Analysis of the MAT1-1 and MAT1-2 Gene Ratio in Black Koji Molds Isolated from Meju

Aspergillus luchuensis is known as an industrially important fungal species used for making fermented foods such as awamori and shochu in Japan, makgeolli and Meju in Korea, and Pu-erh tea in China. Nonetheless, this species has not yet been widely studied regarding mating-type genes. In this study, we examined the MAT1-1 and MAT1-2 gene ratio in black koji molds (A. luchuensis, Aspergillus niger, and Aspergillus tubingensis) and in Aspergillus welwitschiae isolated from Meju, a fermented soybean starting material for traditional soy sauce and soybean paste in Korea. The number of strains with the MAT1-1 locus was 2 of 23 (A. luchuensis), 6 of 13 (A. tubingensis), 21 of 28 (A. niger), and 5 of 10 (A. welwitschiae). Fungal species A. tubingensis and A. welwitschiae showed a 1 : 1 ratio of MAT1-1 and MAT1-2 mating-type loci. In contrast, A. luchuensis revealed predominance of MAT1-2 (91.3%) and A. niger of MAT1-1 (75%). We isolated and identified 2 A. luchuensis MAT1-1 strains from Meju, although all strains for making shochu in Japan are of the MAT1-2 type. These strains may be a good resource for breeding of A. luchuensis to be used in the Asian fermented-food industry.

The Mycobiota of Air Inside and Outside the Meju Fermentation Room and the Origin of Meju Fungi

The fungi on Meju are known to play an important role as degrader of macromolecule of soybeans. In order to elucidate the origin of fungi on traditional Meju, mycobiota of the air both inside and outside traditional Meju fermentation rooms was examined. From 11 samples of air collected from inside and outside of 7 Meju fermentation rooms, 37 genera and 90 species of fungi were identified. In outside air of the fermentation room, Cladosporium sp. and Cladosporium cladosporioides were the dominant species, followed by Cladosporium tenuissimum, Eurotium sp., Phoma sp., Sistotrema brinkmannii, Alternaria sp., Aspergillus fumigatus, Schizophyllum commune, and Penicillium glabrum. In inside air of the fermentation room, Cladosporium sp., Aspergillus oryzae, Penicillium chrysogenum, Asp. nidulans, Aspergillus sp., Cla. cladosporioides, Eurotium sp., Penicillium sp., Cla. tenuissimum, Asp. niger, Eur. herbariorum, Asp. sydowii, and Eur. repens were collected with high frequency. The concentrations of the genera Aspergillus, Eurotium, and Penicillium were significantly higher in inside air than outside air. From this result and those of previous reports, the origin of fungi present on Meju was inferred. Of the dominant fungal species present on Meju, Lichtheimia ramosa, Mucor circinelloides, Mucor racemosus, and Scopulariopsis brevicaulis are thought to be originated from outside air, because these species are not or are rarely isolated from rice straw and soybean; however, they were detected outside air of fermentation room and are species commonly found in indoor environments. However, Asp. oryzae, Pen. polonicum, Eur. repens, Pen. solitum, and Eur. chevalieri, which are frequently found on Meju, are common in rice straw and could be transferred from rice straw to Meju. The fungi grow and produce abundant spores during Meju fermentation, and after the spores accumulate in the air of fermentation room, they could influence mycobiota of Meju fermentation in the following year. This could explain why concentrations of the genera Aspergillus, Eurotium, and Penicillium are much higher inside than outside of the fermentation rooms.

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.

Erratum: Mycoflora of Soybeans Used for Meju Fermentation

There was a spelling error in the Table 2.

An Integrated Database and Web Service for Microbial Resources at KACC

The Korean Agricultural Culture Collection (KACC) has developed a web-based system to provide an integrated database with information updates about microbial resources. This integrated database consists of 5 major functions and contains general information, which includes identification numbers, culture media composition, image information, DNA sequences, patent information, and general forms for ordering and depositing microorganisms. In 2008, KACC started providing characterization information. KACC maintains 9,801 cultures of microorganisms, including 3,296 strains of bacteria, 4,734 fungi, 784 actinomycetes, 64 yeasts, and 923 others.

PCR Assays for Detection of Pseudomonas tolaasii and Pseudomonas agarici

PCR assays were developed to detect Pseudomonas tolaasii and Pseudomonas agarici using primer sets, PTOF/PTOR and PAGF/R23-1R. The primer set, PTOF and PTOR, was designed from the nucleotide sequence of pPTOF2 that showed specificity for P. tolaasii in dot blot previously. For development of primers specific for P. agarici, ITS I regions of seven P. agarici strains were analyzed. P. agarici strains contained from one up to three putative ITS I regions, which were different in size and nucleotide sequence from each other. From the sequence of the band (PaI-III) common to all P. agarici strains, primer PAGF was designed. PAGF was used for forward primer, and R23-1R as reverse primer to detect P. agarici. Multiplex PCR with two primer sets, PTOF/PTOR and PAGF/R23-lR, successfully produced two fragments (PTSF and PASF) specific for P. tolaasii and P. agarici with the mixture of DNA of P. tolaasii and P. agarici.

Erratum: PCR Assays for Detection of Pseudomonas tolasii and Pseudomonas agarici

Volume 28, No. 2, pp.89-92, Table 1 is missing.