Fungal Contamination and Toxigenic Aspergillus flavus Strains on
Dried Anchovies Sold in North Sumatra
Jesica Simanullang and Kiki Nurtjahja
Department of Biology, Faculty of Science and Technology, Universitas Medan Area, Medan, Indonesia
Keywords: Aspergillus flavus, Fungal population, Anchovies, Toxigenicity
Abstract: Dried anchovies at Medan traditional markets commonly sold unpacked and stored in open air, as the result,
the dried-salted fish was easily spoiled and contaminated by mycotoxigenic fungi. The aims of the study
were to enumerate fungal population and to characterize A. flavus strains. Each fungal species were isolated
from medan anchovies at five traditional markets. The percentage of fungal contamination was determined
by a direct plating using dichloran 18% glycerol agar medium. Fungal population was determined using a
serial dilutions and pour plate in DG18 medium. Toxigenic Aspergillus flavus strains were characterized by
the production of yellow pigment on the reverse side of petri dishes containing coconut agar medium 10%.
The presence of regulatory (aflR) and structural genes (nor-1, ver-1 and omt-1) genes determined aflatoxin
biosynthesis was determined by amplifying four sets of specific primers (aflR, nor-1, ver-1 and omt-1).
Results showed that medan anchovies were contaminated by storage fungi. Aspergillus niger was the most
common found followed by A. flavus and A. chevalieri. Eighty strains of A. flavus were isolated from all
samples, they are consisted 69 toxigenic and 11 strains were non-toxigenic.
1 INTRODUCTION
Anchovies is dried-salted fish that one of fish
products that contain high protein content. Medan
anchovies commonly produced traditionally by local
fisherman. During processing the fish product was
preserved conventionally by adding some salt and
dried under the sunlight. Moisture content anchovies
between 32 and 34% with salt content between 13
and 17% (Yusra 2017). During storage and high
relative humidity the dried-stored anchovies might
increase their water activity (a
w
) and contaminated
by storage fungi.
About 14 of 150 dried-salted fish samples stored
at a
w
0.73 to 0.80 were contaminated by Aspergillus
flavus (Indriati et al. 2017). In addition, Aspergillus
flavus was the most predominant found on dried
salted fish (Youssef et al. 2003) Storage fungi such
as Aspergillus flavus, A. candidus, A. niger, A.
wentii, A. chevalieri and Penicillium islandicum
commonly xerophilic and was able to grow at low a
w
(Pitt and Hocking 2009).
Fungal contamination occured n dried fish at
moisture content 12.3 to 32% and genus Aspergillus
particularly A. niger, A. fumigatus, A. ochraceus
and A. flavus were the most common found (Sam et
al. (2015). Fungal contamination on food and
feedstuffs may cause change taste, colour, reduce
nutrition (Hassan et al. 2011). The purpose of recent
study was to enumerate fungal contamination and to
determine toxigenic A. flavus strains on anchovies
sold by retailers at traditional markets in North
Sumatera, Indonesia.
2 MATERIALS AND METHOD
2.1 Anchovies Sampling
Anchovies as sample were bought from five
traditional markets and as mush as 100 g anchovies
obtained from five retailers at each market. All
samples were packed in sterile polyethylene bag and
stored in in cold until used.
2.2 Determination of the Percentage of
Anchovies Contaminated by Fungi
The percentage of anchovies contaminated by each
fungal species was detemined by direct plating on
dichloran 18% glycerol agar (DG18) medium in
548
Simanullang, J. and Nurtjahja, K.
Fungal Contamination and Toxigenic Aspergillus flavus Strains on Dried Anchovies Sold in North Sumatera.
DOI: 10.5220/0010613000002775
In Proceedings of the 1st International MIPAnet Conference on Science and Mathematics (IMC-SciMath 2019), pages 548-551
ISBN: 978-989-758-556-2
Copyright
c
2022 by SCITEPRESS – Science and Technology Publications, Lda. All rights reserved
petri dish (9 cm diameter). Five replicates were
madefor each sample.
2.3 Enumeration of Fungal Population
Each fungal population was determined by serial
dilution method followed by pour plate in Dichloran
18% Glycerol Agar ((DG18). One hundred gram of
each sample were ground using Mill Powder RT-04
no serial number 980923 (Mill Powder Tech. Co
LTD, Taiwan) with speed 25 000 rpm for 30 second
and 25 g of fine sample in 500 ml flask was dilute
by sterilized distilled water until the volume up to
250 ml. The suspension then was homogenazed and
1 ml of the aliquot in petri dish (9 cm in diameter)
was pour plate in DG18 medium, three replicates
was made. All plates then were incubated at 28°C
for 5 d. Each fungal population were enumerated,
isolatesd in Potato Dextrosa Agar (PDA) and
identified according to Pitt and Hocking (2009) by
subculturing in Malt Extract Agar (MEA) and
Czapex Yeast Agar (CYA).
2.4 Determination on the Toxigenicity
of Aspergillus flavus by Culture
Technique
The toxigenicity of each A. flavus strains to
produce aflatoxin was determine using coconut agar
medium (CAM) according to Lin and Dianese
(1976). Each strain of A. flavus was culture in CAM
for 5 day at 28°C. The presence of yellow pigment at
the reverse side of petri dish indicate toxigenicity.
2.5 Extraction of A. flavus Genome
Genome of each Aspergillus flavus strain was
obtained from two 2 days old mycelial culture on
PDA (28°C). As much as 40 g mycelia in 1.5 ml
PCR tube containing 600 µl nuclei lysis solution was
homogenated using micropestle. Genome extraction
kit used was Mini Kit (Promega, Madison, WI,
USA). The homogenate then was incubated at 65°C
for 15 minute and cooled 5 minute until the
temperature 29°C, then, 200 µl protein presipitation
was added, vortexed and centrifuged at 13000×g for
3 minute. The supernatan was mixed in 600 µl
isopropanol followed by centrifugation 13000×g for
2 minute, the supernatant was discarded and 600 µl
ethanol 70% was used to wash the pellet. The
mixture was then centrifuged 13000×g for 2 minute.
The pellet obtained then rehydrated by 50 µl DNA
rehydration solution, vortexed for 5 second and 0.5
µl RNAse was add. The DNA mixture was
incubated at 37°C for 15 minute. The DNA
concentration was determined using nanophotometer
(IMPLEN, Munich, Germany). Electrophoresis
DNA was conducted using SCIE-PLAS. Cambridge,
England with 1,2% gel agarose and gel was stained
with 1 µl ethidium bromide. The photograph used
was Gel Doc (Uvitec, Cambridge, Serial) under UV
light 303 nm.
2.6 PCR Amplification of
Aflatoxin-related Genes
Polymeration chain reaction used was GeneAmp
PCR Labcycler Gradient System (Sensoquest,
Germany, serial number 1123280105). Specific
primer for genes aflatoxin biosynthesis used were
nor-1, ver-1, omt-1 and aflR with basepair 400, 895,
1232 dan 1032 bp respectively. Amplification
preparation was conducted as follows: Amplification
mix for each primer consisted of 5 μl GoTaq Green
Master Mix (Promega), 3 μl nuclease free water, 0,5
μl for each primer F (forward) and R (reverse), then
1 μl DNA template and volume of end DNA Mix
reaction on PCR tube was 10 μl. Process of DNA
mix in PCR was preincubation step at 94
o
C for 10
min followed by 35 cycles of denaturation at 94
o
C
for 10 min, annealing at 65
o
C for 2 min and
extension at 72
o
C for 2 min and final extension at
72
o
C for 7 min. The PCR product was analyzed by
electrophoresis (SCIE-PLAS, Ltd, Cambridge,
England) using 1,5 % agarose gel in 1×TAE 40
mM Tris-acetate, 1mM EDTA (pH 8). Gels stained
with ethidium bromide 0.1 mg/l, visualized under
Gel Doc (Uvitec, Cambridge, serial number 13
200263) under UV light (303 nm).
3 RESULTS AND DISCUSSION
3.1
Percentage of Contaminated
Anchovies
Eleven species of storage fungi that contaminated
anchovies were isolated from 5 traditional market
(Table 1).
Fungal Contamination and Toxigenic Aspergillus flavus Strains on Dried Anchovies Sold in North Sumatera
549
Table 1: Percentage of anchovies contaminated by fungi at five traditional markets in Medan, North Sumatera
Fungal species Percenta
g
e of anchovies contaminated b
y
fun
g
i
Market 1 Market 2 Market 3 Market 4 Market 5
A
sper
g
illus candidus 6.70 0 0 0 0
A
. chevalieri 33.30 46.70 0 0 0
A
.
f
lavus 33.30 0 63.30 10.00 3.00
A
.
f
umi
g
atus 0 0 0 0 6.60
A
. ni
g
e
r
30.0 23.3 23.30 100,00 43.30
A
. or
y
zae 0 0 0 0 10.00
A
. tamarii 3.30 46.70 0 10,00 3.00
A
. wentii 3.30 0 0 0 0
Penicillium sp.
1
13.30 0 0 0 10.00
Rhizopus oli
g
osporus 33.30 13.30 3.30 0 10.00
M
y
celia sterilia 3.30 0 0 0 0
Table 2: Fungal population (cfu/g) on anchovies contaminated by fungi at five traditional markets on Medan, North
Sumatera
Fungal species Fun
g
al population (cfu/
g
)
Market 1 Market 2 Market 3 Market 4 Market 5
A
sper
g
illus candidus 1×100000
A
. chevalieri 8.3×10 1.33×10
-2
0 0 0.3×10
A
.
f
lavus 0.7×10 0 1.13×10
-2
1.7×10 0.3×10
A
.
f
umi
g
atus 0 0 4×10 0.3×10 0.6×10
A
. ni
g
e
r
0 0.3×10 3.3×10 3.3×10 0
Cladosporium sp. 0.3×10 0 0 0 0.3×10
Rhizopus oli
g
osporus 0.3×10 0.3×10 0 0 0
CFU = colony forming unit
Table 3: Toxigenicity representative isolates of A. flavus strains isolated from anchovies based on cultural in CAM
(coconut agar medium) medium and molecular based on the presence of regulatory (aflR) gene and structural (nor-1, omt-1,
ver-1) genes
Strain Regulatory
g
ene
Structural
g
enes
The presence of
yellow pigment in
CAM medium
Toxigenicity
a
f
lR no
r
-
I
om
t
-
I
ve
r
-1
Af1 + + - + ne
g
ative non-toxi
g
en
Af2 + + + +
p
ositive toxi
g
en
Af3 + + - + ne
g
ative non-toxi
g
en
Af4 + + + +
p
ositive toxi
g
en
Af5 + + + +
p
ositive toxi
g
en
Af6 + + + +
p
ositive toxi
g
en
Af7 + + - + ne
g
ative non-toxi
g
en
Af8 + + + +
p
ositive toxi
g
en
Af9 + + + +
p
ositive toxi
g
en
Among the fungal species, the highest
percentage anchovies were contaminated by A. niger
followed by A. flavus and A. chevalieri, A. tamarii,
Penicillium sp. and Rhizopus oligosporus. The
presence of A. niger was high and found at all
markets. Our result in line with Wheeler et al. (1986)
who reported that dried salted fish was common
infected by A. niger. Hassan (2011) also reported
that most salted fish was contaminated by A. niger
(73.3%) higher than A. flavus (66.6%), Penicillium
spp. (40%) and Rhizopus spp. (6.6%).
As shown in Table 1 all anchovies sold by
retailers at traditional markets were infected by
molds, the contamination might occur processing
and fungal population incresed during storage. As
reported by Azam (2002) conventional drying on
fish products susceptible contaminated by
microorganisms. In addition, high relative humidity
IMC-SciMath 2019 - The International MIPAnet Conference on Science and Mathematics (IMC-SciMath)
550
leads the dried fish absorb water vapor from air and
increase moisture content.
3.2 Fungal Population on Anchovies
Fungal population per gram anchovies (colony
forming unit or cfu/g) obtained from relailers at five
traditional markets are shown in Table 2). Genus of
Aspergillus was the most dominant (5 species)
followed by Cladosporium and Rhizopus
oligosporus.
Table 2 showed that Aspergillus chevalieri was
the highest population (8.3×10 cfu/g) followed by A.
niger (3.3×10 cfu/g), A. flavus (1.13×10
-2
cfu/g), and
A fumigatus (3.3×10 cfu/g). Among storage fungi A.
chevalieri is xerophilic and grow at a
w
0.71 to 0.74
(Pitt and Hocking 2009). Previous study by Sam
(2015) stated that 23 fungal species were isolated
from dried fish and were predominated by A. flavus
and A. niger.
3.3 Toxigenicity of A. flavus Strains
Eighty strains of A. flavus were successfully
isolated. Based on toxigenicity determination using
CAM medium, they are consisted of 69 strains are
toxigenic (aflatoxin producers) and 11 strains are
non-toxigen. Gene analysis determine aflatoxin
biosynthesis (Table 3) indicate that regulatory gene
(aflR) and structural genes (nor-1, omt-1 and ver-1)
are key enzyme that determine aflatoxin production
as previously reported by Erami et al. (2007) that the
presence of the for keys genes determine
toxigenicity of Aspergillus flavus.
Table 3 showed that the presence of regulatory
and structural genes on A. flavus determined
aflatoxin biosynthesis. The toxigenicity determined
byn culture technique method in CAM medium and
molecular using specific primers showed similar
results.
4 CONCLUSION
High fungal contamination on anchovies are
potential to spoile and contaminated aflatoxin. Good
handling practices was reqiured to reduce fungal
growth during storage.
ACKNOWLEDGEMENT
The research was funded by Universitas Sumatera
Utara, contract TALENTA Reseach grant no.
136/UN5.2.3.1/PPM/KP-TALENTA USU/2019.
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