The Science of Sake Fermentation: A Precise Brewing Process Woven by Koji, Yeast, and Lactic Acid Bacteria

Sake is not just a simple "alcoholic beverage."

First, it is a uniquely complex fermented beverage, starting from the starchy substrate of white rice, where multiple microorganisms divide roles to perform stepwise transformations.

Let's unravel that fermentation process from the perspective of microbiology and biochemistry.

1. Koji-making Process: Building a Platform for Enzyme Production

The first key to sake brewing lies in the koji-making process by Aspergillus oryzae (koji mold).

It begins by scattering koji mold spores onto steamed rice that is firm on the outside and soft on the inside. The steamed rice, inoculated with tane-koji, is then cultivated in a koji room (koji-muro) with precisely controlled temperature and humidity for about 48 hours.
During this time, the koji mold extends its hyphae into the rice grains and secretes a wide variety of hydrolytic enzymes.

Particularly important is the production of amylases (α-amylase and glucoamylase) and proteases.
α-amylase hydrolyzes the α-1,4-glycosidic bonds of starch in an endo-type manner to produce dextrin, and glucoamylase sequentially releases glucose from its ends.
This two-stage saccharification reaction supplies fermentable monosaccharides to the yeast.
Protease breaks down proteins into amino acids and peptides, serving as a nitrogen source for yeast in subsequent processes and acting as a precursor to umami components in sake.

While using technical terms, essentially, koji mold breaks down rice, which is a lump of starch, into sugar, making it easier for the yeast in the next step to break it down into carbon dioxide and alcohol.

2. Shubo (Yeast Starter): Selection of Microbial Flora and Yeast Proliferation

Next, koji obtained from koji-making, steamed rice, water, and yeast are brewed in a small tank to cultivate a high-density yeast culture solution called "shubo" (moto).

In traditional kimoto and yamahai-moto methods, wild lactic acid bacteria (of the Lactobacillus genus, etc.) are naturally propagated, but this is rarely done in modern sake brewing.
Instead, in the modern mainstream sokujo-moto method, instead of propagating lactic acid bacteria like in kimoto or yamahai-moto, brewing lactic acid is directly added.

Homo-lactic acid fermentation by lactic acid bacteria (C₆H₁₂O₆ → 2CH₃CH(OH)COOH) rapidly lowers the pH of the moromi (main mash). This pH drop acts as a selective pressure, eliminating miscellaneous bacteria that are weak to acidic conditions, while giving an advantage to the proliferation of acid-resistant brewing yeast (Saccharomyces cerevisiae). At this stage, the yeast enters its exponential growth phase, increasing cell density in preparation for the subsequent moromi fermentation.

The acidification of the moromi aims to eliminate miscellaneous bacteria that could negatively affect brewing by making the acidic environment unsuitable for their survival.

In this environment, yeast, being strong against acid, can perform its function without being destroyed along with other miscellaneous bacteria.

3. Moromi Fermentation: A Globally Rare Fermentation Method Called Parallel Multiple Fermentation

The greatest characteristic of sake is this concurrent saccharification and fermentation.

Unlike beer, where saccharification and fermentation are separated, in sake, saccharification by koji and alcohol fermentation by yeast proceed simultaneously within the same tank.

This is an extremely rare fermentation method among brewed beverages worldwide.

The gradual addition of koji rice, steamed rice, and water in three stages ("hatsugoe," "nakagoe," and "tomegoe") during "sandan-jikomi" (three-stage preparation) is also to maintain the substrate concentration within an appropriate range relative to the yeast cell density and to prevent a decrease in yeast activity due to osmotic stress.

Furthermore, this operation, combined with low-temperature, long-term fermentation (approximately 20-30 days), promotes the production of aromatic components (isoamyl acetate, ethyl caproate, etc.), forming a complex flavor profile.

The central reaction of alcoholic fermentation is the production of ethanol via glycolysis (Emden-Meyerhof-Parnas pathway): C₆H₁₂O₆ → 2C₂H₅OH + 2CO₂.

The alcohol concentration at the end of fermentation reaches 14-20%, which is close to the upper limit of the yeast's own alcohol tolerance.

Ultimately, the power of yeast transformed it into carbon dioxide and alcohol through fermentation, but as the alcohol concentration increases, the yeast also dies off.

4. Joso and Hi-ire: Termination of Fermentation and Stabilization of Quality

Once the moromi has matured, it undergoes a solid-liquid separation process called "joso" to be separated into sake and sake lees, resulting in clear sake.

Afterward, in most cases, hi-ire (pasteurization) is performed.

Even after separation from sake lees, there are still living yeast cells, so a short-time heat treatment at 60-65℃ is carried out to inactivate residual wild yeasts and lactic acid bacteria, and to deactivate enzyme activity (especially residual amylase).

This concept of pasteurization, a technology uniquely developed in Japan, is said to predate Western Pasteur (named after the person who elucidated the principle of wine making from grapes) by about 300 years.

Sake fermentation is a precise biological process where microbiology, enzyme chemistry, and food engineering intersect.

Koji mold, lactic acid bacteria, and yeast each occupy different spatiotemporal niches and cooperate to create that clear cup of sake.

Although sake is consumed worldwide, the method of sake production is uniquely Japanese, as this story explains.