As I'm sure my questions indicate - I'm struggling to understand the nature of the Koji mold organism.
I've had collegiate microbial coursework but no relevant experience/experimentation. The key question seems to be "when is the thing making enzymes and when is it alive".
You're starting with mold spores, I'm a first-timer with these organisms and have 1 jar going with inoculated rice bought at the store in the refrigerator section.
I like your description of how you germinate the mold and have a wet inoculated Koji [I think of it as a starter].
It seems clear that the spores themselves don't have any enzymatic action--or you'd never need to germinate them. It would also be biologically weird since the whole purpose of this organism posessing enzymes that break starches into sugars is that it can eat these sugars [unless the yeast get's 'em first!].
So the germinated spores turn into a mold--which like every other mold has long tendrils burrowing into what it's living on. It seems like it's an air breathing organism and "drowns" itself once it breaks down the rice starches so much that they collapse and we see the water coming out. But at this point it's got all these free floating enzymes that do their work until yeast comes along and snaps up the sugars for fermentation. But is that true? Does the yeast die when the water levels rise??
That's what I'd like to know more about. I hear folks trying to make their Koji mold produce spores that are capturing the mold on bread--presumably because the bread structure resists breaking down in the way that the moistened rice does. This argues for the mold only being active in a less than soaking wet environment as I never hear folks talk about the top of their Sake being covered in a fuzzy blue mold, but I can google Koji bread and see that same mold when it's fruiting and producing spores. Or for more google fun, try Aspergillus oryzae & EPA.
But, conversely does that mean the stuff isn't alive?
Enzymes made by the mold are secreted into its environment to do the good dissolving work needed. Would reusing the leftover solid matter at the end of a batch be a sufficient experiment to determine if the mold organism made all the enzyme it was ever going to make early on or if it continued to live and grow [and possibly even reproduce asexually] for the whole time--despite not showing evidence of sexual reproduction, i.e. spores? In such an experiment the enzyme proportion would decrease with each transfer if it was true they were made only initially and not replenished.
Now, enzymes themselves are not exactly getting destroyed [heat], but I gather that they don't last forever either--so it's not as if 10 molecules of beta amlyase are going to rip through kilograms of rice. If that was true then all us beer makers would throw 1lb of malt into 10lb of unmalted grain, cover, and leave till next weekend...
Anyway, lets assume the correct proportions of Koji inoculated rice to regular rice has even TEN TIMES the amount of enzymes required to chop the required starches into sugars. If that were the case then I should expect to be able to reuse my leftovers to make [if not great Sake...then at least some successfully fermented product...] no more than 1 more batch. If the amount of enzyme present was really anything less than 10x the minimum necessary amount even producing 1 subsequent batch should not be successful.
If - on the other hand - there's a living mold organism in there, then there should be little difference in the observed rate of rice turning to sugary liquid [except the yeast would be there at the start].
As I've seen with beer, cheese, etc. - you can't always continue forever with leftovers as you tend to set up a real ecosystem. Meaning that there may be more and more other types of organisms coming along for the ride.
I've got enough storebought Koji rice for a few gallons--but will be working on understanding how this organism [or symbiosis] works to better understand what I'm dealing with. If anyone already has tried this or understands the life cycle please point me in the right direction.