It is important for winemakers to understand the factors contributing to lipid deficiency in grape juice and solutions to circumvent this.
Lipids are hydrophobic molecules, such as fats and oils. In grapes, they are found in the outer waxy layer of the berry, in the skin, flesh and seeds at differing concentrations.
These compounds – which are integral components of cell membranes – are used for energy storage, serve as precursors to varietal aroma production, and are involved in the ripening of grape berries. During the winemaking process, they serve as a key nutrient for the growth of yeasts.
While not typically quantified in grape must, a shortage of lipids may cause sluggish fermentations and the production of off-flavours.
Pre-fermentative clarification in white winemaking and the link with lipid content
Lipids form part of the insoluble fraction of grape must. This fraction also contains polyphenol oxidases and esterases.
These two enzymes can be detrimental as they can result in the browning of white grape must and the reduction of esters, respectively. To prevent this, winemakers implement a pre-fermentative clarification step which reduces this risk by eliminating most of these enzymes.
The clarification also lowers the risk of the production of off-flavours caused by excessive concentrations of higher alcohols and sulphur-containing compounds. Due to these benefits, clarification has become a standard step during white winemaking.
However, winemakers should be wary of the consequences of over-clarifying. Indeed, clarification also eliminates lipids, and excessive clarification may result in a stuck fermentation due to lipid deficiency.
Why do wine yeasts need lipids?
Lipids are found in membranes such as the plasma membrane. In addition to the cell wall, membranes are vital for maintaining the structure and functioning of the cell.
Moreover, lipids can serve as energy reserves in yeasts to be utilised during times of starvation. Lipids are also indirectly involved in nutrient transport and the yeasts’ tolerance to stressors, such as ethanol and cold temperatures.
Therefore, alcoholic fermentation cannot progress in the absence of lipids.
The nitty-gritty of lipid production in wine yeasts
Yeasts primarily produce ergosterol (the yeast version of the human cholesterol and the plant phytosterols), and fatty acids – of particular importance are unsaturated fatty acids (the yeast version of omega acids). It is important to note that the main wine yeast, Saccharomyces cerevisiae only produces unsaturated fatty acids with one double bond along the fatty acid chain.
On the other hand, commercially relevant non-Saccharomyces yeasts, such as Metschnikowia pulcherrima and Torulaspora delbrueckii, can also produce fatty acids with more double bonds (known as polyunsaturated fatty acids). The yeast uses all these fatty acids to produce phospholipids and sphingolipids.
Together with ergosterol, they form the backbone of cellular membranes (Figure 1). Proteins such as nutrient transporters are then embedded into the membranes.
It should be emphasised that the production of ergosterol and unsaturated fatty acids requires oxygen, which is in minimal supply during fermentations. While yeasts can produce their own lipids in the presence of oxygen, they can also take up lipids from their environment (i.e., grape juice).
This is especially important in the absence of oxygen when the presence of lipids, mainly unsaturated fatty acids and phytosterols, in grape juice becomes essential.
How do lipids impact yeast stress survival during wine fermentation?
As fermentation progresses, oxygen is rapidly depleted, and the yeasts must rely on the presence of sterols and unsaturated fatty acids in grape juice. These can be taken up to compensate for the lack of oxygen-dependent production in the cells.
These lipids ensure higher viability and the optimal transport of nutrients as the environment becomes increasingly unfavourable due to the rise in ethanol levels. Indeed, ethanol negatively affects yeast viability by disrupting membrane structure and function.
If yeasts are unable to adjust their membrane composition, the accumulation of ethanol will result in an inhibition of sugar uptake and metabolism, ultimately leading to a stuck fermentation. Adjustments in the lipid composition to counteract the detrimental effects of ethanol include the enhanced incorporation of sterols and monounsaturated fatty acids in S. cerevisiae to maintain membrane fluidity (Figure 2).
On the other hand, the presence of lower concentrations of ergosterol and higher amounts of polyunsaturated fatty acids has been linked to ethanol sensitivity in wine-related non-Saccharomyces yeasts indicating variability in the lipid metabolism of wine yeasts. In addition to playing a role in ethanol tolerance, the presence of unsaturated fatty acids is necessary to increase plasma membrane fluidity during fermentations at a low temperature.
Lipids’ impact on the wine bouquet
Acetic acid production can be influenced by the presence of lipids in grape juice. Indeed, if the concentration of lipids in the juice is sub-optimal, yeasts are forced to produce their own lipids as a compensatory mechanism.
To achieve this, the yeasts increase their production of acetyl-CoA (which derives from sugar metabolism), which serves as a substrate for lipid production. This over-production of acetyl-CoA frequently results in the production of acetic acid and, subsequently, ethyl acetate, thereby increasing the wine’s volatile acidity.
Medium chain fatty acid levels tend to increase, because of this overproduction.
On the other hand, a higher concentration of lipids (or oxygen) has been connected to enhanced production of ethyl esters. Moreover, the addition of lipids has also been shown to increase the production of the sought-after thiols 4-mercapto-4-methyl-2-pentanone (4MMP) and 3-mercapto-1-hexanol (3MH) responsible for box tree or fruity aromas in a S. cerevisiae x Saccharomyces kudriavzevii hybrid, but to decrease 3-mercaptohexyl acetate (3MHA) responsible for fruity aromas.
The latter is due to the repression of the enzyme that catalyses the conversion of higher alcohols to acetate esters.
Recommendations to winemakers
Although it can be achieved in a well-equipped analytical laboratory, quantifying lipids is not a straightforward affair. However, the quantity of grape solids (grape juice turbidity) is a satisfactory proxy for lipid availability in white or rosé grape juice.
Turbidity is typically measured by a turbidity meter and expressed in nephelometric turbidity units (NTU). To ensure enough lipids are available for utilisation by yeasts, turbidities of 50 – 150 NTU are recommended, with 100 NTUs being the best compromise to prevent stuck fermentations, whilst minimising the production of off-flavours.
Commercial lipid-rich nutrients can also be used during rehydration to increase yeast viability, especially if fermentation conditions are predicted to be difficult (e.g., cold temperature or high sugar content). Micro-oxygenation/aeration can also be used to induce lipid biosynthesis and increase yeast viability, but should be implemented with caution during white winemaking to prevent oxidation and the appearance of off-flavours.
Current research
It was recently discovered that wine-related non-Saccharomyces yeasts, such as Lachancea thermotolerans, T. delbrueckii and M. pulcherrima, cannot take up sterols. Thus, the poor fermentation performance of non-Saccharomyces yeasts could, in part, be due to an inability of these yeasts to adjust their membrane composition.
Indeed, whereas S. cerevisiae assimilates sterols in addition to fatty acids to reinforce the plasma membrane as ethanol accumulates, it appears that selected non-Saccharomyces yeasts are limited to only utilising fatty acids during fermentation, but this hypothesis remains to be assessed. Overall, there is still much that we do not fully understand about the role of sterols and fatty acids on yeast tolerance to ethanol and cold temperatures, as well as on aroma production.
These aspects require more research.
Abstract
Lipids are essential for yeast cell structure, functioning and energy storage. These compounds are removed from grape juice during clarification, and lipid deficiency may cause stuck fermentations.
Free fatty acids and phytosterols present in grape must are utilised by S. cerevisiae for fermentation performance, ethanol stress resistance and viability. Furthermore, these compounds are used to produce volatile compounds which impact the organoleptic properties of wine.
Therefore, winemakers should ensure that the grape juices they work with have sufficient lipids for successful fermentation. Grape juice turbidities of 50 – 150 nephelometric turbidity units (NTU) are recommended to ensure successful fermentation.
In case of over-clarification, the lipid content of grape juice can be increased by adding commercial lipid-containing nutrients. Alternatively, micro-oxygenation/aeration could be applied to increase lipid biosynthesis and viability in yeast.
Highlights
- Lipids are vital components of yeast cell membranes.
- During fermentation, yeasts must take up lipids from grape juice to handle ethanol and cold temperature stress.
- Lipid deficiency can occur, because of over-clarification in white grape juice.
- Turbidities of 50 – 150 NTU are recommended to avoid deficiency and possible stuck fermentations.
- Alternatively, lipid-rich nutrients can be added, or micro-oxygenation/aeration implemented with caution.
References
Archana, K.M., Ravi, R. & Anu-Appaiah, K.A., 2015. Correlation between ethanol stress and cellular fatty acid composition of alcohol producing non-Saccharomyces in comparison with Saccharomyces cerevisiae by multivariate techniques. Journal of Food Science and Technology 52: 6770 – 6776.
Luparia, V., Soubeyrand, V., Berges, T., Julien, A. & Salmon, J-M., 2004. Assimilation of grape phytosterols by Saccharomyces cerevisiae and their impact on enological fermentations. Applied Microbiology and Biotechnology 65: 25 – 32.
Mbuyane, L.L., Bauer, F.F. & Divol, B., 2021. The metabolism of lipids in yeasts and applications in oenology. Food Research International 141: 110142.
Tesnière, C., Pradal, M. & Legras, J.L., 2021. Sterol uptake analysis in Saccharomyces and non-Saccharomyces wine yeast species. FEMS Yeast Research 21(3), foab020.
Tumanov, S., Zubenko, Y., Greven, M., Greenwood, D.R., Shmanai, V. & Villas-Boas, S.G., 2015. Comprehensive lipidome profiling of Sauvignon blanc grape juice. Food Chemistry 180: 249 – 256.
– For more information, contact Prof Benoit Divol at divol@sun.ac.za.
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