APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1989, p. 1560-1564 Vol. 55, No. 60099-2240/89/061560-05$02.00/0Copyright ©D 1989, American Society for Microbiology

Effect of Yeast Hulls on Stuck and Sluggish Wine Fermentations:Importance of the Lipid Component

EEVA MUNOZ AND W. M. INGLEDEW*

Department of Applied Microbiology and Food Science, University of Saskatchewan, Saskatoon,Saskatchewan S7N OWO, Canada

Received 28 December 1988/Accepted 2 March 1989

The effect of yeast hulls (yeast ghosts) on sluggish or stuck white wine fermentations was studied. Theenhancing effect on yeast growth and fermentation rate displayed by the hulls was shown to be similar to theeffect provided by lipid extract from the same hulls. Unsaturated fatty acids and sterols were incorporated intothe yeast from lipid extracts during fermentation carried out under oxygen-limited conditions. Adsorption oftoxic medium-chain fatty acid (decanoic acid) onto the yeast hulls took place through a dialysis membrane.However, when the hulls were placed inside a dialysis bag, the increase in yeast growth and fermentation rateseen when freely suspended hulls were used did not occur. Accordingly, the effect of yeast hulls in preventingstuck fermentations cannot be attributed only to the adsorption and consequent removal of medium-chain fattyacids from the juice.

Wine fermentations are normally completed by the meta-bolic activity of resting (nongrowing) yeast cells (19). Juicesoften ferment for longer than 30 days, and occasionally thefermentations become stuck. Stuck fermentations are themost serious, yet one of the most common, problems of thewine industry (21). In a stuck ferrnentation, the availablesugar in juice is not totally fermented and residual sugarremains in the finished product. Although high osmoticpressure and the presence of inhibitory substances includingpesticides from grapes have been implicated, the mainreason for stuck fermentation is premature death of restingcells as a result of intolerance to ethanol (8); this phenome-non can be prevented by the addition of nutritional supple-ments to the juice (14, 19).To alleviate stuck or sluggish (excessively slow) fermen-

tations, oxygen or sterol and unsaturated fatty acids (1, 2),called survival factors (14, 19, 27), or assimilable nitrogen inthe form of ammonium salts or amino acids (24), or bothsurvival factors and assimilable nitrogen (8, 9, 14), have beenprovided early in fermentation to allow the production of asufficient number of new cells and to prevent existing cellsfrom dying. Such work has led enologists to examine theanaerobic conditions of wine making, the practice of muistclarification prior to fermentation, the use of higher inocui-lum levels, and the use of active dry yeast (higher sterollevels) (14, 16). Limitation of free amino nitrogen was shownto be serious in worts (9) and in juices (14) in which thefermentation sticks or becomes sluggish.

Yeast ghosts or yeast hulls, the cell wall materidil rematin-ing after yeast extract preparation, have been suggested assupplements to juice to prevent stuck fermentations (27).The action of the hulls was postulated to be due to physicaladsorption and removal of toxic fermentation side products,the medium-chain fatty acids hexanoic, octanoic, and de-canoic acids (17). These fatty acids are formed via a syn-thetic route in Saccharomyces yeasts (28). In the presence ofethanol they completely inhibit yeast growth at concentra-tions found in wines (18). More recently, Larue and Lafon-Lafourcade (21) observed the effect of aeration in reducingthe concentration of medium-chain fatty acids in wine, as

* Corresponding author.

had been previously observed in beer fermentations (6, 28).The effect of oxygen was seen in the chain elongation and inthe increase in the degree of unsaturation in membrane lipidfatty acids (6). Correlation has also been shown betweenfatty acid concentrations in beer and in cell membranes (28).

In a recent paper, Munoz and Ingledew (25) presentedresults indicating that adsorption of toxic by-products wasnot the only enhancing effect displayed by yeast hulls in asluggish wine fermentation. Under oxygen-limited condi-tions (simulating actual conditions in wine making), theeffect of yeast ghosts was the same as the effect of ergosteroland Tween 80 (source of oleic acid) supplements. Both actedas oxygen substitutes, allowing sterol and unsaturated fattyacid production for yeast cell membrane synthesis. In thepresent work, fermentation experiments and yeast lipidanalysis by gas-liquid chromatography and chemical meth-ods were conducted to verify the importance of yeast hulllipids in the stimulation of fermentation and prevention ofstuck and sluggist fermentations. Addition of decanoic acidto uninoculated media was done to reassess fatty acida1dsorption as the mechainism of yeast hull action.

MATI AII,S AND METHODSFermentations. l:ermentations were carried out with re-

constituted juice. l o 2.8 liters of juice concentrate (Sun CalPinot Chardonn.ay. Niagara Vine Products lAtd.. St. Cathe-rines, Ontakrio. Canada) 15.5 liters of sterilized waiter, 1.74 kgof sucrose. 42 g of acid blend (mixture of tartaric. malic, andcitric acids: Harvest Brewing Co, Saskaltoon. Saskatch-ewan, Canaida. atnd 3 g of grape tannin were aidded. Theresulting juice Wals sterilized with 0.1 ml ot diet hylpyrocar-bonate (Sigmai Chemical Co., St. Louis. Mo.) per liter.Active dry Red Star Montrachet yeast was rehydrated insterile 0.1%, peptone-water at 39°C for 20 min, and the juicewas then inoculated at different levels depending on theexperiment. All fermentations were conducted at 14°C in1-liter or 500-ml fermentors (Celstir; Wheaton Industries,Millville, N.J.) with constant stirring at 90 rpm to ensure thathomogeneous yeast and juice samples could be obtained.The 1-liter fermentors contained 1 liter of juice, and theheadspace volumes were 700 ml; the 500-ml fermentorscontained 500 ml of juice, and the headspace volumes were

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240 ml. The juices were saturated with air prior to inocula-tion. After inoculation, a constant flow of nitrogen gas at 10ml/min was introduced through the headspace. A yeast hullpreparation obtained from Fould Springer, Maisons Alfort,France, was used in the experiments. Either yeast hulls weredirectly added in the fermentor at a concentration of 1 g/literor 1 g of hulls, was suspended in 20 ml of the juice and putinside a dialysis bag to physically separate the hulls from thefermenting yeast. Spectrapor (Spectrum Medical Industries,Inc., Los Angeles Calif.) dialysis membranes with a molec-ular weight cutoff of 6,000 to 8,000 (400 mm long, 23 mmwide) or 50,000 (800 mm long, 12 mm wide) were used forthis purpose.

Lipid extract from yeast hulls was prepared by extracting1 g of yeast hulls with 30 ml of chloroform-methanol-5 Mhydrochloric acid (5:6:1, vol/vol) for 20 min at room temper-ature (15). After addition of 6 ml of water and shaking, theorganic layer was collected and dried under nitrogen gas.

The extracts were redissolved in 5 ml of warm 95% ethanoland immediately added to the fermentor. A 5-ml portion ofethanol was added to the control fermentors. Extraction was

performed immediately before the start of the fermentation.Adsorption experiment. Decanoic acid adsorption onto

yeast hulls was studied by using a sterile juice-ethanolmixture (12°Brix, 5% ethanol). Yeast hulls (1 g) were sus-

pended in 20 ml of the juice and put inside a dialysis bag(Spectrapor membrane; molecular weight cutoff, 50,000; 800mm long, 12 mm in diameter). The sac was placed in a 250-mlErlenmeyer flask containing 50 ml of the juice with 118 mg ofdecanoic acid (Sigma) per liter. The Erlenmeyer flasks were

covered with a double layer of Parafilm and shaken in a

water bath shaker (Aquatherm; New Brunswick ScientificCo., Inc., Edison, N.J.) at 200 rpm and 14°C for 24 h. Afterthe experiment, the volumes of the juices inside and outsidethe dialysis bag were measured, and then the juices were

analyzed for their decanoic acid contents. The yeast hullswere separated from the juice inside the bag by centrifuga-tion (10 min at 3,000 x g and 4°C), extracted with chloro-form-methanol-5 M HCl (15), and then analyzed for theirdecanoic acid concentration. Experiments were run in du-plicate.Methods of analysis. During all fermentations, the specific

gravities of the juices were analyzed with a density meter(DMA 45; Anton Paar, Graz, Austria). The results werepresented as specific gravity or converted to Brix by usingprepared conversion tables (1). Viable yeast cells were

counted in triplicate by the membrane filtration techniqueand expressed as millions of CFU per milliliter (14). Yeastcell mass was estimated gravimetrically after a buffer wash(14).The sterol contents of the yeast (collected, after fermen-

tation, by centrifugation for 10 min at 3,000 x g and 4°C andwashed three times with 1/15 M potassium dihydrogenphosphate buffer [pH 4.5]) were analyzed by the Lieberman-Buchard reaction (10). Ergosterol was used as a standard.

Total fatty acids were extracted with chloroform-metha-nol-5 M HCI from 100 ml of final culture containing the yeastby the method of Kemp and White (15). The acids were

methylated at 100 to 110°C for 30 min with 2 ml of sulfuricacid-methanol-toluene (1:20:10, vol/vol/vol) reagent (13).After methylation, the esters were separated in a gas chro-matograph (model 5750; Hewlett-Packard Co., Palo Alto,Calif.) with a stainless steel column (6 ft by 1/8 in. [1.8 m by0.3 cm]) packed with GP 10% SP-2330 on 100/120 Chro-mosorb W/AW (Supelco, Inc., Bellefonte, Pa.). The temper-ature program used was as follows: 1 min at 130°C, rising at

8°C/min to 242°C, followed by a 6-min hold at 242°C. Thecarrier gas (helium) flow rate was 30 ml/min; the air flow ratewas 400 mllmin, and the hydrogen flow rate was 40 ml/min.Medium-chain fatty acids were extracted from 75 to 100 ml

of centrifuged (10 min at 3,000 x g and 4°C) wine which hadbeen passed through a 0.45-,um-pore-size membrane filter.The samples were saturated with NaCl and acidified to pH 1with concentrated HCI. Nonanoic acid (0.1 mg/ml) was usedas the internal standard. The juices were shaken for 15 minwith an equal volume of chloroform-methanol (3:1). Thesamples were then centrifuged to separate the phases, andthe organic phase was evaporated at room temperature in arotary evaporator. After drying, the acids were refluxed for1 h with 50 ml of 6% (wt/vol) potassium hydroxide in 95%ethanol. After the addition of 50 ml of water, the unsaponi-fied material was removed by extraction three times with 40ml of petroleum ether (bp 30 to 60°C; BDH, Toronto,Canada). The aqueous phase was acidified to pH 1 withconcentrated HCI in an ice bath, and the fatty acids wereextracted with petroleum ether (three times with 40 ml).After evaporation, methylation was carried out as above(13). Fatty acid methyl esters were separated in a gaschromatograph (model 5750; Hewlett-Packard) on the col-umn described above, with a temperature program of 6 minat 75°C, a rise of 4°C/min to 225°C, and a 10-min hold at2250C.

RESULTS

Wine fermentation when yeast hulls are separated from thejuice by a dialysis membrane. The 21.7°Brix Chardonnayjuices were air saturated before being inoculated with 2 x106 CFU of yeast per ml. Fould-Springer yeast hulls wereused in this fermentation. They were either freely suspendedin the juice or physically separated from the fermenting yeastby being placed inside a dialysis sac. In both cases, yeasthulls were used at a concentration of 1 g/liter. Figure 1presents the time course of a fermentation in which a dialysismembrane with a 50,000-molecular-weight cutoff was used.The enhancing effect of yeast hulls on fermentation rate andyeast growth, as reported earlier (25, 27), could be seen onlywhen the hulls were in direct contact with the fermentingyeast. When the hulls were contained inside a dialysis bag,fermentation proceeded as in the control without yeast hulls.Similar results were also obtained when a dialysis bag with amolecular weight cutoff of 6,000 to 8,000 was used (data notshown).

Decanoic acid adsorbtion by the yeast hulls. If the increasedgrowth and increased fermentation rate caused by yeasthulls in a sluggish fermentation were due to the adsorption ofmedium-chain fatty acids, the dialysis membrane in theexperiment presented in Fig. 1 acted as a barrier, preventingthese inhibitory substances from being adsorbed from thejuice onto the hulls. This was considered unlikely, becauseof the low molecular weight of the fatty acids. To verify thatmedium-chain fatty acids can be adsorbed from the sur-rounding juice through the dialysis membrane onto yeasthulls, we conducted an experiment by using a sterile juice-ethanol mixture containing 118 mg of decanoic acid per literand 1 g of the hulls located inside a 50,000-molecular-weight-cutoff dialysis sac as described in Materials andMethods. After 24 h at 14°C, the contents of the sac and thejuice outside the sac were reanalyzed for decanoic acid(Table 1).

During the experiment, decanoic acid passed through themembrane, and 2.35 mg was adsorbed onto the hulls. The

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a

25 -

20

.x 15

10

5

0

suspended

0 5 10 15 20 25 30Time(days)

50 b

E

U- 40control

-'---3-* suspendedC30-

0 sac

E 20

0 5 10 15 20

Time(days)FIG. 1. Carbohydrate utilization (a) and cell viability (b) in

21.7°Brix fermentations inoculated at 2 x 106 CFU/ml. Yeast hulls(1 g/liter) were either freely suspended (0) or put inside a 50,000-molecular-weight-cutoff dialysis bag (U). The control experimenthad no yeast hulls ([]).

concentration outside the sac dropped from 118 to 55.6mg/liter (values are means of duplicate determinations).Mass balance shows that only 13% of the original acid in thejuice outside the membrane was not detected in these twofractions. Some acid may have been lost by evaporation or

by adsorption to glass surfaces or to the dialysis membrane.Effect of yeast hull lipid extract on the fermentation and

yeast membrane composition. Lipid extract from Fould

TABLE 1. Decanoic acid adsorption by yeast hulls througha dialysis membrane

Decanoic acid Recovery of decanoic acidlocation mg/liter mg/sample

Before exptJuice 118.0 5.90Yeast hulls 0.0

After exptJuice 55.6 2.78Adsorbed on hulls 2.35Juice inside bag ND' ND

" ND, Not detected.

TABLE 2. Effect of lipid extracts or complete yeast hulls onfermentation rate and yeast growth in a 20.2°Brix

Chardonnay juice fermentation

Maximum yeast growthFermentation rateaSupplement Viable count Cell mass (g/liter per day)

(106 CFU/ml) (mg/ml)

Lipid extract1 g/liter 80 4.8 14.62 g/liter 91 5.5 15.2

Yeast hulls 72 4.7 13.9(1 g/liter)

None 46 3.1 10.3

" Consumption of dissolved solids (in grams per liter per day) measuredduring days 2 to 11 of the fermentation.

Springer yeast hulls was used as a supplement in a series ofChardonnay juice fermentations. After air saturation, juicesat 20.2°Brix were inoculated with 4 x 106 to 5 x 106 CFU ofthe active dry yeast per ml. After inoculation, nitrogen gas at10 ml/min was used for headspace flushing to prevent oxygenfrom entering into the fermentor headspace. Lipid extracts,corresponding to 1 or 2 g of yeast hull supplementation perliter, were prepared immediately before inoculation as de-scribed in Materials and Methods. The prepared extractsenhanced both fermentation rate and yeast growth as muchas or more than the intact yeast hulls did when comparedwith the unsupplemented control. Data obtained from fer-mentations carried out with yeast hull and lipid extractsupplementations, as well as without supplementation, arereported in Table 2.More sugar was consumed during the first 2 days of the

fermentation in the juice in which yeast hulls were used as asupplement instead of the extracts (12 and 9 g/liter, respec-tively). After day 2 of fermentation, when the fermentationrates became linear, lipid extracts increased growth andsugar consumption more than the hulls did. The largeramount of lipid extract was the most effective enhancer.At the end of the fermentation, the yeasts were harvested

and their composition was analyzed in terms of their fattyacid composition and sterol contents. All the yeasts con-tained between 0.21 and 0.23% (wt/wt) sterol (measured asergosterol). When the sterol contents were calculated perliter of culture, the differences between the control fermen-tation and the lipid extract-supplemented fermentationswere considerable (Table 3). Table 3 also contains the datafor yeast fatty acid analysis expressed as a ratio of theamounts of unsaturated and saturated C16 and C18 acids(palmitoleic and oleic acids to palmitic and stearic acids).The yeast inoculum contained 1% sterol or 2 mg of cultureper liter, and the fatty acid unsaturation ratio was 1.1.The degree of fatty acid unsaturation was low in the

unsupplemented control fermentation, indicating that therewas a limited oxygen supply for the yeast. Incorporation of

TABLE 3. Effect of yeast hull lipid extract on the amount ofsterol and degree of unsaturation in yeast membrane fatty acids

Supplement Amt of sterol Degree of(mg/liter of culture) unsaturation"

Lipid extract1 g/liter 8.67 1.432 g/liter 11.44 2.33

None 7.66 0.29

" Ratio between unsaturated and saturated Clf and Cl8 acids.

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TABLE 4. Effect of different inoculum levels on the enhancingeffect of yeast hulls in a 20.7°Brix juice fermentation

Maximum growthInoculum level Fermentation rate"and supplement (g/liter per day) Cell count Cell mass

(106/ml) (mg/ml)

9 x 106 cellsControl 12 83 3.4Yeast hulls 12 96 3.8

4 x 106 cellsControl 10 46 3.1Yeast hulls 14 73 4.7

" Consumption of dissolved solids (in grams per liter per day) during days2 to 11 of the fermentation.

the lipid extract from 2 g of yeast hulls into the juiceincreased the ratio almost 10-fold. A shift to longer-chainfatty acids was also seen with the lipid extract (data notshown). Lipid extract from Fould Springer yeast hulls wasanalyzed previously (25) and found to contain 1% sterol. Itwas high in palmitoleic and oleic acid content. The results inTable 3 show the incorporation of sterols and especiallyunsaturated fatty acids from the lipid extracts into the yeastmembranes. Incorporation of unsaturated acids into yeastmembranes is known to affect the chain length of the acids,shifting the balance toward longer chains (28).

Effect of inoculum size on yeast hull-supplemented fermen-tation. The amount of oxygen or its substitutes (sterol andunsaturated fatty acids) needed in a particular fermentationdepends on the size of the inoculum and the level of thesecompounds in the inoculum. Active dry yeast cells, as usedin these experiments, are aerobically propagated and haveclose to maximum sterol content (1% of dry weight). Theyare also high in unsaturated fatty acids. An experiment wascarried out in which air-saturated, yeast hull-supplementedjuice (concentration, 1 g/liter) was fermented with twodifferent yeast inocula. The effect of inoculum sizes of 4 x106 to 5 x 106 and 9 x 106 CFU/ml on the enhancing effectof the hulls was studied. The results are presented in Table4.When 9 x 106 cells per ml were used as the inoculum, the

yeast hull preparation did not show a stimulatory effect onthe fermentation rate. A slight increase in growth comparedwith that of the unsupplemented control was noticed. Thehigh sterol content and amount of unsaturated fatty acidspresent in the inoculum yeast, in combination with the initialoxygen concentration of approximately 5 mg/liter (juice airsaturation), were sufficient to permit yeast growth to a higherlevel than when the lower inoculation rate was used. Underthese conditions, external sources of unsaturated acids orsterol as provided by the yeast hulls were not needed topromote higher yeast growth or fermentation rate.

DISCUSSION

The theory concerning fermentation enhancement and theprevention of stuck fermentations by yeast hulls is based ontwo observations. The first is the removal of toxic decanoic,octanoic, and hexanoic acids and their esters by adsorptiononto yeast hulls in a sterile (uninoculated) medium (17). Thesecond is the actual decrease in the concentration of theseacids in a wine fermented in the presence of hulls ascompared with a control fermentation (20). The results of thepresent experiments support the claim that yeast hulls ad-sorb decanoic acid as previously shown (17, 18, 27). The

enhancing effect of yeast hulls seen in a sluggish or a stuckfermentation, however, cannot be attributed only to theadsorption of medium-chain fatty acids from the juice ontothe hulls. The hulls promoted vinification only when in closecontact with the fermenting yeast. When a dialysis mem-brane formed a barrier between the yeast and the hulls,fermentation enhancement was not seen. Decanoic acid wasshown above, however, to flow freely through the dialysismembrane.The decrease in the concentrations of medium-chain fatty

acids seen in juices supplemented with Fould-Springer yeasthulls (20) can be explained by their content of unsaturatedfatty acids. When unsaturated fatty acids are provided to thejuice, increased amounts of both unsaturated fatty acids andlonger-chain fatty acids are found in yeast membranes. Adecrease in the concentration of cell-synthesized medium-chain acids follows (6). Incorporation of unsaturated fattyacids from the lipid extracts of Fould-Springer yeast hullsinto the yeast during the fermentation was seen in thepresent work. The fatty acid adsorption theory presented forthe action of yeast hulls is therefore in question. Enhancingeffects shown by yeast hulls in sluggish wine fermentationsare caused by their role as oxygen substitutes, under condi-tions where insufficient oxygen is present to allow sterol andunsaturated fatty acid synthesis to occur.The increase in the amounts of unsaturated fatty acids and

sterol in the yeast when fermented with lipid extracts iso-lated from yeast hulls (Table 3) shows that unsaturated fattyacids and sterol have been incorporated into the yeastmembranes. The control yeast harvested from the unsupple-mented fermentation contained lower levels of unsaturatedfatty acids, and the total amount of sterol present wassmaller. Increasing the supply of sterol and unsaturated fattyacids in the fermentation medium by supplementation withyeast hulls enabled more yeast cells to be formed andenhanced the fermentation rate. Sterol levels regulate thegrowth of the yeast. When sterol levels have fallen to acertain concentration, growth will cease unless oxygen orexternal sterol is present (4). This level was determined to be0.1% of the yeast dry weight for brewers' yeast (4) and 0.3%cof the dry weight for wine yeast (22). In the present work,the final sterol concentrations in the yeast were from 0.21 to0.23%, which can be considered the lowest level for growthfor the wine yeast used. In aerobically grown brewers' yeast,free sterol normally corresponds to only 0.3% of the dryweight; most of the sterol is present as sterol esters (5). Theamount of free sterol present in yeast hulls is large enough tosupply all the sterol accumulated in the yeast during thefermentation.

In the present experiments, contact between the ferment-ing yeast and yeast hulls was essential to obtain improvedgrowth and fermentation rate. The enzymes required forlipid hydrolysis and/or uptake were most probably mem-brane bound. The presence of membrane-bound lipases andphospholipases in bakers' yeast has been reported (26).Under microscopy, the yeast cells were seen to form aggre-gates around yeast hulls (cell debris). The lipid fraction ofmalt spent grain (29) and Aspergillius orvzcae proteolipids (11)have been reported to enhance growth, fermentation rate,and ethanol tolerance in brewery in sake fermentation. Inboth cases, as was seen in the present work, unsaturatedfatty acids were incorporated into the yeast. In the beerfermentation, enough free sitosterol from spent grains waspresent to supply all the sterol incorporated in membranes(29). The presence of lipolytic activity in the yeast was

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1564 MUNOZ AND INGLEDEW

speculated upon (29), or the aggregation of yeast cellsaround phospholipid fragments was seen (12).

Stuck fermentations are a serious problem in the wineindustry. The problem is more acute when white wines (inwhich the lipids of grape skins are not present) or juices withhigh sugar concentrations are fermented. To prevent stuckfermentations, a large number of yeast cells should bepresent in the fermentation tank. This can be achieved byusing large inocula or by providing adequate yeast nutritionto allow the yeast to grow to desired high cell density. Invinification, especially in Europe, where the natural flora isoften used without a pure-culture yeast, high inoculum levelsare not possible. Under wine-making conditions, in which airis introduced to the juice only during the handling of juicebefore inoculation, oxygen easily becomes a limiting nutrientfor subsequent yeast growth, and the lack of oxygen cancause a cessation of fermentation. The use of yeast hulls as

suggested by Lafon-Lafourcade (17), Ribereau-Gayon (27),and others (20, 21) can partly overcome the problem of lackof oxygen. A more economical way would be to provide thefermentation with a higher concentration of oxygen byaeration or oxygenation initially; this practice is carried outin the brewing industry, and the technique has met withsome success in California (23). Assimilable nitrogen (ni-trogen usable by yeasts such as amino acids, ammoniumsalts, or the now banned urea) is the other essential nutrientrequired to increase cell yield. Some juices are very low inassimilable nitrogen (<40 mg/liter [14]), and increasing thelevel of this nutrient, together with provision of an adequateoxygen concentration, enhances both yeast growth andfermentation rate, thus preventing stuck fermentation (14).The quality of the resultant wine after increasing the nitrogenlevels by vine fertilization (7) or after nitrogen supplemen-tation during the fermentation (30) was reported to beimproved.

Provision of oxygen and extra assimilable nitrogen pro-

motes yeast growth, increases the rate of fermentation, andeliminates stuck fermentations (14). It also allows white winefermentation tankage to be used more than once per year.

ACKNOWLEDGMENT

We thank the Natural Science and Engineering Research Councilof Canada for research support for this project.

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