Vol. 14, No. 2 March - April, 1999

Bruce W. Zoecklein

Department of Food Science and Technology

VPI & SU - 0418

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

www.fst.vt.edu/Zoecklein

Table of Contents

I. Practical Issues in Fermentation 1

Role of Nitrogen, Nutritional Deficiency, Inhibitory Substances, Physiological Condition and Technological Practices 1
Practical Considerations and Recommendations 2

II. Methode Champenoise Extension Publication 5
III. Oak from Forest to Glass - Symposium 5
IV. Managing Oxygen During Bottling 7

For those that say they have not experienced stuck or sluggish fermentations, it should be noted that nutrient deficiencies may impact the sensory quality of your products. The information provided in this issue outlines some factors to consider in managing your fermentations and has been adapted from a variety of sources including Dr. Clayton Cone, Lallemand, Inc., Montreal, Quebec, Canada; Wine Analysis and Production, Zoecklein et al., 1995; Wine Microbiology, Fugelsang, 1996; Die Wein Wissen Schaft, 1996; and Van de Water, personal communication.

Role of nitrogen as a yeast nutrient

Nitrogen is one of the essential macronutrients for yeast and is necessary to build all protein-based cell components, including transporter proteins that facilitate the transport of sugars through the cell membrane. Nitrogen is available to yeasts in two forms, as ammonia N (NH₄+) and as free alpha amino acid N (FAN). The amino acids that the yeasts can use plus ammonia nitrogen are collectively referred to as FAN, assimilable N or fermentable N.

Difficulties with fermentations usually involve the features outlined below: nitrogen deficiency, inhibitory substances, poor physiological conditions for yeast development, and technological practices which influence yeast viability.

Nutritional deficiency

Assimilable nitrogen deficiency
Deficiency of oxygen or ergosterol and
unsaturated fatty acids
Vitamin deficiency
Mineral deficiency

Inhibitory substances

Sugar
Ethanol
Fatty acids
Acetic acid accumulation
Killer toxins
High SO₂ / CO₂ / petite yeast induction
Pesticide residues

Physiological condition

Poorly propagated yeast starter cultures

Technological practices

High clarification/low nonsoluble solids
Excessive temperature
Temperature-shocking
Inadequate agitation/premature flocculation

Practical Considerations and Recommendations

Vineyard. Fermentation problems are vineyard specific. Nitrogen deficiency in apparently heathy grapes can be very severe. Drought, grape vine 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. For example, grapes grown on St. George are much higher in total nitrogen than those on AXR1.

Figure 1 is data from my lab which show the change in concentration of alpha amino nitrogen in Cabernet Sauvignon grapes as a function of maturity and crop load. Two important points are illustrated. The concentration increases with fruit maturation and that crop load (possibly by influencing the rate of fruit maturity) influences fruit N.

Figure 1. Alpha amino nitrogen (mM) of Cabernet Sauvignon grapes cluster thinned to low (2.6 kg/vine), medium (4.9 kg/vine) and high (5.3 kg/vine) crop level in 1997.

Table 1 from Henick-Kling et al. (1996) shows the concentration of two important sources of assimilable N of six cultivars at fruit maturity for two seasons. This data illustrates several important points. 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. The amount of assimilable N required for fermentation is 500-900 mg/L. As can be seen in Table 1, none of the varieties listed has an adequate concentration. As reported previously, there is a simple, effective method for determining assimilable N in juice and wines. This procedure requires only a few reagents and a pH meter and is certainly within the capability of all wine producers. The specifics of this analysis are available in Wine Analysis and Production, Zoecklein et al. (1995) or by contacting my office.

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 avoids 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 to a must at 60 F/ 15 C kills about half the cell population.

Table 1: Survey results from 1993 and 1994. Mean content of free ammonia and free amino nitrogen (less free ammonia).

Source: Henick-Kling et al. (1996)

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

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.

Nutrient Addition. Many musts lack sufficient N, vitamins and other ingredients needed by yeasts during their growth phase for healthy fermentations. As suggested earlier, levels of 500-900 mg/L fermentable nitrogen are required for heathy fermentations. Supplementation should be carried out using a balanced source of DAP (diammonia phosphate), amino acids, minerals, and vitamins.

DAP addition of 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 (Zoecklein et al., 1995).

Timing. Amino acids are not taken up equally by the yeast cell. Some are needed at 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 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 of 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 fermentations 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 oleanoloic 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. CO₂ , nitrogen gas and ascorbic acid reduce molecular oxygen.

Additionally, it should be noted that SO₂ inhibits the enzyme polyphenol-oxidase. In the complete absence of sulfur dioxide, this common plant enzyme system conducts the chemical reaction depicted in Figure 2 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 should be added to the fermenter.

Figure 2. Enzymatic Oxidation

(Colorless)	(Brown)

Source: Zoecklein et al., 1995.

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 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 sp. 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 of a Brix above 30 .

Alcohol toxicity. Alcohol is toxic to all yeasts, including Sacccharomyces sp. 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 of 250-500 mg /L is helpful in reducing the affects of alcohol toxicity.

Wild yeast/bacteria, infected grapes, poor sanita-tion, 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 along with the addition of 250-500 mg/L N.

Acetic acid bacteria, Lactobacillus sp., Leuconstoc sp., and ‘wild' yeast can produce inhibitors and deplete must N and vitamins. Acetic acid is a potent inhibitor of Saccharomyces sp. especially in combination with other negative influences such a high alcohol late in the fermentation. 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 sp. 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 sp., 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 250-500 mg of yeast nutrient/L must 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.

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

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

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 residue pesticides, 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.

II. Methode Champenoise Extension Publication

A 30 page review of Methode Champenoise is available through the Virginia Cooperative Extension. To order, be sure to indicate that you want to order VCE Publication Number 463-017, A Review of Methode Champenoise Production. The cost per copy is $5.00 which includes shipping and handling. Make checks payable to Treasurer, Virginia Tech. Send request and check to: Virginia Tech Extension Distribution Center, 112 Landsdowne Street, Blacksburg, VA 24061-0512.

III. Oak from Forest to Glass - Symposium

An American Society for Enology and
Viticulture/ Eastern Section
Conference on Oak in Winemaking

While wine quality is largely the result of grape characteristics, wine aroma and flavor can be influenced by barrel fermentation and storage as well. Ideally, aromas and flavors will be balanced and integrated to give a complex, multidimensional product. On July 14-17, 1999, grape growers, suppliers, winemakers, and research personnel will convene in St. Louis, Missouri for an American Society for Enology and Viticulture/Eastern Section sponsored international discussion titled "Oak From Forest to Glass: Practical Management of Oak and Wine". The conference is presented by the American Society for Enology and Viticulture/ Eastern Section and Southwest Missouri State University in celebration of the 100 year anniversary of the Research Campus at Mount Grove, Missouri.

"The focus of the conference is the practical applications of oak in winemaking. Through numerous presentations and sensory evaluations, it is hoped that the participants will gain a sense of how oak can be used to delicately alter and blend flavors," says meeting chair, Dr. Murli Dharmadhikari of Southwest Missouri State University. Dr. Dharmadhikari goes on to say, "We would like to recognize a number of organizations for their generous support that has made this conference possible. Our thanks go out to Southwest Missouri State University, World Cooperage, the Illinois Grape and Wine Resources Council, Lallemand, Inc., Wine Bottle Packaging, the Arkansas Wine Producers Association, the Indiana Wine Grape Council, the Michigan Grape and Wine Industry, the Missouri Grape and Wine Program and the Virginia Winegrowers' Advisory Board."

The three day meeting will start with a full day tour of the World Cooperage barrel making facility and oak forest. How the barrel is produced is important with respect to how it will potentially influence the wine's aroma and flavor. Production considerations include the origin and age of the wood, the method and location of seasoning, barrel size and stave width, cooperage techniques, and wood toasting. All of these factors can influence the volatile components extracted from the barrel and thus the sensory features of the wine.

This is an important conference designed to provide practical information for the industry. Wine styles differ because of the tremendous number of variables that come into play in grape growing and in the winemaking process. Overlaid on the basic quality of the grape is the mark of the winemaker. In barrel fermented and/or stored wines, it is desirable to have a series of wood ‘notes' to support the aroma/flavor panoply of the wine. It is hoped that this conference will help winemakers develop and hone the skills necessary to produce a well-balanced, integrated product using barrels and barrel alternatives.

A skillful winemaker must be able to adjust production variables in such a way so as to emphasize one or more of the aromas, flavors and textures. Thus it would be helpful to know about wood chemistry and flavors that might effect the wine. July 15 will start with Dr. Vernon Singleton of UC-Davis discussing the usage of barrels in winemaking. Dr. Berry Gump of CSU-Fresno will present how oak wood constituents are extracted as well as their contributions to wine flavor. The next presentation, by Dr. Jim Swan and Mr. Ed Larmie from World Cooperage, will be on the effects of oak wood seasoning and toasting on wine quality with a wine tasting following the comments. Mr. Michael Martini from Martini Wine Cellars, Napa, California, will then present the various wood flavors in wines with a tasting of relevant Martini wines as examples.

There are a number of barrel fermentation considerations that can influence aroma and flavor, including barrel pretreatment, the age of the barrels, the timing of the fill, the fermentation temperature and rate, and whether the fermentation is produced by yeast only or by a mixed culture. Lees contact is a production component that contributes to the complexity of the wine by the integration of yeast characteristics with fruit and wood flavors. Lees contact is frequently used in Burgundian style Chardonnay. The afternoon session will begin with a talk by Dr. Vincent Gerbaux from Institut Technique de la Vigne et du Vin in Dijon on how the famous winemakers in Burgundy conciliate a strong tradition and new technologies. Peter Bell from Fox Run Vineyards in New York will follow with a talk on producing barrel fermented Chardonnay. Next Ms. Christina Benz of the Murphy-Goode Winery will speak on enhancing oak integration via lees stirring as well as provide a tasting of Murphy-Goode Chardonnay wines from yeast lees stirring trials.

Innovative barrel production techniques and their influence on Chardonnay and Cabernet as well as a relevant tasting will be presented by Mr. Michael Schroeter of Geyser Peak Winery. Mr. Duane Wall of Nadalie Cooperage will discuss the terrior of oak and will use wines that have been in contact with oak wood from various states as examples. The last talk of the day will be Mr. Phil Burton of Barrel Builders who will discuss and demonstrate repairing wine barrels.

Friday July 16 will begin with a discussion of current cooperage practices and future development to be presented by Mr. Henry Work of Canton Cooperage. Next Mr. Bob Rogers of Innerstave will talk about barrel alternatives. This will be followed by a presentation from Ms. Evelyn Heraty of Clos Du Bois Winery on Sauvignon blanc wines from barrel alternatives trials and will include a tasting of Clos Du Bois wines. After a break, there will be a panel discussion by four Eastern winemakers on the Eastern perspective of oak barrel usage in premium wine production as well as a tasting. The participants include Dave Johnson of Stonehill Winery in Missouri, Dave Miller of St. Julian Winery in Michigan, Mark Friszolowski of Pindar Vineyards in New York, and Tom Payette of Prince Michel Vineyards in Virginia. This will be followed by a talk on conducting oak trials in the winery from Ms. Barbara Lindbloom, a wine consultant.

After a theme luncheon, the conference will continue with a talk from Professor Ken Fugelsang of California State University-Fresno. He will speak on microbial contamination of barrels and how to evaluate and treat spoiled barrels. According to Professor Fugelsang, "Two of the more controversial yeasts that grow in juice and wine are Brettanomyces sp. and its generally less well known sporulating ("perfect") equivalent, Dekkera sp. Historically, winemakers have viewed Brettanomyces sp. as producing spoilage in wines where it could grow. Despite traditionally negative connotations surrounding the yeast, some are now questioning whether or not subtle "Brett" character, in some cases, may play a positive role in flavor and bouquet development."

This presentation will be followed by one from Mr. Phil Burton of Barrel Builders on barrel management in the cellar and will include information on buying and treating new and used barrels, handling and storage of full and empty barrels, cleaning and sanitizing barrels, as well as ullage, topping and racking. Ms. Evelyn Heraty of Clos Du Bois Winery will present a talk on electronic barrel tracking and automated barrel cleansing. The final topic of the conference will be a panel discussion of general barrel management with Mr. Phil Burton, Ms. Evelyn Heraty, Mr. Michael Schroeter and others in the know.

The conference is being held at the Marriott St. Louis Airport (I-70 at Lambert Airport, St. Louis, Missouri). The conference fee is $185 (plus a $20 late fee if after June 18) and $25 for the theme luncheon on Friday.

Following the conference on July 17 will be the ASEV/ES Annual Conference and Technical Sessions.

For registration information for either the Oak conference or the annual meeting, contact: E. Harkness, Secretary of ASEV/ES, Inc., Dept. of Food Science, Purdue University, W. Lafayette , IN 47907-1160, phone: (765)494-6704, fax: (765)494-7953, email: harkness@foodsci.purdue.edu

IV. Managing Oxygen During Bottling

There have been some recent concerns about the oxygen pick up during bottling. This is an extremely important issue influencing wine quality, stability, and longevity.

The concentration of molecular oxygen should be measured in the wine before bottling begins and should be less than 0.5 mg/L. If the concentration of oxygen is greater than 0.5 mg/L, it can generally be lowered by sparaging with nitrogen gas (see Zoecklein et al., 1995).

Just prior to bottling, air should be eliminated in all hoses, filter housing pumps and the fill bowl by using displacement gas (nitrogen, carbon dioxide, or argon). Feed tanks should be blanketed with nitrogen, CO₂ or lightly CO₂ sprayed. Bottles should be completely free of particular matter, which can occlude oxygen, and flushed with displacement gas just prior to filling. Any oxygen which remains in the bottle will result in an oxygen concentration increase. Any increase above 0.2 mg/L dissolved oxygen indicates excessive pick-up.

The loss of free sulfur dioxide in wine is proportional to the dissolved oxygen content. Producers not using vacuum filters, corkers, or bottle gas flushing can have up to 5 mL of air in the head space of their bottled wine (750 mL). This amounts to approximately 1 mL (1.4 mg) oxygen. Four mg of sulfur dioxide are needed to neutralize the effects of one mg of oxygen. Using this relationship, an additional 5-6 mg of free sulfur dioxide is needed to reduce molecular oxygen in the head space.

Monitoring molecular oxygen in wine during any stage of processing is relatively easy. Several portable, hand-held meters with probes are available for measuring atmospheric and dissolved oxygen in wine. The use of displacement gases and the use of CO₂ as a sparging gas are discussed in Zoecklein et al., 1995, and in the Volume 10, Number 2, March-April 1995 issue of Vintner's Corner. Additional copies are available upon request.