by Molly Kelly, Enology Extension Educator
By managing fermentation, winemakers today have many options to enhance the varietal characteristics of their wines, and to express regional attributes. Winemakers know that temperature is a management tool that affects the rate of fermentation; similarly, the presence of grape solids enhance yeast survival. Very importantly, adequate nitrogen (N) is necessary for a successful fermentation.
Grapes contain a variety of nitrogenous compounds, the sum of which may be affected by viticultural practices. For instance, research has demonstrated that N concentration is 2X greater with application of foliar N and appropriate irrigation use than without foliar N and irrigation. Other research suggests that N application around veraison appears to be an effective way increasing N in the fruit, regardless of water-supply status of the vines. The concentration of nutrients, whether too great or too little, can induce stress and lead to different concentrations of flavor compounds. For instance, H2S formation is a well-known example related to inadequate nutrients leading to nitrogen depletion stress.
A common practice among winemakers is to make a standard addition of diammonium phosphate (DAP), or other N source, to the juice or must (100-300 mg/L) at inoculation without measuring the nitrogen concentration. The objective of this article is to show that N addition has significant flavor (and ultimately, economic) consequences and that measuring the initial nitrogen concentration provides the opportunity to adjust N addition – not only to achieve an adequate fermentation rate, but also to more reliably guide the flavor profile and style of wine intended.
Definition and Measurement of “YAN”
Grapes contain a variety of nitrogenous compounds of which the most important are the primary (alpha) amino acids, ammonium ions, and small peptides. These three nitrogenous compounds – amino acids (excluding proline), ammonium ions, and small peptides – constitute what is commonly referred to as yeast assimilable nitrogen (YAN) or fermentable nitrogen.
YAN measurements, ideally, should be performed directly on juice or must samples at the point of inoculation to avoid over-estimation due to processing losses which inevitably occur between vineyard and the fermenter. Juice samples taken from grape musts can under-estimate total berry YAN due to the disproportionate concentration of amino acids contained in the unsampled grape skins. While an early warning for low YAN may be obtained by sampling in the vineyard one to two weeks prior to harvest, measurement immediately before fermentation is necessary due to the highly variable nature of YAN measurements during those last weeks before harvest.
Favored methods of measurement of YAN are (a) enzymatic assay kits, (b) the method known as the Formol Titration, which consists of neutralizing a juice sample with a base, then adding an excess of neutralized formaldehyde, and re-titrating the resulting solution to an endpoint; and (c) use of expensive equipment such as the HPLC (high-performance liquid chromatography). Typically, wineries use the first two methods; commercial labs may use the third method.
How much YAN
YAN has the most impact on fermentation speed compared to other compounds. It impacts yeast biomass at the beginning of fermentation and sugar transport during fermentation. At the end of growth phase, N is depleted resulting in decreased protein synthesis and sugar transport. A YAN addition at this point reactivates protein synthesis and sugar transport increasing the fermentation rate. Oxygen is rapidly consumed in the beginning of fermentation. Decreased oxygen inhibits sterols and fatty acid synthesis by yeast. This causes decreased yeast growth and viability at the end of fermentation.
Sterols and fatty acids are survival factors needed for the yeast cell membrane to function. As ethanol increases, hydrogen ions accumulate in cell requiring more energy to expel them. The pH decreases inside the cell causing cell death. Oxygen adds at end of growth phase increase sterol production. Therefore, microoxygenation and pump overs would be beneficial at 1/3 of the way through alcoholic fermentation (end of yeast growth phase).
The manner in which N is assimilated by yeast depends on the source. Organic N (amino acids) is actively transported into the yeast cell. Through additional reactions N is incorporated into glutamine and glutamate and eventually used in the synthesis of other amino acids and nitrogenous compounds. This process is gradual and efficient compared to inorganic sources. Ammonium nitrogen (inorganic N) is consumed quickly and is less beneficial. Amino acid mixtures vs single N sources are more efficient because the yeast directly incorporates the amino acids into proteins rather than having to synthesize them.
Ammonia, which exists as ammonium (NH4+) ions in must, is used by yeasts prior to amino acids. The presence of NH4+ delays timing and uptake of amino acids by yeast.
The timing of N supplements and form of supplement will impact fermentation and volatiles. Types of N supplements include Diammonium phosphate (DAP), proprietary blends of DAP and amino acids (e.g. Superfood®, Fermaid K®, Actiferm) and balanced nutritional formulas containing inorganic N (e.g. Fermaid O®), organic N, sterols, yeast cell walls, fatty acids, yeast autolysis products and others. DAP is best used with low N musts. Other balanced nutrients should be added as well. At a rate of 100 mg/L DAP, 20 mg/L YAN is added.
It is common practice for winemakers to make N additions at the following times:
Yeast rehydration to rebuild cell walls (rehydration nutrients consist of inactivated yeast and autolysates. They contain no inorganic N and only 3 mg/L N for every 100 mg/L added).
Six-twelve hours after inoculation (2-3 Brix drop)
End of growth/exponential phase (1/3 sugar depletion)
Note that at ½ sugar depletion the yeast cannot utilize N since alcohol accumulation prevents uptake. This residual N can then be utilized by other organisms such as Brettanomyces spp.
Results of Deficient YAN
From a practical point of view, the problem of juice nitrogen composition is primarily linked to juices with suboptimal concentrations of nitrogen (<150 mg/L), and higher risk of slow or stuck fermentation. Low YAN (< 200 mg/L) is associated with production of sulfur compounds, e.g. hydrogen sulfide, which results from the nitrogen demand for yeast growth. The amount of H2S produced is dependent on the yeast strain, the sulfur precursor compound, the culture growth rate, and the enzymatic activity immediately before nitrogen depletion.
When working with very low YAN juices, researchers have observed that other nutrients can also be low. Therefore, when YAN is low and other nutrient deficiencies are suspected, it may be useful to add a proprietary yeast food that contains more complex forms of N, as well as vitamins, lipids and minerals. Continued H2S production after N addition suggests a general vitamin deficiency, though other causes are also possible. Most yeast suppliers can advise on the use of yeast foods, which are generally produced from inactivated yeast, e.g. GoFerm® or similar additives.
In summary, low must YAN leads to low yeast populations and poor fermentation vigor, increased risk of sluggish/stuck/slow fermentations, increased production of undesirable thiols (e.g. hydrogen sulfide) and low production of favorable sensory compounds including esters and long chain volatile fatty acids.
Results of Excessive YAN
High must YAN leads to increased biomass and higher maximum heat output due to greater fermentation vigor. Overuse of DAP can also stimulate overproduction of acetate esters, especially ethyl acetate, resulting in the perception of volatile acidity (VA) and suppression of varietal character. High YAN (exceeding 450-500mg/L YAN) can stimulate ethyl acetate production by many yeast strains. Increased concentrations of haze-causing proteins, urea and ethyl carbamate and biogenic amines are also associated with high YAN musts. The risk of microbial instability, potential taint from Botrytis-infected fruit and possibly atypical aging character is also increased.
Main Flavor Changes Affected by Nitrogen
In general, YAN can affect TA and the balance of organic acids which can affect flavor. Malic acid consumption increases with increasing DAP concentration, irrespective of yeast strain. When total nitrogen is increased by adding ammonium to a medium containing very low levels of YAN, the production of higher alcohols is initially increased, but then tends to decrease after a peak between 200-300mg/L YAN. This activity depends on various factors, including yeast strain and fermentation conditions. Higher alcohols are characterized by fusel-like odors, and are generally thought to contribute to the complexity of wine fermentation bouquet. However, when present in very high concentrations they can have a negative impact on wine aroma, mainly because they mask fruity characters.
Of course, intermediate must YAN favors the best balance between desirable and undesirable chemical and sensory wine attributes. The key is to have timely and accurate YAN must concentration data immediately before primary inoculation. Recognizing that measurement is difficult in a winery setting, we encourage use of commercial and extension labs that offer YAN measurements, so that the winemaker might make an informed decision regarding supplemental nitrogen additions.
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