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  1. 1. J. Microbiol. Biotechnol. (2012), 22(12), 1740–1748 published online October 5, 2012pISSN 1017-7825 eISSN 1738-8872Isolation and Identification of Fungi from a Meju Contaminated with AflatoxinsJung, Yu Jung1, Soo Hyun Chung2*, Hyo Ku Lee1, Hyang Sook Chun3, and Seung Beom Hong41 Department of Food Science and Technology, Kongju National University, Yesan 340-702, Korea2 Department of Food and Nutrition, College of Health Science, Korea University, Seoul 136-703, Korea3 Food Safety Research Division, Korea Food Research Institute, Sungnam 463-746, Korea4 Korean Agricultural Culture Collection, National Academy of Agricultural Science, RDA, Suwon 441-707, KoreaReceived: July 24, 2012 / Revised: September 17, 2012 / Accepted: September 28, 2012A home-made meju sample contaminated naturally with kochujang [20, 23]. The quality of traditional meju isaflatoxins was used for isolation of fungal strains. Overall, influenced by the metabolism of microorganisms during230 fungal isolates were obtained on dichloran rosebengal the fermentation process. It is known that most of the fungichloramphenicol (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 aremolecular analysis of a partial β-tubulin gene and the usually present inside the meju where the oxygen levelinternal transcribed spacer (ITS) of rDNA were used for is low [5, 39]. The fungi presented in the meju werethe identification of the isolates. The fungal isolates were recognized as effective microorganisms for fermentationdivided 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 productsfound 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 ofproduction 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 fermentationA. oryzae (42 strains, 36.2%), whereas that from DG18 [23], and differences in the quality of the product can occurwas A. candidus (61 strains, 53.5%). Out of the 230 isolates, because of microbial diversity in place and time ofthe most common species was A. candidus (34.3%) followed production [19]. The fungi that grow in meju or soybeanby A. oryzae (22.2%), Mucor circinelloides (13.0%), P. products are generally regarded as safe. However, it ispolonicum (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 mycotoxinslevels of 2.2%, respectively. The remaining isolates of A. under the natural fermentation environment.unguis, P. oxalicum, Eupenicillium cinnamopurpureum, A. Mycotoxin contamination on agricultural commoditiesacidus, E. rubrum, P. chrysogenum, M. racemosus, and C. has attracted worldwide attention because of its adverseinaequalis 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 theIn Korea, the term meju is used to describe dried fermented morphological and biochemical similarities with A. oryzaesoybeans 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 ofsoybean products and strongly determines the quality of fungi has been dependent on the morphological andthe products including soybean paste, soy sauce, and cultural characteristics of the fungi. To date, molecular techniques have greatly improved our understanding of*Corresponding authorPhone: +82-2-940-2854; Fax: +82-2-941-7825; fungal ecology and have revolutionized the tools availableE-mail: for exploring environmental fungal communities [12]. In
  2. 2. 1741 Jung et al.particular, the specific amplification and the sequence of Morphological Classificationthe 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 potatofungi [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 thedifferentiation, and its regional sequence is relatively easy conidial walls were observed under a microscope. Each strain wasto 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]. Experimentsverification of fungi that grow in traditional foods. To date, were conducted twice with 3 replicate plates.there are only limited reports on the aflatoxigenic fungiand fungal flora in meju. Park et al. [25] collected meju Molecular Identificationsamples from the southern area of Korea and performed The fungal isolates were grown on potato dextrose broth for 7 daysdirect competitive enzyme-linked immunosorbent assays at 25oC. The mycelia were harvested from the plates and the totalto show that some Aspergillus isolates had produced genomic DNA was extracted using the DNeasy Plant Mini-Kitaflatoxins. However, the immunochemical methods used (Qiagen, Valencia, CA, USA). A partial sequence of the β-tubulinto detect aflatoxigenic fungi can sometimes give false- gene was amplified using 2 primers: Bt2a (5-GGTAACCAAATC GGTGCTGCTTTC-3) and Bt2b (5-ACCCTCAGTGTAGTGACCpositive results [36]. Molecular approaches to detect CTTGGC-3) [9]. The ITS region (including ITS1-5.8S rRNA-ITS2aflatoxigenic 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-TCCTCCGCTTATimproved simplicity and sensitivity. Previously, we used TGATATGC-3) [38]. Genomic DNA of the β-tubulin and the ITSmultiplex PCR analysis for detecting aflatoxigenic fungi were added to the PCR reactions containing forward and reversefrom meju samples in Korea and found that 4 of the 65 primers, and PCR was performed using the thermal cycler PC708isolates of Aspergillus section Flavi were potentially (ASTEC, Japan). Each PCR reaction was performed in a totalaflatoxigenic 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 eachhome-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 forThe fungal isolates were identified by morphological and 1 min at 95oC; annealing for 1 min at 65oC (β-tubulin) or 1 min atmolecular characteristics, and the phylogenetic positions 54oC (ITS region); extension for 2 min at 72oC (35 cycles); and aof the isolates were obtained from the fungal β-tubulin and final extension for 7 min at 72oC. Amplicons were separated usingITS 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 ethidiumdetermine 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 (KOMAMATERIALS AND METHODS biotech, Korea). Finally, the purified PCR products were sequenced by Solgent Co. Ltd (Daejeon, Korea).Source of Sample and Fungal IsolationA 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 Wstrains. The meju was finely ground using a laboratory blender, and program [34]. These sequences were used with the BLAST program20 g of the sample was added to 180 ml of peptone water 0.1% (w/v) ( to identify the fungi. Theand maintained at room temperature for approximately 30 min. This neighbor-joining (NJ) method was used for phylogenetic analysis, inmixture was then shaken, and serial dilutions were obtained. One which the data were first analyzed using the Tamura–Nei parameterhundred µl of each dilution was spread onto the surface of 2 types distance calculation model with g-distributed substitution rates, andof solid media: DRBC agar [peptone 5 g, glucose 10 g, KH2PO4 then an NJ tree was constructed using MEGA version 4.0 [33]. A0.1 g, MgSO4·7H2O 0.05 g, dichloran (0.2% in ethanol) 1.0 ml, rose bootstrap analysis was performed with 1,000 replications as confirmationbengal 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 Aflatoxigenicity220 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) andof 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 IEach species isolated from the sample was considered as an isolate. (aflR/omtA) and primer set II (omtB/ver-1), were combined for
  3. 3. FUNGAL FLORA IN A MEJU CONTAMINATED WITH AFLATOXIN 1742performing multiplex PCR by considering a consistent amplification determination of each aflatoxin was carried out using a fluorescencepattern 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 gelwith a 1 kb plus DNA size marker (Solgent Co. Ltd, Korea). A. RESULTS AND DISCUSSIONflavus KACC41403 from the Korea Agricultural Culture Collection(Suwon, Korea) was used as a standard strain for aflatoxin production. Morphological Classification of Fungal Isolates fromHPLC Analysis of Aflatoxigenicity MejuThe production of aflatoxins by 56 isolates of the A. oryzae/flavus A total of 230 fungal isolates were obtained from the mejugroup was determined using HPLC. Individual fungal spores were sample that was naturally contaminated with aflatoxin. Ofremoved from the mycelium after 7 days of growth at 25oC on a all the isolated strains, 116 were from the DRBC platesPDA slant medium and suspended in sterile 0.05% (v/v) Tween 80. and 114 were from the DG18 plates. The cultural andA 0.1 ml aliquot of the spore suspension was used to inoculate into microscopic characteristics of the isolates were analyzed to50 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, whichsucrose 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 thethrough Whatman No.1 filter paper, and 10 ml of the filtrate wasthen diluted with phosphate-buffered saline (pH 7.5) to 100 ml. The DG18 plates showed reduced growth when transferred ontomixture was passed through a Whatman GF/A glass filter and 50 ml PDA, and had similar anamorph and cleistothecia inof the filtrate was loaded onto an immunoaffinity column (AflaTest colony morphology and microscopic features. Most of theWB, Vicam Co., USA) at a flow rate of approximately 1 drop per other isolates showed faster growth on PDA, MEA, andsecond for clean-up. After washing the column with 10 ml of water CA plates than Eurotium spp. at 25oC. Each fungal isolateat the same flow rate, aflatoxin was eluted with 2 ml of methanol. classified at the genus level was used in the next molecularThe 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 trifluoroaceticacid, and the mixture was left to stand for 30 min before it was Molecular Analysis for Identification of Fungal Isolatesdiluted with 800 µl of acetonitrile-water [10:90 (v/v)]. This derivatized DNA sequences with concordance analysis can providesample was filtered through a 0.22 µm membrane filter, and the information that enables the identification of a fungalfiltrate 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 geneUSA). 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, andTable 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. 4. 1743 Jung et al.Curvularia spp., respectively. The identity of the isolates amplicon indicated its identity according to the referenceand PCR product sizes are shown in Table 1, and the strains from the GenBank database. Five clusters ofphylogenetic trees produced using the NJ method are Aspergillus were found in the meju sample after β-tubulinpresented 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 sizetubulin gene, with the exception of the A. oryzae/flavus and homology were identical (100%) to the referencegroup, 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 PCRFig. 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 wasconstructed using neighbor-joining analyses of the β-tubulin and rDNA-ITS region gene sequences. The numbers above the nodes represent bootstrapvalues (out of 1,000 bootstrap replications).
  5. 5. FUNGAL FLORA IN A MEJU CONTAMINATED WITH AFLATOXIN 1744Fig. 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 biosynthesiseither reference strains of A. oryzae or A. flavus. E. rubrum pathway could be used to differentiate aflatoxigenicand 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 reportedcluster 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 andobtained from the meju sample, and each cluster showed a sensitive [24]. Previously, we had developed a multiplexhighly consistent sequence (99-100%) when identified PCR assay for the detection of aflatoxigenic Aspergillusfrom their relevant species. The phylogenetic tree constructed isolates from meju samples [16]. The result showed thatfrom rDNA-ITS sequencing showed that 3 clusters of multiplex PCR may be used for detecting the presence ofZygomycetes (M. circinelloides, M. racemosus, and L. ramosa) aflatoxigenic Aspergillus species in meju samples. Weand C. inaequalis were present in the meju sample. M. used this assay to screen the aflatoxigenic strains from thecircinelloides and M. racemosus had same-sized PCR 56 isolates of the A. oryzae/flavus group. Two sets of 2products 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 werewith 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 aflatoxinsection Flavi, and A. flavus could be classified as a biosynthesis regulatory gene and omtA is known to be anseparate species; however, it is almost genetically identical aflatoxin biosynthesis structural A. oryzae. Chang and Ehrlich [2] suggested that A. Among the 56 isolates of the A. oryzae/flavus group, 5oryzae may be a variant morphotype of typical A. flavus; isolates (KUFNM018, 027, 044, 084, and 098) showedand Rank et al. [29] reported that the gene homology of the complete amplification for both primer sets used in thetwo 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 representedmorphological 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 obtainedthey could be divided into A. flavus and A. oryzae from A. flavus KACC 41403 (used as a standard strain foraccording 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 3relationship 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 productionThe aflatoxigenicity of 56 isolates belonging to the A. by using culture filtrates, and the results are shown inoryzae/flavus group from the meju sample was examined Table 3. The 5 isolates that showed 4 amplicon bandsusing multiplex PCR assay and HPLC confirmation. It is including aflR, omtA, omtB, and ver-1 were divided in 2
  6. 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 be084) and aflatoxin non-producers (KUFNM018 and 098), non-producers of aflatoxin. Previously, the aflR gene haswhich were then designated as aflatoxigenic A. flavus and been used to discriminate between aflatoxin-producing andnon-aflatoxigenic A. flavus, respectively. This result was aflatoxin-non-producing fungi [6]. Our result showed thatconsistent 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 aflatoxigenicas like aflatoxigenic strains; Degola et al. [7] showed that A. flavus might produce considerable amounts of aflatoxinsA. flavus isolates containing 5 genes (aflD, aflO, aflaQ, during meju fermentation; the contents of AFB1 and AFB2aflR, 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 soydiscriminate between aflatoxigenic and non-aflatoxigenic sauce fermented for 6 months using meju blocks that hadA. flavus, because only selected genes of the aflatoxin the same origin (same household and same productionsynthetic pathway were analyzed. In addition, an A. flavus time) as the meju sample in this study was 7.1 ng/g andstrain that contains all the genes of the aflatoxin syntheticpathway still requires other various molecular considerationsto be made, such as post-transcriptional level and/orprotein levels. Therefore, for the accurate detection ofaflatoxigenicity, immunological or cultural methods shouldbe applied. In this study, quantitative HPLC results showedthat the production of aflatoxin B1 (AFB1) and aflatoxin B2(AFB2) by aflatoxigenic A. flavus strains was 114 µg/ml and21 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 3aflatoxigenic A. flavus are shown in Fig. 3. In the case ofthe 51 isolates with three bands of PCR amplicons (omtA,omtB, and ver-1), their culture contained no detectableTable 3. Aflatoxin production by 56 Aspergillus oryzae/flavusgroup 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. 7. FUNGAL FLORA IN A MEJU CONTAMINATED WITH AFLATOXIN 17460.4 ng/g of AFB1 (with no detectable amount of AFB2), was isolated on both DRBC and DG18 media. Therespectively (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 coloniesFor reliable mycological analysis of food or environmental and microscopic morphological characters of the 16samples, the choice of culture media is important. In this representative isolates are shown in Fig., 2 types of media (DRBC and DG18) were used to To date, researches performed in Korea on the mycobiotaallow more complete recovery of fungal species present in that colonize meju have shown that the predominant isolatesthe meju sample of which the surface was dried and the were of A. oryzae/flavus group, Mucor, and Penicilliuminside was somewhat wet. A total of 16 fungal species were spp. [5, 21, 22]. Those data were in accordance with thefound in the meju sample after molecular analysis and mycological results found in this study when analysis wasaflatoxigenecity confirmation (Table 4). The use of DRBC conducted with DRBC. However, A. candidus, the mostmedium made it possible to isolate 12 species with the common species in this study, was not reported to be foundthree 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 topresented 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 enablesfrequent species. A. acidus, P. chrysogenum, P. oxalicum, the isolation and enumeration of fungal flora from driedM. 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 onthe 230 isolates, the predominant fungal species was A. the fungal frequency in meju, which is a starter material incandidus (34.3%), which is known to be a xerophile, Korean fermented soybean products. The molecular assaysfollowed by fungi that grow at a relatively high aw, such as of fungal β-tubulin and the ITS sequence were conductedA. 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 allowedflavus had an occurrence level of 2.2%, and this species more complete recovery of fungal species present in theTable 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 100a Dichloran glycerol 18% agar.b Dichloran rose bengal and chloramphenicol agar.
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