A primer on the biochemistry of miso and soy sauce fermentation

This article sets out to explain what happens to miso and soy sauce when fermenting, i.e. the basic biochemical mechanisms that happen during that time. Don’t be put off by the word biochemistry, you’ll see it’s quite simple! :)

Often when I talk about koji, koji spores, miso, soy sauce, the role of koji in the whole process, I often find that my friends and family members are getting a bit confused, so I figured to do a little write-up of the whole thing.

Where to start? Most people know soy sauce and a few know miso, though the latter mostly in the form of miso soup.

Miso is a fermented paste which consists of usually rice koji, mashed cooked soy beans and a good amount of salt. Every ingredient is vital in its own way, but koji is the most vital.


Koji is the japanese name for Aspergillus oryzae. It is a fungus that is traditionally grown on rice, barley or on soy beans. It produces manyfold enzymes, which are needed to make miso and soy sauce.

A lot of confusion arises from the fact that koji is the name for both the fungus and for the substrate (rice, barley, soy, etc.) on which the fungus has been grown on. The latter usually carries the name of the substrate in front of it, i.e. rice koji or barley koji.

Koji Spores – basically the “seeds” of the fungus
Rice Koji – This is rice on which the fungus has been grown.

The basic biochemical mechanisms that are behind ferments involving koji

In some way, fungi are more similar to animals than to plants in their way of “feeding” themselves. Fungi, like animals, use organic matter to generate energy. In biological terms: they are heterotrophes (plants on the other hand are autotrophes, they generate organic matter from inorganic matter using sunlight).

When we create a batch of miso we make use of this fact. Because the fungus can’t just use the complex molecules that plants make (starches, proteins, cellulose, fats, etc.), the fungus has to produce enzymes to digest them. Enzymes are basically “worker molecules” that break down these macro molecules to usable molecules. This is very much analogous to what happens in our digestive tracts.

These enzymes are at the heart of ferments involving koji. There are enzymes that break down starches to sugars, a process that also makes possible the brewing of sake. Starch is basically a very long chain of glucose molecules that are connected. The enzyme amylase (all enzymes have an -ase suffix) severs those connections to yield single glucose molecules.

This is also what happens if you chew white bread for long enough: the amylases in the mouth break down the starch to yield glucose, thus the mush will start to taste sweet, try it! (if you haven’t already at some point in your life)

Actually it "hydrolyses" it. Every time a glucose molecule is cut off, one molecule of water is used.

(This is more an illustration of principle rather than actuality. The starch is not exclusively broken down only to glucose, but also to maltose (two connected glucose molecules) and to other dextrins (2-20 glucose molecules).)


Analogously, proteins are macro molecules made of amino acids. Unlike starch, which is made exclusively of glucose, proteins are made of 22 different kinds of amino acids.  Koji produces enzymes to break down these proteins to amino acids.

Protease breaking down a protein – basically a chain of amino acids – into its constituent parts

Why miso and soy sauce taste the way they do

So Koji is grown in order to get a substrate with lots of enzymes in it. (Well, and also because it tastes amazing) The enzymes are then let loose on whatever the miso is made with, traditionally soy beans.

Soy bean protein by itself tastes very dull in my humble opinion, but if enzymes break that protein down into its constituent amino acids, the taste changes from dull to delicious, imparting an umami taste.

This is because the soy protein contains a lot of glutamic acid, the salt of which you might know as MSG – mono sodium glutatmate. Many people get an “oh but MSG is bad” reflex, but in my opinion the problem with MSG is not the MSG itself, but the fact that it is mostly used by industrial food producers and cheap restaurants to mask the low quality of their foods.

The umami of MSG is very one-dimensional, while the umami of miso is very much multi-dimensional due to the multitude of different amino acids and fermentation products. It’s not a single compound, miso consists of a huge array of different compounds that create a depth of taste that a single isolated compound just can’t. It would be wrong to reduce the magic of miso fermentation to MSG, since there are so many things going on.

The function of salt and salt tolerant microbes

Another very important ingredient is salt. Without salt the result after a few months of fermentation would not be miso, it would be compost. Salt inhibits many unwanted microbes. Actually the koji fungus does not tolerate the levels of salt required for making miso. So once the miso is mixed, the fungus dies but its enzymes (which are salt tolerant) remain.

Some microbes do tolerate these levels of salt (in a miso that’s about 8-13%), for example lactobacilli and some yeasts. The former produce lactic acid (which lowers pH) and other compounds that add to the characteristic taste of miso. Salt-tolerant yeasts produce alcohol and other compounds that add to taste. The alcohol too is turned into other compounds.

It is possible to make soy sauce on the fast track. Some producers do that by chemically breaking down these macro molecules, i.e. proteins and starches, by boiling them with hydrochloric acid.  This process misses the long ageing and the additional microbes described above that make miso and soy sauce to the wonderful foods that they are. The producers know that and make up for it by using aromas, coloring agents and whatnot.

These fast-track sauces always fall short in blind tasting tests. So the message is clear: long term fermentation is the noble path to deliciousness!


If you have questions or if you found some fault with my explanation, please let me know. Thanks for reading!


This Post Has 32 Comments

  1. Hi Viktor,
    Someone sent me miso made by Musu (Japan) and is the unpasteurized genmai branded Yamaki. It traveled overland for 5 days in hot summer conditions in a truck, no dry ice . I am new to miso. It looked dark red, no funny colored colonies, but the entire package smelled a bit like alcohol. Is it still healthy?
    Thanks for your help.

    1. Hi Julia,

      the miso is still perfectly edible. Miso won’t go bad in high temperatures, but unpasteurized misos will start to ferment again, and if it’s really warm, they’ll even develop alcoholic smells. Once you put it into your miso soup, the smell should be gone :)

  2. Hi Viktor
    I stumbled upon this article because I’m trying to find out if dry aging meat with miso paste is somewhat the same as if I did with rice koji, I loved what you wrote and people have very interesting questions. What do you think would be better? Dry aging with miso paste? Dry aging with dry powdered rice koji, with wet rice koji or directy with koji spores? Thank you so mucho for taking the time to explain.

    1. Hi Diego,

      I hardly eat any meat, so naturally I don’t experiment with it much. I know that Jeremy Umansky knows a lot about koji + meat. He recently wrote a book, you’ll find the information you are looking in it for sure :)

      But I feel that dry koji rice is best. With miso you may run danger of introducing too much salt.

  3. Hi Victor,
    Im planning to experiment with making vinegars and would like to incorporate koji into the process.
    I’ve been using your spores to make soy sauce and miso with great success.
    Do you know if vinegar will kill the enzimes in the Koji?
    I’m just wondering for instance if I could add koji and rice to vinegar (or a soy sauce mash to vinegar) and let the koji ferment in the vinegar without salt?

    1. Hi Duncan,

      to be honest I don’t have much experience with using koji at really low pH, but I think it should work nonetheless – soy sauce can be very acidic as well and the enzymes work just fine.
      I believe it could be a worthwhile experiment to add rice koji to vinagar that is almost done fermenting. You might get a sweet vinegar, kind of like balsamic? For sure try it and let us know if it worked :)
      The low pH by the vinegar should protect well enough against bad bacteria.

  4. Hello!
    I’ve noticed some white spots in my shoyu moromi. I have heard that it might be protein crystals. Should I worry about them? I used soy sauce koji spores which have a high level of protease and that causes a lot of protein activity right?

  5. Hi Victor,
    Thanks for the solid info on this site, it`s like a calm island in the stormy ocean of internet opinions.
    Would you be so kind to share with us some of your tried recipes for making the miso paste- it would be great to learn the differences in the process of how you make the sweet miso( 7% salt) and the salty miso( 14-20% salt).

    1. Hi Vladimir,

      Thanks for the kind words!

      I have been planning for some time to make an article on how to make miso. In these I will explain this question with more detail. But to give you a short answer, I usually tend to mix Koji and cooked soybeans at a ratio of 50:50. The sweet miso has 7% salt as you said, and for the Mugi Miso we always give it 12%. Due to the higher salt content, we age it for much longer as the microbiological processes are slowed down. (8 months for the mugi at least, and 2 months for the light miso).

  6. I think it’s a good article. I have corrected some nutritional concepts through it, but the question remains:
    Dose miso contain alcohol?

    1. Hi,
      Miso will contain slight traces of alcohol due to the fermentation. I would estimate about 0.3%. But if you heat it in soup, it should evaporate. And there’s no way anybody would feel its effects, even unheated :)

  7. In Noma fermentation guide, they recommend to use aspergillus luchuensis ALBINOS. Is this one you sell ? Or is it the black one?
    Nelly C.

    1. Hi Nelly,

      yes, it is the albino strain!

  8. is there an optimal temperature range to ferment miso and shoyu?\
    Thanks, your website is very helpful!

    1. Hi Gus,

      It is best to just keep them at room temperature, in my opinion. Warmer than that and your miso and shoyus are prone to become quite yeasty. It is a matter of taste, but room temperature ferments just taste cleaner and are more balanced, in my opinion.

  9. Also, is it harmful to consume the koji itself? You mentioned that whan adding the salt to the miso, it dies off.
    So is there a problem eating it?

    1. And one last thing… If i may… :)
      I heard of “Nama Miso” – unpasteurized miso that continues to ferment in it’s container, after buying and opening it.
      If i’m buying my miso at the store, whould you recommend the unpasteurized kind, over the pasteurized kind, if i want to get as many nutrients, enzymes and probiotics from it?

      I know you said enzimes won’t survive the stomec acidity, but don’t we have enzymes in our stomecs to begin with?

      Also, it’s fair to assume that we lose enzymes, probiotics and nutrients after they are being heated and pasteurized, and there gor changed, right?

      Or maybe pasteurized miso is healthier due to reduction of bad bacteria?

      Tnx so much again.

      1. If you are after probiotics, you should get unpasteurized miso. But I found that most miso is actually unpasteurized, I think it’d be hard to find pasteurized miso.

        I know you said enzimes won’t survive the stomec acidity, but don’t we have enzymes in our stomecs to begin with?

        There are a lot of different enzymes in nature, with very different properties. I don’t know if we have a lot of enzymes in our stomachs. Our digestive enzymes do their work in the small intestine. There the pH is much higher than in the stomach, so the enzymes can do their work there.

        You will lose the enzymes and probiotics if you heat the miso more than 50-60°C. You won’t lose many nutrients, some vitamins, maybe.

        Or maybe pasteurized miso is healthier due to reduction of bad bacteria?

        There are no bad bacteria in miso due to the high amount of salt.

    2. It is not harmful to eat Koji.

  10. Hey Victor and thank you for your post!

    I wanted to ask, in terms of probiotics and enzyme benifets, and in terms of available amino acids and nutrient content, why not just eat the rice koji itself? Is miso better in those terms?
    Whats the difference, regarding probiotics, enzymes and nutrients, between eating the koji by itself (or rice koji), or eating miso?

    Also, whats the difference between dried rice koji and the “wet” one , and which is better activity-wise or better at it’s nutrient availabilty potential?
    Would it be helpfull, health-wise, to consume one of the koji versions, as is?

    Does the enzymes in the koji (or rice koji) “stops working” when dried? And how to reactivate them, if so?
    Does the probiotics in koji (or rice koji) die when dried? And can i revive them, if so?

    Thank you so much and sorry if my english is not the best, it’s not my first language.

    Best wishes!

    1. Hi Sophia,

      I wanted to ask, in terms of probiotics and enzyme benifets, and in terms of available amino acids and nutrient content, why not just eat the rice koji itself? Is miso better in those terms?
      Whats the difference, regarding probiotics, enzymes and nutrients, between eating the koji by itself (or rice koji), or eating miso?

      Consider that miso is (usually) made with soy-beans additionally to the rice koji. Soy has much more protein than rice, so you are getting more free amino acids that taste great, and you get more protein generally. Miso only made from rice wouldn’t work very well since rice is mostly carbohydrates.
      Miso is probiotic, because during fermentation a lot of Lactic Acid Bacteria grow. Koji itself is NOT probiotic – it is, ideally, only A. Orzyae, which can’t live in our guts.

      Also, whats the difference between dried rice koji and the “wet” one , and which is better activity-wise or better at it’s nutrient availabilty potential?

      One still has its water, while the other is dried. The dried one probably has less enzymes since you lose some in the drying process. We dry ours at low temperatures to make sure to keep as much of the enzymatic potential as possible.

      Does the enzymes in the koji (or rice koji) “stops working” when dried? And how to reactivate them, if so?
      Does the probiotics in koji (or rice koji) die when dried? And can i revive them, if so?

      Enzymes need water to function. So the dry koji needs to be rehydrated. Since Koji is not probiotic, there is nothing to revive in that regard.

  11. Hi Viktor,
    I am wondering if any other fungus could have a similar effect as aspergillus oryzae, in particular cultures used for tempeh (typically, rhizopus oligosporus and rhizopus oryzae), so that they could be used to make a similar product as miso.
    The tempeh cultures also have enzymatic activity, both on beans and grains (and also seeds, nuts, etc) but I do not know if it is similar to the activity of koji.
    I also do not know if the presence of salt is sufficient to stop their activity or if heating would be necessary.
    Have you ever tried using a different fungus?

    1. Hi Fred,

      Sorry for the late reply!

      Theoretically you could use Tempeh instead of Koji, but I think the difference in enzymatic activity is going to be quite noticeable, as Rhizopus is going to produce less enzymes than Aspergillus. Generally, Aspergillus molds are exceptionally good producers of enzymes, and A. Oryzae probably especially so as it has been bred for that purpose.
      An acquaintance of mine makes accelerated miso by mixing rice Koji with soy (or chickpea) Tempeh. I have not tried that yet, but according to him it speeds the process up, since there are more enzymes present.

      Most molds are quite salt tolerant, but I don’t know just how salt tolerant the Tempeh molds are. But if you make miso the conditions for growth are pretty bad anyway (no air, layer of salt on top). Heating is not recommended, it deactivates the enzymes.

      Kind regards,

      1. Thanks Viktor, it seems that testing is required to find out how well this could work!

  12. I would like to buy rice koij. Where?
    Do i write you?
    What are the quantities and the price for France?

    1. Hi,

      Rice Koji is not yet available at this point. I will be able to offer rice Koji by April 2019. Until then, you can buy the spores and try to make your own Koji.


      1. Hello Viktor,
        my previous message was actually for you, my mistake.

        Could you kindly answer when possible?

        Thank you very much,

  13. Hi, I would like a conclusive, both traditional and biochemically supported answer on the long-time Q about USE of miso in soup: i do recall well-known late Macrobiotic teacher Michio Kushi recommending adding diluted miso (traditionally fermented &aged) to soup and SIMMER very few minutes on low flame before consuming. Most modern cookbooks instead say miso should be put directly in plate or diluted in broth after TURNING OFF heat so as to not destry enzymes. A miso-maker here (italy) tells me that the first is probably better, as enzymes are heat sensitive enough already around 50°C so they disappear in both cases anyway, while miso re-heated briefly can be more digestible, ditto for Shoyu or Tamari. Also, most makers re-heat miso in the jar to slow down further fermentation…The benefit to our inner gut Flora would then be of a PRE-BIOTIC effect of M. that will stimulate anew healthy bacteria production.
    Can you please tell me your findings or ideas?

    1. Hi Roberto,

      I think for the enzymes there is no hope to survive, first because of the heat of the soup and secondly because of the stomach acid. So when I cook a starchy sauce I make sure to deactivate the enzymes. Or when I make polenta, for example. I once put miso into polenta directly after cooking it, when it was still at about 90°C, and after half an hour to an hour the polenta was really soupy –> the amylases were still active, so the heat is maybe not too big a problem. The stomach acid, however, would denature the enzymes for sure. So I don’t believe that there is a positive effect on our health because of the enzymes.

      The enzymes make a difference in cooking, though. If the food I make is starchy, I cook the miso in the food, if it isn’t (soup for example) I add it later in order to preserve more of the aroma.

      As for prebiotics, I have not read much about it regarding miso. I do recall that there is one compound that is found in miso that was believed to be prebiotic, but I remember reading that it is actually digested by the body to a degree of 70-80% or so. If there are any other prebiotic compounds, I don’t know, but there probably are some.
      Frankly, the reason I am into these foods so much is because like the taste so much :) I don’t stress about healthy eating questions too much. I just make sure to cook often and not to eat too much crap =)

    2. hello Roberto,
      during my research for cultured plant-based cheese, I found an artigianal cheese maker in Berlin (Anderson Santos A GUIDE TO VEGAN CHEESE The Cashewbert® Manual) using rice and bean koji to help the fermentation process (the breaking process you talk about above).

      below some e.g. of their instructions:
      “…Adding up to 10% of the weight of the nuts of koji to a cheese, can help it to break down proteins and fats to create flavours in some types of semihard cheeses…”
      “… put 20g of koji rice in a blender and make it into a powder.
      Place the curds directly into a press cheese form,
      alternating with koji powder and stir gently to mix. Press…”

      As an expert in the field involved, do you think it will work? Can it really be an alternative use of koji?

      I’m a beginner, experimenting the cheese culture at home, and I would like to avoid too much waste (following a recipe on the most advanced book in the market, I double fermented an almond medium with miso, after the first fermentation with probiotics from capsules, I could not go ahead and I had to bin it….).

      It will be great to have your opinion about it, it might be interesting for vegan people…

      Thank you

      1. Hi Daniela,

        I see, I wasn’t sure if it was meant for me or for Roberto :)

        I am pretty sure that that would work! I am not very acquainted with how the vegan cheeses are made, but if there is enough water present (just damp is enough), then the enzymes can do their magic and break down the fats and proteins of the nuts. If the resultant parts (i.e. the amino acids, smaller chain proteins, free fatty acids) then really remind of cheese is doubtful to my mind, as they are quite different from milk-fats and proteins.
        But for sure it is going to make the nut-cheese more flavorful! I have made a chashew miso once, and it got really nice buttery notes. So that’s for sure a thing that’d be great to try.

        Kind regards,

        1. Thank you very much Victor, I will try.
          Pity your Koji rice is not ready yet, in April I might contact you to see whether it is available.
          Best regards,

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