August 2018 Issue of Wines & Vines

Winemaking in the Era of Climate Change

Determine yeast-assimilable nitrogen and gluconic acid for optimum alcoholic fermentation management

by Simone Bellassai

An increase in average temperatures in recent years is profoundly changing climatic parameters that influence agricultural activities, including rainfall patterns, maximum and minimum temperatures and humidity.

The phenomenon, commonly referred to as climate change results in extraordinary weather parameters and an increase in the frequency of vintages marked by high temperatures and water scarcity as well as vintages characterized by low temperatures and high levels of rainfall. With these kinds of extreme vintages, the analyses of yeast-assimilable nitrogen (YAN) and gluconic acid acquire increasing importance in the winemaking process in order to maximize quality in a finished wine.

Temperature anomalies have been measured for decades during the ripening period of grapes in North America and Europe (Figure 1: "Temperature Anomalies in North America, 1960-2015" and Figure 2: "Temperature Anomalies in Europe 1960-2015"). The past 15 years have been characterized by an increase in abnormalities compared to the average temperatures measured since 1910.

Extreme meteorological phenomena are reflected by diametrically opposite weather trends in 2003 and 2014. In 2003, Northern Europe experienced one of the hottest vintages ever, with average temperatures from May to October far above the standard, accompanied by extreme drought. In 2014, France had one of its rainiest years ever, with precipitation up 140% from June through August. In July 2014, Milan, Italy received about 300 mm of rain during a 15-day stretch, compared to a 73-mm average for that period.

Climate impact on must
What are the effects of high temperatures and water shortages on the grape-ripening phase? Conversely, what are the effects of low temperatures and high precipitation? While the characteristics of these situations differ, both have a strong impact on the must and are equally difficult to manage in order to achieve optimum fermentation. Since the enologist is unable to influence meteorological factors, optimal management of the alcoholic fermentation is crucial to preserve the aromas, avoiding organoleptic deviations that are difficult to "recover" in subsequent steps.

Besides the analyses of total acidity, sugar, pH and volatile acidity, which must be performed on an increasingly time-oriented basis, the analyses of YAN and gluconic acid become fundamental. These two analyses are valuable tools in the hands of enologists, who can use them to manage the delicate process of alcoholic fermentation.

High rainfall and low temperature
Grapes harvested in vineyards characterized by high rainfall and low temperatures have a deficit of phenolic and technological maturation. The climatic conditions characterized by low solar radiation coupled with the potential for reduced photosynthetic surface due to downy mildew attacks are common in this type of vintage, resulting in low sugar production and inadequate degradation of organic acids.

The simultaneous action of fungi such as Botrytis cinerea, resulting in mold formation or, worse, the action of fermentative yeasts and acetic bacteria responsible for development of sour rot provides an additional phytosanitary aggravation of a situation that is already difficult to manage.

Botrytis cinerea is a fungus widely found in temperate viticultural regions where moisture is not limiting during the fruiting season. The presence of water on the surface of the grapes, together with temperatures between 15° and 25° C, triggers the mycelium growth. Under conditions of alternating dry and humid periods, the disease proliferates in its "noble" form. The alternation of warm and windy afternoons with cold and humid mornings, particularly with the presence of loose clusters of grapes that are well-ventilated, allow development of so-called "noble rot."

This particular form of B. cinerea infections developing on certain cultivars under a specific set of environmental conditions can result in wines of high quality and commercial value, such as Sauternes-Barsac and Hungarian Tokay. However, this form of mold is an exception that can be achieved only by combining a favorable climate with specific grape varieties. More often, the winemaker is left in the position of vinifying grapes that have ripened under poorly ventilated conditions, resulting in Botrytis infections that produce gray mold.

Grapes affected by this dangerous pathology result in must with the following characteristics:
o Low sugar content: Sugar concentration is weak due to the degradation caused by fungus.
o Clarification difficulty due to increased viscosity and suspended solids.
o Organoleptic and aromatic deviations due to the presence of mold metabolites.
o Greater degradation of L-malic and tartaric acids, resulting in a decrease in total acidity.
o Risk of color alteration due to the action of fungal laccase enzyme.
o Higher-than-normal concentrations of acetic and citric acids.
o An increase, occasionally consistent, in the concentration of gluconic acid, as much as 3-4 g/L.
o A low concentration of nitrogen and presence of toxins that alter yeast metabolism.

The processed must of grapes affected by gray mold might fall within different "criticalities." Among these, there almost certainly will be difficulties with the fermentation, in large part due to depletion of nutrients, with sources of YAN such as ammonium and amino acid nitrogen having been depleted by the fungal pathogen for its own metabolism.

Moreover, the increase in gluconic acid will make it difficult to protect the must from oxidation because the increased total SO2 will be high. Thus, at the same level of total SO2, a must with high gluconic acid will result in a bound SO2 level that is greater than the same must produced from healthy grapes.

The analysis of gluconic acid is an essential tool for the enologist not only to classify the health level of the must but also to recognize the grape juice most affected by gray mold and thus be able to establish the most suitable fermentation protocol.

When ripening grapes are injured by hail, berry cracking or excessive compaction within tight-clustered cultivars and clones, they are also subject to development of sour rot, especially under warm pre-harvest conditions. This disease, now known to result from the fermentation of berry sugars to ethanol by resident yeasts followed by its oxidation by specific bacteria, results in a major increase in acetic and gluconic acid, which can reach levels that make the must completely unusable.

Gluconic acid
Grapes attacked by gray mold - or worse, affected by sour rot - contain variable amounts of gluconic acid, which is a derivative of glucose, oxidized by the enzyme flavin adenine dinucleotide (FAD) glucosidase present in Botrytis, in the absence of phosphorylation agents (see Figure 3: "Enzymatic Glucose Oxidation").

The presence of Gluconobacter bacteria results in the formation of gluconic acid, as well as respective 2-oxo gluconic, 5-oxo gluconic and 2.5-dioxo-gluconic acids, which have a combined effect in terms of SO2 that is even more pronounced than gluconic acid (see Figure 4: "Enzymatic D-gluconic Acid Oxidation").

High temperatures and high stress due to water scarcity
The best vintages for red wines are those characterized by medium-high temperatures and moderate water stress, while the best white wines, which are more delicate aromatically, require cooler vintages and low water stress.2

What are the effects of extreme temperatures and water scarcity on composition of the must? From a meteorological point of view, during a "normal" vintage, the ripening process of the grape involves an increase in sugar content and a decrease in acids, especially L-malic acid, the form most used by the plant for respiration.

Tartaric acid, however, remains almost constant. Analyses of total acidity, pH, sugars and L-malic acid therefore provide indices useful in evaluating grape maturity. The accumulation of anthocyanins in the skins, and their extractability, are highly vintage-dependent, and technological and phenolic maturity often do not occur at the same time.

The situation is aggravated by climate change. High temperatures minimize the growth of destructive forms of mold, reducing the negative resulting aspects but contributing to a loss of nitrogen in the grapes. Under these conditions, YAN in the must is heavily depleted, and this deficiency will be dangerous for both red- and white-wine musts.

Nitrogen, together with sources of carbon in the form of fermentable sugars (glucose and fructose) and growth factors, is a key element for yeast metabolism. Nitrogen is vital to all living organisms, contributing to the formation of peptide bonds, the "support beam" of protein structures.

Fundamental to the metabolic activity of yeast, nitrogen in both its inorganic form (ammonium ion) and organic form (represented by free amino acids) are used by yeasts both for the production of structural proteins and the enzymes that take part, to a varying degree, in the fermentation process. In addition to free amino acids, the must also contains nitrogen as peptides and protein, but these two forms of nitrogen are not used by yeasts due to their lack of proteasic activity.

In the presence of low levels of YAN, the measurement and the consequent integration of inorganic (ammonium) and organic (amino acidic) forms becomes a fundamental requirement for an optimal alcoholic fermentation. Determination of levels of inorganic and organic nitrogen, and correction of deficiencies, is therefore crucial for an optimal fermentation. Nitrogen supplementation becomes even more important with the high-sugar musts typical of warm vintages. It is no surprise that the analysis of YAN has been recognized in recent years as a fundamental analysis to create high-quality wine.

Absorption of YAN and effect on fermentation
Knowledge of the assimilation mechanisms of YAN and the consequences of insufficient amounts is important to correctly plan and manage alcoholic fermentation. Ammonium absorption and amino acids within the yeast cell occur by means of protein transporters. S. cerevisae possesses at least four of them: two for inorganic nitrogen, two for organic nitrogen. For the latter, there are selective transporters according to the type of amino acid and non-selective transporters, commonly called general amino permease (GAP).

For both assimilable nitrogen forms, the mechanism of entry into the cell is active in contrast to what occurs with glucose and fructose, which flow into the cellular cytosol by passive diffusion. In the early stages of alcohol fermentation, the relatively high concentration of ammonium ions inhibits the non-selective GAP that regulates amino acid nitrogen intake. This is why the nitrogen that is first absorbed by yeast is in an inorganic, ammonium form.

The correction of organic nitrogen at the start of fermentation is highly recommended because specific carriers are not inhibited by ammonia. Amino acids can spread within the cell, significantly stimulating the formation of enzymes and proteins. The yeast is able to use the amino acids directly, without modifications, entering them into the protein synthesis process.

Entry of YAN into the yeast cell occurs by means of active transport and therefore involves energy consumption at the expense of adenosine triphosphate (ATP). Alcoholic fermentation, with its corresponding increase in ethanol, alters the plasma membranes. The active transport process is greatly restricted, resulting in a reduction of nitrogen flow into the yeast cell. In other words, it is only at the initial stages of fermentation, when the concentration of ethanol is low that the yeast is able to rapidly assimilate the nitrogen that then will be used throughout the entire fermentation.

Yeast either can use the amino acids in this form or perform deamination, releasing the nitrogen and the respective higher alcohol. The released nitrogen will then be used for synthesis of the amino acids needed for protein synthesis. Addition of amino acids at the initial stage, at the expense of ammonia, can thus increase the deamination process of the yeast, which needs nitrogen for its protein synthesis. In this case, there will be higher alcohol production that can adversely affect the aromatic profile of the wine.

YAN correction prior to the start of alcoholic fermentation, as well as avoiding dreaded stuck fermentations accompanied by sudden increases in volatile acidity, minimizes production of reduced sulfur compounds. Nitrogen deficiency does not, in fact, allow the yeast to produce sulfur-containing amino acids such as cysteine and methionine through the use of sulfates, sulfites and sulfur anions. Thus, with a low YAN value these anions enter into a reduction chain that results in acquiring the degree of minimum oxidation in the form of sulfur ion, causing the characteristic smell of rotten eggs.

What are the stages at which YAN analysis is important? The answer depends on the kinetics of alcoholic fermentation. Generally speaking, there are two crucial points at which YAN analysis must be performed: prior to the onset of fermentation, to evaluate the appropriate supplementation rates, and at the exponential growth stage of the yeast, when the almost complete depletion of nitrogen can become a limiting factor in the fermentation process.

Valuable tools against climate change
Wine and grape-juice analyses are crucial elements to achieve a high-quality wine. The winemaking process has changed drastically over the years thanks to enological research and new analytical techniques. Enzymatic and colorimetric analyses play an important role in improving winemaking management, and the ability to conduct them in "real time" can give a winemaker a more accurate vinification control, besides the "standard" enological analyses, with climate change.

YAN and gluconic acid constitute two important analyses for the management of alcoholic fermentation. In years when weather conditions are extreme, these analyses are even more important to obtain a quality wine. Their execution, in real time, allows an enologist to better design the fermentation protocol, providing appropriate SO2 addition and planning adequate nutrition for the yeasts.

Simone Bellassai is a food and beverage analysis expert with a double degree in chemistry and enology from University of Florence. CDR srl offers CDR WineLab, an innovative analysis system for grape juice and wine.

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