The many roles of nitrogen in alcoholic fermentation
Wine yeast requires essential elements to transform must into wine: sugars, vitamins, minerals, oxygen, and also a sufficient concentration of Yeast Assimilable Nitrogen. This type of nitrogen is composed of amino acids, small peptides and ammonium that can be used by yeast cells to complete fermentation and to fully develop the sensory profile of the wine. The type of nutrient added to deficient must and the timing of addition is crucial in the development of alcoholic fermentation.
What is Nitrogen ?
Nitrogen is a molecule composed of two atoms (N2). Its scientific name is dinitrogen. Nitrogen is the main compound found in the atmosphere, representing 78% of the air that we breathe. It is indispensable since it dilutes oxygen, which would otherwise be so concentrated that it would be detrimental to humans.
Nitrogen is present in grape must in different forms: ammonium, amino acids, peptides, and proteins. The part of nitrogen that can be used by yeast during alcoholic fermentation is called ‘assimilable nitrogen’. Yeast cells also contain nitrogen in the form of proteins, peptides – particularly tripeptides – and amino acids (see Figure 1).
YAN (for Yeast Assimilable Nitrogen) includes free α-amino acids (AA), ammonium and some peptides. It is the part of the nitrogen that can be used by wine yeast to efficiently carry out fermentation.
YAN = free α-amino acids + NH4+ + some small peptides
Proline is the only free α-amino acid (AA) not assimilated by yeasts, even if it’s one of the AAs most abundant in grape musts.
➠ FAN = Free α-Amino Nitrogen, which is equivalent to the free α-amino acids
➠ Main free AAs (in quantity) in grapes: proline, arginine, glutamate
Why a good nutrition is important in wine making ?
Nitrogen is a key factor that has a significant impact on wine fermentation. It is the most important yeast nutrient, influencing both fermentation kinetics and wine quality.
Nitrogen is essential to yeast growth and yeast metabolism. In winemaking, YAN plays a key role at two different levels :
- It represents an important nutritional factor for yeasts during alcoholic fermentation
due to its function in protein synthesis and sugar transport.
- It is essential for the biosynthesis of wine quality markers like higher alcohols,
thiols and esters by wine yeast
YAN concentrations in natural grape musts range from about 60 mg/l to 500 mg/L, depending on grape variety, vintage and microclimate.
In nitrogen deficiency conditions, yeast growth and fermentation speed are limited. A low initial YAN concentration has been shown to cause slow and sluggish fermentations, which is why nitrogen addition to the must has become a necessary action in wine production.
Nowadays, YAN measurement is a good way for wineries to assess the general state of grapes in terms of nitrogen quantity, but not necessarily nitrogen quality, which is another key parameter from the point of view of fermentation and the sensory quality of final wine.
In order to properly manage fermentation, the best fermentation practices recommend the addition of nitrogen in inorganic (ammonium) or organic form. Organic nitrogen has been shown to be the most efficient and complete nutrient for securing fermentation and developing the full aromatic potential of the grape, as will be shown further on.
Where do we find Nitrogen ?
In the must: 1/3 of the nitrogen is found in ammonium form and 2/3 in amino acids. Musts are often deficient in YAN ( i.e. < 150 mg/L).
Nitrogen from external sources can be added to the must to assist with fermentation :
- Inorganic nitrogen: ammonium salts (DAP, DAS), which are added during alcoholic
- Organic nitrogen: proteins, peptides, tripeptides and free amino-acids issued
from yeast (inactivated yeast and yeast autolysate). (Figure 1)
When is Nitrogen addition the most efficient ?
Extensive studies have been done to determine the best time to add nitrogen. It has been shown that a nitrogen addition at 1/3 of the fermentation when the yeast population has reach its maximum – i.e., upon nitrogen depletion of the must (all nitrogen from must has been consumed by yeast for the multiplication phase and build biomass) – has the greatest benefit for fermentation rate and kinetics.
A single addition of nitrogen at the beginning of fermentation is not recommended as it leads to a very high yeast population, a sudden increase in fermentation speed accompanied by an exothermic reaction (heat production), and high nitrogen depletion. This quickly leaves the yeasts without any nitrogen left to convert sugar to ethanol. As shown in Figure 2, sluggish or stuck fermentations can occur with a single addition of DAP (30g/hl, equivalent to 63 mg/l of YAN) at the onset of the fermentation.
The effect of nitrogen on wine fermentation kinetics
YAN content has the most influence on fermentation speed ; it impacts yeast biomass at the beginning of fermentation, as well as sugar transport kinetics during fermentation. As soon as a must has a nitrogen deficiency at the end of the growth phase, there is a decrease in protein synthesis and sugar transport activity. YAN addition to nitrogen deficient must leads to a significant decrease in fermentation length by reactivating protein synthesis and increasing sugar transport speed, which results in an increase in the fermentation rate.
There are more and more studies describing the difference in efficiency between organic and inorganic nitrogen additions with respect to kinetics. In Figure 4, we compared the efficiency of both sources (DAP for inorganic and Fermaid O™ for organic) and applied 2 nutrition strategies using the same amount of YAN : 16 mg/L, in organic versus inorganic form. A third fermentation was performed without nutrition, as a control.
Figure 4 shows that for an equivalent amount of assimilable nitrogen added, the addition of organic nitrogen effectively enables the fermentation kinetics (green line) to achieve complete fermentation. With the inorganic nitrogen source added (purple line), the fermentation is sluggish then stuck, as is the case with the control fermentation (blue line) with no nitrogen added.
For an equivalent dosage of YAN, an organic nitrogen source is much more efficient than an inorganic nitrogen source.
On a highly nitrogen deficient must (100 mg/L de YAN), an appropriate organic nutrition strategy is efficient enough to complete the alcoholic fermentation.
Impact of different nitrogen sources on the wine sensory profile
The metabolism of nitrogen, notably amino acids, generates the formation of numerous aroma compounds involved in the aroma matrix of wine: higher alcohols and their acetates. The yeast metabolism also influences the revelation or preservation of certain aroma precursors of an amino nature (cysteinylated precursors or glutathionylated precursors of varietal thiols). As a result, the nitrogen composition of the must can modulate the aroma profile of the wine. The use of organic nutrients has also been shown to influence the formation of aroma compounds when used during alcoholic fermentation.
The metabolism of amino acids (anabolism and catabolism) by yeast leads to the formation of higher alcohols, esters acetate and ethyl esters.
Modulating the esters profile by adding nitrogen has been studied (AWRI-Lallemand project, 2012). DAP additions versus organic nutrient additions were done in Chardonnay grapes. Some results are reported below (Figure 5), comparing the synthesis of esters compounds with an addition of 50mg/L of YAN under DAP form versus 24mg/L of YAN under organic nutrient form. A significant increase for all aromatic compounds is observed with the organic nutrient, underlying the greater efficiency of organic nitrogen compared to inorganic nitrogen on the formation of esters.
During fermentation it has been shown that excessive ammonium levels or addition at the beginning of AF limits the release of varietal thiols by the yeast (Subileau et al. 2008). This phenomenon can be explained by the catabolic repression by ammonium of the synthesis of amino acid transporters in fermenting yeast. This limits the entry of thiol precursors of the cysteinylated type into the cell, and, consequently, their intracellular conversion into volatile thiols (Figure 6).
In terms of thiol production and as shown in Figure 7, the thiols 3-MH and 3-MHA are lower when DAP is used. This observation, made in the laboratory as well as on a pilot scale, has led to the implementation of a strategy for nitrogen nutrition during AF, with the addition of organic nutrient only, divided between the beginning of the AF and 1/3 of the AF.
Two different formulas of organic nutrients have been tested on Colombard grapes, which are very rich in thiol precursors. Looking at the results on the 3-MH release and its acetate of 3-MH, we confirmed the positive impact of organic nutrition on thiol conversion and revelation. (Figure 8a et 8b).
3. Impact of nitrogen source on the wine sensory profile
In all trials, we were able to highlight the positive influence of organic nutrient on the wine sensory profile, mainly when compared to inorganic nutrition such as DAP addition.
In Figure 9 below, the differences are very significant: a professional tasting panel preferred by far the organic nutrition, which produced a more complex and intense wine with greater aromatic length.