Enology Notes

Enology Notes #105, August 12, 2005

To: Regional Wine Producers

From: Bruce Zoecklein, Head, Wine/Enology-Grape Chemistry Group, Virginia Tech

Subjects: Producing a Healthy Fermentation; Current Topics in Fermentation Workshop

Producing a Healthy Fermentation.

Figure 1. Environmental and processing factors influencing viability and fermentation performance of wine yeast. Source: Gump, Zoecklein and Fugelsang (2001).

The following is a review of some of the important fermentation issues depicted in Figure 1.

Factors/Considerations                 Recommendations/Comments

Vineyard. Fermentation problems are often vineyard specific. Fermentable nitrogen and yeast-required micronutrient deficiency in apparently healthy grapes can be severe. Drought, grapevine nutrient deficiencies, high incidences of fungal degradation, and fruit maturity all influence must nitrogen and micronutrients, as do cultivar, rootstock, crop load, season, and winemaking practices.

The combination of seven alpha-amino acids (called free amino nitrogen or FAN) and ammonia are referred to as fermentable nitrogen, assimilable nitrogen, or yeast assimilable nitrogen (YAN), the nitrogen required by yeast. Both nitrogen sources in grapes can change as a function of maturity and crop load. The concentration increases with fruit maturation, then plateaus (see posted slide show from the Volatile Sulfur Compound Workshop at www.vt.wines.info), and decreases with crop load.

There are large variations from one season to the next, and among cultivars in both ammonia and free amino nitrogen. The minimum amount of assimilable N required for fermentation is greater than 140 mg/L (even higher concentrations are need for some yeasts, and if there are stress conditions such as low pH, high Brix, etc).

As reported previously in Enology Notes, there is a simple, effective method of determining assimilable N in juice and wines. This procedure requires only a few reagents and a pH meter, and is within the capability of all wine producers. The specifics of this analysis are available on our website at www.vt.wines.info.

Yeast Preparation. Hydration of fresh culture in warm water at exactly the supplier’s stated temperature is critical for maximum viability. A large percentage of the cells die if rehydration is done at cooler or warmer temperatures, resulting in a significant loss of activity. After rehydration, the yeast should be added to the must within 20-30 minutes, or a source of sugar added to the culture. If this is not done, cells go into a premature decline phase, resulting in an inoculum of low cell concentration.

Avoid temperature shock (no more than 5 - 7ºC differential between culture and must temperature). Temperature shock kills great numbers of yeasts. For example, adding a yeast culture at 104ºF/40ºC to a must at 60ºF/15ºC kills about half the cell population.

Yeast Strains. There are large differences among strains, in ability to ferment to dryness and their fermentable nitrogen and micronutrient requirements.

Yeast Population. Yeast population should be large enough to overwhelm indigenous microflora and grow to 2 - 5 x 106 yeast cells/mL of must (1 to 3% vol/vol of an active starter). These concentrations apply when the Brix is below 24, the pH is above 3.1, and the temperature is above 55ºF.

Increases in inoculum volume should be made when the must is outside these parameters.

Nutrient Addition. Some musts lack sufficient N needed by yeasts during their growth phase for healthy fermentations. As suggested, levels of greater than 140 mg/L fermentable nitrogen are required for healthy fermentations. Too high a concentration of fermentable N can also be a problem, resulting in loss of aroma and flavor.

Therefore, it is important to test the N status of the juice prior to fermentation. If the fermentable N is low, it is likely that micronutrient concentration is also low. Supplementation, if needed, therefore should be made using a balanced source of nitrogen in the form of FAN amino acids, ammonia and micronutrients.

Additions of nutrient cocktails can be made, such as Fermaid K at 2 lb/1,000 gal (25 g/hL = 25 mg N/L), GoFerm (25 g/hL =7.5 mg N/L), or DAP (diammonium phosphate, 25 g/hL = 50 mg N/L). As indicated, fermentable nitrogen concentration in juice or wine can be easily estimated, and should be measured due to the detriment of too little or too much fermentable N.

Timing. Ammonia is consumed preferentially by yeast to FAN amino acids. Therefore, timing of nutrient additions is important. One large addition of DAP at the beginning of fermentation may delay/inhibit uptake of amino acids. Multiple additions of multiple sources are preferred. First addition should be a nutrient mix, such as Super Food or Fermaid K, followed by DAP. Adding nutrient supplements all at once can lead to too fast a fermentation rate, and an imbalance in uptake and usage of nitrogen compounds.

Supplements added too late (after mid-fermentation) may not be used by the yeasts, in part because the alcohol prevents their update. For the same reason, adding nutrients to a stuck fermentation seldom does any good at all.

Nutrient Addition. Musts can be deficient in nutrients and often will be when there is a low concentration of fermentable N, and a high incidence of microorganisms (mold, yeast and/or bacteria). Addition of sulfur dioxide tends to inactivate thiamine, which is necessary for yeast growth. It is usually desirable to add a mixed nutrient supplement along with a nitrogen supplement.

It grapes are degraded by Botrytis and/or Kloeckera, add extra thiamine.

Oxygen/SO2. Oxygen should be considered an essential yeast nutrient. Slight aeration during yeast stationary and growth phases increases the production of lipids (principally oleanoloic acid) and sterols (ergosterol and zymosterol) which are important cell membrane constituents.

It has been shown that yeast propagated aerobically contain a higher proportion of unsaturated fatty acids and up to three times the steroid level of anaerobically-grown yeast. This correlates positively with improved yeast viability and fermentation.

Because yeasts are not able to synthesize membrane compounds in the absence of oxygen, existing steroids must be distributed within the growing populations. Without initial oxygen, yeast multiplication is usually restricted to 4 to 5 generations, due largely to diminished levels of steroids, lipids and unsaturated fatty acids. CO2, nitrogen gas, and ascorbic acid reduce molecular oxygen.

Additionally, it should be noted that SO2 inhibits the enzyme polyphenoloxidase. In the complete absence of sulfur dioxide, this common plant enzyme system converts diphenols to quinones using a large concentration of available oxygen.

As indicated, sulfur dioxide also inactivates thiamine. If additions of more than 50 mg/L SO2 occur, extra thiamine (nutrient cocktails) should be added to the fermenter.

pH. Maintain pH as high above 3.1 as wine style permits. Musts which have a pH below 3.1 should receive an increased yeast inoculum.

Non-Soluble Solids. Reduction of the non-soluble solids content to below 0.5% prior to white wine fermentation can result in nutrient deficiencies and odor-defect volatile sulfur compounds. Too high a level may cause fermentation rates to proceed too quickly, and may also produce volatile sulfur compounds. Fermentation in contact with bentonite is occasionally done to help obtain white wine protein stability. Bentonite additions in the fermenter can reduce must N and should only be done in conjunction with supplemental nutrient additions.

Sedimentation. Yeast cells at the bottom of a fermenter can die prematurely. To help avoid this problem, large tanks should be mixed.

CO2 Toxicity. Carbon dioxide in concentrations of up to 0.2 atm stimulates yeast growth. Above this level, carbon dioxide becomes toxic to the yeasts. Agitation to prevent supersaturation of CO2 can minimize this problem.

Sugar Toxicity. High sugar concentrations can inhibit yeast growth due to osmotic pressure. Saccharomyces spp. are more tolerant than most others. High sugar musts start fermentation slowly and are likely to stick. There is a synergism between alcohol and sugar concentration. Inoculation with greater than 5 x 106 yeast cells/mL should occur if the must is 25 - 30º Brix. Inoculate with an additional 1 x 106 yeast cells for each degree increase in a Brix above 30º.

Alcohol Toxicity. Alcohol is toxic to all yeasts, including Saccharomyces spp. Alcohol has a profound effect on all aspects of yeast metabolism, from membrane integrity to nitrogen uptake and sugar transport. There are many factors which are synergistic with alcohol, including pH, high temperature, acetic acid, sugar, short-chained fatty acids, nitrogen depletion, and deficiency of sterols and vitamins.

As indicated, light aeration during growth phase of the yeast helps to produce lipids needed by the yeast cell wall. Nitrogen supplementation is helpful in reducing the affects of alcohol toxicity.

Native yeast/bacteria, fruit rot, poor sanitation, long settling, and late inoculation. Native yeast/bacteria, fruit rot, poor sanitation, long settlings, and delayed inoculation can deplete must nutrients, and may produce toxins. In such cases, the level of yeast inoculum should be increased, along with the fermentable N supplementation to a level of 250 mg/L N or more.

Acetic acid bacteria, Lactobacillus spp., Leuconstoc spp., and native yeast can produce inhibitors and deplete must N and vitamins. Acetic acid is a potent inhibitor of Saccharomyces spp., especially in combination with other negative influences, such as high alcohol late in the fermentation. A stuck wine with more than about 0.8 g/L acetic acid may need to go through an R.O. filter to reduce the acetic acid content before attempting refermentation.

Some Saccharomyces spp. and strains, and some non-Saccharomyces yeasts can produce killer toxins that inhibit sensitive strains. These killer toxins can play a roll in stuck fermentations. It is suggested that vigorous strains be used for high-risk fermentations.

Uninoculated Musts. Usually non-Saccharomyces from the vineyard and Saccharomyces from the winery dominate the initial fermentation of uninoculated musts, possibly resulting in a significant depletion of N and vitamins, such as thiamine. Kloeckera spp., which may dominate the early portion of uninoculated fermentations, are cold and sulfur dioxide tolerant and can produce high levels of ethyl acetate. Kloeckera can also significantly deplete N and thiamine. It is desirable to supplement uninoculated fermentations with nitrogen and vitamins.

Temperature. Increase inoculum when fermenting at low temperatures. Decrease inoculum slightly for uncontrolled high temperatures, and select a slower fermenting strain of yeast. Add yeast nutrient to protect the yeast at each end of the temperature range.

Fructose. Grape juice is usually composed of equal concentrations of glucose and fructose sugars. Stress can affect the yeast’s ability to metabolize the last residual fructose. Add small amounts of glucose to a small portion of the wine to determine if this is the cause of a stuck fermentation. This problem seems to occur more with the S. bayanus strains which are more glucophilic and, therefore, unable to ferment fructose.

Yeast Hulls. Yeast hull additions (0.2 g/L) can stimulate fermentation not simply by detoxification as was previously believed, but by supplying unsaturated fatty acids (C-16, C-18) as an oxygen substitute and preventing deficiencies of this nutrient. Also, yeast hulls add some amino acids and facilitate the release of CO2.

Pesticides. Pesticides can influence fermentation by causing production of stress metabolites such as reductive compounds, as well as inhibiting and/or preventing fermentation. Not all yeasts and bacteria are affected the same way by pesticides. There is a significant difference between systemic and contact fungicides in regard to residues. Vinification style influences pesticide residue concentrations. For example, contact pesticide residues are influenced by preclarification of whites and the addition of bentonite. To help prevent the problem of residual pesticides, be aware of spray schedules, use less than the maximum permitted when possible, and avoid late season spraying. Late season copper sulfate sprays (Bordeaux mix) can significantly increase the production of reductive odor defect and the rate of wine oxidation.

Adapted from: Dr. Clayton Cone, Lallemand, Inc., Montreal, Quebec, Canada; Wine Analysis and Production, Zoecklein et al., 1999; Wine Microbiology, Fugelsang, 1996; Die Wein Wissenschaft, 1996; and Lisa Van de Water, personal communication.

Current Topics in Fermentation Workshop. The Enology-Grape Chemistry Group will conduct a pre-harvest workshop on current fermentation topics, and a sensory evaluation of our training systems research wines on Monday, August 22nd beginning at 1:00 p.m. at Horton Vineyards.

Invited speakers to the meeting include:

  • Lisa Van de Water, Director, Pacific Rim Oenology
  • Patricia Roca, Technical Manager, Vinotec, Chile

Lisa and Patricia will discuss the latest practical information on conducting healthy fermentations.

I will present our Traminette, Viognier and Cabernet Franc wines produced from our training system trials: VSP, GDC and Smart-Dyson (Up vs. Down) from 2003 and 2004.

Registration: To reserve a spot, send an email message to Terry Rakestraw, at with the word “Pre-harvest” in the subject line. Space is limited.

Cost: $25 per person. The complete registration fee is due NO LATER THAN August 18, 2005. Checks are to be written payable to Virginia Tech Foundation and mailed to Terry Rakestraw, Department of Food Science and Technology (0418), Virginia Tech, Blacksburg, VA 24061. Course fee is non-refundable.

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Dr. Bruce Zoecklein
Professor and Enology Specialist Head Enology-Grape Chemistry Group
Department of Food Science and Technology, Virginia Tech
Blacksburg VA 24061
Enology-Grape Chemistry Group Web address: http://www.vtwines.info/
Phone: (540) 231-5325
Fax: (540) 231-9293