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In search of yeasts for low-alcohol wine

Climate change, consumer preferences or even taxes are all reasons for producing wine with less alcohol. However, it is more difficult than it seems to produce low-alcohol wine without residual sugar, and with an aromatic profile that meets the requirements of a quality wine. The use of yeasts that produce less alcohol seems obvious, but are they the solution?

Controlling the amount of alcohol in the wine starts in the vineyard. The amount of sugar in the grapes can be reduced by reducing the leaf area per cluster, increasing the yield, or making adjustments to the type of rootstock, the grape variety, the location of the vineyard or the water management1. If the grape must still contains too much sugar after these adjustments, there are a number of (modern) cellar techniques that can be used. The simplest way is to blend it with wine with a lower alcohol percentage, or with water (!). In addition, the glucose content in the must can be reduced by filtration, or by adding the enzyme glucose oxidase. The latter converts glucose into gluconic acid so that it can no longer be converted into alcohol. It does however require oxidative conditions to work, which can be unfavourable for the wine2. Finally, reverse osmosis can be applied to (partially) dealcoholize the final wine. However, these cellar techniques are not always allowed, and can also have a major influence on the sensory properties of the wine. Low-alcohol producing yeasts may offer an alternative solution.

The following sections provide an overview of how the molecular conversion of sugar to alcohol takes place in the yeasts, and which experimental ways are used to limit the amount of alcohol in the wine with the help of yeasts.

The conversion of glucose by yeast

The carbon metabolism

Yeasts take in the sugar (glucose) in the must, which is then converted into alcohol in the yeast cell by various metabolic steps (see Figure 1). Glucose and alcohol (ethanol) are carbon compounds, they consist of a carbon structure to which oxygen and hydrogen atoms are bound. In the yeast cell, glucose is first converted to pyruvate. This process is called “glycolysis”. Pyruvate then serves as a raw material for the citric acid cycle in the mitochondria – the cell’s power plants. The degradation and conversion of carbon compounds such as glucose and pyruvate in the cell is called the carbon metabolism. The glycolysis and the citric acid cycle together form a large part of the carbon metabolism of the yeast cell, and produce the energy-rich compounds ATP and NADH. These compounds are the “fuel” for the cell and are necessary for many metabolic processes, allowing the yeast cell to grow and multiply. When there is no longer any supply of glucose – for example, because all of the glucose in the must has been used up – the processes in the yeast cell fall silent and the cell will eventually die.

Carbon metabolism yeast cell

FIGURE 1. The carbon metabolism in the yeast cell. Glucose is converted to pyruvate via glycolysis. Pyruvate then serves as a raw material for the citric acid cycle in which the high-energy compounds ATP and NADH are produced, or can be converted to lactic acid or acetaldehyde. The most important (by) products of carbon metabolism are shown in turquoise. Proteins in the cell ensure that the conversion from one reaction product to the other can take place. The proteins that have been genetically modified in various studies to make the yeast produce less alcohol are indicated in blue. (Figure based on references 4-6).

By-products       

The carbon metabolism produces various by-products in the conversion of glucose (and pyruvate) to energy (see Figure 1). Scientists have long wondered why yeasts produce these products – such as ethanol, acetic acid, and glycerol – and do not break them down to the less energetic CO2. A theory has recently been developed by Groningen scientists who state that it is likely to “relieve” the cell’s metabolism. In the case of an excess of glucose, the carbon metabolism of the yeast cell starts off as a madman to convert this into energy. However, there is a maximum that the cell can handle. When this maximum is reached, glucose – to prevent cell overload – is quickly converted to other by-products, even though these products still have energetic value3.

Saccharomyces cervisiae yeasts are naturally good at converting glucose, with alcohol as a primarily “waste product” of carbon metabolism. They are therefore extremely suitable for a pure alcoholic fermentation, and produce few by-products – such as glycerol, lactic acid, acetic acid, acetoin and 2,3-butanediol – that can have an adverse sensory effect on the wine (see Table 1). However, for the production of low-alcohol wine, yeasts are needed that produce alcohol less efficiently, but that do use up all the glucose in the must (otherwise only sweet wines can be made). Nevertheless, yeast cells with this adjusted carbon metabolism should still produce sufficient energy-rich compounds to maintain the yeast cell, and the alternative end products should not adversely affect the quality of the wine5,9. The latter in particular is a huge challenge.

Table 1.Common by-products of the alcoholic fermentation and their influence on the wine4,5,7,8
By-productSensory effect on the wine
2,3-butanediolVirtually no sensory effect, at higher concentrations it is viscous and gives a slightly bitter taste
AcetaldehydeAt low concentration fruity aromas, at higher concentrations aromas of crushed apple, and oxidized, nut-like and sherry-like aromas.
AcetoinButter-like aromas
Acetic acidA weak acid with vinegar aromas
GlycerolSlightly sweet and gives a rounder mouthfeel
Lactic acidA mild acid with milk aromas

Finding the right yeast for low-alcohol wine

Genetic modification of yeasts

By making (enzymatic) steps in the carbon metabolism work faster or slower, the production of the different by-products can be changed. Many metabolic steps are regulated by proteins. By increasing or decreasing the amount of these proteins, the speed of the reaction steps can be adjusted. With a higher production of the proteins, the reaction step will proceed faster, and with a reduced production, it will be lower. Using genetic techniques, the DNA part – the gene – that codes for a protein can be modified, so that more or less of this protein is produced in the yeast cell. With an “overexpression” of the gene, the protein is produced more, and with a “deletion”, the protein is produced less. The gene can also be modified, giving the protein an improved or reduced function.

Table 2 gives an overview of the proteins whose production or function in the yeast cell has been modified with the help of genetic modification. These proteins are also shown in Figure 1 in the reaction steps that they regulate in the carbon metabolism. The aim of all these studies was to deflect carbon metabolism away from alcohol production and towards the production of other by-products. Many of these genetic modifications were unsuccessful for the production of yeasts for low-alcohol wine. For example because they inhibited the growth of the yeast cells, had too little effect on the alcohol percentage, or produced too many unwanted by-products such as acetaldehyde, acetic acid or acetoin.

Table 2.Genetic adaptations in the carbon metabolism of yeasts to directly or indirectly produce less alcohol5,6
Intended effectGenetic adaptation *
Restriction of glucose uptakeHXT adjustment
Breakdown of glucoseTPS1 overexpression;
GOX1 overexpression
Increase glycerol productionGPD1 or GPD2 overexpression;
TPI1 deletion;
FPS1 adjustment
Increase lactic acid productionLDH overexpression
Reduce ethanol productionPDC2 deletion;
ADH1, ADH3 or ADH4 deletion
Reduce acetic acid productionALD6 deletion
Increase 2,3-butanediol productionBDH1 overexpression
Accelerate the citric acid cycleMDH2 overexpression;
FRD1 overexpression

* The proteins whose production is blocked (deletion) or increased (overexpression) by genetic modification are also shown in Figure 1. The glucose transporter HXT is adjusted so that less glucose is transported into the cell, and the protein FPS1 is adjusted so that it transports glycerol faster out of the yeast cell.

Almost successful genetic modification

Steering the carbon metabolism towards the production of glycerol appears to be the most promising method of modifying the yeasts for a low-alcohol production. Depending on the concentration, the effects of glycerol on the wine are acceptable and sometimes even beneficial. The increased production of the proteins GPD1 and GPD2 ensures an increased production of glycerol (up to 548% higher) and a maximum reduction of the alcohol percentage with 3.6% REF5. However, in addition to increased glycerol levels, this low-alcohol wine also contains high concentrations of acetaldehyde and acetoin. These compounds have a negative influence on the wine taste10. To counter this, a new yeast has been made in which the enzyme ALD6 has also been eliminated to reduce the metabolic pathway to acetaldehyde and acetic acid. This adjustment reduces the production of acetic acid, but the amount of acetaldehyde in the wine is still too high. The wines therefore have unwanted aromas of “crushed apples” 10-12. To compensate for this too, the BDH1 gene has been overexpressed. In this way the excess of acetaldehyde is converted to acetoin and then to the neutral 2,3-butanediol. These yeasts, with modified GPD1, ALD6 and BDH1, produce acceptable quantities of acetaldehyde, acetic acid, acetoin and 2,3-butanediol, but unfortunately also less glycerol and more alcohol compared to the yeast cells with only the BDH1 adjustment. This is probably because these adjustments have too great an effect on the energy balance of the cell. As a result, too little NADH is available for all the reaction steps in the carbon metabolism that depend on NADH10,13.

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All in all, the genetic modification of wine yeasts has provided a great deal of insight into the development of yeasts for low-alcohol wine. Unfortunately, current genetic modification studies are on a dead end, because the modified yeasts produce too many unwanted by-products. In addition, the use of genetic modification is not accepted for commercial low-alcohol wine production. It is possible that the CRISPR technology can still offer a solution here and makes it possible to use modified yeasts. But still a low-alcohol-producing yeast still needs to be developed that does not have a sensory disadvantage for the wine. Anyway, the knowledge gained about the balance of the carbon metabolism in the yeast cell can be used for the development of yeasts without the use of genetic modification.

Evolution in the laboratory

Yeasts are rapidly dividing organisms and can therefore quickly adapt evolutionarily to new circumstances. This principle can be used to cultivate new Saccharomyces yeasts that, for example, produce more glycerol and therefore less alcohol. One of the ways in which this is done is by adding salt to the growth medium of the yeasts, causing osmotic stress. This means that the higher salt concentration in the medium means that the yeasts have to work harder to keep the salt out of the cell. Glycerol helps the yeast cell to keep the salt out, and is therefore a natural remedy for reducing osmotic stress. Yeasts with a high glycerol production are therefore more resistant to osmotic stress and better able to survive in a salt growing medium. By allowing these yeasts to grow for hundreds (!) of generations under increasingly saltier and therefore more stressful conditions, an evolutionary pressure is exerted on the yeasts. Of every generation of yeasts, only the yeasts with the greatest resistance to this osmotic stress survive, and thus the yeasts with an adapted carbon metabolism that produce more glycerol. In this way yeasts have been developed that produce 41% more glycerol and ensure a reduction of the alcohol percentage by 1.3% REF9,14.

Saccharomyces_cerevisiae_SEM

Saccharomyces cerevisiae yeast cells under the electron microscope
Adapted from Mogana Das Murtey & Patchamuthu Ramasamy via CC BY-SA 3.0

The above method takes a long time and is very laborious. Mutagenic techniques have therefore been developed that accelerate the evolution of yeast cells. With these techniques it is possible to develop a suitable yeast with the desired properties within a few yeast generations. For example, yeast populations can be treated with UV radiation, alkylating or deaminating agents, resulting in small mutations in the DNA of the yeast cells. The DNA of these yeasts has changed, but they are – because a mutagenic technique does not provide a targeted adaptation of the DNA – not regarded as genetically modified organisms. The treatment gives the yeasts in the yeast population all different mutations in their DNA. The trick is now to find the yeasts from this mutated yeast population that have received mutations that produce less alcohol, but for example more glycerol. As above, this can be done by increasing the salt content in the growth medium of the yeasts. A new strain of yeast can then be grown from the surviving yeast cells in which the entire yeast population contains the alcohol-lowering mutation. Erbslöh Geisenheim AG holds the patent (number WO 2016/128296 AI) to develop new types of yeasts in this way. Currently, Erbslöh has marketed a yeast under the Oenoferm brand that produces less alcohol (up to 1% less) and more glycerol. However, very specific rules are also provided for the vinification, and one is warned that an increase in the amount of volatile acids may occur15.

The use of “wild” yeasts

Why do all that effort if there is also a whole arsenal of “wild” yeasts that naturally produce less alcohol? In contrast to the Saccharomyces cerevisiae yeasts, these wild yeasts have a carbon metabolism that results in less alcohol production, but – minor detail – also a higher production of acetic acid, acetaldehyde and other undesirable by-products. Nevertheless, at low concentrations, these by-products can give the wine more complexity and thus have a positive influence. However, the disadvantage is that these “wild” yeasts are generally not resistant to alcohol percentages above 5-7% REF16, which means that they cannot convert all sugars in the must. To make low-alcohol wine with the help of these wild yeasts, they can be inoculated sequentially or together with a classical Saccharomyces wine yeast that completes the fermentation16,17.

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Various strains of yeasts have been identified, including from the yeast species Saccharomyces uvarum, Saccharomyces kudriavzevii, Candida membranaefaciens, Hanseniaspora uvarum, Lachancea thermotolerans, Metschnikowia pulcherrima, Pichia kudriavzevii, Pichia kluyverii, Torulaspora delbrueckii and Zygosaccharomyces bailii that are able to produce a reduced amount of alcohol6,16,18-21. In most cases, these yeasts reduced the alcohol content of the wine by less than 1%. Only with the yeast M.pulcherrima and the P. kluyveri were successful in reducing the alcohol percentage by more than 3% REF21. In all cases (at least, when a sensory analysis was carried out) there was an increase in unwanted by-products that made the wine more acidic, bitter, less fruity or caused unwanted aromas of vinegar and oxidation. In addition, these yeasts were often tested in the lab in a sugar solution and only in a few cases on an industrial scale with real grape juice. It is possible that the yeast will behave differently due to an increase in scale from the lab to the large wine tanks in the wine cellar. Therefore, more research is still needed into yeasts that are capable of producing low-alcohol wine on an industrial scale and that have no sensory adverse effects.

The future of low-alcohol wine

The development of yeasts for the production of low-alcohol wine is still developing enormously. None of the methods used have led to a wine yeast producing a wine with spectacularly lower alcohol percentages. In the best case, it is possible to reduce the alcohol percentage by a few percent, but a price must always be paid for this. Current studies show that a (substantial) reduction in the amount of alcohol (regardless of the method used) has a negative influence on the quality of the wine.

The widely used Saccharomyces cerevisae wine yeasts have the great advantage that their carbon metabolism is aimed at a fairly pure conversion of glucose into alcohol. Adjustments in this carbon metabolism ensure the formation of by-products that have an adverse effect on the quality of the wine. It is therefore highly questionable whether it is at all possible to develop a (combination of) yeast(s) with which a complete alcoholic fermentation can be carried out, the alcohol percentage remains (for example) below 5%, and whose sensory influences on the wine will be acceptable. Therefore, for the time being, to produce a really low-alcohol wine, one has to put some water in the wine.

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References
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