Mycoflora of soybeans used for meju fermentation.

Diverse fungi are present in Korean traditional meju and they are known to play an important role in fermented soybean products. To determine the origin of the fungi in meju, we examined the mycoflora of soybeans from 10 traditional meju factories. The samples were untreated or treated with sodium hypochlorite, and placed on malt extract agar (MEA), dichloran 18% glycerol agar (DG18), and dichloran rose bengal chloramphenicol agar (DRBC) medium. A total of 794 fungal strains were isolated and they were identified as 41 genera and 86 species. From sodium hypochlorite untreated soybeans, the genera, Cladosporium (55%), Eurotium (51%), Fusarium (33%), Penicillium (22%), and Aspergillus (exclusion of Eurotium) (20%), were mainly isolated, and Eurotium herbariorum (22%), Eurotium repens (18%), Cladosporium tenuissimum (18%), F. fujikuroi (18%), Aspergillus oryzae/flavus (7%), and Penicillium steckii (6%) were the predominant species. In case of sodium hypochlorite-treated soybeans, Eurotium (31%) and Cladosporium (5%) were frequently isolated, but Aspergillus (excluding Eurotium), Penicillium and Fusarium which were frequently isolated from untreated soybeans, were rarely isolated. Eurotium herbariorum (21%), Eurotium repens (8%), and Cladosporium tenuissimum (3%) were the predominant species. Of the 41 genera and 86 species isolated from soybeans, 13 genera and 33 species were also found in meju. These results suggest that the fungi on soybeans may influence the mycoflora of meju.

The soybean is native to the Korean Peninsula and the Manchurian area [1] and soybean has been one of the major sources of protein in Korean food [2]. Numerous foods are made by soybeans, including doenjang (soybean paste), ganjang (soybean source), cheonggukjang (fast-fermented bean paste), bean curd, soybean milk, bean-curd dregs, etc. [3]. Soybean consumption is effective for the prevention of osteoporosis, arteriosclerosis, strokes and dementia, and can reduce the risk of cancer and obesity [2]. Furthermore, fermented soybean foods are effective in preventing and curing adult diseases [3].

Korean traditional meju is an important food ingredient in Korean cuisine. Meju is made by soaking and boiling soybeans, and then ferment them with various microorganisms such as bacteria, fungi, and yeast [4]. The fungi related to meju fermentation are Aspergillus, Cladosporium, Eurotium, Lichtheimia, Mucor, Penicillium, Rhizopus, Scopulariopsis, etc. [5-7]. Mycoflora of meju could be influenced by its environmental factors such as air, rice straw and soybeans. In particular, soybeans, which are the main component of meju, are contaminated by various fungi, such as Cercospora, Diaporthe, Colletotrichum, Alternaria, Aspergillus, Fusarium, Chaetomium, and Penicillium [8]. The genera, Aspergillus, Penicillium, Fusarium, and Cladosporium have been isolated not only from soybeans but also from meju. Therefore, it seems that there is a relationship between the fungi found on soybeans and those found in meju. The aims of this study are 1) to examine the mycoflora of soybeans used for meju fermentation and 2) to compare these fungal species with those found in meju.

MATERIALS AND METHODS

From November 2011 to February 2012, we collected soybeans used for meju from 10 traditional meju factories. The information of collected soybeans is listed in Table 1.

Table 1

Information of the soybeans used in this study

To elucidate whether fungi exist on the surface or inside of soybeans, soybean samples were either untreated or treated with sodium hypochlorite (0.5%) for 1min. Sodium hypochlorite treated soybeans were washed with distilled water then dried. Fifty kernels were placed (5 kernels per plate) on malt extract agar (MEA), dichloran rose bengal chloramphenicol agar (DRBC), and dichloran 18% glycerol agar (DG18) [9], respectively. After 5~7 days of incubation at 25℃ in the dark, fungi growing on the soybeans and media were transferred to new MEA or DG18 (for xerophilic fungi). The grown fungi were examined with a light microscope and were maintained at 4℃ after transferring to MEA or DG18 slant.

Molecular and morphological methods were used to identify the fungi. To extract genomic DNA, all strains were cultured in malt extract broth or DG18 (for xerophilic fungi). After harvesting the mycelium, genomic DNA was extracted using the DNeasy Plant Mini Kit (69106; Qiagen, Hilden, Germany) according to the manufacturer's instructions, and then stored at -20℃ until use. For identification, several genes were analyzed depending on the genus: the partial sequence of the beta-tubulin gene [10] for Aspergillus, Eurotium and Penicillium; the partial sequence of the actin gene [11] for Cladosporium; the partial sequence of the elongation factor 1-alpha gene [12] for Fusarium, and an internal transcribed spacer of nuclear ribosomal DNA [13] for the other genera. Morphological characteristics of all strains were analyzed according to the methods of Samson et al. [14] and Pitt and Hocking [15].

RESULTS AND DISCUSSION

A total of 794 fungal strains were isolated from 93% of sodium hypochlorite untreated soybeans and from 37% of sodium hypochlorite-treated soybeans, and were identified as 41 genera and 86 species (Table 2). There was no correlation between mycoflora with the region of collection or the soybean cultivar (Daewonkong and Taekwangkong).

Table 2

List of fungal species from soybeans and their isolation frequencies

aThe RDA numbers are DNA sequence accession number of Korean Agricultural Culture Collection (KACC). Readers can access to the sequence from information of corresponding KACC No. in KACC homepage (http://www.genebank.go.kr).

bThe species were isolated from meju, with ***high frequency, **medium frequency, or *low frequency.

cThe frequency indicates the number of soybeans from which the species were isolated from 500 soybeans.

dThe number indicates the number of factories from which the species were isolated from 10 factories.

The occurrence and frequency of fungi differed according to the media. Most fungi grew well on DRBC, and Pitt and Hocking [16] suggested that DRBC is adequate for the numeration of fungi on food and feed. In this study, the number of isolated fungi and the incidence on soybeans were based on fungi grown on DRBC. However, Eurotium did not grow well on DRBC, but did grow well on DG18. Therefore, the number and incidence of Eurotium were from fungi grown on DG18, which is medium for xerophilic fungi [9].

In the case of untreated soybeans, 32 genera and 68 species were isolated (Table 2). Cladosporium was isolated from 273 of 500 soybeans (54.6%), and from 9 of 10 factories. Eurotium was isolated from 254 of 500 soybeans (50.8%) and from 7 of 10 factories. Fusarium, Penicillium, and Aspergillus (excluding Eurotium) were isolated from all 10 factories and their incidences on soybeans were 32.8%, 22.4%, and 20%, respectively. All other genera were isolated from less than 4% of soybeans. Although Alternaria (3.4%), Rhizopus (3%), Phoma (2.8%), Phomopsis (1%), Cheatomium (1%), and Epicoccum (1%) occurred infrequently, they were isolated from several factories. Lichtheimia (3.8%) and Monascus (0.6%), each were isolated from 2 factories. All other genera were isolated from only 1 factory. Eurotium herbariorum (22.4%), E. repens (18.4%), F. fujikuroi (anamorph of Gibberella fujikuroi) (18.4%), and Cladosporium tenuissimum (18.2%) were major species isolated from untreated soybeans, followed by C. pseudocladosporioides (9.4%), C. cladosporioides (9%), Eurotium manginii (8.4%), Paecilomyces sp. (7.6%), A. oryzae/flavus (7%), A. westerdijkiae (6.2%), and A. versicolor (5.4%).

A total of 28 genera and 52 species were isolated from the sodium hypochlorite-treated soybeans (Table 2). Among these, the most frequent genus, Eurotium, was isolated from 153 of 500 soybeans (30.6%) on DG18, and from 9 of 10 factories. The second most frequent genus was Cladosporium, which was isolated from 27 of 500 soybeans (5.4%) on DRBC, and from 7 of 10 factories. The next frequent fungi were Fusarium (from 8 factories) and Aspergillus (excluding Eurotium) (8), which were isolated from 2.2% and 2% of soybeans, respectively, followed by Cercospora (3) and Botryosphaeria (3). Other genera, such as Alternaria, Chaetomium, Penicillium, Phoma, and Phomopsis, were isolated from less than 1% of soybeans, but were isolated from quite a few factories. E. herbariorum (21.2%), E. repens (8%), and C. tenuissimum (2.6%) were the major species isolated from sodium hypochlorite-treated soybeans, followed by Cercospora sp. (1.2%), B. dothidea (1%), and C. pseudocladosporioides (1%). Other species were isolated from less than 1% of soybeans.

Fourteen species of Aspergillus (excluding Eurotium) were isolated from untreated soybeans, and A. oryzae/flavus, A. westerdijkiae, and A. versicolor were the major species isolated from soybeans (Table 2). Of these, A. oryzae/flavus is one of the major fungi for meju fermentation, whereas A. westerdijkiae and A. versicolor are considered simple contaminants of meju that are found very infrequently (unpublished data). Eurotium, the teleomorph of Aspergillus section Aspergillus, were frequently isolated from both sodium hypochlorite untreated and treated soybeans on DG18 (Table 2). E. herbariorum, E. repens and E. manginii were the major species. From meju, E. repens and E. chevalieri were the predominant species [5]. Five species of Cladosporium were isolated from both untreated and sodium hypochlorite-treated soybeans on DRBC (Table 2). The Cladosporium spp. from soybeans were not isolated from many factories, but although these species were found on soybeans from a factory, the incidence rate was high. Cladosporium sp., C. tenuissimum, C. pseudocladosporioides, and C. cladosporioides were mainly isolated from soybeans, whereas C. tenuissimum and C. sphaerospermum were predominant in meju (unpublished data). In case of Fusarium, F. fujikuroi (anamorph of Gibberella fujikuroi) and Fusarium sp. were frequently isolated from untreated soybeans (92 and 61 out of 500 soybeans, respectively) on DRBC, however they were rarely isolated from sodium hypochlorite-treated soybeans (Table 2). This means that the species mainly exist on the surface of soybeans and are not pathogens of soybeans. In meju, Fusarium asiaticum is a predominant species (unpublished data). Although 13 species of Penicillium were isolated from soybeans, and P. steckii, Penicillium sp., P. polonicum, and P. oxalicum were the major species, almost of them were isolated from untreated soybeans, and the incidence rates were not high (Table 2). In case of P. polonicum, it is also frequently isolated from meju [4]. Meju is a nutrient rich material for zygomycota, and Mucor, Lichtheimia, and Rhizopus grow well on/in it [6]. However, soybeans are dry, and zygomycota cannot utilize its nutrients. Therefore, zygomycota were rarely isolated from untreated soybeans on MEA (Lichtheimia, 16 of 500 soybeans; Mucor, 1 of 500; and Rhizopus, 15 of 500) and were not isolated from sodium hypochlorite-treated soybeans. Among the other genera, Paecilomyces, Alternaria, Phomopsis, and Phoma were quite frequently isolated from untreated soybeans, and Alternaria, Cochliobolus, Botryosphaeria, and Cercospora, which could be soybean pathogens, were isolated from sodium hypochlorite-treated soybeans.

The fungi isolated from sodium hypochlorite-treated soybeans may be endophytic or pathogenic fungi. Fusarium oxysporum and F. solani were reported as pathogens causing Fusarium wilt, Fusarium blight and root rot of soybeans in Korea [17]. Phomopsis longicolla and Corynespora cassiicola were reported as pathogenic fungi to soybeans in other countries [18, 19]. Additionally, B. dothidea, F. fujikuroi, Khuskia oryzae, and P. oxalicum, were reported as pathogenic fungi in other plants [17]. Therefore, fungi isolated from sodium hypochlorite-treated soybeans may have a potential to cause disease or affect the growth of soybeans. Research about the role of fungi on soybeans will be necessary.

Yum and Park [8] isolated 16 fungal genera from sodium hypochlorite-treated yellow soybeans collected from factories or markets in various regions of Korea, and Cercospora, Diaporthe, Alternaria, Aspergillus, Chaetomium, Fusarium, Colletotrichum, Penicillium, and Cladosporium were frequently isolated. Of these genera, Alternaria, Aspergillus, Cercospora, Chaetomium, Cladosporium, Colletotrichum, Fusarium, and Penicillium were also isolated in this study. However, Diaporthe was not isolated in this study. In contrast, 19 genera, including Eurotium, were only isolated in this study. Pitt et al. [20] isolated 26 genera and 59 species from 49 samples of surface-disinfected soybeans collected from markets in Thailand. Penicillium restrictum, Aspergillus penicilloides, A. restrictus, Cladosporium cladosporioides, Eupenicillium cinnamopurpureum, Eurotium amstelodami, Eur. chevalieri, Eur. Rubrum, and P. citrinum were frequently isolated. Among the 26 genera from the study by Pitt et al. [20], 12 genera, including Alternaria, Arthrinium, Aspergillus, Chaetomium, Cladosporium, Colletotrichum, Epicoccum, Eurotium, Fusarium, Penicillium, Pestalotiopsis, and Phoma, were also isolated in the present study. However, 14 genera, including Curvularia, Eupenicillium, Lasiodiplodia, Macrophomina, Nigrospora, Rhizopus, and Syncephalastrum, were not isolated in the present study. In contrast, 16 genera, including Botryosphaeria, Cercospora, Cochliobolus, Khuskia, Phomopsis, and Stemphylium, were isolated in the present study, but were not isolated in the study by Pitt et al. [20]. The mycoflora isolated in this study were more similar to that of Pitt et al. [20] than that of Yum and Park [8], even though the former used Thai soybeans and the latter used Korean soybeans. In particular, Eurotium was one of the most frequently isolated fungi in the present study and in Pitt et al. [20] however, Yum and Park [8] did not isolate the genus. This is likely caused by the use of different media. Yum and Park [8] only used potato sucrose agar containing streptomycin sulfate in which Eurotium rarely grow, whereas Pitt et al. [20] used DG18, DRBC, dichloran chloramphenicol peptone agar, and Aspergillus flavus and parasiticus agar.

Among the 41 genera and 86 species isolated from soybeans, 13 genera and 33 species were isolated from both soybeans and meju. The fungi on soybeans cannot be directly transferred into meju, because soybeans are sterilized during the boiling process during which producers usually boil the soybeans in water for more than 4hr. Only Bacillus could survive this step (Fig. 1). However, the fungi on the soybeans may move into the air during the washing process, and may be re-inoculated on the meju during or after meju forming process. This study suggests that some fungi on soybeans, such as Aspergillus, Eurotium, and Penicillium, may influence the mycoflora of meju fermentation.

Fig. 1

The incubation results of boiled soybeans on MEA after 7 days at 25℃ in the dark. The soybeans were boiled in an iron pot for 6 hr in a meju factory in Icheon, Korea. Fungi could not grow and only Bacillus strains could grow.

Selected strains from this study have been preserved in the Korean Agricultural Culture Collection (KACC; http://www.genebank.go.kr) and are accessible for future research.