Tuesday, May 5, 2015

Nitrogen as a yeast nutrient in alcoholic fermentation

Wine is a result of using yeasts in an anaerobic (oxygen-free) environment to convert sugars from pressed grape juice into ethanol in the two-step process illustrated in the figure below. The first step -- glycolysis -- results in the 6-carbon glucose being split into two 3-carbon pyruvate molecules. In the next step --- alcoholic fermentation (AF) - four atoms of oxygen and two atoms of carbon leave the pyruvate, resulting in acetaldehyde, which is subsequently converted into ethanol.

Alcoholic fermentation (Source: http://alcoholicfermentation.net/)
According to Fugelsang (Overview of yeast selection and malolactic fermentation on aroma, flavor and phenols), the yeasts (i) extract compounds from the solids in the must/juice in order to form the "characteristic metabolites of fermentation (alcohols, esters, fatty acids, carbonyls, etc.) and (ii) cleave cysteine-containing precursors such that volatile thiols (aroma component of several varieties) can be released. The yeast that receives most of the credit -- and does most of the work -- in alcoholic fermentations is a species called Saccharomyces cerevisiae (SC) which is "specialized in metabolizing media with high sugar content and small quantities of nitrogenous compounds" (Suárez-Lepe and A. Marota, New trends in yeast selection for winemaking, Trends in Food Science and Technology 23 (2012), 39-50.). Yeasts require nutritive support to allow the performance of the above functions in the hostile environment (ethanol-rich, acidic) of the fermentation tank. It is the nitrogenous aspect of that support that is the focus of this blog post.

Proteins are used by the yeast as (i) enzymes for the glycolytic pathway (indicated above), (ii) permeases in the cell membrane responsible for the transportation of compounds into the cells, (iii) cellular constituents (Kennedy and Reid, Yeast nutrient management in winemaking, The Australian and New Zealand Grapegrower and Winemaker, 537, October 2008). These proteins are synthesized by the yeast and nitrogen (N) is a key component in that process. According to Kennedy and Reid, "Efficient protein synthesis is needed for efficient sugar transport and overall yeast metabolism."

According to Schwarcz and Schoeninger (Stable Isotope Analysis in Human Nutrition, Yearbook of Physical Anthropology 34, pp. 293-321), almost 100% of exchangeable nitrogen is found in the atmosphere or dissolved in the world's oceans and is transferred from these environments into the biological system through the processes illustrated in the figure below.  Grape vine plants receive their nitrogen through this terrestrial nitrogen cycle.

Source: http://tolweb.org/notes/?note_id=3920
The nitrogen content of grapes are affected by variety, rootstock, climatic conditions, soil composition, vineyard management practices, fertilization, irrigation, rot incidence, and grape maturity (Kennedy and Reid). The yeast cells extract yeast assimilable nitrogen (YAN) from the grape must in the form of ammonia (preferred source of nitrogen for yeast growth as most easily assimilated) and amino acids and these are stored in the cell walls for later use. This extraction and storage of YAN is front-loaded in the AF process.

Nitrogen is required throughout the fermentation process with larger amounts being utilized during the exponential growth phase of the yesats and small amounts during the stationary phase. In some cases the grape must does not provide adequate amounts of assimilable nitrogen and is supplemented by added nitrogen in the form of diammonium phosphate (DAP). Juice levels of < 25 mg/L ammonia or < 150 mg N/L (measured as YAN) is considered nitrogen-deficient (UCDavis). Insufficient nitrogen can result in sluggish/stuck fermentations or sulfide formation (sulfur-like off-odors, mercaptans, and sulfur-containing acetic esters; the less assimilable nitrogen in the must, the more hydrogen sulfide will be produced). Supplements are best added incrementally and proportional to yeast growth (UCDavis).

Excessive nitrogen in the must can lead to elevated levels of ethyl carbamate (a supposed carcinogen) or urea excretion. The levels of nitrogen required for a successful AF is dependent on (Kennedy and Reid):
  • Initial must YAN
  • Yeast strain
  • Fermentation temperature
  • Initial grape sugar
  • Other factors.
Amino acids are the building blocks of proteins and, when brought into the yeast cell, can be incorporated as is, transformed into a different amino acid, or broken down as a source of nitrogen or sulfur. The amino acids taken up by the yeasts from the grape must is primarily stored in the vacuole to be used for protein synthesis during yeast growth. Once access to inorganic nitrogen becomes difficult, the yeast begins to break down the stored amino acids to provide nitrogen for protein synthesis. The most important amino acids taken up by the yeasts are shown in the table below.

Amino Acid Characteristics
Proline Not metabolized appreciably by yeasts under winemaking conditions
One of the predominant amino acids along with Arginine and Glutamine
Main amino acid from low-fertilization vineyards
Arginine One of the predominant amino acids
Breakdown results in formation of urea and ammonia (During wine storage, urea can react with ethanol to form ethyl carbamate, a carcinogen).
Located mostly in grape skin so processing practices could influence content in juice
Main amino acid from low-fertilization vineyards
Glutamine One of the predominant amino acids
Favored by yeasts because it can be broken down to glutamate and ammonia

The less-important amino acids taken up by the yeast cells are alanine, serine, and theronine.

©Wine -- Mise en abyme

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