Koji Molds for Japanese Soy Sauce Brewing: Characteristics and Key Enzymes

Soy sauce is a traditional Japanese condiment produced from the fermentation of soybeans, wheat, and salt by three types of microorganisms, namely koji molds, halophilic lactic acid bacteria, and salt-tolerant yeast. The delicate balance between taste, aroma, and color contributes to the characteristic delicious flavor imparted by soy sauce. In soy sauce brewing, protein and starch of the raw materials are hydrolyzed into amino acids and sugars by enzymes derived from koji molds. These enzymatically hydrolyzed products not only directly contribute to the taste but are further metabolized by lactic acid bacteria and yeasts to most of organic acids and aromatic compounds, resulting in its distinctive flavor and aroma. The color of the soy sauce is also due to the chemical reactions between amino acids and sugars during fermentation. Therefore, koji mold, which produces various enzymes for the breakdown of raw materials, is an essential microorganism in soy sauce production and plays an essential role in fermenting the ingredients. In this review, we describe the manufacturing process of Japanese soy sauce, the characteristics of koji molds that are suitable for soy sauce brewing, and the key enzymes produced by koji molds and their roles in the degradation of materials during soy sauce fermentation, focusing on the production of umami taste in soy sauce brewing.

Koji Starter and Koji World in Japan

Koji is made by culturing koji mold on grains. Koji has wide-ranging applications, for example, in alcoholic beverages and seasonings. The word ‘mold’ generally has a bad image, but in Japan, koji mold is valued for its usefulness, and over the years, efforts have been made to make safe, stable, and delicious food products from it. Koji mold spores, essential when making koji, are called koji starter in the industry. From the many available strains, those suitable for the production of each fermented food are chosen based on indicators such as growth rate and enzyme production capacity. In manufacturing using microorganisms, purity and yield are prioritized. However, the production of fermented foods using koji is more complex, with focus not only on the degree of decomposition of raw materials but also on factors influencing overall product design, including palatability, color, smell, and texture. Production can be facilitated by the variety of koji brought about by the diversity of koji mold combined with the solid culture method which increases the amount of enzyme production. In this report, we introduce the history of koji starter in Japan, the characteristics of koji mold in practice, and various fermented foods made from it. In addition, the factors affecting the quality of koji in solid culture are described.

The ancient koji mold (Aspergillus oryzae) as a modern biotechnological tool

Aspergillus oryzae (A. oryzae) is a filamentous micro-fungus that is used from centuries in fermentation of different foods in many countries all over the world. This valuable fungus is also a rich source of many bioactive secondary metabolites. Moreover, A. oryzae has a prestigious secretory system that allows it to secrete high concentrations of proteins into its culturing medium, which support its use as biotechnological tool in veterinary, food, pharmaceutical, and industrial fields. This review aims to highlight the significance of this valuable fungus in food industry, showing its generosity in production of nutritional and bioactive metabolites that enrich food fermented by it. Also, using A. oryzae as a biotechnological tool in the field of enzymes production was described. Furthermore, domestication, functional genomics, and contributions of A. oryzae in functional production of human pharmaceutical proteins were presented. Finally, future prospects in order to get more benefits from A. oryzae were discussed.

Dietary fermented products using koji mold and sweet potato-shochu distillery by-product promotes hepatic and serum cholesterol levels and modulates gut microbiota in mice fed a high-cholesterol diet

It has been reported that fermented products (FPs) prepared from sweet potato-shochu distillery by-product suppressed weight gain and decreased serum cholesterol levels in mice under normal dietary conditions. Furthermore, from the information gained from the above data regarding health benefits of the FPs, the aim of this study was evaluating the effects of dietary FPs on lipid accumulation and gut microbiota in mice with or without cholesterol-load in the diet. C57BL/6N mice were fed normal (CO) diet, CO with 10% FPs (CO + FPs) diet, cholesterol loaded (HC) diet, or HC with 10% FPs (HC + FPs) diet for 8 weeks. The mice were then euthanized, and blood samples, tissue samples, and feces were collected. The adipose tissue weight and liver triglyceride levels in the HC + FPs diet groups were significantly reduced compared to that in the HC diet groups. However, FPs significantly increased the serum non-high-density lipoprotein cholesterol (HDL-C) levels, the ratio of non-HDL-C to HDL-C and hepatic total cholesterol levels in mice fed cholesterol-loaded diet compared with that of the HC diet group. Since dietary FPs significantly decreased the protein expression levels of cholesterol 7 alpha-hydroxylase 1 in the HC + FPs diet groups, the cholesterol accumulation in FPs group may be explained by insufficient catabolism from cholesterol to bile acid. In addition, the dietary FPs tended to increase Clostridium cluster IV and XIVa, which are butyrate-producing bacteria. Related to the result, n-butyrate was significantly increased in the CO + FPs and the HC + FPs diet groups compared to their respective control groups. These findings suggested that dietary FPs modulated the lipid pool and gut microbiota.

Modulation of Fatty Acid Composition of Aspergillus oryzae in Response to Ethanol Stress

The koji mold Aspergillus oryzae is widely adopted for producing rice wine, wherein koji mold saccharifies rice starch and sake yeast ferments glucose to ethanol. During rice wine brewing, the accumulating ethanol becomes a major source of stress for A. oryzae, and there is a decline in hydrolysis efficiency. However, the protective mechanisms of A. oryzae against ethanol stress are poorly understood. In the present study, we demonstrate that ethanol adversity caused a significant inhibition of mycelium growth and conidia formation in A. oryzae, and this suppressive effect increased with ethanol concentration. Transmission electron microscopy analysis revealed that ethanol uptake triggered internal cellular perturbations, such as irregular nuclei and the aggregation of scattered vacuoles in A. oryzae cells. Metabolic analysis uncovered an increase in fatty acid unsaturation under high ethanol conditions, in which a large proportion of stearic acid was converted into linoleic acid, and the expression of related fatty acid desaturases was activated. Our results therefore improve the understanding of ethanol adaptation mechanisms in A. oryzae and offer target genes for ethanol tolerance enhancement via genetic engineering.