To: Regional Wine Producers

From: Bruce Zoecklein

Subject: Practical Considerations and Recommendations for Fermentation, Survey of All Virginia Vineyards

Practical Considerations and Recommendations for Fermentation.

In advance of our Pre-harvest Workshop at White Hall Vineyard on August 5 (course is full no additional registration available) the following is an outline of some important fermentation considerations.

Vineyard. Fermentation problems are often vineyard specific. Nitrogen deficiency in apparently heathy grapes can be very severe. Yeast require both ammonia N and certain amino acids. Drought, grapevine nutrient deficiencies, high incidences of fungal degradation and level of fruit maturity all influence must nitrogen and vitamins, as do cultivar, rootstock, crop load, season and winemaking practices. For example, some varieties (such as Chardonnay) have a greater tendency towards deficiency. Some rootstocks produce more nutritious grapes than others with regard to total nitrogen.

The concentration of amino nitrogen increases with fruit maturation. Additionally, high crop load (possibly by influencing the rate of fruit maturity) lowers fruit N.

There is a large variation from one season to the next in both free ammonia and free amino nitrogen. There is also a significant difference in the concentration of both sources of N among cultivars.

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. After rehydration, the yeast should be added to the must within 20-30 minutes, or a source of sugar should be added to the culture. If this is not done, cells go into a premature decline phase resulting in an inoculum of low cell concentration.

It is imperative that one avoid temperature shock (no more than 5-7°C differential between culture and must temperature). Temperature shock kills great numbers of yeasts. For example, adding yeast hydrated at 100ºF to a must at 60°F kills about half the cell population.

Yeast Strains. There are large strain differences in terms of nitrogen requirements and ability to ferment to dryness

Yeast Population. Yeast populations should be large enough to overwhelm indigenous micro-flora and grow to 2 to 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 55°F. Increases in the inoculum volume should be made when the must is outside these parameters and when cold soaking of reds is conducted.

Nutrient Addition. Many musts lack sufficient N, vitamins and other ingredients needed by yeasts during their growth phase for healthy fermentations. As suggested in previous editions, levels of greater than 140 mg/L fermentable nitrogen are required for healthy fermentations. Supplementation should be carried out using a balanced source of DAP (diammonium phosphate), amino acids, minerals, and vitamins.

DAP addition at 1 g/L (8.3 lb/1,000 gal) provides about 258 mg/L fermentable N. This is greater than the suppliers' recommended level. In the US, the legal limit of DAP is 960 mg/L, which is equal to 202 mg N/L. As indicated, fermentable nitrogen concentration in juice or wine can be easily estimated (see Formol procedure and reduced volume formol procedure on our web site at www.vtwines.info).

Timing. Amino acids are not taken up equally by the yeast cell. Some are needed at the beginning of the growth cycle, some later, some not at all. Ammonia, on the other hand, is consumed preferentially to amino acids. Therefore, timing of DAP (25.8% ammonia, 74.2% phosphate) addition is important. One large addition of DAP at the beginning of fermentation may delay/inhibit the uptake of amino acids. Multiple additions (at beginning, at 16°Brix, and 1/3 at 10°Brix) are preferred. 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 half the fermentation) may not be used by the yeasts, in part because the alcohol prevents their uptake. For the same reason, adding nutrients to a stuck fermentation seldom does any good at all. Do not wait until you have a sluggish or stuck fermentation to add nutrients.

Vitamin Addition. Musts can be vitamin deficient, and often will be when there is 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 vitamin supplement along with a nitrogen supplement. If grapes are degraded by Botrytis and/or Kloeckera, add extra thiamine.

Fermentation Rate. The rate of fermentation should be monitored by the use of Brix hydrometers and/or an analysis of residual sugar. What is desired is a steady fermentation that gradually declines.

Oxygen/SO2. Oxygen should be considered an essential yeast nutrient. Slight aeration during yeast stationary and growth phases increases the production of lipids (principally oleanolic acid) and sterols (ergosterol and zymosterol), 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 components 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. Carbon dioxide, nitrogen gas and ascorbic acid reduce molecular oxygen.

Additionally, it should be noted that sulfur dioxide inhibits the enzyme polyphenyloxidase. In the complete absence of sulfur dioxide, this common plant enzyme system conducts the chemical reaction using a large concentration of available oxygen.

As indicated, sulfur dioxide also inactivates thiamine. If additions of more than 50 mg/L sulfur dioxide occur, extra thiamine 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.

Nonsoluble Solids. Reduction of the nonsoluble solids content to below 0.5% prior to white wine fermentation can result in nutrient deficiencies. Too high a level may cause fermentation rates to proceed too quickly. 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 be done in conjunction with measurement of fermentable N and 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.

Carbon Dioxide 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 carbon dioxide 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 25-30°Brix. Inoculate with an additional 1 x 106 yeast cells for each increase in Brix above 30°.

Alcohol Toxicity. Alcohol is toxic to all yeasts, including Sacccharomyces 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 the 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.

‘Wild’ Yeast/Bacteria, Rot-degraded Grapes, Poor Sanitation, Long Setting, and Late Inoculum. Wild yeast/bacteria, infected grapes, poor sanitation, long setting, and late inoculum deplete must nutrients and may produce toxins. In such cases, the level of yeast inoculum should be increased.

Acetic acid bacteria, Lactobacillus spp., Leuconstoc spp., and ‘wild' 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 a high alcohol. A stuck wine with more than about 0.8 g/L acetic acid may need to go through a R.O. to reduce the acetic acid content before attempting refermentation.

Some Saccharomyces spp. and strains and some non-Sacccharomyces 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 temperature. Decrease inoculum slightly for uncontrolled high temperature 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 (see Zoecklein et al., 1999). This problem seems to occur more with the S. bayanus strains which are more glucophilic and, therefore, unable to ferment fructose.

Use fructose syrup as a last choice for amelioration. Fermentation rate can be easily measured by the use of Durham or fermentation tubes.

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

Pesticides. Pesticides can influence fermentation by producing stress metabolites such as reductive compounds, as well as by 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 with regard to residues. Vinification style influences pesticide residue concentrations. For example, contact pesticide residues are influenced by preclarification of whites and by the addition of bentonite.

To help prevent the problem of pesticide residues, be aware of spray schedules, use less than the maximum permitted when possible and avoid late season spray. Late season copper sulfate sprays (Bordeaux mix) can significantly increase the production of hydrogen sulfide and mercaptans.

Survey of All Virginia Vineyards

Researchers at Virginia Commonwealth University (VCU), working in conjunction with the Virginia Winegrowers Advisory Board, are conducting a survey of all vineyards and wineries in the state. The results of this survey will be used by VCU to estimate the economic importance of Virginia's wine industry and to develop an industry profile.

It is very important that each of the state’s vineyards be included in this study. A survey was mailed to the owners (or a key contact) at each vineyard on July 17. If your site has not received a survey, or if you are unsure, please contact Mr. Billy Kinsey at VCU. You may reach him at (804) 278-0961 or by e-mail at bwkinsey@vcu.edu.

Subscription to Enology Notes. All past Enology Notes and Vintner's Corner newsjournals are posted on the Enology-Grape Chemistry Group's web site at: http://www.fst.vt.edu/zoecklein/index.html or http://www.vtwines.info/. Enology Notes are slightly different in content from the subscription based Vintner's Corner newsjournal.

To be added to the Enology Notes list serve send an email message to bzoeckle@vt.edu with the word "ADD" or "REMOVE" in the subject line.

Dr. Bruce Zoecklein
Associate 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: www.vtwines.info or www.fst.vt.edu/zoecklein/index.html
Phone: (540) 231-5325
Fax: (540) 231-9293
Email: bzoeckle@vt.edu