Screening of Toxigenic Aspergillus flavus Strains and

Aflatoxin Content from Agricultural Commodities in Indonesia

Anidah

1,2

, Winiati P. Rahayu

1,3

and Siti Nurjanah

1,3

Department of Food Science and Technology, IPB University, Darmaga Campus Bogor 16680, Indonesia

SEAMEO BIOTROP, Jl Raya Tajur Km. 6 Bogor 16134, Indonesia

SEAFAST Center, IPB University, Darmaga Campus, Bogor 16680, Indonesia

Keywords: Aspergillus flavus, Aflatoxin, Toxigenic A. flavus, HPLC.

Abstract: Infection of toxigenic A. flavus in agricultural commodities may result in production of aflatoxin, a mycotoxin

which is genotoxic carcinogenic for humans and animals. The aims of this study were to screen toxigenic A.

flavus strains and to determine aflatoxin content of six agricultural commodities in Indonesia. A total of 50 A.

flavus strains were obtained from Phytopathology Laboratory, SEAMEO BIOTROP. The strains were isolated

from nutmeg, corn, cacao, white pepper, coffee bean, ground peanut and peanut-cropped soil. The toxigenicity

of A. flavus were determined bfy growth simulation on aflatoxin-inducing medium (10% coconut agar

medium) followed by observation of their fluorescence using 365 nm UV light. AFB and AFG toxin produced

were quantified using HPLC. The results showed that 18% (9 strains) A. flavus were toxigenic, which derived

from nutmeg (5 strains), ground peanut (2 strains), cacao (1 strain), and peanut-cropped soil (1 strain). Six

toxigenic strains produced AFB1 exceeding the Indonesian-regulatory maximum level (15 ug/kg). A. flavus

from peanut-cropped soil (BIO 3352) produced the highest AFB1 content (90.94 ug/kg), while the other from

nutmeg (BIO 3345 and BIO 33212), ground peanut (BIO 3313 and BIO 3338), and cacao (BIO 33404) had

AFB1 content of 89.53, 84.24, 70.26, 40.27, and 69.06 ug/kg respectively. The producing aflatoxin capability

of these strains can be potentially hazard if contaminated in agricultural commodities.

1 INTRODUCTION

Aflatoxins are secondary metabolites that produced

by Aspergillus section Flavi, particulary A. flavus and

A. paraciticus (Ellis et al, 1991). Natural occurrence

of aflatoxin in agricultural product lead to severe

health problems for human and livestock. Aflatoxins

confirmed as a Group-1 agent which is carcinogenic

to humans (IARC, 2012). Exposure to higher levels

of aflatoxin increases cancer incidence, including risk

of hepato-cellular carcinoma and neural tube defect

(Sun et al, 2011 and Woo et al, 2011). The Food and

Agricultural Organization (FAO) has been estimated

that approximately 25% of crops worldwide get

contaminated by mycotoxin producing fungi

including A. flavus, that contributing to global losses

of 1000 million metric tons foodstuffs each year

(Bhat et al, 2010). The contamination by

mycotoxigenic fungi can occur during harvest,

postharvest, storage and transportation and causes

significant economic losses yearly (Hedayati et al,

2007 and Nurtjahja et al, 2017).

Aflatoxins have a high occurrence in tropical and

subtropical regions due to optimal humidity and

temperature conditions for toxin production (Bhat et

al, 2010). Contamination of aflatoxin in agricultural

commodities was reported in many countries. Mandel

(2005) had reported that A. flavus was the dominant

fungi in contaminated nutmeg imported from India,

Sri Lanka, Indonesia and Brazil. Aflatoxins and

fumonisins were reportedly widespread in major

dietary and export targeted crops such as maize and

peanuts in Southern Africa (Hove et al, 2016;

Mwalwayo et al, 2016). According to Davari et al.,

(2015), out of 28 strains of A. flavus and A.paraciticus

isolated from 110 feed samples in northeastern Iran,

10 strains were toxigenic.

Indonesia’s agricultural commodities including

maize, peanut, pepper, nutmeg, and cacao have been

reported contaminated by aflatoxin (Nurtjahja et al,

2017; Dharmaputra, 2002; Dharmaputra et al, 2013).

About 54% of isolated fungi from stored nutmeg in

North Sulawesi, was identified as A. flavus with highest

Anidah, ., Rahayu, W. and Nurjanah, S.

Screening of Toxigenic Aspergillus ﬂavus Strains and Aﬂatoxin Content from Agricultural Commodities in Indonesia.

DOI: 10.5220/0009981200002964

In Proceedings of the 16th ASEAN Food Conference (16th AFC 2019) - Outlook and Opportunities of Food Technology and Culinary for Tourism Industry, pages 221-226

ISBN: 978-989-758-467-1

221

level of AFB1 and total aflatoxin content of 1.63 ppb and

1.83 ppb respectively (Dharmaputra et al, 2015).

According to Indonesian Food Security Agency

(Badan Ketahanan Pangan/BKP) from unpublish data

noted there was still rejection for Indonesia’s nutmeg

commodities until 2019 due to aflatoxin

contamination (Figure 1).

Figure 1: Notification and rejection of Indonesia’s nutmeg

export commodity (BKP, unpublish data).

The data from annual reports of Rapid Alert

System for Food and Feed (RASFF) in the last decade

showed mycotoxin alert notification in food which

aflatoxin were predominantly notified each year

(Table 1). Nuts and nut products were addressed as

notified contaminated food product every year.

Table 1: RSAFF notification on mycotoxin and aflatoxin in

food from 2010 – 2017.

Year

Notification

Mycotoxin Aflatoxin

2010 688 649 (94.3%)

2011 635 585 (92.1%)

2012 528 484 (91.7%)

2013 410 341 (83.2%)

2014 359 314 (87.5%)

2015 475 421 (88.6%)

2016 489 360 (73.6%)

2017 529 416 (78.6%)

There were many researches have been conducted

biological control strategy recently involving

toxigenic and atoxigenic A. flavus to reduce aflatoxin

contamination (Doner et al, 2003; Yin et al, 2009).

The information about characterization toxigenic as

well as atoxigenic were required as prelude for

choosing suitable strains for biological control. There

is minimum information about diversity of A. flavus

isolated from agricultural product in Indonesia. The

aims of this study were to screen toxigenic A. flavus

strains and to determine aflatoxin content from six

agricultural commodities in Indonesia. The

information about molecular characterization can be

used as further information for controlling aflatoxin

contamination using screened A. flavus.

2 MATERIAL AND METHODS

2.1 Aspergillus flavus Strain

The total of 50 strains of A. flavus were selected

randomly and were kindly provided by Fitophatology

Laboratory of SEAMEO BIOTROP, Indonesia

(Table 2). The strains were isolated from nutmeg,

corn, cacao, white pepper, coffee bean, ground peanut

and peanut-cropped soil from various regions in

Indonesia. BIO 747 strain was used as positive

control that can produced both AFB and AFG from

previous study (Nagur et al, 2014). Fungal cultures

were routinely subcultured on potato dextrose agar

(PDA: 39 g l-1, Difco Laboratories, Sparks, USA)

every two years.

2.2 Screening of Toxigenic A. flavus

Strains

For screening toxigenicity of A. flavus, all strains

were cultured on aflatoxin-inducing medium, 10%

(v/v) coconut agar medium (CAM, 100 mL fresh

shredded coconut endosperm, 900 mL distilled water,

15 g bacto agar, pH 7.0). A small amount of A. flavus

mycelium transferred into the centre of CAM and

incubated at 27

C for 5 days in the dark condition.

Observation of presence or absence of blue

fluorescence in the agar surrounding the A. flavus

colonies was determinated by exposing the petri dish

to long-wave (365 nm) UV light and expressed as

positive or negative toxigenicity. An uninoculated

plate was used as reference (Nurtjahja et al, 2017;

Davis et al, 1987). All the positive toxigenic strain

was further confirmed for aflatoxin quantification by

HPLC, along with positive control (BIO 747) and one

atoxigenic strain from screening as reference.

2.3 Aflatoxin Extraction and

Quantification by HPLC

Aflatoxin production simulated on 10% (v/v) coconut

broth (CB, 100 mL fresh shredded coconut

endosperm, 900 mL distilled water, pH 7.0) medium.

As much as 2 inoculum (ϕ 5mm) of each strains were

inoculated on 50 mL 10% (v/v) CB medium with

continuous shaking at 100 rpm (27

C, 10 days) in the

dark condition.

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222

Table 2: Expression of fluorescence and contents of aflatoxin from 50 A. flavus strains.

(na) not applicable; (+) fluorescence observed; (-) no fluorescence observed

(<) below the LoQ, for AFB1 = 1.42 ug/kg, AFB2 = 6.72 ug/kg, AFG1 = 5.09 ug/kg, and AFG2 = 0.66 ug/kg.

Commodities

(origin)

A. flavus

Strains

Fluorescence

in CAM

Aflatoxin (ug/kg)

AFB1 AFB2 AFG1 AFG2 AF-total

Nutmeg

(Manado – North Sulawesi)

BIO 3316 - na na na na na

BIO 3345 + 89.53 < < < 89.53

BIO 33184 - na na na na na

BIO 33212 + 84.24 < < < 84.24

BIO 33402 - na na na na na

BIO 33403 + 4.48 3.02 2.82 0.82 11.14

BIO 33211 + 5.03 < < < 5.03

BIO 3376 + 6.47 < 26.71 < 33.18

BIO 33185 - na na na na na

BIO 35102 - na na na na na

Coffee bean

(Jember – East Java)

BIO 3314 - na na na na na

Coffee bean

(Toraja – South Sulawesi)

BIO 3384 - na na na na na

BIO 3393 - na na na na na

BIO 3394 - na na na na na

BIO 3396 - na na na na na

Corn

(Bogor – West Java)

BIO 3382 - na na na na na

BIO 3311 - na na na na na

BIO 35111 - na na na na na

Cacao

(Makasar – South Sulawesi)

BIO 3312 - na na na na na

BIO 33404 + 69.06 < < < 69.06

BIO 33405 - na na na na na

White pepper

(Bogor, West Java)

BIO 3383 - na na na na na

BIO 3316 - na na na na na

BIO 25119 - na na na na na

Ground peanut

(Bogor – West Java)

BIO 3313 +

70.26

6.59 < 1.01 77.86

BIO 3381 - < < < < <

BIO 3346 - na na na na na

BIO 3348 - na na na na na

Ground peanut

(Wonogiri – Central Java)

BIO 3342 - na na na na na

BIO 3324 - na na na na na

BIO 3334 - na na na na na

BIO 3338 + 40.27 < 97.28 < 137.55

BIO 3340 - na na na na na

BIO 3341 - na na na na na

BIO 3322 - na na na na na

BIO 3325 - na na na na na

BIO 3324 - na na na na na

Peanut-cropped soil

(Wonogiri – Central Java)

BIO 3352 + 90.94 < < < 90.94

BIO 3362 - na na na na na

BIO 3364 - na na na na na

BIO 3367 - na na na na na

BIO 3374 - na na na na na

BIO 3378 - na na na na na

BIO 3386 - na na na na na

BIO 3387 - na na na na na

BIO 3390 - na na na na na

BIO 3357 - na na na na na

BIO 3391 - na na na na na

BIO 3392 - na na na na na

BIO 3357 - na na na na na

Toxigenic strain BIO 747

74.01 < 54.05 < 128.06

Screening of Toxigenic Aspergillus ﬂavus Strains and Aﬂatoxin Content from Agricultural Commodities in Indonesia

223

Total aflatoxin was extracted from ten-days-old 10%

(v/v) CB medium cultures of toxigenic strains, using

AOAC method 991.3125,26. A 25 ml of filtered

extract was pipetted and extracted with 5 g NaCl and

125 ml of methanol:water (70:30) ratio into blender

jar, and blended for 2 minutes at maximum speed.

The filtered extract (15 ml) was diluted with 30 ml of

purified water into a clean vessel. The diluted extract

was filtered through glass microfiber filter. A 15 ml

filtered diluted extract passed completely through

AflaTest affinity column (VICAM, USA) at a rate of

about 1-2 drops/second and washed with 2 x 10 ml of

purified water at a rate of 2 drops/second. Total

aflatoxin was eluted from column with addition of 1

ml HPLC grade methanol (Merck, Germany) at rate

of 1 drop/second. Eluted sample was collected in a

glass cuvette and added with 1 ml deionized water.

Afterward, 20 ul of eluate were injected onto HPLC.

Chromatographic analyses were performed with

an Agilent 1260 Infinity Isocratic LC (Agilent

Technologies, USA), equipped with Photochemical

Reactor Derivatization (AURA Industries).

Excitation and emission wavelengths were 365 and

465 nm respectively. A Bonclone 10u C18 Column

(Phenomex, 3.9 x 150 mm) was used. The mobile

phase was methanol: water (60:40) and the flow rate

was 1.3 ml/min. Injection volume was 20 ul.

Quantification of aflatoxin was perfomed by

comparing the peak areas with the calibration curves

of each aflatoxin.

3 RESULT AND DISCUSSION

The screening on aflatoxin-induced medium (CAM)

was initially used to identify the aflatoxin production

from 50 A. flavus strains by fluorescence observation,

revealed that nine strains (18%) were toxigenic

(Table 2).

Figure 2: Fluorescence of A. flavus strains on 10% CAM.

(Left – right); uninoculated CAM, atoxigenic strain (BIO

3381), toxigenic strain (BIO 3313).

Toxigenic strains were originated from nutmeg

(56%), cacao (22%), ground peanut (11%), and

peanut-cropped soil (11%). Atoxigenic strains was

obtained from coffee bean, corn, and pepper. Blue

fluorescence was observed from outside of the colony

in CAM from eight strains, meanwhile fluorescence

was not seen either from uninoculated media or

atoxigenic strain (Figure 2). The naturally presence of

toxigenic and atoxigenic A. flavus have been reported

in many studies. Wei et al, (2014) found by UPLC

detection, 76% of the 323 A. flavus strains isolated

from peanut field in four provinces in China, were

aflatoxins producer with limit of detection method

was 1 µg/kg.

All the toxigenic strains, also positive and

negative control strains were further confirmed by

measuring AFB dan AFG content by HPLC from

growth simulation in CB medium. BIO 3381 was

chosen as negative control as no fluorescent observed

in CAM. During the incubation process, mycelia

grew on the surface of the media, while the toxin

produced was dissolved in the media. The aflatoxins

content determined as AFB1, AFB2, AFG1, AFG2

and total aflatoxin. The result showed all toxigenic

strains produced AFB1 (Table 3). Six toxigenic

strains produced AFB1 exceeding the Indonesian-

regulatory maximum level (15 ug/kg). A. flavus from

peanut-cropped soil (BIO 3352) produced the highest

AFB1 content (90.94 ug/kg), while the other strains

from nutmeg (BIO 3345 and BIO 33212), ground

peanut (BIO 3313 and BIO 3338), and cacao (BIO

33404) had AFB1 content of 89.53, 84.24, 70.26,

40.27, and 69.06 ug/kg respectively.

Table 3: Summary of aflatoxin content of 9 toxigenic A.

flavus strains.

Commodities

A. flavus

Strains

Aflatoxin (ug/kg)

AFB1 AFB2 AFG1 AFG2

Nutmeg BIO33212 84.24 < < <

BIO33403 4.48 3.02 2.82 0.82

BIO33211 5.03 < < <

BIO3376 6.47 < 26.71 <

BIO3345 89.53 < < <

Cacao BIO33404 69.06 < < <

Ground

peanut

BIO3313 70.26 6.59 < 1.01

BIO3338 40.27 < 97.28 <

Peanut-

cropped soil

BIO3352 90.94 < < <

(<) below the LoQ, for AFB1: 1.42 ug/kg, AFB2: 6.72

ug/kg, AFG1: 5.09 ug/kg, and AFG2: 0.66 ug/kg.

There was only one strain (BIO 33403) that

produced all aflatoxins types. Meanwhile one strains

(BIO 3313) produce all aflatoxins types except

AFG1, and two strains (BIO 3338 and BIO 3376)

produce AFB1 and AFG1. Five strains observed

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which only produced AFB1 were BIO 3345, BIO

3352, BIO 33211, BIO 33212, and BIO 33404. A.

flavus had known as AFB producer and A. paraciticus

as AFG producer which were determined by the color

of fluorescence of the colony on 10% CAM

(Nurtjahja et al, 2017). This study found that 44.4%

strains of toxigenic A. flavus can produce either AFB

or AFG.

In this study, strain isolated from peanut-cropped

soil was the higher production of AFB1. According

to Pitt (1989) in Dharmaputra et al., (2001), A. flavus

and A. paraciticus are present in high numbers in

cultivated soils. They are able to grow as commensals

in developing peanut plants, and start to invade

developing peanuts (Pitt et al., 1991). The study of

soil isolates and the correlation with toxigenicity

potential was reported by Dharmaputra et al., (2002).

She reported that 44% of toxigenic A. flavus were

identified from 48 soil sample during wet season, and

51% during dry season, in Pati regency (Central

Java). Most of the toxigenic A. flavus produced AFB1

and AFB2 and some of them produced AFB1, AFB2,

AFG1, and AFG2. Toxigenic A. flavus also found as

much as 27.5% from 66 strains isolated from corn

field soil in Iran, and only produce AFB1 or AFB1

and AFB2 (Razzaghi-Abyaneh et al., 2006).

4 CONCLUSIONS

Nine strains of toxigenic A. flavus were obtain from

screening of 50 strains from 6 agriculture

commodities and 1 peanut-cropped soil in Indonesia

which can produced aflatoxin. It was assumed that

soil from plantation could be a media for A. flavus

infection to the plant. The result of this study gave

information that toxigenic A. flavus strains have

ability to produce aflatoxin and could be used as

positive control in biological control. Further studies

are needed to characterize the diversity in DNA level

among the toxigenic strains. The information about

molecular characterization could help to develop

more effective biological control strategy.

ACKNOWLEDGEMENTS

The authors would like to acknowledge SEAMEO

BIOTROP for providing financial support through

Daftar Isian Pelaksanaan Anggaran (DIPA) 2019.

Thanks, are also to Prof. Dr Okky Setyawati

Dharmaputra and Ms. Ina Retnowati, S. Si for their A.

flavus collection isolates.

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