Fermentation management is one of the most important processes in winemaking. Depending on how this fermentation proceeds, the resulting wine will vary in both taste and aroma.
When discussing fermentation management, attention tends to focus on what temperature to set for fermentation, what speed to maintain, or what type of yeast to use. These factors are certainly important and cannot be ignored in fermentation management. However, there is an even more crucial point in managing fermentation: not letting the fermentation stop.
Fermentation is a change driven by yeast metabolism. Some may think that simply adding commercial dry yeast to grape juice will ensure smooth progress, but it's actually not that simple.
Yeast, along with many other microorganisms, exhibit unpredictable behavior. No matter what methods are employed, there is no absolute means that is 100% safe and secure. Though it's an inconvenient truth, fermentation always carries the risk of stopping.
There are several causes for fermentation stopping midway. One of these causes involves yeast itself.
Yeast should be the protagonist that drives fermentation forward, yet that same yeast can become the cause of fermentation stopping. This may sound contradictory, but it's a fact. This article will explain this seemingly contradictory existence known as killer yeast.
What Is Killer Yeast?
There are numerous types of yeast, and there are several criteria for their classification. For example, the classification into Saccharomyces and non-Saccharomyces yeasts is based on the genus of the yeast.
Killer yeast is not classified based on such biological genus or species, but rather on whether it possesses the characteristic of killing other yeasts. If a yeast has the property of killing surrounding yeasts, it is equally treated as killer yeast, whether it is Saccharomyces or non-Saccharomyces yeast. Yeasts that are killed by killer yeast are called sensitive yeasts, while yeasts that are not killed by killer yeast are called neutral yeasts.
The majority of yeasts are said to be sensitive yeasts. Sensitive yeasts are often simultaneously killer yeasts, and there are reports that approximately 50% of Saccharomyces cerevisiae strains showed killer activity. The proportion of killer yeasts is known to vary depending on the environmental conditions of testing, and this figure should be understood as one example.
Killer yeast was discovered in Britain in 1963. While research and application are currently said to be advancing primarily in the wine industry, killer yeast itself has been found in fruits, flowers, trees, mushrooms, soil, and other sources, making it a field still under research including the existence of undiscovered strains.
Classification and Types
Killer yeasts are classified not by their yeast strain types, but by the causative substances called killer toxins that kill other yeasts.
Killer toxins are primarily antifungal compounds that exist as diverse molecular clusters including proteins, enzymes, cyclic peptides, surfactants, and volatile low-molecular-weight compounds. Each yeast produces these compounds within its cells and secretes them outside the cell to attack target yeasts. The characteristic of these substances is that they act specifically on yeasts and their related fungi. Yeasts that produce killer toxins have immunity against the toxic substances they create, but even if they lacked immunity, yeasts that are not targets of that killer toxin would not be affected. In this respect, their mode of action differs from antibiotics, which do not discriminate between attack targets.
The types of killer toxins vary depending on the yeast that produces them. Currently, they are classified by type. However, this classification is still limited to major types, and comprehensive classification and systematization including killer toxins produced by non-Saccharomyces yeasts has not yet been achieved.
Killer Toxins Produced by Saccharomyces Yeasts
Among current classifications, four types called K1, K2, K28, and Klus are killer types that act particularly strongly on yeasts of the Saccharomyces genus. Their production is also carried out by yeasts of the Saccharomyces genus.
The K1 type has particularly high killer activity and has been isolated from beer and sake yeasts. However, the killer toxin produced by this type of yeast shows its strongest activity at pH ranges above 4, making it a type of low importance in wine.
In contrast, the killer type mainly found in wine is called K2. The killer toxin produced by this type has an optimal pH of 2.8 to 4.8, which matches wine pH, and is considered a major cause of fermentation stopping in wine.
Target Specificity and Degree of Impact of Killer Toxins
An important point in understanding killer yeast is that yeasts of the same killer type do not kill each other. Killer yeasts have immunity against the killer toxins they produce. Therefore, for example, if yeast A and yeast B belonging to the same K1 killer type exist in the same liquid, the killer toxins produced by each will not act on both.
It should also be noted that each killer type does not necessarily kill all other killer types.
While there are exceptions to the target range of each killer type, for example, K1 and K2 types act on Saccharomyces sensitive yeasts but hardly act on non-Saccharomyces sensitive yeasts. However, killer types with very broad effect ranges that act on sensitive yeasts belonging to almost all killer types except their own have also been reported.
Action Range and Threat Level Do Not Coincide
The action range and action intensity of killer toxins do not necessarily coincide. Both K1 and K2 types act on Saccharomyces sensitive yeasts, but K1 type has higher action intensity than K2 type and shows strong killer activity. Similarly, there are killer yeasts that act on almost all types but produce killer toxins that pose little threat to Saccharomyces sensitive yeasts belonging to K1 or K2 types due to low action intensity against these types.
Just because a killer yeast produces killer toxins that act on its own type does not necessarily mean the risk is high.
It has been reported that killer toxins produced by non-Saccharomyces yeasts tend to show higher killer activity than those produced by Saccharomyces yeasts. The killer toxins produced by these yeasts are often highly stable and may act over long periods. Killer toxins produced by non-Saccharomyces yeasts often show killer activity against Saccharomyces sensitive yeasts, making them characteristics that easily become risks in fermentation management.
One reason why alcoholic fermentation by Saccharomyces yeasts tends to stop after spontaneous fermentation dominated by non-Saccharomyces yeasts is thought to be that killer toxins produced and secreted by non-Saccharomyces yeasts inhibit the growth of Saccharomyces yeasts.
Mechanism and Content of Action
The details of how killer toxins secreted by killer yeasts act are not yet clearly understood. Among these, the mechanism of action of the K1 type is particularly well-researched.
K1 killer toxin is known to bind to (1-6)-β-D-glucan contained in the cell walls of sensitive yeasts as a receptor. Since this binding is selective, whether or not the cell wall contains (1-6)-β-D-glucan as a binding target becomes one selection criterion for whether the K1 type will act.
After binding to the cell wall, the toxin is then introduced into the cell membrane and forms ion-permeable channels within the cell membrane. Through these permeable channels, intracellular substances such as potassium ions and ATP begin to leak out, causing cell death.
While the K1 type primarily kills target sensitive yeasts through cell membrane function disruption, other killer types are known to kill target yeasts through different mechanisms. Such methods include cases of inhibiting the activity of specific enzymes, cases of inhibiting glucan synthesis in cell walls, and cases of inhibiting DNA synthesis.
Effects Are Not Consistent
The action of killer toxins is known to be extremely pH-dependent. In addition to the existence of optimal pH that affects activity levels, it has been pointed out that the structure and function of the toxins themselves may change depending on pH. Due to these various conditions, the proportion of killer yeasts needed for them to become the dominant species is also thought to change.
To secure an effective concentration of killer toxins, the cell density of killer yeasts must reach a certain level or higher. However, the degree of initial cell concentration required to secure such dominance has strong condition dependency, and details are not understood.
In the case of S. cerevisiae, there are reports that killer yeasts became dominant species at initial concentrations of 0.01 to 10%, while other reports indicate that killer effects are not exhibited at initial ratios of about 1%, with 2 to 6% being the minimum initial ratio. There are also cases where killer strains became dominant only when they comprised 50% or more. While determining what degree of initial concentration killer yeasts require to overwhelm other yeasts remains a future challenge, it is necessary to recognize that killer yeasts may always potentially become the dominant species as an uncertain factor in fermentation management.
Killer Properties Are Virus-Derived
While killer toxins are produced within yeast cells themselves, it is known that their origins include several types.
It is known that two types of dsRNA plasmids are involved in the production of killer toxins mainly related to Saccharomyces yeasts. These are LdsRNA and MdsRNA, which are dsRNAs derived from M virus and L-A virus, respectively. Functionally, MdsRNA plays a role directly related to killer phenotype, while LdsRNA plays a role in coding coat proteins.
Non-infectious virus-like particles (VLPs) exist within yeast cells, and these VLPs are thought to produce killer toxins. These VLPs are propagated through yeast proliferation, and no new infections from external sources have been observed. Since the existence of killer toxins plays an important role in the survival strategy of yeasts themselves, it is thought that a neutral symbiotic relationship with viruses was established during evolution and continues today.
It should be noted that killer types controlled directly by nuclear genes independent of dsRNA plasmids have also been confirmed, and new killer types that lack MdsRNA despite being detected from S. cerevisiae have been discovered, suggesting that many undiscovered killer types likely exist at present.
Methods to Prevent Killer Toxins
There are several methods to avoid the effects of killer toxins. Since killer toxins are often unstable to pH and heat, killer toxins can be inactivated by adjusting pH or heating. Also, since many killer toxins are protein-based compounds, their effects can be suppressed by adding substances like bentonite for adsorption.
However, since killer toxins are secreted during yeast metabolism, temporary inactivation or adsorption alone is insufficient. To eliminate risks throughout the entire fermentation process, continuous treatment is necessary while yeasts are metabolizing.
This is where resistant yeasts are being developed.
For example, commercial yeasts such as VIN13 have acquired resistance to specific types of toxins through breeding. Through such adjustments, high fermentation stability can be secured even during fermentation processes where similar types of killer yeasts are likely to exist. Recently, there has been an expanding trend in the distribution of products with killer plasmids introduced into existing yeasts, as well as products given resistance.
Using neutral yeasts that are not affected by killer types is also effective for avoiding the influence of killer toxins. However, since the proportion of sensitive yeasts is relatively high among yeasts overall, attempting to use only neutral yeasts will likely narrow the available options.
The Significance of Killer Yeast in Wine Production
The impact of killer yeast in wine production can be considered from two perspectives. One is the contamination of killer types that strongly act on K2 types into the fermentation process.
This includes, for example, killer yeasts such as Wickerhamonyces anomalus (formerly Hansenula genus), which are mainly non-Saccharomyces yeasts. Since this type of killer yeast has high killer properties against Saccharomyces yeasts, if contamination occurs, the risk of fermentation by Saccharomyces yeasts stopping becomes high.
The second is the impact on fermentation in secondary fermentation cases such as sparkling wines.
Killer toxins secreted by killer yeasts during primary fermentation remain in the wine unless removed by some method. Therefore, if killer yeasts of a type that acts on K2 were used during base wine fermentation, secondary fermentation of that base wine with K2 type yeasts becomes difficult. Recently, methods that deliberately choose such combinations are being considered, but fermentation management risks become higher.
In both cases, to restart fermentation that has stopped, it becomes necessary to use yeasts of different types or neutral yeasts unaffected by killer types, or to remove killer toxins from the liquid.
Will the Use of Killer Yeast Advance?
While killer yeast may seem like a villain when we hear that it inhibits yeast growth and destabilizes fermentation, there is actually a background of successful utilization in brewing sites for some time. For example, in sake brewing, killer yeasts have been used to suppress wild yeasts. Killer yeasts have both the risk of killing desired yeasts and the benefit of eliminating unwanted yeasts.
The utilization of killer toxins is also being considered outside brewing sites. For example, killer toxins produced by killer yeasts are expected to play a role in achieving food security by avoiding antifungal resistance.
In food crop cultivation, crop losses due to fungi are said to exceed 20%. While control measures are currently being implemented, conventional chemicals have been criticized for problems such as the development of resistant strains and off-target effects. In contrast, killer toxins that act more selectively on specific targets are thought to have significant advantages as new biological control agents.
At present, utilization has not advanced due to poor production efficiency and significant constraints in usage and storage conditions including pH and thermal stability. However, depending on future research and development, this is viewed as a field with the potential for significant expansion of application scenarios.
In alcoholic beverage fermentation sites as well, changes in fermentation behavior may emerge due to changes in brewing environments. In such circumstances, the active utilization of killer yeasts may be required as one means of achieving more stable fermentation. In such cases, if utilization expands in related industries, the possibility of use as preparations rather than necessarily as yeasts is expected to emerge.
Future developments in research and development cannot be overlooked.
