INTRODUCTION

Meju is a fermented soybean block used as a key material for preparing soy sauce (ganjang) and soybean paste (doenjang), the popular Korean traditionally fermented foods [1]. Meju is produced from soybeans rich in nutrients, including proteins, carbohydrates, and vitamins; hence it has been a good source of proteins and fat for a long time. Furthermore, it contains numerous essential amino acids and functional materials that are essential for physiological regulation of the body and prevent various diseases such as cardiovascular disorders, cancer, and atopic dermatitis [2].

Meju produced using the traditional procedure is generally prepared by washing soybeans, steeping, steaming, cooling, mashing, molding, drying, and fermentation. During the natural drying and fermentation process of meju, various microorganisms such as bacteria, yeasts, and mold, present in soil or rice straw, are involved in fermentation. These microorganisms can produce a variety of compounds that affect the unique taste and flavor of meju [3]. Therefore, the microbial community is important for the quality of meju and other fermented foods. Traditionally produced meju has the advantage of enabling the production of unique aroma and taste; however, its disadvantage is that the quality is not constant and depends on the fermentation conditions, including temperature, humidity, and time [4]. When meju is contaminated with foodborne bacteria or toxin-producing fungi during fermentation, it can cause various diseases, including food poisoning and cancer. Therefore, it is important to control the microbial community during meju fermentation.

Fungi play a key role in the fermentation of meju [5,6]. Fungi produce and secrete various hydrolytic enzymes such as amylase, protease, and lipase, decomposing macromolecules such as starch and proteins of soybeans to produce the unique taste and flavor [7]. According to previous studies, the major fungal genera involved in the fermentation process include Aspergillus, Rhizopus, Mucor, and Penicillium [6-9]. In particular, Aspergillus species belonging to Aspergillus section Flavi, including A. oryzae and A. sojae, are predominant and key microorganisms for meju fermentation [6,9]. These fungi are nontoxigenic and detoxify during the fermentation process. A. oryzae is dominant during the fermentation of meju, producing reducing sugars, amino acids, and metabolites to improve aroma and taste [6]. A. oryzae is nontoxigenic and cannot produce the carcinogenic mycotoxin aflatoxin B1 [10,11], which is because it lacks the aflatoxin gene cluster. Moreover, A. oryzae detoxifies various mycotoxins, including aflatoxins and ochratoxins [12,13]. Therefore, A. oryzae, also known as yellow koji mold, has been used for meju fermentation and might act as a starter for fermentation.

In this study, we examined the distribution of fungal communities in meju samples during fermentation and final production using metagenomic and morphological analyses. Among the fungal isolates, we investigated the ability to produce amylase, protease, and aflatoxin and propose several candidates that can be used as starters for meju fermentation.

MATERIALS AND METHODS

RESULTS

DISCUSSION

In this study, fungi were isolated and identified from Meju M and Meju F samples obtained from traditionally fermented soybean paste producers in Gyeongsangbuk-do to identify fungal colonies and dominant fungi. Through metagenomic analysis and cultured-based analysis, we found that Aspergillus strains, especially A. flavus/oryzae, are dominant during meju fermentation. Pichia spp., including P. kudriavzevii, were found in Meju M samples but disappeared from the final product. The final meju product contained Rhizopus spp. and Lichtheimia spp.. The results of TLC and PCR showed that the representative strains lack some genes of the aflatoxin biosynthesis gene cluster and may not produce aflatoxin B1, suggesting that the fungi in meju do not produce mycotoxins. Moreover, the amylase and protease activities of these strains revealed that several A. flavus/oryzae strains produce amylase or protease. In conclusion, our results demonstrate the changes in fungal distribution during meju fermentation, and various fungi produce various enzymes such as protease and amylase to promote fermentation. Our findings also confirmed that the meju samples examined in this study were safe from aflatoxin-producing fungi.