Koji mould that has been cultured and processed into an easy-to-use "seed" for making koji.

~11,000 years ago — wheat farming may have begun around western Iraq (?!).

Cold, dry climate — suited to wheat → cattle and sheep were raised.

~10,000 years ago — rice farming may have begun around Hunan, in China’s Yangtze basin.

Hot, humid climate — suited to rice → water-rich land → fish thrive.

Fallen, crushed grapes fermented by chance. From ~5000 BC it was made in the Caucasus (Georgia), spreading to Mesopotamia, Egypt, Greece, southern Europe and China. ~70% of world grape consumption is for wine.

~5000 BC, on the Central Asian steppe: milk from sheep domesticated by nomads; stored sheep’s milk fermented by chance.

~4000 BC: Mesopotamia had flatbread (unleavened); Egypt had leavened bread (left and forgotten?) → spread to Greece and Europe. Kimuraya’s sake-yeast anpan (using rice koji, 1874) is famous.

~4000 BC, Mesopotamia: a barley porridge left out fermented by chance. Later barley was milled, baked into bread, torn up, added to water and left to ferment naturally.

First recorded in the early-Nara "Osumi-no-kuni Fudoki" (present-day eastern Kagoshima). cf. the film "Your Name."

Starchy food (rice) is chewed until sweet, then spat into a vessel. Saliva’s saccharifying enzyme (amylase) turns starch into glucose; airborne wild yeast enters and ferments it into sake.

The word "kamosu" (to brew) is said to derive from "kamusu" (to chew).

First recorded in the "Harima Fudoki" (present-day southwestern Hyogo). Rice farming (short-grain japonica) reached Japan from southern China (mid/lower Yangtze) around the 4th century BC (late Jomon).

Countless microbes float in the air (koji mould, Rhizopus, Mucor, yeasts, etc.).

For people who ate rice and habitually steamed it, the cake-koji method naturally never took hold. Onto steamed rice the air’s microbes settle, and koji mould — favoured by that environment — likely germinated and spread ahead of the rest.

Modeled on China’s "hishio" (a thick paste seasoning), brought to Japan in the Nara period. Its liquid form came after the Kinzanji-miso method was learned from China at Yuasa around the Kamakura/Muromachi periods.

More: the Soy Sauce Information Center. https://www.soysauce.or.jp/

The Taiho Code (701) records "mishou" alongside "hishio." Around the Kamakura/Muromachi periods it was eaten as "name-miso." Favoured by Warring-States warlords (bean miso, Sendai miso, etc.).

More: the Miso Health Promotion Committee archive. https://miso.or.jp/

The Harima Fudoki (713) records mould growing on rice offered to the gods, used to brew sake for a banquet — the "natural seeding" method. Rice koji is thought to have entered sake brewing around the early 8th century.

The Engishiki (927) mentions "yone-no-moyashi." The "tomo-tane" method was used — reusing part of the last batch as seed (~10% added to white rice).

The 3rd shogun Ashikaga Yoshimitsu exempted the Nishi-kyo guild (tied to Kitano Tenmangu) from the koji tax. In 1419 the 4th shogun Yoshimochi granted a production/sales monopoly. In 1444 the "Bun’an Koji Riot" destroyed it.

The three groups — moulds, yeasts and bacteria — and the fermented foods each is involved in.

Living things divide into animals, plants, protists and viruses; protists further into higher microbes (→ true fungi) and lower microbes (→ bacteria).

In 1876 (Meiji 9) Hermann Ahlburg, a natural-history professor at the Tokyo Medical School newly arrived from Germany, isolated a mould from rice koji and named it Eurotium oryzae. It was later reclassified — lacking an ascocarp — as an imperfect fungus and renamed Aspergillus oryzae (Ahlburg) Cohn.

The genus name Aspergillus derives from "aspergillum," the Catholic holy-water sprinkler.

The koji mould is made up of spores (conidia), a vesicle, sterigmata (phialides), basal sterigmata, hyphae and so on.

Koji mould has long been used in Japanese brewing and foods, enriching the food culture; it was also the source of Dr. Takamine’s Taka-diastase. In 2005 its full genome was sequenced, and it is expected to grow ever more important industrially.

Dr. Eiji Ichishima, who proposed it, said transmitting koji-mould science and culture from Japan would greatly impact the 21st-century world. The Brewing Society of Japan certifies koji mould as the "national fungus."

October 12, 2006, Brewing Society of Japan (partially revised November 28, 2013).

Koji mould refers to the following fungi widely used in Japanese brewing and foods.

"Koji mould" (the useful set) is the group of the world’s koji moulds that benefit humans (good for health, make tasty miso, etc.).

The harmful counterpart is mycotoxins — a general term for mould metabolites toxic to humans/animals; over 300 are known.

Research in Japan took off with the post-war "yellowed-rice" incidents (imported rice carrying liver-damaging fungi), and the 1960 British case where 100,000+ turkeys died from a mycotoxin in their feed.

These events raised mycotoxins as a new concern beyond the usual fermentation/spoilage view.

Named after the causative Aspergillus flavus in the 1960 turkey deaths; A. parasiticus was later found to produce it too. Types include B1, B2, G1, G2, M1.

Made by A. ochraceus, A. niger, Penicillium viridicatum, etc. Ochratoxin A is nephro- and hepatotoxic; in mice it reportedly caused liver and kidney cancers.

A. flavus (one α-amylase gene) → ancestor? → A. oryzae: 99% identical genome; three α-amylase genes; ~12,000 genes on 8 chromosomes (humans ~20,000).

A. parasiticus (aflatoxin-producing) → ancestor? → A. sojae: named in 1944 by Sakaguchi & Yamada (Univ. of Tokyo); one α-amylase gene.

Koji mould is a microbe too small to see; its spores (conidia) are 3–10 μm across — 10 billion weigh 1 g.

Given suitable temperature, oxygen and nutrients they germinate and spread vigorously by tip growth (germination 30–33°C, growth ~35–38°C, ~98% humidity).

It has three forms — basal hyphae, aerial hyphae and conidia. Hyphae grow ~1 μm/min; after extending 2–300 μm they begin producing protease.

Takamine (1854–1922), born to a physician father and a sake-brewer’s daughter, worked in R&D in the US. In 1894 he invented the digestive enzyme "Taka-diastase"; in Japan he founded Sankyo (now Daiichi Sankyo) to make and sell it.

Wheat bran was inoculated with seed-koji and cultured; cold water dissolved out the enzymes, which were dried to a powder. Using koji instead of malt in whisky drew the maltsters’ anger — a distillery was even burned down.

"Taka-diastase" appears in Soseki’s "I Am a Cat." In 2015 the National Museum of Nature and Science registered it as Essential Historical Material for Science and Technology.

Koji mould cultured and processed into an easy-to-use "seed" for making koji.

A sieving machine used to separate the spores.

Koji is steamed rice, barley/wheat or soybeans scattered with seed-koji and left for the mould to grow at suitable temperature and humidity.

Types include rice, barley/wheat and bean koji. Soy-sauce koji mixes soybeans and cracked wheat equally. Koji holds many enzymes; culturing takes 48 h.

Basic idea: balance the drying rate of the steamed rice against the growth rate of the koji mould.

Product-temperature over time: turning ~35°C → heaping 40–42°C → naka-shigoto 38°C → shimai-shigoto 40°C → max 41°C → out.

As time passes after seeding, α-amylase, glucoamylase and neutral protease all increase (U/g).

Product examples: Tokusen Ginjo, Gekka Hyogin (Hikami-zukuri), No.430, Byakuya, Byakuya 70/50, No.560, Marugin. The catalogue lists each product’s enzyme activity and use.

A catalogue of miso and soy-sauce strains, listing enzyme activity, use and packaging.

An enzyme is something made inside living things that mediates chemical change in substances (dictionary definition).

From this, the meat-dissolving substance in the stomach was named "pepsin."

Enzymes are proteins of 20 kinds of L-amino acids joined by many peptide bonds, with a three-dimensional (not merely planar) structure.

An enzyme is like special-purpose scissors whose target is fixed; the optimal temperature and pH sharpen its edge.

Substrate specificity: in 1894 Fischer likened enzyme and substrate to a "lock and key." An enzyme acts only on its specific substrate.

Starch consists of amylose and amylopectin. Non-glutinous rice is 17–23% amylose; glutinous rice is almost all amylopectin.

Proteins are many amino acids joined together; proteases hydrolyse their peptide bonds.

The cooperative action of acid proteinase and acid carboxypeptidase (ACP) on soy protein.

A. oryzae’s α-amylase peaks at 55–60°C, the Bacillus one at 80°C; A. luchuensis’s resists deactivation even at low pH.

Food tastes comprise the five: sweet, sour, salty (kanmi), bitter and umami.

5′-guanylic acid (GMP) was found in shiitake by Kuninaka (1957). Mixed with MSG, umami surges — the "synergy of taste" (kombu × katsuobushi, kombu × shiitake).

Taste buds (mirai) in the tongue’s papillae sense taste, relayed via nerves to the cerebrum.

To observe amino-acid generation by the mould’s proteases, soy milk (not milk casein) was used as substrate. Rice and barley koji made with our white-spore seed-koji were saccharified overnight at 60°C with water or soy milk, then analysed.

Amino acids in fermented soy milk (rice koji × soy milk) vs rice-koji amazake (rice koji × water), mg/100 ml. Using soy milk gives more, both in total and per amino acid.

Amino-acid analysis of barley koji × soy milk (strains Cho-haku, improved Cho-haku, Mugijiro, SR-108). Totals reach roughly double those of rice koji.