Toxigenic and Non Toxigenic Aspergillus Flavus Strains Isolated

from Candlenut (Aleurites Moluccana) in North Sumatera

Ngalo Putri Ginting and Kiki Nurtjahja

Department of Biology, Universitas Medan Area, Medan, Indonesia

Keywords: Aspergillus flavus, Aflatoxin, Candlenut, Toxigenic

Abstract: This study was conducted to investigate contamination of Aspergillus flavus strains in distribution chains of

candlenut (Aleuritaes moluccana). Dried-stored unshelled candlenut were collected from farmers, collectors

and distributors at 3 Ragencies in North Sumatra (Regency of Karo, Deli Serdang and Langkat) during

harvest period (February to April 2019). One and half kilogram of candlenut was taken from each of the

distribution chains. Kernel moisture content were determined by distillation. Population each of A. flavus

strain was enumerated by a dilution method followed by a pour plate in dichloran 18% glycerol agar

(DG18) medium. Culture method in agar medium containing 10% coconut milk was used to determine

toxigenicity of A. flavus. Their toxigenicity also was determined molecularly using specific primers for

amplifying regulatory (aflR) and structural genes (nor-1, ver-1, omt-1) that determine aflatoxin biosynthesis.

Results showed that moisture content of candlenut kernels at the level of farmers, collectors and distributors

in Regency of Karo, Deli Serang and Langkat was above 5%. A total of 38 strains of A. flavus were isolated

and 37 of the strains were toxigenic (aflatoxin producers). The average of A. flavus population (cfu/g) on

candlenut at farmers was the lowest and the highest population was at distributor chain.

1 INTRODUCTION

Candlenut (Aleurites moluccana) is one of the

important commodity in North Sumatera. The main

area of candlenut producers in North Sumatera are at

Deli serdang, Langkat, Binjai, Karo, Tapanuli, Dairi,

Nias, and Asahan (Sihombing, 2015). Most of the

candlenut are produced by farmers. Preharvest and

postharvest handling of the nut such as harvesting,

drying, cracking and storing were conducted

conventionally.

Drying using sun light in open air and high

relative humidity during inappropriate storage,

therefore, dried-stored candlenut is susceptible

infected by fungi and contaminated by mycotoxins.

In addition, high lipid content in the nut commonly

contaminated by fungi (Krishnawati et al. 2011).

Among storage fungi that common contaminate

unshelled candlenut are Aspergillus flavus, A.

niger, A. wentii, A. tamarii, Aspergillus rubrum, A.

chevallieri and Penicillium citrinum (Pitt et al. 1998

; Lambaga, 2005).

Aspergillus flavus is one of toxigenic fungi that

commonly contaminated on corn, peanut, spices,

nuts, cotton seeds, pistachio, etc. However, not all

strains of A. flavus are toxigenic (aflatoxin producer)

(Ehrlich (2014). Toxigenicity of A. flavus is

determined by strains, substrates, geography and

culture technique (Perrone et al. 2014). The purpose

of the recent study was to enumerate A. flavus

strains and their toxigenicity that contaminated on

shelled candlenut at farmers, collectors and

distributors chains in North Sumatera.

2 MATERIALS AND METHOD

2.1 Sample Collection

As much as 1500 g samples of unshelled candlenut

were obtained at farmers, collectors and distributors

chain at 3 Regencies in North Sumatera i.e Karo,

Deli Serang and Langkat. Tree replicates were

conducted for each sample Sample then was placed

into a sterile polyethylene bag and stored in a

refrigerator at ±12°C for further use.

Ginting, N. and Nurtjahja, K.

Toxigenic and Non-toxigenic Aspergillus Flavus Strains Isolated from Candlenut (Aleurites Moluccana) in North Sumatera.

DOI: 10.5220/0010613200002775

In Proceedings of the 1st International MIPAnet Conference on Science and Mathematics (IMC-SciMath 2019), pages 555-557

ISBN: 978-989-758-556-2

555

2.2 Determination of A. flavus

Population

Aspergillus flavus population on each sample was

enumerated by a dilution method and followed by

pour plate method in dichloran 18% glycerol agar

(DG18) medium. Each sample was ground and 25 g

were ground nut was placed onto a 500 ml flask and

suspended with 250 ml of sterile distilled water and

then homogenized to obtain a 10

-1

suspension. The

dilution was carried out on 10

-2

, 10

-3

and 10

-4

. One

ml of the suspension was placed on DG18 medium

in petri dish (9 cm in diameter).. Each dilution was

replicate 3 times. All plates were incubated for 5

days (29

C). Population of A. flavus per gram of

candle nut (cfu/g) was determined using the formula:

A. flavus population =

X.Y

. Z (cfu/g)

X = volume of suspension transferred to each petri

dish (1 ml)

Y = dilution which gives the A. flavus colonies

separately

Z = average number of colonies of A. flavus from 3

petri dishes

2.3 Morphological Identification of

Aspergillus flavus

All of A. flavus colonies was isolated on potato

dextrose agar (PDA) medium then identified

according to Pitt and Hocking (2009).

2.4 Determination on the Toxigenicity

of A. flavus Strains

The potential aflatoxin production of each A. flavus

strains was determined by culture method in 10%

coconut agar medium (CAM) in petri dish (9 cm in

diameter) according to the procedure of Lin and

Dianese (1978). The aflatoxin producers were

indicated by the presence of yellow pigment at the

reverse side of the medium.

2.5 Extraction of Genomic DNA

About 40 mg A. flavus mycelia, harvested from 4

days old in a microtube containing 600 µl nuclei

lytic was homogenised and extracted following

procedure of Mini Kit (Promega, Madison, WI,

USA). The DNA concentration obtained was

determined using nanophotometer (IMPLEN,

Munich, Germany) and followed by electrophoresis

using 1.2% agarosa gel (SCIE-PLAS, Cambridge,

England), stained by 1µl ethidium bromide (EtBr)

and visualised by Gel Doc (Uvitecc, Cambridge,

Serial) under UV light (303 nm).

2.6 PCR Amplification

Specific primers used to amplify genes determine

aflatoxin biosynthesis were regulatory gene (aflR)

and structural genes (omt-1, nor-1, ver-1,) with the

length of the base 1032, 895, 400, 1232 bp

respectively. Amplification reaction was conducted

according Erami et al. (2007). PCR was performed

as follows: as much as 12,5 µL amplification mix

[dNTP, Taq DNA polymerase (Promega

Corporation Madison WI), MgCl2, Sybr Green 1

dye], 1 µM on each primer and 75 ng DNA template

were added until the volume up to 25 µL. PCR was

conducted 35 cycle consisted of preincubation for 10

min (94 ℃), denaturation 1 min (94 ℃), annealing

for 2 min (65 ℃), extension for 2 min (72 ℃), final

extension for 7 min (72 ℃). All amplification

process was 3 h 20 min. The PCR products were

continued by electrophoresis (SCIE-PLAS. Ltd,

Cambridge, England) using 1.5% agarose gels in 1 ×

TAE [40 mM Tris-acetate, 1 mM EDTA (pH 8)].

Gels were stained with 0.5 µg µl

-1

ethidium bromide

and visualized using Gel Doc (Uvitec, Cambridge,

Serial no. 13200263) under UV light (260 nm) and

1 kb DNA ladder (Promega, Madison, WI) was used

as standard.

3 RESULTS AND DISCUSSION

3.1

Moisture Content of Candlenut

The moisture content of all candlenut obtained at

farmers, collectors and distributors chain in Regency

of Karo, Deli Serang and Langkat found that the

highest moisture occured farmers (Table 1).

According to Indonesia National Standard (SNI 01-

1684-1998) the maximum moisture content of

candlenut up to 5%. Most candlenut in all

distribution chains above standard, it means that the

commodity are potential contaminated by fungi.

Most of the storage fungi are xerophilic and grow at

low water activity (Pitt and Hocking 2009).

Therefore, unproper postharvest handling might

increase A. flavus population.

IMC-SciMath 2019 - The International MIPAnet Conference on Science and Mathematics (IMC-SciMath)

556

Table 1: Candlenut moisture content of candlenut obtained

at farmers, collectors and distributors at Regency of Karo,

Deli Serdang and Langkat

Regencies candlenut moisture content

(

)

Farmers Collectors Distributors

Karo 5.49 5.19 5.02

Deli Serdang 5.33 4.69 5.09

Lan

kat 5.19 5.09 5.02

3.2 Population of A. flavus

All samples of candlenut were contaminated by A.

flavus (Table 2). In general, the lowest

contamination occur at farmers and it become

increase at collectors and at distributors the

ontamination were highest.

Table 2: Population of A.flavus (cfu/g) isolated from

candlenut on distribution chains at farmers, collectors and

distributors at Regency of Karo, Deli Serdang and Langkat

flavus

code

Primers CAM

aflR omt-1 nor-1 ver-1

+ + + + +

+ + + - -

D + + + + +

L + + + + +

+ = amplicon were amplified

- = no amplification

+/- = the presence of yellow pigment in coconut agar

medium (CAM)

The presence of regulatory and structural genes

in each of A. flavus determine aflatoxigenicity. The

results was similar to the study of Criseo et al.

(2001) Erami et al. (2007) and Nurtjahja et al.

(2019). It was found that more toxigenic A. flavus

strains than that of non toxigenic on candlenut.

4 CONCLUSION

The presence of toxigenic Aspergillus flavus at

candlenut on distribution chains were potential to

spoil and contaminated by aflatoxin. Good handling

practices on candlenut were required to prevent the

fungal growth and produce aflatoxins during storage.

ACKNOWLEDGEMENT

The research was funded by Universitas Sumatera

Utara, contract DRPM Reseach grant no.

152/UN5.2.3.1/PPM/KP-DRPM/2019.

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