Understanding the impact and differences of amino acids versus ammonium to produce aroma compounds can help winemakers optimise their processes and produce high-quality wines with desired aromatic profiles. Yeasts’ metabolism of some nitrogenous compounds can contribute aromas to the wine that include chemical and delicate fruit – think varnish versus a summer fruit salad.
Nitrogen is a key element necessary for synthesising amino acids, nucleic acids, vitamins and various other compounds in living organisms, including wine yeasts. It is present in various compounds in grape juice, including ammonium, amino acids, nitrates, peptides and proteins. Nevertheless, wine yeasts can only make use of two sources of yeast assimilable nitrogen (YAN) for their metabolism and growth, namely, amino acids (organic nitrogen) and ammonium (inorganic nitrogen). The uptake of these nitrogen sources is subject to complex regulatory processes, and the timing and order of uptake vary significantly depending on the yeast species, strains and also on environmental conditions.
During wine fermentation, the skeleton of selected amino acids may be used by the yeasts to produce aromas (i.e., the so-called “fermentative” or “secondary” aromas). Alternatively, yeasts can make some of these aromas “from scratch” (i.e., from the breakdown of sugars).
Differences in transport between amino acids, proteins and ammonium
Amino acids are the building blocks of peptides and proteins that are essential as enzymes, transporters and structural components, amongst other functions. One key difference between amino acids, proteins and ammonium is their assimilability by the yeast. Under fermentation conditions, proteins do not contribute to the YAN in the main wine yeast, Saccharomyces cerevisiae, as it lacks transporters able to take in such large molecules, whereas amino acids are already in a form that can be incorporated by various transporters. Some of these transporters can take up all amino acids, while others are much more specific and only facilitate the uptake of one or a limited number of amino acids. Once in the cell, amino acids can be stored or used directly to produce proteins, but are most often broken down to liberate the nitrogen that may be used similarly to ammonium (see below). The remaining skeleton may then be used to produce higher alcohols, fatty acids and esters.
Ammonium is transported into the cell via permeases, where it must be converted into organic forms, such as amino acids, via the process of nitrogen assimilation. These organic forms can then be used by the cell to produce any of the other amino acids. The uptake of nitrogenous compounds from the grape juice differs for each amino acid and ammonium, with each requiring different regulatory mechanisms for incorporation. The mechanisms for nitrogen metabolism are complex as its intermediates are also shared between other metabolic pathways, including carbon metabolism.
Effect on aroma production
Amino acids and ammonium differ in their impact on the production of specific aromatic compounds. Studies have found that supplementing certain amino acids enhances the production of aroma compounds, including esters, which can contribute to fruity and floral aromas in wine. Most aroma compounds in wine arise from sugar metabolism. In studies where yeasts were provided with ammonium only, they were limited to producing aroma compounds arising only from sugar metabolism. These aromas include acetic acid (vinegar off-aroma), medium-chain fatty acids, and certain higher alcohols and esters. The most common ester present in wine is ethyl acetate (nail polish remover off-aroma) which is formed through the reaction of acetic acid with ethanol, but other more sought-after esters include phenyl-ethyl acetate (rose/honey) and ethyl isovalerate (fruit).
In a study of amino acids added to a wine fermentation, it was noted that the yeasts produced a more diverse range of aroma compounds. Indeed, important aromatics are produced from certain amino acids in the so-called Ehrlich pathway (Figure 1). This pathway is linked with sugar metabolism to produce certain amino acids, which result in the production of a corresponding aroma compound. This means that some aroma compounds are produced from both sugar and amino acid metabolism, while others can only be produced via the breakdown of specific amino acids, as in the Ehrlich pathway.
The Ehrlich pathway (Figure 1) is responsible for breaking down certain amino acids into aromatic compounds that can have unpleasant odours. Depending on the redox status of the cell, these compounds can be short-chain fatty acids formed by the oxidation of an aldehyde. Examples of fatty acids produced through this pathway include isovaleric acid, which can cause rancid cheese odour, and propionic acid, which can create a pungent aroma. Higher alcohols, such as isobutanol with a savoury/wine-like aroma or isoamyl alcohol that is pungent, are also produced. Higher levels of amino acids in grape juice have been observed to result in an increased production of volatile fatty acids and higher alcohols, as these are substrates for aroma compounds through the Ehrlich pathway. The yeasts can then metabolise these higher alcohols and fatty acids further into acetate esters or fatty acid ethyl esters, which impart desirable aromas to the wine.
Common acetate esters produced from amino acids are isoamyl acetate (banana aroma), isobutyl acetate (fruity/floral) and propyl acetate (pear). Common fatty acid ethyl esters produced in this pathway include ethyl decanoate (grape/brandy aroma) and ethyl butyrate (fruity pineapple). Off-odours on their own might be off-putting, but in small amounts and/or in interaction with other aroma compounds, they contribute to aromatic complexity and can greatly enhance the bouquet of the wine.
FIGURE 1. The Ehrlich pathway leading to the production of fermentative aroma compounds.
Yeast species and strain selection for aroma production
The production of aromatic compounds from amino acids and ammonium can vary greatly depending on the metabolic flux of the species and the strain of yeast in the fermentation. Some yeasts may be more efficient at utilising different amino acids as nitrogen sources. Similarly, some might channel more amino acids into fermentative aromas, while others will mostly produce these compounds from sugars. This partly explains why so many active dry yeast products are available on the market. These yeasts have been specifically selected for their aroma compound production to suit any requirement.
Impact of winemaking conditions
Winemaking conditions, such as temperature, oxygen levels and nutrient availability, can also impact the production of aroma compounds. Higher temperatures result in increased evaporation rates and loss of sensitive aroma compounds. This includes the esters, which are especially sensitive to high temperatures. Indeed, at 24°C, most esters produced during the fermentation will be lost due to evaporation.
The presence of oxygen limits the production of acetate esters from higher alcohols. Higher levels of oxygen lead to an accumulation of higher alcohols. Fermentations that have been oxygenated, such as red wine fermentations, also result in lower levels of fatty acids and ethyl esters.
The levels of lipids in the grape must also affect the production of esters. Higher levels of lipids have been correlated with lower concentrations of acetate esters as the enzymes converting higher alcohols to acetate esters are repressed. On the other hand, lipid deficiency can also result in slow or stuck fermentations.
Besides the source of nitrogen, the levels of yeast assimilable nitrogen (YAN) also play a large role in producing aromatic compounds. Grape musts with higher levels of YAN tend to produce higher levels of fruity esters and lower levels of undesirable higher alcohols and hydrogen sulphide (H2S) during fermentation. If the YAN is too low, yeasts must produce their own amino acids, including the sulphur-containing amino acids methionine and cysteine. To obtain the sulphur necessary to produce the latter two amino acids, the yeasts take up sulphate from grape juice. During the reduction of sulphate to sulphur, they produce SO2 and H2S. Excess accumulation of these intermediates results in their liberation into the fermenting grape juice, with the latter generating the well-known rotten egg off-odour in the wine. Differences in SO2 and H2S production between yeast strains remain poorly understood, but are typically connected to their differing nitrogen requirements.
YAN supplementation in nitrogen-deficient musts will ensure that (1) yeasts produce sufficient biomass and avoid stuck fermentation and (2) produce sought-after aroma compounds. It is now well understood that a single addition in grape juice of a large amount of nitrogen (especially ammonium) is likely to induce very high biomass production coupled with a very high, but short-lived, fermentation rate accompanied by excessive temperature increase. A sluggish second phase of fermentation that may even become stuck is common. A better solution is to split the addition as follows: before fermentation in the grape juice (a source of organic nitrogen) and the second addition when yeasts stop growing after around one-third of the sugar has been depleted (organic and/or inorganic nitrogen). Indeed, adding an organic source of YAN at the beginning of growth will ensure that sufficient biomass is produced, while not increasing the fermentation rate excessively.
YAN supplementation during the yeast growth phase during fermentation is also observed to increase fatty acid ethyl esters, acetate esters, as well as ethyl acetate, but decrease levels of higher alcohols. The supplementation of YAN at the beginning of the stationary phase will “boost” fermentation kinetics and the production of aroma compounds. Nevertheless, significant differences are observed in the production of specific higher alcohols. This is mainly because, as mentioned above, some of the compounds mostly originate from sugar metabolism, while others mostly originate from amino acid metabolism in a yeast species/strain-dependent manner. Finally, the timing of addition seems to play a role, but once again, the impact differs depending on the aroma compound. Indeed, recent results showed that the addition of nitrogen during the stationary phase increases the production of sought-after acetate esters by inducing transcription of the genes coding for the enzyme that converts higher alcohols to acetate esters. It is, however, advised not to add excessive amounts too late, because the fermenting yeasts will not be able to take them up fully, and the remaining amounts can be used by spoilage organisms after the completion of fermentation.
YAN addition to grape must
It is permissible in most wine regions to raise the YAN of grape juice with the addition of ammonium salts in various forms. This includes the regularly utilised diammonium phosphate (DAP) and ammonium sulphate, which should be used when confronted with severe YAN deficiencies. However, whereas the addition of specific amino acids to grape must to “flavour” the final wine by influencing the production of specific aromatic compounds is not permitted, that of inactive yeasts (containing ammonium, amino acids, peptides and proteins) is allowed and has shown evidence of affecting the production of aroma compounds positively. Nevertheless, while most metabolic pathways leading to aroma production are known, there is much that is not understood. Research is ongoing regarding the complex interactions between nitrogen sources, yeasts and other nutrients, and the impact thereof on the production of aromatic compounds in winemaking.
Abstract
Nitrogen is an essential element for yeast metabolism, including the production of aroma compounds. Wine yeasts mainly make use of ammonium and amino acids to meet their nitrogen requirements, but these two differ in their availability to the yeast and their impact on the production of specific aroma compounds. The source of nitrogen also plays a role in the production of aroma compounds, including esters, higher alcohols and fatty acids. The Ehrlich pathway significantly contributes to the formation of aroma compounds in wine. During the fermentation, environmental conditions, nitrogen levels, and the selection of yeast species and strains have a significant impact on the production of aroma compounds. Therefore, understanding the impact and differences of the nitrogen source during fermentation can help winemakers optimise their processes to produce high-quality wines with the desired aromatic profiles.
Highlights
- Nitrogen is a key element necessary for the synthesis of amino acids, nucleic acids, vitamins, and proteins in living organisms.
- During grape juice fermentation, yeasts mainly utilise sugars, but can also use amino acids and ammonium salts to produce compounds that contribute to the aroma profile of the wine.
- While most aroma compounds arise from sugar metabolism, supplementing certain amino acids and ammonium salts can impact the production of certain aroma compounds, including esters, higher alcohols and fatty acids.
- Winemaking conditions, including temperature, pH, oxygen and availability of nitrogen and other nutrients, all influence the production of aroma compounds from nitrogen sources.
- The production of aroma compounds varies greatly between species and strains.
- It is permissible to increase the YAN of grape must by using ammonium salts only or yeast-derived products.
References
Barbosa, C., Mendes-Faia, A. & Mendes-Ferreira, A., 2012. The nitrogen source impacts major volatile compounds released by Saccharomyces cerevisiae during alcoholic fermentation. International Journal of Food Microbiology 160(2), pp. 87 – 93.
Burdock, G.A., 2010. Flavor Ingredients, 6th Edn Boca Raton. FL: Fenaroli’s Handbook, CRC Press.
Fairbairn, S., McKinnon, A., Musarurwa, H.T., Ferreira, A.C. & Bauer, F.F., 2017. The impact of single amino acids on growth and volatile aroma production by Saccharomyces cerevisiae strains. Frontiers in Microbiology 8, p. 2554.
Mouret, J.R., Camarasa, C., Angenieux, M., Aguera, E., Perez, M., Farines, V. & Sablayrolles, J.M., 2014. Kinetic analysis and gas-liquid balances of the production of fermentative aromas during winemaking fermentations: Effect of assimilable nitrogen and temperature. Food Research International 62, pp. 1 – 10.
Rollero, S., Mouret, J.R., Bloem, A., Sanchez, I., Ortiz‐Julien, A., Sablayrolles, J.M., Dequin, S. & Camarasa, C., 2017. Quantitative 13C‐isotope labelling‐based analysis to elucidate the influence of environmental parameters on the production of fermentative aromas during wine fermentation. Microbial Biotechnology 10(6), pp. 1649 – 1662.
Vilanova, M., Pretorius, I.S. & Henschke, P.A., 2015. Influence of diammonium phosphate addition to fermentation on wine biologicals. In: Processing and impact on active components in food (pp. 483 – 491), Academic Press.
For more information, contact Benoit Divol at divol@sun.ac.za.
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