Vol. 13, No. 4 July - August, 1998

Bruce W. Zoecklein

Department of Food Science and Technology

VPI & SU - 0418

Blacksburg, VA 24061, E-mail: bzoeckle@vt.edu

Table of Contents

I. Nitrogen Compounds 1

II. Oxygen and Yeast Metabolism 2

III. Sulfur Containing Compounds 3

IV. Control of Hydrogen Sulfide and Mercaptans in Wine 4

V. Estimation of Fermentable Nitrogen 5

I. Nitrogen Compounds

Nitrogen is taken up by the plant in the form of nitrate (NO₃^-), ammonia (NH₄⁺), or urea. Nitrate is reduced to ammonia, incorporated into the amino acids glutamate and glutamine, and subsequently converted to other amino acids needed for protein synthesis.

Ammonia (present as NH₄⁺) serves as the primary form of nitrogen available for yeast metabolism. As grapes mature, ammonia decreases with increases in protein and peptide nitrogen.

Grape variety, rootstock, fertilization, grape maturity, soil, climate, and potential fungal degradation may affect nitrogen content of the juice and finished wine.

Free Amino Nitrogen (FAN) and Nitrogen Deficiency

Nitrogenous compounds in the grape which are available to yeast (amino acids and ammonia) are referred to collectively as free amino nitrogen or FAN. Dukes and Butzke (1998) conducted a survey of the primary amino acids and ammonium ion concentration in fruit from a number of west coast vineyards in 1996. Primary amino acids ranged from 29-370 mg/L, average 135; ammonium ranged from 5 to 32.5 mg/L, with an average of 70. There was no statistical different between the nitrogen status from different varieties, and no correlation between the primary amino nitrogen concentration and the concentration of ammonium nitrogen. We have linked slow or incomplete fermentations to FAN deficiency in Virginia. The concentration of nitrogen available for yeast growth varies among regions and vineyards. Mold growth on the fruit may dramatically change the qualitative and quantitative distribution of amino acids. For example, decreases in the amino acid content caused by Botrytis cinerea range from 7 to 61% (Sponholz, 1991) and may cause levels to fall below that needed for complete fermentation. Little or no prefermentation addition of SO₂ may encourage the growth of native yeast populations which compete for and deplete assimilable nitrogen levels needed by Saccharomyces.

Bely et al. (1990) found that a minimum FAN concentration of 140 mg/L was required for satisfactory fermentation of table wine must. However, a maximum fermentation rate may require 400-500 mg N/L as assimilable N (Henschke and Jiranek, 1993). FAN deficiency in the juice is often corrected by the addition of ammonia in the form of diammonium phosphate (DAP). Vos et al. (1980) established an optimal FAN to ^oBrix ratio of 43.9. Assuming a 20 ^oBrix must, this corresponds to a FAN level of 878 mg/L.

I have recommended that musts be supplemented with assimilable nitrogen to help prevent nitrogen deficiencies and fermentation problems. In the United States, the maximum addition of ammonium salts (such as DAP) for correction of nutritional deficiencies is 968 mg/L.

Several alternative nitrogen supplements are available including various "yeast foods." However, many may be of limited value (in correcting FAN deficiency) due to the variable concentrations of assimilable nitrogen. The involvement of urea in ethyl carbonate formation has led to its elimination as an approved wine additive in many countries. Yeast hulls or ghosts are thought to stimulate fermentation not simply by release of assimilable nitrogen and adsorption of toxic fatty acids (produced by lactic acid bacteria), but by stimulating the mechanism involved in yeast cell membrane sterol formation as well.

In nitrogen-deficient musts, low levels of sulfur-containing acids (cysteine and methionine) force the yeast to synthesize these from carbon/nitrogen precursors and sulfide. Sulfate and sulfite are reduced to sulfide (which accumulates as H₂S) as part of this synthesis. Although the addition of DAP corrects for nitrogen deficiency in the juice, it also stimulates an increase in cell biomass and may not completely prevent the production of perceptible hydrogen sulfide.

Before amino acids and ammonia can be used by the yeast they must be transported across the cell membrane. This transport is dependent on the sterol concentration in the plasma membrane and occurs via oxygen dependent synthesis. Therefore, oxygen is as important as nitrogen for healthy yeast growth, allowing transport of solutes such as amino acids and ammonia across the cell membrane.

II. Oxygen and Yeast Metabolism

Oxygen plays important roles in the physiological status of yeast. Molecular oxygen is required for the synthesis of lipids and steroids needed for functional yeast cell membranes. Steroids play a structural role in membrane organization, interacting with and stabilizing the phospholipid component of the membrane which helps to protect the membrane against alcohol. It has been shown that yeasts propagated aerobically contain a higher proportion of unsaturated fatty acids and up to three times the steroid level of conventionally prepared cultures. This increase correlates well with improved yeast viability during the fermentative phase.

As fermentation begins, oxygen present in must is rapidly consumed, usually within several hours. After utilization of initial oxygen present, fermentations become anaerobic. Because yeasts are not able to synthesize membrane components in the absence of oxygen, existing steroids must be redistributed within the growing population. Under such conditions, yeast multiplication is usually restricted to 4 to 5 generations, due largely to diminished levels of steroids, lipids and unsaturated fatty acids. This can result in fermentation sticking.

Methodology of starter propagation is important with respect to subsequent requirements for oxygen. Aerobic propagation has been demonstrated to significantly enhance subsequent fermentative activity. Yeast populations reach higher final cell numbers and fermentations proceed at a faster rate and are more likely to complete.

There is also evidence that exogenous addition of yeast "ghosts" or "hulls" may overcome the oxygen limitation, possibly by providing a fresh source of membrane components. Regardless, yeast should be grown aerobically to help assure successful fermentations.

The group of enzymes responsible for catalyzing oxidative reactions in juice are the polyphenol- oxidases, also referred to as phenolases or tyrosinases. Polyphenoloxidases catalyze the oxidation of dihydroxyphenols to their corresponding quinones (brown products shown below).

Traditionally, winemakers added sulfur dioxide to inhibit this reaction. Currently, sulfur dioxide is generally not added until long after the fermentation is completed. As the above reaction proceeds, phenols polymerize and precipitate from solution. The result is a wine with a lower tannin level and frequently in the case of whites, a nicer finish that is less harsh and less astringent.

The popularity of no prefermentation sulfur dioxide addition may be contributing to sluggish and/or stuck fermentations. As stated, unsaturated fatty acids and sterols help to protect the yeast cell membrane from the affects of alcohol. These can only be produced if molecular oxygen is present. Therefore, winemakers should have some air present during the first 30-72 hours of the fermentation. If no SO₂ is present, the grape's polyphenoloxidase will greatly reduce the oxygen available for the yeast (see reaction above). Sulfur dioxide will inactivate the enzyme and thus allow a greater concentration of oxygen to be available for yeast lipid production. As a reducing or antioxidizing agent, sulfur dioxide can react or bind with oxygen. This reaction, however, is relatively slow and much slower than polyphenoloxidase utilization of available oxygen.

Some wineries are adding a small amount of SO₂ (15-20 mg/L total) before fermentation of white wines to help avoid oxygen depletion. Oxygen should be considered an essential 'nutrient' required for proper yeast cell growth. It's management in the initial stages of fermentation may be an important factor in determining if a wine will complete fermentation.

III. Sulfur-Containing Compounds

Volatile sulfur containing compounds are known to impart distinctive aromas to wines such as rubbery, skunky, or like onion, garlic, cabbage, kerosene, etc. The objectionable odor of hydrogen sulfide, generally described as rotten-egg-like, also has been observed. If no correction is made, hydrogen sulfide may undergo reactions with other wine components to yield mercaptans, which can have detrimental effects on wine palatability and may be difficult to remove.

Sulfur, an essential element for yeast growth, is utilized in the formation of cell components such as protein and vitamins. Available as sulfate (SO₄^2-) in grape juice, it can be reduced to hydrogen sulfide (H₂S). As H₂S is an integral part of yeast metabolism, it is not possible to completely prevent its formation. However, vineyard management including selection and timing of spray applications and wine processing techniques, may effectively minimize its detrimental effects. The following is a review of some of the causes and solutions to the production of sulfur containing compounds.

Elemental Sulfur: Elemental sulfur is used as a fungicide in vineyards throughout the world. Because of increasing awareness of the problems associated with sulfur in winemaking, most viticulturists are using micronized sulfur, which consists of very small particles, ranging from 6 to 8 µm in size, which are readily miscible in water. An advantage of micronized sulfur is that the application rate is less than one-third the normal dusting sulfur rate for the same measure of fungal control. Only 5 mg/L of elemental sulfur in the must is enough to produce H₂S concentrations which cannot be removed. Therefore, sulfur sprays should not occur less than 35 days prior to harvest.

An additional source of elemental sulfur in juice is sulfur candles, used by some vintners to disinfect barrels. These candles may not burn completely, so that unburned sulfur enters the wine or juice. The use of dripless sulfur sticks and/or sulfur cups may effectively overcome this problem.

Redox State and Temperature: Hydrogen sulfide formation also is a function of the oxidation-reduction (redox) state of the must during fermentation. Higher levels of H₂S are produced from fermentations carried out in tall (height to diameter) tanks. The design of such fermentors is conducive to a rapid drop in redox potential. The fermentation temperature also affects the overall formation of H₂S; generally, less H₂S is produced at lower temperatures. However, at lower temperatures, less H₂S is lost through entrainment with carbon dioxide.

Yeast and Yeast Physiology: Yeast differ significantly in their ability to form hydrogen sulfide. One of the problems traditionally associated with native yeast is the production of high levels of H₂S.

Among commercially available strains, Pasteur Champagne (UCD 595), Epernay 2, and Prise de Mousse are known to be low H₂S producers, whereas Montrachet (UCD 522) and some strains of Steinberg are known to produce high levels of H₂S. The reasons for these differences are not clearly understood, but some yeasts are known to have deficiencies in their sulfur metabolism that promote increased production of H₂S. Such yeasts appear to have an absolute requirement for the vitamins pantothenate and/or pyridoxine (vitamin B₆). Although grape juices normally are not deficient in these two vitamins, must treatment, seasonal variations, rot, etc. may result in the depletion of one or both.

In nitrogen deficient musts, yeast extracellular proteolytic enzymes attack protein and other nonassimilable nitrogen sources to yield assimilable forms (Vos and Gray, 1979). In the process, H₂S can be produced from sulfur-containing amino acids. Free amino nitrogen (FAN) components of must, therefore, play a role in subsequent H₂S formation. Specifically, assimilable free amino nitrogen content is inversely related to H₂S levels. Deficiencies in total yeast assemilable nitrogen are not always correlated with the formation of H₂S at the end of fermentation. Although ammonium nitrogen additions may cause H₂S formation in the early stage of fermentation, they cannot prevent the more important second peak of sulfide production near the end of fermentation.

Yeast Autolysis: Upon yeast cell death, degradation and rupture of cell membranes release cytoplasmic components including free amino acids, peptides, and polypepetides. Other degradation products include fatty acids, as well as components of the yeast nucleic acids, and vitamins. Yeast autolysate may play a role in the character and complexity of wine. However, the process of sur lie with heavy lees (particularly in the absence of stirring or oxygen) can occasionally result in the production of 'off' flavors and aromas, including H₂S and mercaptans.

Sulfur Compounds and Metals: Copper, manganese, and zinc are components of many vineyard fungicides. Late-season application of metal-containing fungicides to the grapes is known to increase the production of H₂S and possibly other sulfur containing compounds. Because of the nature of the growing season, many have applied Bordeaux mix fairly late in 1996 in an attempt to help minimize downy mildew. There is a definite relationship between the use of copper-containing fungicides, like Bordeaux mix, and increased incidences of H₂S formation in wines.

Questions as to how late Bordeaux mix can be applied and how much copper stimulates H₂S formation are not resolved. Copper ions are constituents of both certain enzyme systems as well as known inhibitors of respiration. Yeast grown in the presence of copper adopt a protective mechanism of H₂S formation and consequently copper sulfide formation. The advantage of late season copper sprays for mildew control must be balanced with concerns for the production of sulfur containing compounds. If late season fungicides are applied, juice settling of whites and the addition of a yeast nutrient is advisable.

Organic Sulfur-Containing Compounds: In addition to H₂S there are several organic sulfur containing compounds which can form in wine. These are significant because, like H₂S, they have very low sensory thresholds. For example, threshold values for dimethyl sulfide, dimethyl disulfide, diethyl sulfide, diethyl disulfide, and ethaneothiol are 25, 29, 0.92, 4.3, and 1.1 µg/L (parts per billion), respectively (Goniak and Nobel, 1987).

Many terms have been used to describe these sulfur compounds including garlic, cabbage, onion, rubber, and so on. For example, the aroma of dimethyl sulfide is reminiscent of asparagus, corn, or molasses, whereas ethanethiol is more onion - or rubber-like.

The mercaptans are another group of sulfur-containing compounds that are also important from a sensory standpoint. Although only a few such compounds exist in nature, those present are very odorous. For example, n-butyl mercaptan is responsible for the objectional odor of the skunk. The odor of ethyl mercaptan is reported to be detectable in air at levels of 50 parts per billion.

IV. Management of Hydrogen Sulfide and Mercaptans in Wine

Suppression of H₂S has been obtained by exogenous addition of nitrogen in the form of both assimilable nitrogen and vitamins. As stated, this is of particular importance in rot degraded fruit.

A key to minimizing H₂S formation is the maintenance of assimilable nitrogen in the fermentor and avoiding yeast stress. Some simply add DAP at levels of up to 1 g/L (8 pounds per 1000 gallons). The monitoring of assimilable nitrogen in the form of ammonia can be accomplished with an ammonium ion-selective electrode, similar in operation to a pH electrode (Zoecklein et al., 1995).

Formation of excessive H₂S in white wines often can be minimized by settling, centrifuging, or filtering the juice prior to fermentation. These practices all accomplish the same goal, that is, removal of high-density solids along with associated elemental sulfur and possibly metals when present. In the case of red wine fermentation, some winemakers deal with excessive H₂S by aeration at first racking, thus volatilizing the H₂S. Increased H₂S production will occur, however, if aeration is carried out during or too soon after the completion of alcoholic fermentation. In these cases, elemental sulfur is believed to act as a hydrogen acceptor, forming H₂S.

Coincidental with H₂S formation are increases in yeast populations arising as a result of transient exposure to oxygen.

Additional techniques for controlling H₂S include sparging problem wines with nitrogen gas shortly after the completion of alcoholic fermentation. This practice may be relatively effective in eliminating minor quantities of H₂S, but desirable volatile wine components also may be swept away during excessive sparging. In cases where methyl mercaptan appears to be the problem carefully controlled aeration may bring about oxidation of methyl mercaptan to the less objectionable compound dimethyl disulfide.

Winemakers can remove objectionable H₂S and mercaptans from a 'still' (non-fermenting wine) wine by direct contact with copper. The addition of 4 g copper (II) sulfate (CuSO₄ 5H₂0) per 1000 gallons raises the copper content by 0.2 mg/L. Although governmental regulations permit additions of up to 0.5 mg/L (as copper), residual levels in the wine cannot exceed 0.2 mg/L (as copper). It should be noted that although mercaptans react with copper, dimethyl disulfide does not. Thus if the wine in question has undergone any oxidation, it may be necessary to reduce dimethyl disulfide back to the reactive species, methyl mercaptan. This can be accomplished by addition of ascorbic acid (Zoecklein et al., 1995 or Vintner's Corner, Vol. 3, No. 5, 1987). Generally, addition levels of 50 mg/L or more of ascorbic acid are used, and such additions usually are made several days prior to the addition of copper. (The sulfur dioxide analysis by Ripper titration cannot be performed accurately in wines containing ascorbic acid, as the latter also reacts with the iodine titrant). Copper should not be added until the fermentation is complete and the yeast titer reduced by racking, filtration, and so on. Yeast cells will bind copper ions to cell surfaces and may reduce reactivity with H₂S.

Lastly, the addition of SO₂ to wines may reduce H₂S levels. The addition results in an SO₂-induced oxidation of H₂S to yield elemental sulfur, which after precipitation, may be removed by centrifugation or filtration.

SO₂ + 2H₂S - 3S^o + 2H₂O

V. Estimation of Fermentable Nitrogen

The following procedure is adapted from Zoecklein et al. (1995), and is a quick, easy method for determining the fermentable nitrogen in juice or wine.

I. Equipment

pH meter

200 mL beaker

200 mL volumetric cylinder

Funnel

Filter paper, Whatman No. 1

II. Reagents

Sodium hydroxide (NaOH) 1 N and 0.1 N

*Barium chloride (6.5g/L)

Hydrochloric acid (HCI)

Formaldehyde, 40%

III. Procedure

1. Place 100 mL of sample in 200 mL beaker, adjust to pH 8.0 with 1 N sodium hydroxide and add 10 mL of barium chloride.

2. Wait 15 minutes then transfer solution into a 200 mL volumetric flask. Bring to volume with deionized water.

3. Stir and filter solution through Whatman No. 1 filter paper.

4. Place 100 mL of the solution into a 200-mL beaker and readjust pH to 8.0.

5. Add 25 mL of formaldehyde solution and titrate with 0.1 N NaOH to a pH of 8.0.

6. The concentration of fermentable nitrogen is given as follows: Fermentable nitrogen (mg N/L) = (mL of 0.1 N NaOH titrated x 28.

IV. Supplemental Note

Barium chloride is included to precipitate sulfur dioxide. If juice is sulfited, this addition may be omitted.

References

Bely, M., Sablayrolles, J.M., and Barre, P. 1990. Automatic detection of assimilable nitrogen deficiencies during alcoholic fermentation in enological conditions. J. Ferm. and Bioengineering 70:246-252.

Dukes, B.C. and Butske, C.E. 1998. Rapid determenation of primary amino acids in grape juice using a o-pthaldi-aldehyde/N-acetyl-L-cysteine spectophotometric assay. Am. J. Enol. Vitic. 49:125-134.

Henschke, P.A. and Jiranek, V. 1993. Yeasts - Metabolism of Nitrogen Compounds. In: Wine Microbiology and Biotechnology. Graham H. Fleet (ed.). Harwood Academic Publishers. pp. 77-164.

Monk, P. 1994. Nutrient requirements of wine yeast. Practical Winery and Vineyard. July/August p. 24.

Ough, C.S. 1969. Substances extracted during skin contact with white must. I. General wine composition and quality changes with contact time. Am. J. Enol. and Vitic. 20:93-100.

Radler, F. 1965. The main constituents of the surface waxes of varieties and species of the genus Vitis. Am. J. Enol. and Vitic. 16:159-167.

Sponholz, W.R. 1991. Nitrogen compounds in grapes must and wine. In: Proceedings of the International Symposium on Nitrogen in Grapes and Wine. Seattle, WA. Am. J. Enol. Vitic. J. Rantz (ed.) pp. 67-77.

Vos, P.J.A., Crous, E., and Swart, L. 1980. Fermentation and the optimal nitrogen balance of musts. Wynboer. 582:58-62.

Zoecklein, B.W., Fugelsang, K.C., Gump, B.H. and Nury, F.S. 1995. Wine Analysis and Production. Chapman & Hall. Thomson Publishing.