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The Australian Wine Research Institute

Yeast assimilable nitrogen article

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.

References
Agenbach, W.A. (1977) A study of must nitrogen content in relation to incomplete fermentations, yeast production and fermentation activity. In: Beukman, E. F., ed. Proceedings of the South African Society for Enology and Viticulture; (Cape Town, South Africa, Nov., 1977). Stellenbosch, South Africa: South African Society for Enology and Viticulture. pp 66–88.

Bely, M.; Sablayrolles, J.-M.; Barre, P. (1990) Automatic detection of assimilable nitrogen deficiencies during alcoholic fermentation in oenological conditions. J. Ferment. Bioeng. 70: 246–252.

Dukes, B.C.; Butzke, C.E. (1998) Rapid determination of primary amino acids in grape juice using an o-phthaldialdehyde/N-acetyl-L-cysteine spectrophotometric assay. American Journal of Enology and Viticulture 49: 125–134.

Dukes, B.; Goldspink, B.; Elliott, J.; Frayne, R. (1991) Time of nitrogenous fertilization can replace fermentation time and improve wine quality. In: Rantz, J. ed. Proceedings of the international symposium on nitrogen in grapes and wines; 18–19 June 1991; Seattle, Washington, USA. Davis, CA: American Society for Enology and Viticulture. pp. 249–254.

Gockowiak, H.; Henschke, P.A. (1992) Nitrogen composition of grape juice and implications for fermentation: results of a survey made in N-E Victoria. Australian Grapegrower and Winemaker 340: 131, 133–138.

Grubb, J. (1997) Varietal differences in the composition and biosynthesis of free amino acids in berries of Vitis vinifera, and the distribution of amino acids between berry components. Adelaide, SA: University of Adelaide. 81 p. Honours thesis.

Hebbard, S.; Goodman, K.; Jin, H.N. (1993) Chemical analysis of ammonium and amino acids in Chardonnay juices. Australian Grapegrower and Winemaker 353: 19–21.

Henschke, P.A. (1997a) Preparation of a yeast starter culture: fresh or dried yeast. In: Allen, M.; Leske, P.; Baldwin, G., eds. Advances in juice clarification and yeast inoculation. Australian Society for Viticulture and Oenology, World Congress Centre, Melbourne; 15 August 1996; Adelaide, SA: Winetitles. pp. 17–21.

Henschke, P.A. (1997b) Stuck fermentation: causes, prevention and cure. In: Allen, M.; Leske, P.; Baldwin, G., eds. Advances in juice clarification and yeast inoculation. Australian Society for Viticulture and Oenology, World Congress Centre, Melbourne; 15 August 1996; Adelaide, SA: Winetitles. pp 30–38, 41.

Henschke, P.A.; Jiranek, V. (1993) Yeasts – metabolism of nitrogen compounds. In: Fleet, G. H., ed. Wine microbiology and biotechnology. Chur, Switzerland: Harwood Academic Publishers. pp 77–164.

Jiranek, V.; Eglinton, J.M.; Gockowiak, H.; Langridge, P.; Henschke, P.A. (1993) Nitrogen: a critical regulator of fermentation. In: Stockley, C. S.; Johnston, R. S.; Leske, P. A.; Lee, T. H., eds. Proceedings of the eighth Australian wine industry technical conference; 25–29 October 1992; Melbourne, Victoria. Adelaide SA: Winetitles. pp. 133–141.

Jiranek, V.; Langridge, P.; Henschke, P.A. (1995) Amino acid and ammonium utilization by Saccharomyces cerevisiae wine yeasts from a chemically defined medium. Am. J. Enol. Vitic. 46: 75–83.

Lie, S. (1973) The EBC-ninhydrin method for determination of free alpha amino nitrogen. J. Inst. Brew. 79: 37–41 .

McWilliam, D.J.; Ough, C.S. (1974) Measurement of ammonia in musts and wines using a selective electrode. Am. J. Enol. Vitic. 25: 67–72 .

Monk, P.R.; Hook, D.; Freeman, B.M. (1987) Amino acid metabolism by yeasts. In: Lee, T. H., ed. Proceedings of the sixth Australian wine industry technical conference; 14–17 July 1986; Adelaide, S A. Adelaide, S A: Winetitles. pp. 129–133.

Monteiro, F.F.; Bisson, L.F. (1991) Biological assay of nitrogen content of grape juice and prediction of sluggish fermentations. American Journal of Enology and Viticulture 42: 47–57.

Ough, C.S.; Amerine, M.A. (1988) Nitrogen compounds. Methods for analysis of musts and wines. New York: Wiley-Interscience. pp 172–195.

Rantz, J. M. ed. (1991) Proceedings of the international symposium on nitrogen in grapes and wine; 18–19 June 1991; Seattle, Washington, USA. Davis, CA: American Society for Enology and Viticulture.

Sablayrolles, J.-M.; Dubois, C.; Manginot, C.; Roustan, J.-L.; Barre, P. (1996) Effectiveness of combined ammoniacal nitrogen and oxygen additions for completion of sluggish and stuck wine fermentations. J. Ferment. Bioeng. 82: 377–381.

Salmon, J.M. (1989) Effect of sugar transport inactivation in Saccharomyces cerevisiae on sluggish and stuck enological fermentations. Appl. Environ. Microbiol. 55: 953–958.

Treeby, M.; Holzapfel, B.P.; Walker, R.R.; Nicholas, P.R. (1998) Profiles of free amino acids in grapes of grafted Chardonnay grapevines. Australian Journal of Grape and Wine Research 4: 121–126.

Vos, P.J.A.; Gray, R.S. (1979) The origin and control of hydrogen sulfide during fermentation of grape must. American Journal of Enology and Viticulture 30: 187–197.

Vos, P.J.A.; Crous, E.; Swart, L. (1980) Fermentation and the optimal nitrogen balance of musts. Die Wynboer 582: 58–59, 60, 63.

Zoecklein, B.W.; Fugelsang, K.C.; Gump, B.H.; Nury, F.S. (1995) Wine analysis and production. New York: Chapman & Hall.

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This article was published in the

Australian & New Zealand Wine Industry Journal 14 (1) : 53-54; 1999

(Institute publication #587).


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