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I want to understand the process by which koji rice is not effected by Bacillus Cereus et.al.

The basic instructions for fermenting your own koji rice is:

  • Steam rice
  • Let it cool to around 35℃
  • Inoculate with Aspergillus Oryzae
  • Keep the rice warm (34-36℃) for ~5 days
    • Occasionally separate and aerate the rice grains during this time.

Some government food safety sites report issues with bacterial-caused toxicity if cooked rice is not cooled quickly before storage. Obviously the fermentation procedure above purposefully keeps the cooked rice at a high room temperature.

Koji rice is often used in situations where it is not re-cooked (e.g.: Amazake, Sake). Millions of people eat koji rice derived products every day without issue, so one must infer that it is a safe product.

Is there some biochemical process caused by the fungal growth of A.Oryzae that is preventing the growth of B.Cereus ?

Further reading: https://www.sciencedirect.com/topics/food-science/koji

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    I'd guess that it's a combination of acidification and breakdown of the rice starches, but I don't have references.
    – FuzzyChef
    Sep 19, 2022 at 15:38
  • Why are you asking for "a biochemical process"? You seem to be hunting for a single reason, which likely doesn't exist. If you throw around a few orange seeds into a beech forest, you won't get an orange grove, not because there is a biochemical reaction, but because the environment is not suitable to be overtaken by the new species. This is how all fermentation works, you just fill the environmental niche with whatever you need, and so the usual pathogens can't get a foothold.
    – rumtscho ♦
    Sep 20, 2022 at 7:05
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    @rumtscho "Outcompetition by the desired culture which fills the environmental niche" certainly sounds like the "bio" part of a "biochemical process" to me.
    – dbmag9
    Sep 20, 2022 at 12:12
  • @dbmag9 the word "biochemical" has a set meaning, actually. An example for a biochemical process would be glycolysis. I don't know if some biochemical process is prominent in the competition between A.Oryzae and B.Cereus, but statistically, that would be a rather unlikely case. The competition itself is, of course, matter of biology, as you pointed out :) but it would be classically studied by microbiologists and food technologists, not by biochemists.
    – rumtscho ♦
    Sep 20, 2022 at 12:22
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    @rumtscho - Well if we were discussing fermentation of say wine/beer, I would answer with something like: "The yeast removes oxygen an acidifies the liquid, making the environment unsuitable to other microorganisms.". To me, that's a biochemical process. If "biochemical" is a reserved word, we can edit it to something else. Do you have a suggestion?
    – Kingsley
    Sep 20, 2022 at 23:57

1 Answer 1

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The biochemical processes for A. oryzae inhibiting the growth of B. cereus involves the production of a range of metabolites that exert antimicrobial properties [1,2,3,4]:

"The exAP-AO17 protein strongly inhibited pathogenic microbial strains, including pathogenic fungi, Fusarium moniliform var. subglutinans and Colletotrichum coccodes, and showed antibacterial activity against bacteria, including E. coli O157 and Staphylococcus aureus." [1] Park et al.

"Besides its potent secretion machinery, A. oryzae is a generous source of various secondary metabolites [...]. Many of the metabolites secreted by A. oryzae have different reported bioactivities such as anticancer, cytotoxicity, antimicrobial, antihypertensive, and antiviral activities (Table 2)." [2] Daba et al.

A. oryzae metabolites
Table of metabolites from Daba et al.

A mutated strain of A. oryzae also produced an unknown compound with antimicrobial effect, suggesting antimicrobial potential outside of the stable domesticated strains:

"Over 3000 EMS-treated, putative mutant A. oryzae cultures were tested in the described screening assay for antibacterial activity. A single EMS-treated A. oryzae isolate, named CAL220, exhibited antibacterial activity in the screening assay as indicated by complete lack of visible MRSA growth in the assay." [3] Leonard et al.

As you're aware, Koji itself is an inoculum rich in enzymes for further fermentation of soy and grains, but also contains easily digestible sugars derived from A. oryzae breaking down the rice [4]. Combined with the near-neutral pH (>6) throughout and increasing at the end of fermentation [5], Koji would be very hospitable to B. cereus without the antimicrobial metabolites. As only a relatively small amount of Koji is used for the second stage of fermentation, the concentration and effect of these metabolites is diluted, hence the need for added salt for pathogen inhibition for this stage.

pH of koji fermentation
Figure of pH over time from Chancharoonpong et al.

Given the innumerable regional strains of A. oryzae in koji manufacture, and other species of fungi involved, there are countless other undocumented metabolite compounds that we're only beginning to identify - and biochemists outside of the field of food technology are very interested in these as well. [6]

But to simplify things, yes, there is a single (group of) biochemical process responsible for A. oryzae outcompeting B. cereus, and it's the production of antimicrobial compounds.

[1] Isolation and Characterization of an Extracellular Antimicrobial Protein from Aspergillus oryzae.
Seong-Cheol Park, Nae Choon Yoo, Jin-Young Kim, Hae Kyun Park, Byung Jo Chae, Song Yub Shin, Hyeonsook Cheong, Yoonkyung Park, and Kyung-Soo Hahm.
https://doi.org/10.1021/jf802373h

[2] The ancient koji mold (Aspergillus oryzae) as a modern biotechnological tool.
Ghoson M. Daba, Faten A. Mostafa & Waill A. Elkhateeb.
https://doi.org/10.1186/s40643-021-00408-z

[3] Random Mutagenesis of the Aspergillus oryzae Genome Results in Fungal Antibacterial Activity. Cory A. Leonard, Stacy D. Brown, and J. Russell Hayman.
https://doi.org/10.1155%2F2013%2F901697

[4] Fermentation and the microbial community of Japanese koji and miso: A review.
Joanne G. Allwood Lara T. Wakeling David C. Bean.
https://doi.org/10.1111/1750-3841.15773

[5] Enzyme Production and Growth of Aspergillus oryzae S. on Soybean Koji Fermentation.
Chuenjit Chancharoonpong, Pao-Chuan Hsieh, Shyang-Chwen Sheu
https://doi.org/10.1016/j.apcbee.2012.06.011

[6] Medical Application of Substances Derived from Non-Pathogenic Fungi Aspergillus oryzae and A. luchuensis-Containing Koji.
Hiroshi Kitagaki.
https://doi.org/10.3390%2Fjof7040243

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  • Great Answer! Reference [4] is a good read too.
    – Kingsley
    Sep 21, 2022 at 0:03

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