Once the koji substrates are prepared, there are essentially two means by which they can be inoculated. One is simply to use a pure culture containing spores of Aspergillus oryzae and/ or Aspergillus sojae. Alternatively, the mixture can be inoculated with 0.1% to 0.2% of a seed culture called tane koji.Tane koji is made by inoculating soaked, steamed, and cooled rice (usually brown rice) with spores of A. oryzae or A. sojae. The inoculated rice is incubated overnight, then transferred and distributed evenly into shallow trays and incubated at 30°C for five to seven days. Intermittent mixing provides aeration necessary for fungi growth. Eventually, the moldy rice mixture is dried and packaged. This tane koji material is then used to inoculate other koji mixtures.

Ordinarily, multiple strains are used to make tane koji, depending on the final product that is manufactured. In general, koji mold strains should produce proteolytic, peptidolytic, and amylolytic enzymes with high activity, and they should sporulate and grow well on their substrate. Some species of Aspergillus are known to produce aflatoxin and other mycotoxins. However, none of the koji strains that have been isolated and studied produce these toxins under production conditions. Finally, koji strains should be genetically stable and produce products having consistent flavor and color properties.

After the koji substrate (rice, soybeans, wheat, barley) has been inoculated with either a pure spore culture or the tane koji, it is mixed and incubated in large rectangular trays or boxes (5 X 12 meters) with a depth of about 30 to 40 cm.Very large producers may use vats that can accommodate even greater quantities. Perforations in the trays enhance air and moisture circulation. Temperature control is particularly important because some proteolytic enzymes tend to be produced at lower temperatures (25°C to 30°C), and amylases are produced at higher temperatures (>30°C to 35°C). Modern manufacturers now rely on temperature programs to maximize enzymatic activities. In fact, the incorporation of continuous cookers, automated mixers, and other modern devices has made it possible to automate the entire koji-making operation so that as much as 3,000 kg of koji can be produced per hour.After two to four days at 30°C, the mass should be completely covered throughout with mold growth.

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