Traditional Starter Koji Making Procedures

a. Koji Room The beds in the koji room for making koji should be made of stainless steel with the exception of black bean soy sauce. The frames should be made of stainless steel or other corrosion-resistant materials. The double-walls of the koji room can be made of wood, bricks, concrete, lime panels, or thick panels, and can be filled with rice bran to maintain the temperature. The insulation materials can also be asbestos or aluminum foils. The floor in the room should be made of concrete. On one side of the room is a 1-meter-wide opening with easily opened double-sliding-doors. The room ceiling has 2-3 large easily opened ventilators for the convenience of air exchange. The height of these windows (ventilators) should be 2.2 m. The ceiling temperature is maintained by electrical heating. Temperature of the incubation beds is maintained by passing steam through pipes on both sides of the beds. Lower racks are placed along the walls of the room for easy placements of koji trays.

b. Koji Trays The dimensions of the koji trays vary among locations. Wooden trays 60 cm long, 30 cm wide, and 7 cm deep are common. In recent years, large trays six times the size of the traditional trays or saran sheets are also used. They have the advantage of cleanliness and easy operation.

c. Mold Starter When the raw material for mold starter (cooked barley and wheat bran at ~1:1 ratio, or cooked brown rice and potash) is cooled to about 40°C, it is inoculated and mixed thoroughly. In order to ensure thorough mixing of raw material with small quantity of starter inoculum, a small quantity of broken and roasted wheat is mixed with the starter inoculum thoroughly before spreading onto the raw material. The inoculum is 0.01%. (The larger the inoculum, the shorter the starter preparation time, whereas the smaller the quantity, the longer the incubation time.) The inoculated mixture is then placed on starter trays or in the starter room to incubate, with spore number reaching 8 x 108/g. Two hours after inoculation, about half of the spores will germinate, with mycelia visible after 20 hr of incubation. The optimum incubation temperature is 37-38 °C. The soya starter is different from rice starter: it can be used directly without sieving.

d. Preparation of Soya Koji After the mixing of the raw materials with the starter (ratio of starter to raw material is 0.1% for 4-day-old starter, and 0.2-0.3% for 3-day-old starter), the mixture is then piled on the koji trays. The piles should have the center being higher, about 20-30 cm. Pile height too high may cause temperature too high inside and is not easy to cool down. Therefore, the amount has to be suitable, with less in the summer and more in the winter. The piled koji mixture is then moved to the koji room previously adjusted to 28 °C. Also, the room should have good air circulation, with even moisture and temperature control.

The temperature of the koji mixture will drop at the beginning. This is due to the moisture evaporation, drawing the latent heat out of the mixture. With the progression of mold multiplication, the temperature will increase again. After 20 hr of piling, the temperature reaches 38-40°C, and the mixture requires the first turnover. The purpose is to lower the temperature of the koji mixture, replenish the air, remove the carbon dioxide and other undesirable gases produced in the incubation, loosen up the caked mixture, provide more even distribution of the inoculum, and evaporate off the excess moisture. This process is usually conducted manually; at the same time several shallow horizontal trenches are created to provide more surface to lower the temperature. In order to provide even temperature for each tray, it is necessary to exchange the position of the trays, the upper and inside trays having higher temperatures and the outer and lower trays having lower temperatures. During the process, the ventilators at the ceiling are opened to adjust the moisture and temperature inside the room and to introduce fresh air. The temperature of the koji mixture is about 31-33 °C.

During the initial koji starter-producing period, the temperature is maintained to encourage rapid mold growth. This fungal growth also at the same time produces heat that has to be removed to maintain the temperature not higher than 40 °C. At temperatures higher than 45°C, growth of Rhizopus, Mucor, and Bacillus subtilus may occur and overproduce the koji starter. This will initiate the production of black koji, burnt koji (due to excessive temperature increase with inadequate turnover), natto koji (excess temperature increase causing the natto koji to produce natto odor and ammonia) and results in failure of the soya koji production. Therefore, the bacteria number has to be controlled at 1000 organisms per gram or lower.

Five to six hours after the first turnover, the temperature will increase again to about 40 °C. The mycelia on the surface will turn white with production of characteristic soya koji odor. This is the time for the second turnover. The purpose is the same as the first turnover, but with extra consideration of the air circulation and moisture evaporation. The koji mixture is again shallowly trenched. The temperature will increase again, but most of the moisture will be evaporated. Holding the koji mixture after the second turnover will not increase the temperature that much. When the temperature is over 43 °C, additional turnover is needed to overcome the problem.

e. Finished Koji Twenty hours after the second turnover, the elongation of the mycelia will stop with the appearance of spores. This is the time to terminate the soya koji production process. Holding the koji mixture overnight will evaporate off additional moisture. The soya koji is ready and is called the finished koji. It took three evenings starting from the beginning step to the end, and therefore the finished koji is also called 4-day-koji. Visibly, the surface of the koji is covered with gray mycelia without spores on the surface. However, there are many yellowish green spores inside, with the mycelia extended into the soy beans. The better koji should be drier, without offensive odor. The moisture content is about 26%. The weight of finished koji should be the same as the original weight of the raw materials.

The purpose of making soya koji is to produce strong protease enzymes without offensive odor or taste, with minimum contamination by foreign microorganisms (especially Bacillus subtilis), and minimal loss in raw materials. The soya koji making process should be limited to 45 hr to achieve strong protease activity. It is known that the protease activity can be increased when the soya koji is produced at about 25°C (In general, koji produced at higher temperature has higher saccharifying ability, but lower temperature can produce koji with higher acidic protease activity). However, the optimum growth temperature for the mold is about 33 °C. In the past, koji production took 72 hr (4-day-koji). In order to reduce contamination from foreign microorganism, the soya koji making time is thus reduced to 45 hours (3-day-koji) at an increased temperature of 30°C.

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