Sake Yeast Part 1
The definitive, technical guide to those little beasties.
The Mother of Sake Mothers.
A number of really important things were happening around 1880 (Meiji Era) to 1920 in Japan. This was an incredible period of cultural exchange with the outside world, following hundreds of years of being isolated on an island nation. This had a profound impact on technology and fast tracked the sciences in ways that forever changed the sake industry.
Mostly notably, was the ability to isolate strains of yeast through the use of microscopes. Prior to this advance, sake brewing relied on so-called kuratsuki “蔵付き”, which directly translates to “attached to the brewery” or… to put it in more succinct terms, “native organisms that inhabit the brewery”. This microflora was composed of everything needed to create sake and in many cases it was cultivated over hundreds of years. Some might even go back further and point out the thousands of years in which adaptation and evolution drove these single celled creatures to specialize in brewing.
For the scope of this article we will not be diving that far back, because within the topic of modern sake yeast, there is plenty to talk about in terms of their evolution and specialization in just the past 100 years. So let’s start with how kuratsuki biseibutsu “蔵付き微生物” or “organisms attached to the brewery” came to be there and then describe how we arrived at the modern sake yeast.
Discovering Yeast
Yeast cells are huge (yuuuuuge) in comparison to other microorganisms. You can easily observe them and some of their moving parts with a basic microscope. But, then again, that does require a microscope. We have Antonie van Leeuwenhoek to thank for that discovery in the 1600s.
One might ask, how did we know that bacteria, yeast, and other microscopic organisms existed at all? If you stop for a second and think about your experience with these lifeforms, the answer is really quite obvious: People observed fermentation, spoilage, disease, and decomposition. We knew something was “growing”. We just didn’t know what these organisms looked like as individuals, we could only see the collective effect they had on their macro-environment.
Using these observations, a very important figure, Carl Linnaeus, also known as Carl von Linné, categorized all life on earth into a system of “binomial nomenclature”... such as “Saccharomyces Cerevisiae”. Mr. Linné used this system of observable comparisons of physical traits such as color, movement, shape, smell, etc. to categorize the differences of the living world.
Due to the limitations of technology at the time, Mr. Linné did not extensively study microbiology. However, in his book “Systema Naturae” (1758) he did include a group he called “Chaos”, which was composed of microscopic organisms we know as algae and protozoa.
The nomenclature he developed would later be used to characterize the differentiations that would lay the groundwork for the “tree of life”[1], which we have perhaps come to take for granted, given the thousands of years of observation that got us to this level of understanding.
This system of classification led to further study and categorization, especially as technology evolved. Eventually, someone was able to see one of these tiny organisms with the use of a microscope. That person was Franz Julius Ferdinand Meyen in 1837 [2]. Of course, this organism wasn’t given its latin name until 1883 by Emil Christian Hansen, around the same time (1881) that another scientist, by the name of Robert William Atkinson, observed sake yeast for the first time at the University of Tokyo. [3]
Atkinson’s presence in Japan was part of a much larger effort by the Japanese Government to modernize their scientific capabilities with the research they were not privy to during the hundreds of years of isolation. Of course, it should not be forgotten that while this technology might have existed in the western world, all of humanity was just beginning to embark on the journey into microbiology.
What makes Japan unique in this story is that the organisms on this island had evolved in ways completely different than that of their western cousins and at the same time, they would share similar traits as well.
Kuratsuki Biseibutsu
We like to give ourselves grand titles in the sake world like “Toji” which means “Brew Master”. Perhaps it makes us feel better to think that we are in control. But, the real masters of their craft are the organisms that inhabit the fermentation itself. These are far too numerous to list all of them today, but the ones we usually hear about are:
Saccharomyces Cerevisiae (Bread/Beer/Wine/Sake Yeast)
Lactobacillus Sakei (Sourdough/Sausage/Kimoto Sake)
The yeast cells observed by Atkinson were not “pure”. Under a microscope, sake fermentation is full of koji spores, bacteria, yeast, and other organic matter. To be able to study the traits of a single organism, you must first isolate it. This is where two very famous scientists by the name of Kikuji Yabe (founder of the NRIB) and Yoshinao Kozai ( Professor at Tokyo Imperial University of Agriculture) enter this story. In 1897 (conflicting dates also say 1895), Mr. Yabe and Mr. Kozai isolated what is now known as “Saccharomyces Cerevisiae var. Sake” RIB0001, designated GIB, B-5 [4]. This is the first yeast to bear the name “Saccharomyces Sake” and it was isolated from sake-rice koji. Later, these yeast were proved to be the same strain with little genetic variation.
But, why were they looking at rice-koji for yeast?
When koji is made in a brewery, the environment is hot and humid for 2 full days. The rice that koji spores grow on is a breeding ground for much more than just the intended organism. Yeast cells are floating all over the brewery and attach themselves to the brewer's hands and clothes. (Bacteria too). So, it makes a lot of sense that koji had a bunch of yeast growing on it. Then again, they might have just been curious to study koji and stumbled upon this oasis of life.
The general term for these Native Organisms inhabiting breweries is Kuratsuki Biseibutsu “蔵付き微生物”. They were originally invasive species (Read more about this on the Yeastcidin article). In order to survive in their new environments, living on rice grains, stalks, chaff, etc., organisms found ways of working together to provide symbiotic nutrients or protective mechanisms.
It might be worthwhile taking a moment to look at how these organisms work together to survive in their environments before we dive into the isolation of various strains.
Symbiotic Relationships Ensure Survival
Sake breweries are like little ecosystems. An ideal environment for many of these symbiotic relationships to thrive, but with a few side-effects that are essential to a successful fermentation. Here are some places you might find these organisms living in a brewery:
Cracks and surfaces of wood beams and walls.
Inside the pores of wooden fermentation tanks
On brewers clothes and skin
Tools and machinery
Bags of Rice
Inside of hoses or pumps (more modern)
The daily cycle of washing, soaking, steaming, and cooling rice throughout the brewery sends water filled with bran, or steam full of starch particles all over the place. Food is replenished upon every batch. Even if a brewery closes down for the summer, many of these organisms can go dormant, waiting for moisture and food to return again the following season. Surviving the cold winters adapted some yeast to perform well even in suboptimal temperatures. Well below their European cousins.
There was a study conducted in 2014 [5] on Kimoto bacteria and it found a link between why yeast are so much stronger in naturally grown lactic acid bacteria environments as opposed to Sokujo starters where cultured lactic acid is used. (Hint: it’s a symbiotic relationship)
Quick aside:
If you do a sokujo starter, you’ll have about 2x10^8 yeast cells per milliliter by the time you prepare for "san-dan”. But, when you drop the temp for the purpose of killing off all the other baddies in the tank (karashi), you need to use that starter within about 3-5 days, otherwise the yeast cell count will start to decline rapidly. (like 20-50%). However in a kimoto starter, you can let it sit at 10°C for 5-7 WEEKS before you see that kind of drop in cell count.
In fact… when you extend this further, you can see in the graphic below, that even after 10 weeks, the kimoto starter has gone from 125,000,000 cells / ml, to 20,000,000 cells/ml , but the sokujo starter is below 200,000, if there are any left at all.
Why is that?
Well, during the Kimoto-kei process there is a secretion by lactic acid bacteria (specifically those species typically involved in Kimoto production) that produces a chemical, let's call it GAR+, which tells the sake yeast to basically not be as gluttonous.[6-8] When that happens, the Yeast switches back from "halloween candy mode" to "broccoli mode" and as a result, the bacteria survive longer due to the consumption of more nutrients instead of simple sugars. Essentially, they’re all jacked up on protein supplements and more complex carbon sources.
This is just one of the reasons why Kimoto yeast is more well-conditioned than standard yeast, and much more suited for long-term fermentations than kobojikomi (pure-pitch yeast). However, this doesn't mean that it inherently is a better sake flavor.** Kimoto yeast are basically hardened, gritty beasts that can endure extremely long fermentations in rigorous conditions.
** There are more symbiotic relationships that exist here, specifically with lactic acid bacteria which are known to produce higher amounts of amino acids, including glutamic acid. If you recall, glutamic acid is the chemical responsible largely for “umami” flavor. In addition to that, LAB has an ability to augment the structure of L-Amino Acids into D-Amino Acids through a process called Racemazation. Glutamate Racemase is an enzyme produced by LAB that plays an important role in its metabolism, but has the additional benefit of turning “bitter” tasting amino acids into their D-form “sweet” tasting Isomer. If you ever drink a kimoto sake that is +10 SMV and but has fairly heavy mouth feel and is quite sweet… its probably due to the racemase enzyme vis-à-vis Lactic Acid Bacteria.
Why is this cool? Few things:
This is advantageous to yeast because their growth and long-term viability is improved by consuming complex carbon sources. Also alcohol is poisonous to yeast, so the slower it produces alcohol, the better.
This is advantageous to the bacteria, because the reduction in ethanol production means that more will survive.
This effect only seems to happen with specific strains of yeast and bacteria that are common to sake brewing. For instance, Leuconostoc mesenteroides and Latilactobacillus sakei produce the effect, but spoilage bacteria, such as Lentilactobacillus hilgardii, does not create the inhibitive effect.
It may be surprising, but even with all of our modern technology, we are still finding out how sake fermentation really works and constantly identifying new relationships between all the organisms involved in the brewery.
Finding
“Saccharomyces Sake”
Now, up until about 1910, all sake fermentations were essentially “spontaneous”. Take that word with a grain of salt, because as this article has been repeating over and over, the brewery was basically inhabited by thousands or even millions of generations of yeast and bacteria that have adapted so well to their environment that it substantially increased the chances of the microflora in the tank being ideal and reduces the chances of contamination.
If you have ever heard one of the stories of a brewery relocating to a new building and then suddenly their sake is terrible, this is the reason why. In fact, there are some which took extra care to relocate wood from one building to the next in order to ensure continuity in their specific biological cocktail.
Just like in lambic, open-fermentations, the same logic applies. You would be hard pressed to create such an environment at home unless you actually clean a space quite thoroughly and then literally spray it with chosen bacteria and yeast in order to ensure the “good” organisms will have the best chance of dominating the local microflora.
Before cultured yeast, Kimoto sake fermentations would acquire their yeast and bacteria from the environment, especially when the steamed rice was cooling, from brewers touching the koji, from the humid koji room itself, the actually wooden tanks, and finally, the air in the brewery.
After Mr. Yabe and Mr. Kozai successfully isolated a yeast strain from koji, the idea evolved toward locating specific strains of sake yeast. If you are unfamiliar with the naming conventions, here is how we talk about it:
Kingdom: “Fungi”
Genus: “Saccharomyces”
Species: “Saccharomyces cerevisiae”
Variety: Saccharomyces cerevisiae var. sake.
Strain: Saccharomyces cerevisiae var. sake strain K7
Isolate: Saccharomyces cerevisiae var. sake strain K7 isolate 1234
A specific sample taken from a sake brewery environment
** NOTE: While this structure, known as “Binomial Nomenclature” with “infraspecific ranks” such as “var. sake” or “isolate 1234” are common in organism identification, the NRIB tends to use a different classification of “Saccharomyces Cerevisiae Kyokai-7” substituting the standard intraspecific ranks with a more simple designation. Sometimes the isolate is used in place of the strain name, for instance, “Sake yeast AA-6” which is a mutant of another yeast. This can be confusing if you are used to more standardized system, so we will refer to them in the standard way, but ensure you know what we are referring to. **The idea was to start isolating different strains, comparing their traits until they found some ideal brewing characteristic.
Yabe and Kozai would identify several varieties, but none were considered ideal for sake brewing. Actually, there were 7 yeast strains cataloged before Kyokai #1 would be officially named. When Kozai’s successor at the University, Professor Teizo Takahashi, took over the project, he decided to take a more aggressive approach. He worked with the Tax Agency to have moto and moromi samples from the best breweries in Japan be sent to his lab.
From the samples, Mr. Takahashi isolated more than 60 strains of yeast. They were analyzed for cell shape, endospore formation, film formation, fermentation ability, amount of acid and alcohol production, amount of amino acid consumption, and other variables.
One particular yeast, from Sakura Masamune in Nada, Hyogo, significantly improved the flavor. This yeast would become “Kyokai Strain No. 1” The first pure-cultured, commercial sake yeast. Kyokai strain #2 would come from Gekkeikan in Fushimi, Kyoto and then Strains 3 through 5 would come from breweries in Hiroshima.
Now if you know the geography of Japan, you might notice that all of these came from warmer regions. Areas like Hyogo were gifted with temperatures and water that specifically benefited strong fermentations. However, these strains didn’t particularly perform well as you started to go farther north.
This all changed in 1935, when a new strain was isolated at Aramasa brewery. The northern location had adapted this yeast to the cold temperatures and it allowed fermentations to take place throughout the winter in previously difficult brewing areas. In addition, the cold temperatures prevented bacterial contamination, and produced a cleaner flavor, with the yeast also metabolizing differently.
Thus, Kyokai #6 (Aramasa Yeast), also known as RIB 1001, entered the history books. Due to its brewing characteristics it immediately put all other strains out of work. During WWII, only Kyokai #6 was distributed throughout Japan.
Beyond this point in time, the isolation of new strains and regional isolates becomes relatively routine. The process is extremely tiresome and the advancement of biology and chemistry research lends itself to new procedures that help to distinguish between different strains. From a technical perspective, it just isn’t that interesting for a while.
However, the production side of sake was absolutely jolted by the introduction of Yeasts #7 and #9. While their discovery might have been routine, the yeast characteristics were anything but ordinary. These two especially became the workhorses of the brewing industry, with traits that produce incredible aroma and lasting fermentation power in all temperatures and other stressful conditions.
Most modern yeast that we think of as “high aroma producing” or specialty yeast strains from Kyokai like 1101, 1404, 1501, 1601, 1701, 1801, and 1901 are all descendants of these two main yeast in addition to Meiri Yeast, which is Kyokai #10. Yeasts 7, 9 and 10 only began production in 1946, 1968, and 1977 (star wars: a new hope) respectively, so this is all relatively new in the history of sake.
Advances in Yeast Isolation and Culturing
Mutations
As science reaches new understanding, cell mutations are not only identified, but can be forced through UV light and other genetic procedures. Researchers began to realize that these mutations augment the metabolism in different ways. In some cases, the yeast produce huge amounts of beautiful aroma and in other cases, they can’t survive beyond 10% alcohol concentration.
Cross-Breeding
It was also discovered that yeast are capable of sexual reproduction and laboratories began to cross-breed different strains and varieties, hoping to create new flavor and aroma profiles. With the Ginjo boom in full swing toward the end of the 1900s, the search for “Ginjo-Ka”, a smell often characterized by high ethyl-caproate, or various levels of isoamyl-acetate and ethyl-acetate, seems to completely preoccupy the yeast laboratories.
Trait Selection
One major advancement was the identification of hydrophobic yeast, colloquially, but incorrectly named “non-foaming” yeast. This was identified as being caused by the aptly named “AWA1” gene, which is responsible for creating a polar charge on the cell surface that in turn forms a bond with the water, trapping the CO2 released within the mash. When the AWA1 gene is present, we call it “foaming yeast” or hydrophilic yeast. When it is absent, the yeast is hydrophobic and will not prevent CO2 from escaping. [9]
*As Arline Lyons recently reminded me, during a discussion on recipe creation, water ratios, etc. that when dealing with a hydrophobic yeast (foaming) you may need a higher water ratio than with hydrophilic yeast. This is because of the physical difference in % of the yeast which are above the ferment, vs % of the yeast that are actually IN the ferment. In other words, during high-foam, the additional water actually keeps the yeast in the tank, where as with hydrophobic yeast (less-foaming), the fermentation is prone to accelerate if the water ratio is higher, so you might reduce the ratio early on and correct with Oimizu (water added during fermentation) later.
Sake-Yeast-Specific Traits
The search for new yeast continues, mostly as a desire to diversify the market and create new and exciting reasons for consumers to try a bespoke product. “Local Yeast” from famous sources like Aramasa and Masamune, was eagerly sought by other breweries trying to make their mark and as a result of a huge breakthrough in the 1980s, another technological advancement was about to make that a reality.
If you skipped the Yeastcidin article, make sure you circle back to it, because that’s where the real meat of the science for this item is described.
It was determined that when a specific strain of Aspergillus Oryzae (koji spores) is grown in a liquid medium, it will produce a substance that has a Symbiotic relationship to Sake Yeast. If this substance was concentrated and placed in a yeast starter, it would kill all the other yeast except Saccharomyces Sake. Not the genus of Cerevisiae, but specifically the Variety: Saccharomyces cerevisiae var. sake. This is particularly interesting because this Variety ONLY exists in Japan. In a phylogenetic tree it has been isolated from the rest of the world for over 3000 years.
Growing Yeastcidin - brewsake.org
With this new found trait (Yeastcidin resistance), every place that yeast was growing in Japan (flowers, fruit, leaves, etc.) suddenly became a cornucopia of potential new strains of Saccharomyces Sake, complete with their own unique adaptations and metabolisms to exploit and hopefully create a new, exciting sake profile.
Now, when you look at the market, you’ll see breweries with brands like Amabuki, that produce sake with Strawberry Yeast, Olive Yeast, Bougainvillea Yeast, etc. And it's important to note that what they mean is that it was isolated from those fruits, vegetables, and flowers. They do not taste or smell like those things. If they do, that’s more of a coincidence of the yeast selection process.
For instance, the flavor and aroma for “strawberry” is often associated with minor levels of butyric acid. In small amounts it has a sweet and beautiful bouquet of aroma. However, in large amounts, it is the unfortunate taste and aroma of vomit. Not really appealing. So, was the yeast from a strawberry? Was it a flower yeast that produced a balanced amount of butyric acid? Was it both? Most of that is marketing. What matters is that you don’t go around thinking that The Snozberry Yeast tastes like Snozberries.
Genetic Manipulation
Now, while different countries have different policies on this next one, there are other ways to generate new varieties and that is CRISPR. With the advent of gene editing, and the full sequencing of the Saccharomyces Cerevisiae genome, we can identify positive and negative traits in a strain and simply slice and dice until it does what we want.
Some countries allow these to be used in fermentations and others see it as a public health concern. But regardless of where you or your nation stand on this one, genetics are the next wave of customization and there are ways to make a yeast do incredible things with flavor and aroma by just switching a few things around.
Adjacent Technology
Rice Polishing
While yeast is the topic of this article, it is extremely important to note the impacts of the vertical rice polishing machine, which was created in the 1930s. Besides removing the outer layers of protein from the rice that contributed to excess acidity and amino acidity, it also removed lipids, which are converted to fatty acids by lipase during the fermentation. By 1979, research had concluded that the presence of fatty acids have an inverse relationship to the formation of isoamyl acetate, which you might know as “banana” and is one of the core components of “ginjo aroma”. [10]
Aroma Component Byproducts of the TCA Cycle Click for image source
Yeast Metabolism
The ability to produce higher levels of ginjo aromas was also studied. In addition to new strains of yeast, there are also methodologies or environmental factors that were found to play a large role in yeast performance. For instance, how the mash progresses and is managed can affect the type and quality of esters produced by the yeast. In high glucose, semi-anaerobic conditions, there is a significant (2-3x) increase in isoamyl acetate production in the first 72 hrs, with very little gain after that point. However, in similar conditions, ethyl acetate will see a similar 2x gain in the first 72hrs, but if it can be maintained for 10 days, the potential is a 6x increase in aromatic compound production (relatively, 2-3x a low glucose environment). It was also found that too much oxygen or totally anaerobic conditions reduced the production of each compound. [11]
If you ever see a ginjo temperature course, this is the main reason why. By keeping the yeast in a high glucose environment, their metabolism, via enzymatic activity (in this case AATase), will change to produce different amounts of flavor compounds.
Changing the fermentation course can increase the amount of time yeast spend in a high-glucose environment, which increases the production of isoamyl acetate and ethyl acetate.
Koji Spore Metabolism
Another technological development we’ll mention is the influence of koji spores on this complex system. Just as choosing your yeast can have profound effects, each strain of koji spores can have an impact on the yeast metabolism and therefore final flavor. This occurs because koji’s enzymes and its own metabolic processes can produce what are called “precursors”. These are essentially compounds that the yeast cell can consume to improve or complete various parts of their own metabolism.
If you look at the left side of the metabolic map below. If you supercharge the Leucine pathway [12], for example, you can potentially create more isoamyl acetate. On the flipside, you would also end up with more isovaleraldehyde… so you need to be careful and understand which strains or environmental factors influence the production of these under different circumstances.
You can kind of think of a precursor as something like Caffeine. Your body already produces things to stimulate your brain and keep you active. But, when you feed it something specialized, it will then perform specific tasks that benefit you in some ways, but probably send you to the restroom more often… Let’s call that the human form of isovaleraldehyde.
Leucine acid is a precursor to desirable and undesireable compounds. Click for image source
Water Profile
Finally, we’ll discuss the influence of water. While this is truly a giant topic deserving of not 1, but perhaps 100 or so in depth articles, we’ll just give you the headline for now. Basically, just as precursors are important to the metabolic rate and compound production of yeast, the various nutrients in water, as you may now assume, are incredibly important.
Even just the presence of more hydrogen ions, (did you know pH stands for Power of Hydrogen?) can play a substantial role in how performant yeast is. Enzymes are proteins and proteins are made of amino acids. With the presence or absence of extra hydrogen ions, it can actually change the structure of the enzymes, causing them to malfunction. This would have significant effects on the production of compounds and subsequent aroma and flavor.
Other impacts would include the speed of alcohol production, maximum alcohol concentration, ester and phenol production, higher-alcohols and fusel alcohols production, and more. Sometimes it just overwhelms the flavor due to the concentration of elements like sulfur, which can present as various compounds.
Summary
We know that a more direct route would be to simply describe each yeast and its properties, but that felt disingenuous. The history and attributes of yeast are a major part of what you see in the market today.
There are so many interesting aspects of yeast. The relationships it has to the bacteria and Aspergillus are fascinating and the sheer scale of research that has gone into this field is remarkable.
We’re almost afraid that we did not cover enough information, but we hope you have learned some new things and can help others appreciate the awesome world of sake brewing!
Checkout the next article for a full write up of all the Kyokai Yeasts and what is so special about each one.
Sources
History, lineage and phenotypic differentiation of sake yeast (Yoshikazu Ohya, Mao Kashima)
https://academic.oup.com/bbb/article/83/8/1442/5938654Atkinson, R. W. The chemistry of saké-brewing. Tokio: Tokio Daigaku. Retrieved from https://doi.org/10.5479/sil.248402.39088006703755
(Tables that show different yeasts and their isolation dates and locations)
The yeasts, a taxonomic study Taxonomic characterization of sake yeasts based on the Key to species in the 5th edition
https://www.jsmrs.jp/journal/No36_2/No36_2_25.pdfSake yeast symbiosis with lactic acid bacteria and alcoholic fermentation
https://academic.oup.com/bbb/article/88/3/237/7450476Cross-Kingdom Chemical Communication Drives a Heritable, Mutually Beneficial Prion-Based Transformation of Metabolism
https://www.cell.com/cell/fulltext/S0092-8674(14)00975-1A history of research on yeasts 9: regulation of sugar metabolism
https://onlinelibrary.wiley.com/doi/pdf/10.1002/yea.1249Feasting, fasting and fermenting glucose sensing in yeast and other cells
https://sci-hub.st/https://doi.org/10.1016/S0168-9525(98)01637-0The AWA1 Gene Is Required for the Foam-Forming Phenotype and Cell Surface Hydrophobicity of Sake Yeast
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC123892/Effects of Cellular Fatty Acids on the Formation of Flavor Esters by Sake Yeast
https://www.jstage.jst.go.jp/article/bbb1961/43/1/43_1_45/_pdfInfluence of glucose and oxygen on the production of ethyl acetate and isoamyl acetate by a Saccharomyces cerevisiae strain during alcoholic fermentation
https://pismin.com/10.1007/s11274-004-2780-5Ginka Spores
https://www.akita-konno.co.jp/products/shuzo/seishu/
