For correspondence. E-mail: funguy@korea.kr; Tel.: +82-31-299-1866; Fax: +82-31-299-1869 Seung-Beom Hong 1 * , Dae-Ho Kim 1 , Mina Lee 1 , Seong-Yeol Baek 2 , Soon-wo Kwon 1 , Jos Houbraken 3 , and Robert A. Samson 3 1 Korean Agricultural Culture Collection, and 2 Fermentation and Food Processing Division, National Academy of Agricultural Science, RDA, Suwon 441-707, Republic of Korea 3 CBS-KNAW Fungal Biodiversity Centre, 3508AD Utrecht, The Netherlands (Received August 31, 2011 / Accepted November 7, 2011) The Journal of Microbiology (2012) Vol. 50, No. 3, pp. 386–393 Copyright 2012, The Microbiological Society of Korea DOI 10.1007/s12275-012-1437-6 Zygomycota Associated with Traditional Meju, a Fermented Soybean Starting Material for Soy Sauce and Soybean Paste Various zygomycota species were detected during a study of the mycobiota of meju, a brick of dried fermented soybeans, used in Korean cuisine. Two hundred and sixty-eight strains were isolated from 98 finished meju products collected in various regions of Korea from 2009 to 2011, and 96 strains were isolated from in-process meju on various farms from 2010 to 2011. The isolated zygomycota were identified using phenotypic characteristics combined with DNA sequences of the internal transcribed spacer regions of ribosomal DNA and the D1/D2 nuclear ribosomal large subunit. Of 364 zy- gomycota strains, 108 were identified as Mucor circinelloides, 96 as M. racemosus, 60 as Lichtheimia ramosa, 22 as Rhizopus stolonifer, 16 as Lichtheimia corymbifera, and the other 62 strains comprised 10 other species. The psychrotrophic species, Mucor circinelloides and M. racemosus were predominantly present during low temperature fermentation (LTF) and the thermotolerant species Lichtheimia ramosa and Rhizomucor species were predominant during high temperature fermen- tation (HTF). The results suggest that temperature has a large influence on the zygomycota composition during the fer- mentation process of meju. Keywords: meju, zygomycota, Mucor circinelloides, Mucor racemosus, Lichtheimia ramosa Introduction Korean traditional meju (hereinafter meju) is a brick of dried fermented soybeans, which serves as the basis of sev- eral Korean condiments, such as doenjang (soybean paste), ganjang (soy sauce) and gochujang (hot pepper paste), which are essential sauces of authentic Korean cuisine. Meju is naturally fermented and various microorganisms, such as bacteria, yeasts and fungi, are involved in the process. Fungi play an important role in the fermentation and degrade macromolecules of the soybeans into small nutrient mole- cules (Lee, 1995; Lee et al., 1995). However, the fungi of meju have not been determined exactly, and neither have they been preserved in any microbial culture collection (Hong et al., 2011). Therefore, people who work on meju farms, name the fungi by their colors, such as white fungus, yellow fungus, green fungus, black fungus, etc. Recently, the mycobiota of meju was elucidated and Euro- tium and Penicillium were isolated, identified, and preserved (Hong et al., 2011; Kim et al., 2012). However, the zygomy- cota were not treated in those studies, although zygomycota species are known to be important organisms in the produc- tion in various traditional Asian foods. Rhizopus oligosporus is the main producer of tempeh, a traditional soy product in Indonesia. R. chinensis, R. oryzae, and Mucor indicus are also found in tempeh (Dijksterhuis and Samson, 2006). Actino- mucor repens, A. taiwanensis, Mucor circinelloides, M. hiemalis, M. racemosus, and Rhizopus microsporus were isolated from Sufu Pehtze, Chinese molded tofu (Han et al., 2004). Three species of Rhizopus (Yihn and Lee, 1968) and 12 species of zygomycota were reported from meju (Lee et al., 1993, 1995). In the study of Lee et al. (1995), Mucor hiemalis, M. circi- nelloides f. griseo-cyanus, Absidia corymbifera, Rhizopus or- yzae, R. stolonifer, Absidia glauca, and A. spinosa were the main microorganisms in meju fermentation, together with Bacillus spp. and Scopulariopsis brevicaulis, and the zygo- mycota played an important role in the early stage of meju fermentation when a high moisture level was maintained. The objectives of this study were: 1) to elucidate which zygomycota occur and which are the main species in meju, 2) to elucidate during which meju process they grow, and 3) to secure zygomycota strains in biological resource col- lections to provide a starting point for future meju research. Materials and Methods To examine which fungi occur in meju products, we col- lected 98 meju loaves from various regions in Korea as fol- lows: Gangwon (n=6), Gyeonggi (n=30), Gyeongnam (n=3), Gyeongbuk (n=15), Jeonnam (n=5), Jeonbuk (n=28), Jeju (n=4), Chungnam (n=3), and Chungbuk (n=4) from 2008 to 2011. Two isolation methods, namely direct plating and dilution plating (Hong et al., 2011), were used to isolate zy- gomycota from the meju. To determine during which meju process the fungi grow, we visited four meju farms in Gyeonggi province every week from molding process (the end of November, 2010) to the submergence of meju in brine (the middle of February, 2011). Additionally, we visited