1. J. Microbiol. Biotechnol. (2012), 22(12), 1740–1748
http://dx.doi.org/10.4014/jmb.1207.07048
First published online October 5, 2012
pISSN 1017-7825 eISSN 1738-8872
Isolation and Identification of Fungi from a Meju Contaminated with Aflatoxins
Jung, Yu Jung1, Soo Hyun Chung2*, Hyo Ku Lee1, Hyang Sook Chun3, and Seung Beom Hong4
1
Department of Food Science and Technology, Kongju National University, Yesan 340-702, Korea
2
Department of Food and Nutrition, College of Health Science, Korea University, Seoul 136-703, Korea
3
Food Safety Research Division, Korea Food Research Institute, Sungnam 463-746, Korea
4
Korean Agricultural Culture Collection, National Academy of Agricultural Science, RDA, Suwon 441-707, Korea
Received: July 24, 2012 / Revised: September 17, 2012 / Accepted: September 28, 2012
A home-made meju sample contaminated naturally with kochujang [20, 23]. The quality of traditional meju is
aflatoxins was used for isolation of fungal strains. Overall, influenced by the metabolism of microorganisms during
230 fungal isolates were obtained on dichloran rosebengal the fermentation process. It is known that most of the fungi
chloramphenicol (DRBC) and dichloran 18% glycerol grow predominantly on the dried and air-contacted surface
(DG18) agar plates. Morphological characteristics and of the meju during its fermentation, whereas bacteria are
molecular analysis of a partial β-tubulin gene and the usually present inside the meju where the oxygen level
internal transcribed spacer (ITS) of rDNA were used for is low [5, 39]. The fungi presented in the meju were
the identification of the isolates. The fungal isolates were recognized as effective microorganisms for fermentation
divided into 7 genera: Aspergillus, Eurotium, Penicillium, and composed mostly of Aspergillus oryzae, Mucor spp.,
Eupenicillium, Mucor, Lichtheimia, and Curvularia. Three and Penicillium spp. [21, 22].
strains from 56 isolates of the A. oryzae/flavus group were Currently, large quantities of fermented soybean products
found to be aflatoxigenic A. flavus, by the presence of the are manufactured commercially, and inoculation with A.
aflatoxin biosynthesis genes and confirmatory aflatoxin oryzae is used for mass fermentation [25]. In the case of
production by high-performance liquid chromatography home-made meju, manufactured by traditional methods,
(HPLC). The predominant isolate from DRBC plates was the quality of the meju depends on natural fermentation
A. oryzae (42 strains, 36.2%), whereas that from DG18 [23], and differences in the quality of the product can occur
was A. candidus (61 strains, 53.5%). Out of the 230 isolates, because of microbial diversity in place and time of
the most common species was A. candidus (34.3%) followed production [19]. The fungi that grow in meju or soybean
by A. oryzae (22.2%), Mucor circinelloides (13.0%), P. products are generally regarded as safe. However, it is
polonicum (10.0%), A. tubingensis (4.8%), and L. ramosa possible for home-made meju to be contaminated with
(3.5%). A. flavus and E. chevalieri presented occurrence mycotoxin-producing fungi, which can produce mycotoxins
levels of 2.2%, respectively. The remaining isolates of A. under the natural fermentation environment.
unguis, P. oxalicum, Eupenicillium cinnamopurpureum, A. Mycotoxin contamination on agricultural commodities
acidus, E. rubrum, P. chrysogenum, M. racemosus, and C. has attracted worldwide attention because of its adverse
inaequalis had lower occurrence levels of < 2.0%. effects on human health, poultry, and livestock [4, 13].
Keywords: Meju, fungi, aflatoxigenicity, fungal frequency Some mycotoxins are carcinogenic, mutagenic, teratogenic,
nephrotoxic, and immunosuppressive agents [8]. In particular,
it is possible for meju to be contaminated with aflatoxins
produced by A. flavus or A. parasiticus-those have the
In Korea, the term meju is used to describe dried fermented morphological and biochemical similarities with A. oryzae
soybeans that have been formed into a block. The meju or A. sojae [20, 25, 29].
is an important starter material for Korean fermented The method used for the detection and identification of
soybean products and strongly determines the quality of fungi has been dependent on the morphological and
the products including soybean paste, soy sauce, and cultural characteristics of the fungi. To date, molecular
techniques have greatly improved our understanding of
*Corresponding author
Phone: +82-2-940-2854; Fax: +82-2-941-7825; fungal ecology and have revolutionized the tools available
E-mail: chungs59@korea.ac.kr for exploring environmental fungal communities [12]. In

2. 1741 Jung et al.
particular, the specific amplification and the sequence of Morphological Classification
the internal transcribed spacer (ITS) region of fungal DNA To observe macro- and microscopic characteristics of the colonies,
(rDNA) are widely used for the phylogenetic study of the fungi were grown on CA, malt extract agar (MEA), and potato
fungi [10, 14, 38]. The β-tubulin gene also has high dextrose agar (PDA) plates for 5 days at 25oC. Next, the conidial
heads, conidiophores, vesicles, conidia shapes, and roughness of the
differentiation, and its regional sequence is relatively easy
conidial walls were observed under a microscope. Each strain was
to obtain [9, 15]. identified in genus level according the standard methods provided
Currently, there is an increasing demand for the safety by Pitt and Hocking [28] and Samson et al. [31, 32]. Experiments
verification of fungi that grow in traditional foods. To date, were conducted twice with 3 replicate plates.
there are only limited reports on the aflatoxigenic fungi
and fungal flora in meju. Park et al. [25] collected meju Molecular Identification
samples from the southern area of Korea and performed The fungal isolates were grown on potato dextrose broth for 7 days
direct competitive enzyme-linked immunosorbent assays at 25oC. The mycelia were harvested from the plates and the total
to show that some Aspergillus isolates had produced genomic DNA was extracted using the DNeasy Plant Mini-Kit
aflatoxins. However, the immunochemical methods used (Qiagen, Valencia, CA, USA). A partial sequence of the β-tubulin
to detect aflatoxigenic fungi can sometimes give false- gene was amplified using 2 primers: Bt2a (5'-GGTAACCAAATC
GGTGCTGCTTTC-3') and Bt2b (5'-ACCCTCAGTGTAGTGACC
positive results [36]. Molecular approaches to detect
CTTGGC-3') [9]. The ITS region (including ITS1-5.8S rRNA-ITS2
aflatoxigenic fungi, including polymerized chain reaction region) of the rDNA were amplified using primers ITS1 (5'-
(PCR) and gene sequencing, have been used recently with TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTAT
improved simplicity and sensitivity. Previously, we used TGATATGC-3') [38]. Genomic DNA of the β-tubulin and the ITS
multiplex PCR analysis for detecting aflatoxigenic fungi were added to the PCR reactions containing forward and reverse
from meju samples in Korea and found that 4 of the 65 primers, and PCR was performed using the thermal cycler PC708
isolates of Aspergillus section Flavi were potentially (ASTEC, Japan). Each PCR reaction was performed in a total
aflatoxigenic strains [16]. volume of 50 µl, containing 5 µl of 10× PCR buffer, 3 µl of
In this study, 230 fungal isolates were selected from a deoxyribonucleotide triphosphate (dNTP; 2.5 mM), 0.4 µl of each
home-made meju sample manufactured using traditional primer (100 pmol), 0.3 µl of Taq DNA polymerase (Solgent, Korea),
methods, in which aflatoxins were contaminated naturally. 39.9 µl of sterile deionized water, and 1 µl of DNA template. The
PCR conditions were as follows: 4 min at 95oC; denaturation for
The fungal isolates were identified by morphological and
1 min at 95oC; annealing for 1 min at 65oC (β-tubulin) or 1 min at
molecular characteristics, and the phylogenetic positions 54oC (ITS region); extension for 2 min at 72oC (35 cycles); and a
of the isolates were obtained from the fungal β-tubulin and final extension for 7 min at 72oC. Amplicons were separated using
ITS sequence. The fungal composition of the meju sample the Mupid-2 Plus submarine electrophoresis system (Advance, Japan)
was presented, and we used PCR assay and HPLC to on 1.5% (w/v) agarose gels. The gels were stained with ethidium
determine which isolates were potential aflatoxigenic strains. bromide and bands were visualized with a UV transilluminator. The
above-mentioned amplicons were also used for sequencing analysis
and were purified with the EzWay PCR Clean-up kit (KOMA
MATERIALS AND METHODS biotech, Korea). Finally, the purified PCR products were sequenced
by Solgent Co. Ltd (Daejeon, Korea).
Source of Sample and Fungal Isolation
A home-made meju sample naturally contaminated with aflatoxins DNA Sequence and Phylogenetic Analysis
(aflatoxin B1 and B2: 210 ppb) was manufactured by the traditional DNA sequences were edited using the DNASTAR computer package,
method in Chungnam Province and used for the isolation of fungal and sequence alignment was performed using the CLUSTAL W
strains. The meju was finely ground using a laboratory blender, and program [34]. These sequences were used with the BLAST program
20 g of the sample was added to 180 ml of peptone water 0.1% (w/v) (http://www.ncbi.nlm.nih.gov/BLAST) to identify the fungi. The
and maintained at room temperature for approximately 30 min. This neighbor-joining (NJ) method was used for phylogenetic analysis, in
mixture was then shaken, and serial dilutions were obtained. One which the data were first analyzed using the Tamura–Nei parameter
hundred µl of each dilution was spread onto the surface of 2 types distance calculation model with g-distributed substitution rates, and
of solid media: DRBC agar [peptone 5 g, glucose 10 g, KH2PO4 then an NJ tree was constructed using MEGA version 4.0 [33]. A
0.1 g, MgSO4·7H2O 0.05 g, dichloran (0.2% in ethanol) 1.0 ml, rose bootstrap analysis was performed with 1,000 replications as confirmation
bengal 0.025 g, chloramphenicol 0.1 g, agar 15 g, and distilled water of each clade.
1 L] and DG18 agar [peptone 5 g, glucose 10 g, KH2PO4 0.1 g,
MgSO4·7H2O 0.05 g, dichloran (0.2% in ethanol) 1.0 ml, glycerol Multiplex PCR Analysis for Screening Aflatoxigenicity
220 g, chloramphenicol 0.1 g, agar 15 g, and distilled water 1 L] In order to screen for aflatoxigenic isolates from the A. oryzae/flavus
[28]. The plates were incubated for 1 week at 25oC. On the last day group, 4 pairs of primers were used. One regulatory gene (aflR) and
of incubation, the fungal colonies were transferred to Czapek agar 3 structural genes (omtA, omtB, and ver-1) were amplified using the
(CA) slants, and were incubated for 7 days at 25oC for further study. appropriate primers, and 2 different sets of 2 primers, primer set I
Each species isolated from the sample was considered as an isolate. (aflR/omtA) and primer set II (omtB/ver-1), were combined for

3. FUNGAL FLORA IN A MEJU CONTAMINATED WITH AFLATOXIN 1742
performing multiplex PCR by considering a consistent amplification determination of each aflatoxin was carried out using a fluorescence
pattern for every expected amplicon. The PCR mixture reactions detector (excitation: 360 nm; emission: 440 nm).
and condition were performed as described in a previous report [16].
PCR products were electrophoresed on a 1.5% (w/v) agarose gel
with a 1 kb plus DNA size marker (Solgent Co. Ltd, Korea). A. RESULTS AND DISCUSSION
flavus KACC41403 from the Korea Agricultural Culture Collection
(Suwon, Korea) was used as a standard strain for aflatoxin production.
Morphological Classification of Fungal Isolates from
HPLC Analysis of Aflatoxigenicity
Meju
The production of aflatoxins by 56 isolates of the A. oryzae/flavus A total of 230 fungal isolates were obtained from the meju
group was determined using HPLC. Individual fungal spores were sample that was naturally contaminated with aflatoxin. Of
removed from the mycelium after 7 days of growth at 25oC on a all the isolated strains, 116 were from the DRBC plates
PDA slant medium and suspended in sterile 0.05% (v/v) Tween 80. and 114 were from the DG18 plates. The cultural and
A 0.1 ml aliquot of the spore suspension was used to inoculate into microscopic characteristics of the isolates were analyzed to
50 ml of sterile Czapek yeast-extract (CY; NaNO3 3 g, KH2PO4 1 g, classify the fungal genus using adequate keys [28, 31, 32].
KCl 0.5 g, MgSO4·7H2O 0.5 g, FeSO4·H2O 0.01 g, yeast extract 5 g, Most isolates had typical morphological features, which
sucrose 30 g, and distilled water 1 L), which was used for the classified them into 1 of 7 fungal genera: Aspergillus,
production of aflatoxins by the fungi. The inoculated flasks were Eurotium, Penicillium, Eupenicillium, Lichtheimia, Mucor,
incubated for 14 days at 25oC. The fungal culture broth was filtered
and Curvularia. The isolates of Eurotium spp. from the
through Whatman No.1 filter paper, and 10 ml of the filtrate was
then diluted with phosphate-buffered saline (pH 7.5) to 100 ml. The
DG18 plates showed reduced growth when transferred onto
mixture was passed through a Whatman GF/A glass filter and 50 ml PDA, and had similar anamorph and cleistothecia in
of the filtrate was loaded onto an immunoaffinity column (AflaTest colony morphology and microscopic features. Most of the
WB, Vicam Co., USA) at a flow rate of approximately 1 drop per
other isolates showed faster growth on PDA, MEA, and
second for clean-up. After washing the column with 10 ml of water CA plates than Eurotium spp. at 25oC. Each fungal isolate
at the same flow rate, aflatoxin was eluted with 2 ml of methanol. classified at the genus level was used in the next molecular
The eluate was evaporated at 40oC under a stream of N2 until dry. analysis for further identification.
The dry residue was derivatized by adding 200 µl of trifluoroacetic
acid, and the mixture was left to stand for 30 min before it was Molecular Analysis for Identification of Fungal Isolates
diluted with 800 µl of acetonitrile-water [10:90 (v/v)]. This derivatized DNA sequences with concordance analysis can provide
sample was filtered through a 0.22 µm membrane filter, and the
information that enables the identification of a fungal
filtrate was used for HPLC analysis. Separation of aflatoxins B1, B2,
G1, and G2 from the injected 50 µl of samples was carried out using
species [27]. To identify the species of the 230 isolates,
a Nova-Pack C18 column (150 mm, 3.9 mm i.d., 4 µm; Waters, molecular analysis was conducted using the β-tubulin gene
USA). The mobile phase was acetonitrile-methanol-water [17:17:66 for Aspergillus, Eurotium, Penicillium, and Eupenicillium
(v/v/v)], pumped at a constant flow rate of 0.5 ml/min. The quantitative spp. and rDNA-ITS gene for Mucor, Lichtheimia, and
Table 1. Molecular identification of fungal isolates from the meju sample using gene sequencing of β-tubulin and the ITS region.
Products No. of base differences
Fungal species Gene regions GenBank accession no.
(bp) (Identity, %)
Aspergillus acidus 510 0 (100) JF450869
Aspergillus candidus 517 0 (100) EU014092
Aspergillus oryzae/flavus 497 0-1 (99-100) EF661486, EF661483
Aspergillus tubingensis 506 0 (100) HE577808
Aspergillus unguis 402 0 (100) EF652333
Eurotium chevalieri β-Tubulin 412 0 (100) EF651913
Eurotium rubrum 403 1 (99) EF651922
Eupenicillium cinnamopurpureum 432 0 (100) EF506216
Penicillium chrysogenum 431 0 (100) EF198568
Penicillium oxalicum 492 5 (99) JF521520
Penicillium polonicum 436 0 (100) EU128570
Lichtheimia ramosa 816 2 (99) HQ285692
Mucor circinelloides 599 0 (100) DQ118989
ITS1–5.8S–ITS2
Mucor racemosus 597 0 (100) HQ285603
Curvularia inaequalis 560 2 (99) AF313409

4. 1743 Jung et al.
Curvularia spp., respectively. The identity of the isolates amplicon indicated its identity according to the reference
and PCR product sizes are shown in Table 1, and the strains from the GenBank database. Five clusters of
phylogenetic trees produced using the NJ method are Aspergillus were found in the meju sample after β-tubulin
presented in Fig. 1. gene analysis, including A. acidus, A. candidus, A.
Ten species were identified by the sequencing of the β- tubingensis, and A. unguis, and their PCR amplicon size
tubulin gene, with the exception of the A. oryzae/flavus and homology were identical (100%) to the reference
group, and 4 species were identified by the sequencing of strains from the GenBank database. In the case of the A.
the rDNA-ITS region. The sizes of each fungal PCR oryzae/flavus group, their cluster had a same-sized PCR
Fig. 1. Phylogenetic tree of the fungal genera isolates from the meju sample.
(A) Aspergillus, Eurotium, Penicillium, and Eupenicillium: β-tubulin gene. (B) Mucor, Lichtheimia, and Curvularia: rDNA-ITS region. The tree was
constructed using neighbor-joining analyses of the β-tubulin and rDNA-ITS region gene sequences. The numbers above the nodes represent bootstrap
values (out of 1,000 bootstrap replications).

5. FUNGAL FLORA IN A MEJU CONTAMINATED WITH AFLATOXIN 1744
Fig. 2. Agarose gel electrophoretic pattern of PCR products obtained from genomic DNA of the Aspergillus oryzae/flavus group.
(A) Primer set I. (B) primer set II. M (bp): 1 kb plus DNA ladder.
amplicon (497 bp) and high homology (99-100%) with known that genes involved in the aflatoxigenic biosynthesis
either reference strains of A. oryzae or A. flavus. E. rubrum pathway could be used to differentiate aflatoxigenic
and E. chevalieri were identified with 99-100% homology Aspergillus fungi from non-aflatoxigenic Aspergillus spp.
within each cluster. Three clusters of Penicillium and 1 [3, 6, 35]. Molecular approaches such as PCR are reported
cluster of Eupenicillium—P. chrysogenum, P. oxalicum, P. to be efficient for the detection of aflatoxigenic fungi,
polonicum, and Eupenicillium cinnamopurpureum—were because PCR can be conducted in vitro and is specific and
obtained from the meju sample, and each cluster showed a sensitive [24]. Previously, we had developed a multiplex
highly consistent sequence (99-100%) when identified PCR assay for the detection of aflatoxigenic Aspergillus
from their relevant species. The phylogenetic tree constructed isolates from meju samples [16]. The result showed that
from rDNA-ITS sequencing showed that 3 clusters of multiplex PCR may be used for detecting the presence of
Zygomycetes (M. circinelloides, M. racemosus, and L. ramosa) aflatoxigenic Aspergillus species in meju samples. We
and C. inaequalis were present in the meju sample. M. used this assay to screen the aflatoxigenic strains from the
circinelloides and M. racemosus had same-sized PCR 56 isolates of the A. oryzae/flavus group. Two sets of 2
products and 100% sequence consistency with the reference primers were used to amplify the genes, including aflR,
strains, and L. ramosa and C. inaequalis had 99% homology omtA, omtB, and ver-1. The genes of aflR and omtA were
with reference strains from the GenBank database. used to successfully detect aflatoxigenic strains by other
Both A. flavus and A. oryzae belong to the Aspergillus researchers because aflR is known to be an aflatoxin
section Flavi, and A. flavus could be classified as a biosynthesis regulatory gene and omtA is known to be an
separate species; however, it is almost genetically identical aflatoxin biosynthesis structural gene.
to A. oryzae. Chang and Ehrlich [2] suggested that A. Among the 56 isolates of the A. oryzae/flavus group, 5
oryzae may be a variant morphotype of typical A. flavus; isolates (KUFNM018, 027, 044, 084, and 098) showed
and Rank et al. [29] reported that the gene homology of the complete amplification for both primer sets used in the
two species was ca. 99.5%. Although the isolates of the A. multiplex PCR assay, and the remaining 51 isolates (91%)
oryzae/flavus group from the meju sample showed similar showed only 3 bands after multiplex PCR, which represented
morphological characteristics (conidial heads in the shades the amplified omtA, omtB, and ver-1 genes, respectively;
of yellow-green to brown, usually metulated vesicles and the regulatory gene aflR was deleted (Fig. 2 and Table 2).
dark clerotia) and β-tubulin gene homology (99-100%), In Fig. 2, the lanes with 4 amplicon bands were obtained
they could be divided into A. flavus and A. oryzae from A. flavus KACC 41403 (used as a standard strain for
according to the difference in aflatoxigenicity. It is known aflatoxin production) and 5 isolates (KUFNM018, 027,
that A. oryzae does not produce aflatoxins despite its close 044, 084, and 098) of A. oryzae/flavus; the lanes with 3
relationship with A. flavus [1, 18, 37]. bands were from the remaining 51 isolates (showing 5
representative isolates: KUFNM119–123).
Aflatoxigenicity of Aspergillus oryzae/flavus Group The 56 isolates were then tested for aflatoxin production
The aflatoxigenicity of 56 isolates belonging to the A. by using culture filtrates, and the results are shown in
oryzae/flavus group from the meju sample was examined Table 3. The 5 isolates that showed 4 amplicon bands
using multiplex PCR assay and HPLC confirmation. It is including aflR, omtA, omtB, and ver-1 were divided in 2

6. 1745 Jung et al.
Table 2. Genetic patterns of 56 Aspergillus oryzae/flavus group isolates from the meju sample.
Gene presence detected by multiplex PCR
Genetic pattern Number of isolates Primer set I Primer set II
aflR omtA omtB ver-1
Four bands 5 +a + + +
b
Three bands 51 – + + +
a
+: Amplification in PCR.
b
–: No amplification in PCR.
subgroups: aflatoxin producers (KUFNM027, 044, and amount of aflatoxins; therefore, they were confirmed to be
084) and aflatoxin non-producers (KUFNM018 and 098), non-producers of aflatoxin. Previously, the aflR gene has
which were then designated as aflatoxigenic A. flavus and been used to discriminate between aflatoxin-producing and
non-aflatoxigenic A. flavus, respectively. This result was aflatoxin-non-producing fungi [6]. Our result showed that
consistent with results published by other researchers. the 51 aflR-lacking isolates were non-aflatoxigenic fungi,
Criseo et al. [6] reported that 36.5% of 134 non-aflatoxigenic and they were classified as A. oryzae for further study.
A. flavus contained 4 genes (aflR, omtA, ver-1, and nor-1) In addition, it is assumed that the 3 isolates of aflatoxigenic
as like aflatoxigenic strains; Degola et al. [7] showed that A. flavus might produce considerable amounts of aflatoxins
A. flavus isolates containing 5 genes (aflD, aflO, aflaQ, during meju fermentation; the contents of AFB1 and AFB2
aflR, and aflS) were separated into aflatoxin producers and in the meju sample were 206.2 ng/g and 3.8 ng/g, respectively.
non-producers. However, these studies did not clearly However, the aflatoxin content in soybean paste and soy
discriminate between aflatoxigenic and non-aflatoxigenic sauce fermented for 6 months using meju blocks that had
A. flavus, because only selected genes of the aflatoxin the same origin (same household and same production
synthetic pathway were analyzed. In addition, an A. flavus time) as the meju sample in this study was 7.1 ng/g and
strain that contains all the genes of the aflatoxin synthetic
pathway still requires other various molecular considerations
to be made, such as post-transcriptional level and/or
protein levels. Therefore, for the accurate detection of
aflatoxigenicity, immunological or cultural methods should
be applied. In this study, quantitative HPLC results showed
that the production of aflatoxin B1 (AFB1) and aflatoxin B2
(AFB2) by aflatoxigenic A. flavus strains was 114 µg/ml and
21 µg/ml (KUFNM027), 55 µg/ml and 3 µg/ml (KUFNM044),
and 25 µg/ml and 6 µg/ml (KUFNM084), respectively.
The chromatograms for the aflatoxin production by the 3
aflatoxigenic A. flavus are shown in Fig. 3. In the case of
the 51 isolates with three bands of PCR amplicons (omtA,
omtB, and ver-1), their culture contained no detectable
Table 3. Aflatoxin production by 56 Aspergillus oryzae/flavus
group isolats from the meju sample.
Isolates of Aspergillus Aflatoxin (µg/ml)
oryzae/flavus group B1 B2 G1 G2
5 isolates with 4 amplicons
KUFNM018 – – – –
KUFNM027 144 21 – – Fig. 3. HPLC chromatograms showing aflatoxin production: (A)
KUFNM044 55 3 – – A. flavus KUFNM027; (B) A. flavus KUFNM044; (C) A. flavus
KUFNM084; and (D) Aflatoxin standard mixture.
KUFNM084 25 6 – – Czapek yeast-extract (CY; NaNO3 3 g, KH2PO4 1 g, KCl 0.5 g,
KUFNM098 – – – – MgSO4·7H2O 0.5 g, FeSO4·H2O 0.01 g, yeast extract 5 g, sucrose 30 g,
and distilled water 1 L) was used for the production of aflatoxins by each
51 isolates with 3 amplicons – – – –
fungal isolate. The inoculated flasks were incubated for 14 days at 25oC,
–: Not detected and culture filtrates were used for the aflatoxin analysis.

7. FUNGAL FLORA IN A MEJU CONTAMINATED WITH AFLATOXIN 1746
0.4 ng/g of AFB1 (with no detectable amount of AFB2), was isolated on both DRBC and DG18 media. The
respectively (data not shown). It is well known that aflatoxins isolates of E. chevalieri, A. unguis, P. oxalicum, and E.
are degraded during the fermentation and ripening process cinnamopurpureum had occurrence levels of 2.2%, 1.7%,
of traditional soybean paste and soy sauce production [26]. 1.3%, and 1.3%, respectively. The remaining isolated
species—A. acidus, E. rubrum, P. chrysogenum, and M.
Frequency of Fugal Isolates from the Meju Sample racemosus—had occurrence levels of <1.0%. The colonies
For reliable mycological analysis of food or environmental and microscopic morphological characters of the 16
samples, the choice of culture media is important. In this representative isolates are shown in Fig. 4.
study, 2 types of media (DRBC and DG18) were used to To date, researches performed in Korea on the mycobiota
allow more complete recovery of fungal species present in that colonize meju have shown that the predominant isolates
the meju sample of which the surface was dried and the were of A. oryzae/flavus group, Mucor, and Penicillium
inside was somewhat wet. A total of 16 fungal species were spp. [5, 21, 22]. Those data were in accordance with the
found in the meju sample after molecular analysis and mycological results found in this study when analysis was
aflatoxigenecity confirmation (Table 4). The use of DRBC conducted with DRBC. However, A. candidus, the most
medium made it possible to isolate 12 species with the common species in this study, was not reported to be found
three major occurrences of A. oryzae (18.3%), M. circinelloides in meju samples before, because the authors of previous
(10.0%), and A. candidus (7.8%), whereas DG18 medium studies used standard media, which allowed the fungi to
presented 11 species isolated with A. candidus (26.5%), A. grow at a relatively high aw, such as PDA, MEA, and CY.
oryzae (3.9%), and P. polonicum (3.9%) being the 3 most The water activity (aw: 0.95) of the DG18 medium enables
frequent species. A. acidus, P. chrysogenum, P. oxalicum, the isolation and enumeration of fungal flora from dried
M. racemosus, and C. inaequalis only grew on the DRBC and semidried foods [11]; thus, xerophilic fungi, such as A.
agar, and A. unguis, E. chevalieri, E. rubrum, and E. candidus and Eurotium spp., were present on this medium.
cinnamopurpureum only grew on the DG18 agar. Of all To the best of our knowledge, this is the first report on
the 230 isolates, the predominant fungal species was A. the fungal frequency in meju, which is a starter material in
candidus (34.3%), which is known to be a xerophile, Korean fermented soybean products. The molecular assays
followed by fungi that grow at a relatively high aw, such as of fungal β-tubulin and the ITS sequence were conducted
A. oryzae (22.2%), M. circinelloides (13.0%), P. polonicum for the identification of 230 isolates from the meju sample,
(10.0%), A. tubingensis (4.8%), and L. ramosa (3.5%). A. using 2 types of media. The DRBC and DG18 allowed
flavus had an occurrence level of 2.2%, and this species more complete recovery of fungal species present in the
Table 4. Number and frequency of fungal species isolated from the meju sample on DRBC and DG18 agar plates.
Number Frequency (%)
Fungal species a b
DRBC DG18 Total DRBC DG18 Total
Aspergillus acidus 1 0 1 0.4 0 0.4
Aspergillus candidus 18 61 79 7.8 26.5 34.3
Aspergillus flavus 3 2 5 1.3 0.9 2.2
Aspergillus oryzae 42 9 51 18.3 3.9 22.2
Aspergillus tubingensis 3 8 11 1.3 3.5 4.8
Aspergillus unguis 0 4 4 0 1.7 1.7
Eurotium chevalieri 0 5 5 0 2.2 2.2
Eurotium rubrum 0 2 2 0 0.9 0.9
Penicillium chrysogenum 1 0 1 0.4 0 0.4
Penicillium oxalicum 3 0 3 1.3 0 1.3
Penicillium polonicum 14 9 23 6.1 3.9 10.0
Eupenicillium cinnamopurpureum 0 3 3 0 1.3 1.3
Mucor circinelloides 23 7 30 10.0 3.0 13.0
Mucor racemosus 2 0 2 0.9 0 0.9
Lichtheimia ramosa 4 4 8 1.7 1.7 3.5
Curvularia inaequalis 2 0 2 0.9 0 0.9
116 114 230 50.4 49.6 100
a
Dichloran glycerol 18% agar.
b
Dichloran rose bengal and chloramphenicol agar.

8. 1747 Jung et al.
and 2 non-aflatoxigenic A. flavus were successfully
differentiated from 56 isolates of the A. oryzae/flavus
group, using cultural experiments after multiplex PCR
assay of aflatoxigenic genes (aflR, omtA, omtB, and ver-1).
Cultural and quantitative HPLC analyses ascertained the
potential production of aflatoxins by the 3 strains of A.
flavus isolated from the sample.
Acknowledgments
This research was supported by a grant from Korea
University and a grant (11162KFDA995) from the Korea
Food and Drug Administration.
REFERENCES
1. Barbesgaard, P., H. P. Heldt-Hansen, and B. Diderichsen. 1992.
On the safety of Aspergillus oryzae: A review. Appl. Microbiol.
Biotechnol. 36: 569-572.
2. Chang, P. K. and K. C. Ehrlich. 2010. What does genetic
diversity of Aspergillus flavus tell us about Aspergillus oryzae?
Int. J. Food Microbiol. 15: 189-199.
3. Chang, P. K., B. W. Horn, and J. W. Dorner. 2005. Sequence
break points in the aflatoxin biosynthesis gene cluster and
flanking regions in nonaflatoxigenic Aspergillus flavus isolates.
Fungal Genet. Biol. 42: 914-923.
4. Chelkowski, J. 1991. Mycological quality of mixed feeds and
ingredients, pp. 217-227. In J. Chelkowski (ed.). Cereal Grain,
Mycotoxins, Fungi and Quality in Drying and Storage. Elsevier,
Amsterdam, London, New York.
5. Cho, D. H. and W. J. Lee. 1970. Microbiological studies of
Korean native soy-sauce fermentation; A study on the microflora
of fermented Korean maeju loaves. J. Kor. Agric. Chem.
Soc. 13.
6. Criseo, G., C. Racco, and O. Romeo. 2008. High genetic
variability in non-aflatoxigenic A. flavus strains by using
quadruplex PCR-based assay. Int. J. Food Microbiol. 125:
341-343.
7. Degola, F., E. Berni, C. Dall’Asta, E. Spotti, R. Marchelli,
I. Ferrero, and F. M. Restivo. 2006. A multiplex RT-PCR
approach to detect aflatoxigenic strains of Aspergillus flavus. J.
Appl. Microbiol. 103: 409-417.
8. Fraga, M. E., F. Curvello, M. J. Gatti, L. R. Cavaglieri, A. M.
Dalcero, and C. A. da Rocha Rosa. 2007. Potential aflatoxin
and ochratoxin A production by Aspergillus species in poultry
feed processing. Vet. Res. Commun. 31: 343-353.
9. He, C. H., Y. H. Fan, G. F. Liu, and H. B. Zhang. 2008.
Fig. 4. Colony surface and conidial head of fungal species Isolation and identification of a strain of Aspergillus tubingensis
isolated from the meju sample on potato-dextrose agar.
A, a: Aspergillus acidus; B, b: A. candidus; C, c: A. oryzae; D, d: A. flavus; with deoxynivalenol biotransformation capability. Int. J. Mol.
E, e: A. tubingensis; F, f: A. unguis; G, g: Eurotium rubrum; H, h: E. Sci. 9: 2366-2375.
chevalieri; I, i: Penicillium chrysogenum; J, j: P. oxalicum; K, k: P. 10. Hillis, D. M. and M. T. Dixon. 1991. Ribosomal DNA:
polonicum; L, l: Eupenicillium cinnamopurpureum; M, m: Mucor circinelloides; Molecular evolution and phylogenetic inference. Q. Rev. Biol.
N, n: M. racemosus; O, o: Lichtheimia ramosa; P, p: Curvularia inaequalis. 66: 411-453.
11. Hocking, A. D. and J. I. Pitt. 1980. Dichloran-glycerol medium
meju sample, which consisted of 16 species, including for enumeration of xerophilic fungi from low moisture foods.
xerophilic fungi. Three strains of aflatoxigenic A. flavus Appl. Environ. Microbiol. 39: 488-492.

9. FUNGAL FLORA IN A MEJU CONTAMINATED WITH AFLATOXIN 1748
12. Horton, T. R. and T. D. Bruns. 2001. The molecular revolution 26. Park, K.-Y., K.-B. Lee, and L. B. Bullerman. 1988. Aflatoxin
in ectomycorrhizal ecology: Peeking into the black box. Mol. production by Aspergillus parasiticus and its stability during the
Ecol. 10: 1855-1871. manufacture of Korean soy paste (doenjang) and soy sauce
13. Hussein, S. H. and J. M. Brasel. 2001. Toxicity, metabolism, (kanjang) by traditional method. J. Food Prot. 51: 938-945.
and impact of mycotoxins on humans and animals. Toxicology 27. Peterson, S. W. 2008. Phylogenetic analysis of Aspergillus
167: 101-134. species using DNA sequences from four loci. Mycologia 100:
14. Jumpponen, A. 2009. Analysis of rhizosphere fungal communities 205-226.
using rRNA and rDNA, pp. 29-40. In A. Varma and A. C. 28. Pitt, J. I. and A. D. Hocking. 2009. Fungi and Food Spoilage,
Kharkwal (eds.). Symbiotic Fungi, Soil Biology, 18th. Ed. 3rd Ed. Springer, New York.
Springer-Verlag, Berlin. 29. Rank, C., M. L. Klejnstrup, L. M. Petersen, S. Kildgaard, J. C.
15. Kim, D. H., S. H. Kim, Y. K. Kim, S. O. Kim, S. J. Kim, and Frisvad, C. H. Gotfredsen, and T. O. Larsen. 2012. Comparative
S. B. Hong. 2009. Reidentification of Aspergillus spp. isolated chemistry of Aspergillus oryzae (RIB40) and A. flavus (NRRL
from clinical specimens of patients suspected as pulmonary 3357). Metabolites 2: 39-56.
aspergillosis in Korea. Kor. J. Med. Mycol. 14: 133-144. 30. Reddy, B. N. and C. R. Raghavender. 2007. Outbreaks of
16. Kim, D. M., S. H. Chung, and H. S. Chun. 2011. Multiplex aflatoxicoses in India. Afr. J. Food Agric. Nutr. Devel. 7: 1-15.
PCR assay for the detection of aflatoxigenic and non- 31. Samson, R. A., E. S. Hoekstra, and J. C. Frisvad. 2004.
aflatoxigenic fungi in meju, a Korean fermented soybean food Introduction to Food and Airborne Fungi, 7th Ed. Centraalbureau
starter. Food Microbiol. 28: 1402-1408. voor Schimmelcultures, Utrecht, The Netherlands.
17. Kim, J. Y., S. H. Yeo, S. Y. Baek, and H. S. Choi. 2011 32. Samson, R. A., K. A. Seifert, A. F. A. Kuijpers, J. A. M. P.
Molecular and morphological identification of fungal species Houbraken, and J. C. Frisvad. 2004. Phylogenetic analysis of
isolated from bealmijang meju. J. Microbiol. Biotechnol. 21: Penicillium subgenus Penicillium using partial β-tubulin
1270-1279. sequences. Stud. Mycol. 49: 175-200.
18. Kiyota, T., R. Hamada, K. Sakamoto, K. Iwashita, O. Yamada, 33. Tamura, K., J. Dudley, M. Nei, and S. Kumar. 2007. MEGA4:
and S. Mikami. 2011. Aflatoxin non-productivity of Aspergillus Molecular evolutionary genetics analysis (MEGA) software
oryzae caused by loss of function in the aflJ gene product. J. version 4.0. Mol. Biol. Evol. 24: 1596-1599.
Biosci. Bioeng. 111: 512-517. 34. Thompson, J. D., T. J. Gibson, F. Plewniak, F. Jeanmougin, and
19. Kwon, D. J. 2002. Comparison of characteristics of koji D. G. Higgins. 1997. The CLUSTAL X Windows interface:
manufactured with Bacillus subtilis B-4 and Aspergillus oryzae Flexible strategies for multiple sequence alignment aided by
F-5. Kor. J. Food Sci. Technol. 34: 873-878. quality analysis tools. Nucleic Acids Res. 25: 4876-4882.
20. Lee, C. H. and S. S. Lee. 2002. Cereal fermentation by fungi. 35. Tominaga, M., Y. H. Lee, R. Hayashi, O. Suzuki, K. Tamada,
Appl. Mycol. Biotechnol. 2: 151-170. K. Skamoto, K. Gotoh, and O. Akita. 2006. Molecular analysis
21. Lee, S. S., K. H. Park, K. J. Choi, and S. A. Won. 1993. of an inactive aflatoxin biosynthesis gene cluster in Aspergillus
Identification and isolation of Zygomycetous fungi found on oryzae RIB strains. Appl. Environ. Microbiol. 72: 484-490.
maeju, a raw material of Korean traditional soysauces. Kor. J. 36. Tsai, G. J. and S. C. Yu. 1997. An enzyme-linked immunosorbent
Mycol. 21: 172-187. assay for the detection of Aspergillus parasiticus and Aspergillus
22. Lee, S. S., K. H. Park, K. J. Choi, and S. A. Won. 1993. A flavus. J. Food Prot. 60: 978-984.
study on Hyphomycetous fungi found on maejus, a raw 37. Wei, D. L. and S. C. Jong. 1986. Production of aflatoxins by
material of Korean traditional soysauce. Kor. J. Mycol. 21: strains of the Aspergillus flavus group maintained in ATCC.
242-272. Mycopathologia 93: 19-24.
23. Lee, S. W., S. K. Park, and H. C. Kim. 2001. Characteristics of 38. White, T. J., T. Bruns, S. Lee, and J. W. Taylor. 1990.
red mold isolated from traditional meju. Kor. J. Post-harvest Amplification and direct sequencing of fungal ribosomal RNA
Sci. Technol. 8: 199-205. genes for phylogenetics, pp. 315-322. In M. A. Innis, D. H.
24. Niessen, L. 2008. PCR-based diagnosis and quantification of Gelfand, J. J. Sninsky, and T. J. White (eds.). PCR Protocols: A
mycotoxin-producing fungi. Adv. Food Nutr. Res. 54: 81-138. Guide to Methods and Applications. Academic Press Inc., NY.
25. Park, J. H., S. J. Kang, S. S. Oh, and D. H. Chung. 2001. The 39. Yoo, J. Y. and H. G. Kim. 1998. Characteristics of traditional
screening of aflatoxin producing fungi from commercial meju mejus of nation-wide collection. J. Kor. Soc. Food Sci. Nutr. 27:
and soybean paste in western Gyeongnam by immunoassay. J. 259-267.
Food Hyg. Safety 16: 274-279.