Miso is a traditional Japanese seasoning paste produced by fermenting soybeans using the power of koji mold. A recent Japanese cohort study has shown that increased consumption of fermented soybean products is associated with a reduced risk of death in both men and women. In this review, we briefly explain what miso means in the Japanese culture and food industry, varieties of miso available today, and steps involved in miso making. Then, we review early and latest scientific researches in koji mold species, their safety, and beneficial enzymes they produce during fermentation and maturation processes, which play a major part in determining the quality and sensory profile of miso.

Aims: Prolyl aminopeptidase (PAP) degrades only amino‐terminal proline from peptides. The food‐grade fungus Aspergillus oryzae produces this enzyme only in small amounts. In this paper, we present efficient production of recombinant PAP with an overexpression system of A. oryzae and characterization of its biochemical properties. Methods and Results: The gene encoding PAP was overexpressed as a His‐tag fusion protein under a taka‐amylase gene (amyB) promoter with a limited expressing condition in A. oryzae. The PAP activity in the mycelia grown in rich medium containing glucose (repressing condition) was twice that in starch (inducing condition). The enzyme prepared as cell‐free extract was partially purified through two‐step column chromatography. The PAP was estimated to be a hexameric protein and exhibited salt tolerance against NaCl of up to 4 mol l⁻¹. Conclusions: Aspergillus oryzae PAP was produced under the repressing condition of amyB promoter in a PAP‐overexpressing strain and purified 1800‐folds. Overproduction of PAP under promoter‐inducing conditions led to an increase in inactive PAP, possibly because of irregular folding. Significance and Impact of the Study: PAP with a high specific activity and salt tolerance may be used effectively in the manufacturing processes of fermented foods.

Aims: Aspartyl aminopeptidase (DAP) has a high degree of substrate specificity, degrading only amino‐terminal acidic amino acids from peptides. Therefore, attention is focused here on the efficient production of this enzyme by a recombinant Aspergillus oryzae and characterization of its biochemical properties. Methods and Results: The gene encoding DAP was overexpressed under a taka‐amylase gene promoter, with His‐tag linker in A. oryzae, during cultivation in a Co²⁺‐containing medium. The enzyme was extracted from the mycelia and purified with immobilized nickel ion absorption chromatography using a buffer containing cobalt ion and imidazole. The active fraction was further purified with gel filtration chromatography. The resultant, electrophoretically pure enzyme displayed a molecular mass of 520 kDa. This enzyme displayed high reactivity towards peptide substrate rather than synthetic substrates. Conclusions: Recombinant A. oryzae DAP was purified to homogeneity with an increased specific activity, when cultivated in a Co²⁺‐rich medium. Moreover, the use of suitable metal ions in microbial cultivation and purification processes may help in increasing the specific activity of other metalloproteases and their functional analysis. Significance and Impact of the Study: Recombinant DAP produced using a cobalt ion in culture media of A. oryzae and purification process allow high yield of the enzyme activity.

Koji mold, classified in the genus Aspergillus, is used to produce traditional Japanese fermented foods such as miso, soy sauce, and sake. In recent years, the application of koji mold to cheese ripening has attracted attention, and cheese surface-ripened with koji mold (koji cheese) has been studied. In this study, to evaluate the taste characteristics of koji cheese, an electronic tongue system was employed to measure the taste values of cheese samples ripened using 5 strains of koji mold in comparison with commercial Camembert cheese. All koji cheese samples exhibited lower sourness and greater bitterness, astringency, saltiness, and umami richness than the Camembert cheese samples. The intensity of each taste characteristic differed depending on the koji mold strain. These results indicate that koji cheese has a different taste value than conventional mold-ripened cheese. Furthermore, the results also indicate that various taste characteristics can be achieved by selecting different koji molds.