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

) and contaminated

by storage fungi.

About 14 of 150 dried-salted fish samples stored

at a

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

(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

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

C for 10

min followed by 35 cycles of denaturation at 94

for 10 min, annealing at 65

C for 2 min and

extension at 72

C for 2 min and final extension at

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

e of anchovies contaminated b

fun

Market 1 Market 2 Market 3 Market 4 Market 5

sper

illus candidus 6.70 0 0 0 0

. chevalieri 33.30 46.70 0 0 0

lavus 33.30 0 63.30 10.00 3.00

umi

atus 0 0 0 0 6.60

. ni

30.0 23.3 23.30 100,00 43.30

. or

zae 0 0 0 0 10.00

. tamarii 3.30 46.70 0 10,00 3.00

. wentii 3.30 0 0 0 0

Penicillium sp.

13.30 0 0 0 10.00

Rhizopus oli

osporus 33.30 13.30 3.30 0 10.00

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

al population (cfu/

)

Market 1 Market 2 Market 3 Market 4 Market 5

sper

illus candidus 1×100000

. chevalieri 8.3×10 1.33×10

-2

0 0 0.3×10

lavus 0.7×10 0 1.13×10

-2

1.7×10 0.3×10

umi

atus 0 0 4×10 0.3×10 0.6×10

. ni

0 0.3×10 3.3×10 3.3×10 0

Cladosporium sp. 0.3×10 0 0 0 0.3×10

Rhizopus oli

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

ene

Structural

enes

The presence of

yellow pigment in

CAM medium

Toxigenicity

lR no

-1

Af1 + + - + ne

ative non-toxi

Af2 + + + +

ositive toxi

Af3 + + - + ne

ative non-toxi

Af4 + + + +

ositive toxi

Af5 + + + +

ositive toxi

Af6 + + + +

ositive toxi

Af7 + + - + ne

ative non-toxi

Af8 + + + +

ositive toxi

Af9 + + + +

ositive toxi

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

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.

REFERENCES

Azam, K., 2002. Fisherman community of Kuakata

Bangladesh: Fisherman Activities and Quality of Fish.

Sufer Project (DFID- UGC), Khulna University.

Erami, M., Hashemi S.J., Pourbakhsh S.A., Shahsavandi

S., Mohammadi S., Shooshtari S., Jahanshiri Z. 2007.

Application of PCR on detection of aflatoxigenic fungi

[short communication]. Arch Razi Institute 62(2):95-

100.

Hassan, A.A., Hassan M.A., Howayda M., Shafei E.I.,

Rasha M.H., El-Ahi H.S Abd Ei-Dayem R.H., 2011.

Detection of aflotoxigenic moulds isolated from fish

and their products and its public health significance.

Nature and Science, 9(9): 106-114.

Indriati, N., Hermana I., Hidayah I., Rahayu E. 2017.

Prevalence of aflatoxin B1 in commercial dried fish

from some regions of Java. Squallen Bull. of Mar.And

Fish. Postharvest and Biotechnology 12:107-115.

Lin, M.T., Dianese J.C. 1976. A coconut-agar medium for

rapid detection of aflatoxin production by Aspergillus

spp. Phytopathology. 66:1466-1469.

Pitt, J.I., Hocking A.D. 2009. Fungi and Food Spoilage.

New York (US): Springer.

Sam, J.F., Jeyasanta K.I., Edward J.K.P. 2015 . Aflatoxin

investigation on dried fishes of Tuticorin, South East

Cost of India. Journal of Foodborne and Zoonotic

Diseases. 3:49-62.

Yusra, 2017. Analisis kandungan formalin ikan asin

kering di Gasan Gadang, Kabupaten Padang

Pariaman Sumatera Barat. Jurnal Katalisator, 2(1):20-

28.

Youssef, M.S., Abo-Dhab NF, Farghaly R.M. 2003.

Studies of mycological status of salted fish Moloha

in Upper Egypt. Mycobiology, 31:166-172.

Yuliar, Abidin Z., Mangunwardoyo W. 2011. Potency of

biocontrol agents isolated from compost and peat soil

of tropical peat swamp forest in Kalampangan zone,

Central Kalimantan. Journal of Forestry Research

8(2): 144-157.

Warren, W., Hergoualch, Kauffman J.B Murdiyarso D.,

Kolka R. 2017. Ann appraissal of Indonesia’s

immense peat carbon stockusing national peatland

maps: uncertainties and potential lossesfrom

conversion. Carbon Balance and Management 12 (12):

1-12.

Wheeler, K.A., Hocking A.D., Pitt J.I., Anggawati A.M.

1986. Fungi Associated with Indonesian Dried Fish.

Food Microbiology, 3:351-357.

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