Sake koji starts with rice. Brewers have spent centuries learning how Aspergillus oryzae behaves on that grain. At Blue Marble, we grew koji on hundreds of feedstocks, including pink peppercorns, spent brewer’s grain, ginger, and rice blended with spices. We kept asking what the fungus would do with something different. On rice, its hyphae grow between and inside the cells. Yasui and colleagues measured penetration of about half a millimeter in sake rice polished to half its original mass. The fungus grows into the material while producing enzymes that help break it down. [1]
α-Amylase cuts starch into shorter chains, and glucoamylase releases glucose. Acid proteases break down glutelin, the main protein in rice, and peptidases break the resulting fragments down further, releasing amino acids. These enzymes work outside the fungal cells. By the time the koji moves on, the grain is already partly digested, and the enzymes go with it. [1]
Ken Oda and colleagues compared the proteins A. oryzae released during growth on a solid substrate and in liquid culture. Glucoamylase B and an alanyl dipeptidyl peptidase were detected only in the solid culture. α-Amylase was released into the surrounding material in solid culture but remained associated with the fungal cell wall in liquid. Growing conditions changed where the enzymes ended up and which ones the researchers detected. [2]
In sake, the stages overlap. In the mash, koji enzymes keep turning starch into sugar while yeast turns sugar into alcohol. Brewers call this multiple parallel fermentation. [1] For a staged process, start with what the next organism needs from the first one. Measure the available sugar or the enzyme activity that carries into the next stage, then compare the result with the same process run without the fungus. Oda’s work shows that growing conditions change enzyme secretion. Whether that helps a later organism has to be tested for each feedstock and process.
At Blue Marble, one surprise was finding peptides in the fermentations that acted on the calcium-sensing receptor, CaSR. Published work links CaSR-active peptides to kokumi, including mouthfulness and lingering taste, and to enhancement of sweet, salty, and umami tastes. [3] In our work, we observed taste effects beyond what we were describing as kokumi. That made the peptides worth following. What the fungus left behind could matter to how the finished product tasted.
Sources
[1] Yasui et al. (2020). Invasive growth of Aspergillus oryzae in rice koji and increase of nuclear number. Fungal Biology and Biotechnology.
https://doi.org/10.1186/s40694-020-00099-9
[2] Oda et al. (2006). Proteomic analysis of extracellular proteins from Aspergillus oryzae grown under submerged and solid-state culture conditions. Applied and Environmental Microbiology.
https://doi.org/10.1128/AEM.72.5.3448-3457.2006
[3] Ohsu et al. (2010). Involvement of the calcium-sensing receptor in human taste perception. Journal of Biological Chemistry.
https://pmc.ncbi.nlm.nih.gov/articles/PMC2801228/