the case of fermented fish, the main source of LAB is the gastrointestinal tract of the fish.
Ringø [23] reported a minor number of LAB from the Arctic charr, Salvelinus alpines, approximately
10 % from the gastrointestinal microflora of Arctic charr, Salvelinus alpines. In contrary, Buntin at al
[21] reported the high number of LAB from warm-water or tropical fish, approximately 72.5 % of the
total isolated LAB. In this study, the low number of LAB (52.7 %) may be due to the high
concentration of salt employed to produce the peda fermented fish. The addition of salt during peda
fermentation reduces the water activity of its food matrix. This condition inhibits the growth of Gram-
negative bacteria and promotes the growth of Gram-positive bacteria such as LAB. Therefore LAB
plays a central role in fish fermentation along with the addition of salt [24]. Salt is used to control the
growth of desirable bacteria such as lactic acid bacteria [25].

3.2 Antagonistic activity of isolated LAB

Among 57 isolates, 27 isolates exhibited inhibition activity (Table 1) and only one isolate exhibited
strong activity against S. aureus ATCC 6538, namely NJ-20 with the inhibition zone of 7.6 ± 1.35
mm. The inhibition of pathogenic bacteria by LAB is due to the acidification of the medium. Organic
acids are substances produced by LAB that inhibit Gram-negative bacteria such as Yersinia
enterocolitica, Salmonella sp., Escherichia coli, Pseudomonas sp. [16,10,6]. The inhibition of
pathogenic bacteria may be due to more than one mechanism, which includes suppression of growth,
production of organic acids (such as lactic acid), and active compounds (bacteriocins) [24,26].

3
ASEAN-FEN INTERNATIONAL FISHERIES SYMPOSIUM – 2017 IOP Publishing
IOP Conf. Series: Earth and Environmental Science 137 (2018)
1234567890 ‘’“” 012060 doi:10.1088/1755-1315/137/1/012060

Table 1. Antagonistic acticity of isolated lactic acid bacteria from Peda against S. aureus ATCC 6538
and P. aeruginosa ATCC 27853.

Inhibition Zone (mm)a

Isolates
S. aureus ATCC 6538 a P. aeruginosa ATCC 27853 a
NJ-15 2.9 ± 0.46a 0±0
NJ-17 2.2 ± 1.46 0±0
NJ-18 2.6 ± 0.75 0±0
NJ-20 7.6 ± 1.35 0±0
NJ-21 4.7 ± 1.38 1.1 ± 0
KD-1 2.3 ± 1.25 0±0
KD-3 2.3 ± 1.35 0±0
KD-6 1.3 ± 0.40 0.6 ± 0.55
GR-2 0±0 4.5 ± 0.51
GR-3 0±0 1.8 ± 0
GR-8 0±0 3.5 ± 0
GR-10 0.9 ± 0 0±0
GR-11 0±0 1.5 ± 0
GR-13 0±0 2.3 ± 0.2
GR-14 0.9 ± 0 0.7 ± 0.75
SD-18 0±0 0.9 ± 0
SD-19 0±0 2.2 ± 0
SD-20 0±0 0.8 ± 0
SD-21 0±0 1.1 ± 0
SD-22 0±0 1.2 ± 0
SD-24 0±0 2.7 ± 0
SD-25 0±0 0.7 ± 0
SD-28 3.1 ± 0.36 0±0
SB-1 1.7 ± 0 0±0
SB-4 2.8 ± 0.83 0±0
SB-19 0±0 1.7 ± 0
SB-20 0±0 3.4 ± 0.87
a
Mean ± SD

The LAB NJ-20 isolate was then selected for the detection of bacteriocin producing activity against
S. aureus ATCC 6538 due to its inhibitory zone of ≥ 6 mm [18]. Gram-negative is susceptible to
organic acids because the cytoplasmic membrane of Gram-negative bacteria is damaged by organic
acids [27]. In this study, the strong activity of LAB NJ-20 isolate against Gram-positive bacteria (S.
aureus ATCC 6538) indicates the potential for bacteriocin production.

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ASEAN-FEN INTERNATIONAL FISHERIES SYMPOSIUM – 2017 IOP Publishing
IOP Conf. Series: Earth and Environmental Science 137 (2018)
1234567890 ‘’“” 012060 doi:10.1088/1755-1315/137/1/012060

3.3 Bacteriocin-producing test of isolate LAB NJ-20

The bacteriocin-producing test employed an Agar Well Diffusion Assay (AWDA) of LAB NJ-20
isolate which was obtained from peda. However, the neutralized-cell free supernatant of the NJ-20 did
not exhibit a clear zone around the well against S. aureus ATCC 6538. This finding indicates that the
neutralized-cell free supernatant did not contain an antimicrobial substance such as bacteriocin.
Bacteriocin is an antimicrobial peptide or protein that is heat-stable, resistant to pH, sensitive to
protease and inactivated by the digestive enzyme [12].
The incubation temperature and pH play an important role in the production of bacteriocin, because
an optimum temperature and pH are required for bacterial growth so as to produce bacteriocin. The
optimal range of temperature and pH to produce bacteriocins depend on the strains of the producer
bacteria [4,28]. The optimal temperature and pH stimulate LAB to produce bacteriocin at the
beginning of the exponential phase. According to Ponce et al. [29] bacteria produces bacteriocin
exponentially in the initial phase and is inactivated at the stationary phase. The phase of lactic acid
bacteria in producing bacteriocin varies depending on the strains of the producer bacteria [29]. The
long incubation time resulted in the bacteriocin being degraded by a bacterial secreted enzyme [30].
However, LAB is known to produce a wide range of antimicrobial compounds (organic acids,
bacteriocin, diacetyl and hydrogen peroxide) [10,24,26] that can still be applied as biopreservation.
LAB had been granted Generally Recognized As Safe (GRAS) status by the US Food and Drug
Administration [24]. Meat-borne LAB generally inhibits other LAB species as well as Gram-positive
bacteria such as Staphylococcus aureus, Listeria monocytogenes, Clostridium perfringens, Clostridium
botulinum, Enterococcus faecalis, and Bacillus spp. due to the formation of pores in the cytoplasmic
membrane of sensitive strains [31].

Table 2. Identfication of NJ-20 isolate.

Isolate LAB
Identification Aerococcus
NJ-20
Shape Cocci Cocci
Shape tetrad + +
Gram-positive + +
Catalase - -
CO2 from glucose - -
Growth at 10 oC + +
Growth at 45 oC - -
Growth in NaCl 6.5 % + +
Growth in NaCl 18 % - -
Growth at pH 4.4 - -
Growth at pH 9.6 + +

3.4 Identification of LAB NJ-20

The genus identification based on morphology and physiology tests indicate that NJ-20 belong to the
Aerococcus genus. The results of identification of NJ 20 isolates are shown in table 2. These results
were different with the previous study on the identification of LAB from peda which found Lactic acid
bacteria isolated from peda consist of Lactobacillus plantarum, L. curvatus, L. murinus, Streptococcus
thermophilus. The difference may be due to the different sampling locations as well as the raw
materials used. Aerococcus is included in the Lactobacillales order which is closely related to the
Streptococcus, Lactococcus, Carnobacterium, Vagococcus and Enterococcus species. The Aerococcus
species lives in a wide range of environments including air, vegetation, meat-curing brines, soil, and
marine sources [32].

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ASEAN-FEN INTERNATIONAL FISHERIES SYMPOSIUM – 2017 IOP Publishing
IOP Conf. Series: Earth and Environmental Science 137 (2018)
1234567890 ‘’“” 012060 doi:10.1088/1755-1315/137/1/012060

4. Conclusion
The antagonistic activity of lactic acid bacteria isolated from peda, a traditional fermented fish from
Indonesia, suggested that 27 isolates exhibited an antagonistic effect against S. aureus ATCC 6538
and/ or P. aeruginosa ATCC 27853. Strong inhibition to S. aureus ATCC 6538 was performed by the
LAB NJ-20 isolate. The inhibitory activity of Aerococcus NJ-20 was not due to the production of
bacteriocin. This finding suggests that Aerococcus NJ-20 may be applied as a biopreservation
ingredient for fishery products as well as a culture starter of peda fermentation. Nevertheless, further
studies are needed to evaluate the enzyme activity, safety, growth of isolates on the fermentation
process and biogenic amine production prior to applications.

5. References
[1] Tanasupawat S and Visesssanguan W 2014 Seafood processing: technology, quality and
safety 1st edn.(United Kingdom: John Willey and Sons) pp 177-207
[2] Huda, N 2012 Handbook of animal-based fermented food and beverage technology
Indonesian fermented fish product 2nd, ed Y H Hui (New York: CRC Press) p 717-737
[3] Albarracin W, Sanchez IC, Grau R, and Barat J M 2011 Int. J. Food Sci and Technol 46
1329–36
[4] Akkoc N, Ghamat A, and Akcelik M 2011 Int J. of Dairy Tech 64 3
[5] O’Bryan C A, Crandall P G, Ricke S C, and Ndahetuye J B 2015 Handbook of natural
antimicrobials for food safety and quality: Lactic acid bacteria (LAB) as antimicrobials in
food products: types and mechanisms of action ed T M Taylor (United Kingdom: Woodhead
Publishing,) pp 117-129
[6] Hwanhlem N, Buradaleng S, Wattanachant S, Benjakul S, Tani A, and Maneerat S 2011 Food
Control 22 401-7
[7] Rahayu ES 2003 Agritech 23 75-84
[8] Sukmarini L, Mustopa A Z, Normawati M, and Muzdalifah I 2014 J. Biosci 21 144-150
[9] Udomsil N, Rodtong S, Tanasupawat S, and Yongsawatdigul J 2010 Int J. Food Microbio 141
86-194.
[10] Cizeikiene D, Juodeikiene G, Paskevicius A, and Bartkiene E 2013 Food Control 31 539-5
[11] Sonomoto K, Nagao J I, and Nishie N (2012) Biocontrol Sci. 17(1) : 1-16
[12] Hwanhlem N, Chobert J M, and H-Kittikun A H 2014 Food Control 41 202-211
[13] H-Kittikun AH, Biscola V, El-Ghaish S, Jaffres E, Dousset X, Pillot G, Haertle T, Chobert
JM, Hwanhlem N 2015 Food Control 54 126-134
[14] Desniar, Rusmana I, Suwanto A, and Mubarik NR 2012 J. Akuatika 3 135-145
[15] Cho GS and Do HK 2006 Ocean Sci J. 41 9-113
[16] Angmo K, Kumari A, Monika, Savitri, Bhalla TC 2016 Food Biosci. 13 26-31
[17] Pramono H, Suciati P, and Andriyono S 2015 Mar. Sci. 20 33-37 (In Indonesia)
[18] Heredia-Castro PY, Romero JIM, Mendoza AH, Félix EA, Córdova AFG, and Cordoba BV
2015 J. Dairy Sci 98 1-9
[19] Nuryadi M M, Istiqomah T, Faizah R, Ubaidillah S, Mahmudi Z, and Sutoyo 2013 J. UNEJ 1
1-11 (In Indonesia)
[20] Pringsulaka O, Thongngam N, Suwannasai N, Atthakor W, Pothivejkul K, Rangsiruji A 2012
Food Control 23 547-551
[21] Buntin N, Chanthachum S, and Hongpattarakere T 2008 J. Sci Technol 30 141-8
[22] Maeda M, Shibata A, Biswas G, Korenaga H, Kono T, Itami T, and Sakai M 2014 Mar.
Biotechnol. 16 181–192
[23] Ringø E 1993 Aquac. Fish. Man. 24 133-5
[24] Françoise L 2011 Food Microb. 27 698-709
[25] Yang E, Fan L, Jiang Y, Doucette C, and Fillmore S 2012 EMB express 2 48
[26] Sofyan A, Aswari AN, Purwoko T, and Damayanti T 2013 Vet. Med. 36 216-223 (In
Indonesia)

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[27] Schirru S, Favaro L, Mangia NP, Basaglia M, Casella S, Comunian R, Fancello F, Franco
BDGdeM, Oliveira RPdeS, Todorov SD 2013 Microbiol.
[28] Mandal V, Sen SK, and Mandal NC 2008 J. Biochem & Biophy 45 106-110
[29] Ponce AG, Moreira MR, del Valle CE, and Roura SI 2008 LWT 41 432-441
[30] Pal A, Ramana KV, and Bawa AS 2010 J. Food Sci Technol 47 258-265
[31] Ammor MS and Nayo B 2007 Meat Sci. 76 138–146
[32] Zhang H And Cai Y 2014 Lactic acid bacteria : fundamental and practice (London: Springer)