Yeast assimilable nitrogen
S.M. Weeks and P.A. Henschke, The Australian Wine Research Institute
Grape juice/must provides the main source of nitrogen necessary for yeast growth and fermentation activity, and can influence wine flavour and composition (Rantz 1991; Henschke and Jiranek 1993). Because the nitrogen is present in chemically complex forms, including amino acids, ammonium ion, amines, purines, pyrimidines, peptides, and proteins, chemical analysis has proven somewhat problematical with many chemical and biological methods being proposed over recent years (Lie 1973; Ough and Amerine 1988; Monteiro and Bisson 1991; Hebbard et al. 1993; Henschke and Jiranek 1993; Zoecklein et al. 1995; Dukes and Butzke 1998). The major sources of nitrogen for yeasts are the primary amino acids and ammonium ion (Jiranek et al. 1995), referred to as FAN (free amino nitrogen) or YAN (yeast assimilable nitrogen (YAN); proline, an abundant secondary amino acid in many grape juices, is not metabolised appreciably under winemaking conditions.
Surveys of juices for nitrogen content from various regions of Australia have shown that a significant number of vineyards produce grapes with sub-optimal concentrations (see for example Monk et al. 1987; Dukes et al. 1991; Gockowiak and Henschke 1991; Henschke and Jiranek 1993; Treeby et al. 1998). Low nitrogen is associated with lagging and incomplete fermentation, and sulfide evolution (Agenbach 1977; Vos and Gray 1979; Dukes et al. 1991; Jiranek et al. 1993). In order to predict these problems, it is necessary to measure the initial concentration of YAN in the juice or must. A value less than approximately 150 mg N/L is associated with a greater chance of a fermentation problem (reviewed by Henschke and Jiranek 1993). It is recommended to supplement such juices with diammonium phosphate (DAP) at the start of fermentation to ensure an adequate population of yeast (Vos et al. 1980; Salmon 1989; Bely et al. 1990).
Juice analysis of grapes intended for fermentation on skins can underestimate YAN. Research undertaken by Julian Grubb, Holger Gockowiak, Robyn van Heeswijck and Peter Høj has shown that up to 30% of the amino acid content of berries is associated with the skin (Grubb 1997); this nitrogen, at least in red grapes, is believed to be liberated during the maceration process of fermentation. This mechanism may be highly beneficial to yeast by providing a more constant but low source of nitrogen. A nitrogen extraction method for the analysis of whole berries has been developed.
Recent research has also identified the need for YAN throughout the stationary phase (non-growth associated glycolysis) of fermentation (Bely et al. 1990; Sablayrolles et al. 1996). At this stage of fermentation nitrogen acts as a fermentation stimulant but only a small concentration is needed compared with that needed during the growth phase of the yeast. Again, an assay should be made at the mid point of fermentation (after yeast budding has ceased) to verify the presence of some nitrogen. A small addition of oxygen made after yeast growth has ceased is also beneficial, and appears to reduce the likelihood of incomplete fermentation (Sablayrolles et al. 1996). If the wine, immediately after fermentation, is intended for maturation or packaging without further microbial intervention, such as malolactic fermentation or secondary fermentation, it is desirable to add the minimum amount of nitrogen at the mid point stage of fermentation such that only a detectable amount of nitrogen is present at the completion of fermentation. Such nitrogen additions, when made with DAP, can be conveniently monitored with an ammonia selective electrode (McWilliam and Ough 1974). This will help to ensure that the wine contains low residual nitrogen after fermentation for better microbial stability.
It is important that winery prepared yeast starter cultures, including that intended for restarting a stuck fermentation, are never subjected to nitrogen starvation stress (Salmon 1989; Henschke 1997a, b). This reduces the fermentation activity of the yeast. Again, monitoring with an ammonia selective electrode is convenient.
The Australian Wine Research Institute uses amino acid analysis for research purposes, but this method is too slow and expensive for routine measurement. Recently, a convenient method was developed by Bruce Dukes while undertaking his masters degree at UC Davis with Christian Butzke (Dukes and Butzke 1998). The assay relies on the derivatisation of primary amino groups with o-phthaldialdehyde/N-acetyl-L-cysteine reagent to form an isoindole derivative which can be conveniently measured at the near ultraviolet wavelength of 335 nm. The assay is claimed to be insensitive to proline, a non-assimilable secondary amino acid, has low sensitivity to ammonium and correlates well with HPLC estimates of amino nitrogen. The flavonoids (catechin) present in grape juice, however, absorb at 335 nm, necessitating the use of a reagent blank in the assay.
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This article was published in the
Australian & New Zealand Wine Industry Journal 14 (1) : 53-54; 1999
(Institute publication #587).