APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1989, p. 1560-1564 0099-2240/89/061560-05$02.

00/0 Copyright D 1989, American Society for Microbiology

Vol. 55, No. 6

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 O WO, 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. The enhancing effect on yeast growth and fermentation rate displayed by the hulls was shown to be similar to the effect provided by lipid extract from the same hulls. Unsaturated fatty acids and sterols were incorporated into the yeast from lipid extracts during fermentation carried out under oxygen-limited conditions. Adsorption of toxic 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 rate seen when freely suspended hulls were used did not occur. Accordingly, the effect of yeast hulls in preventing stuck fermentations cannot be attributed only to the adsorption and consequent removal of medium-chain fatty acids from the juice.

Wine fermentations are normally completed by the metabolic activity of resting (nongrowing) yeast cells (19). Juices often ferment for longer than 30 days, and occasionally the fermentations become stuck. Stuck fermentations are the most serious, yet one of the most common, problems of the wine industry (21). In a stuck ferrnentation, the available sugar in juice is not totally fermented and residual sugar remains in the finished product. Although high osmotic pressure and the presence of inhibitory substances including pesticides from grapes have been implicated, the main reason for stuck fermentation is premature death of resting cells as a result of intolerance to ethanol (8); this phenomenon can be prevented by the addition of nutritional supplements to the juice (14, 19). To alleviate stuck or sluggish (excessively slow) fermentations, oxygen or sterol and unsaturated fatty acids (1, 2), called survival factors (14, 19, 27), or assimilable nitrogen in the form of ammonium salts or amino acids (24), or both survival factors and assimilable nitrogen (8, 9, 14), have been provided early in fermentation to allow the production of a sufficient number of new cells and to prevent existing cells from dying. Such work has led enologists to examine the anaerobic conditions of wine making, the practice of muist clarification prior to fermentation, the use of higher inocuilum levels, and the use of active dry yeast (higher sterol levels) (14, 16). Limitation of free amino nitrogen was shown to be serious in worts (9) and in juices (14) in which the fermentation sticks or becomes sluggish. Yeast ghosts or yeast hulls, the cell wall materidil rematining after yeast extract preparation, have been suggested as supplements to juice to prevent stuck fermentations (27). The action of the hulls was postulated to be due to physical adsorption and removal of toxic fermentation side products, the medium-chain fatty acids hexanoic, octanoic, and decanoic acids (17). These fatty acids are formed via a synthetic route in Saccharomyces yeasts (28). In the presence of ethanol they completely inhibit yeast growth at concentrations found in wines (18). More recently, Larue and LafonLafourcade (21) observed the effect of aeration in reducing the concentration of medium-chain fatty acids in wine, as
* Corresponding author.
1560

had been previously observed in beer fermentations (6, 28). The effect of oxygen was seen in the chain elongation and in the increase in the degree of unsaturation in membrane lipid fatty acids (6). Correlation has also been shown between fatty acid concentrations in beer and in cell membranes (28). In a recent paper, Munoz and Ingledew (25) presented results indicating that adsorption of toxic by-products was not the only enhancing effect displayed by yeast hulls in a sluggish wine fermentation. Under oxygen-limited conditions (simulating actual conditions in wine making), the effect of yeast ghosts was the same as the effect of ergosterol and Tween 80 (source of oleic acid) supplements. Both acted as oxygen substitutes, allowing sterol and unsaturated fatty acid production for yeast cell membrane synthesis. In the present work, fermentation experiments and yeast lipid analysis by gas-liquid chromatography and chemical methods were conducted to verify the importance of yeast hull lipids in the stimulation of fermentation and prevention of stuck and sluggist fermentations. Addition of decanoic acid to uninoculated media was done to reassess fatty acid a1dsorption as the mechainism of yeast hull action.
MATI AII,S AND METHODS Fermentations. l:ermentations were carried out with reconstituted juice. l o 2.8 liters of juice concentrate (Sun Cal Pinot Chardonn.ay. Niagara Vine Products lAtd.. St. Catherines, Ontakrio. Canada) 15.5 liters of sterilized waiter, 1.74 kg of sucrose. 42 g of acid blend (mixture of tartaric. malic, and citric acids: Harvest Brewing Co, Saskaltoon. Saskatchewan, Canaida. atnd 3 g of grape tannin were aidded. The resulting juice Wals sterilized with 0.1 ml ot diet hylpyrocarbonate (Sigmai Chemical Co., St. Louis. Mo.) per liter. Active dry Red Star Montrachet yeast was rehydrated in sterile 0.1%, peptone-water at 39C for 20 min, and the juice was then inoculated at different levels depending on the experiment. All fermentations were conducted at 14C in 1-liter or 500-ml fermentors (Celstir; Wheaton Industries, Millville, N.J.) with constant stirring at 90 rpm to ensure that homogeneous yeast and juice samples could be obtained. The 1-liter fermentors contained 1 liter of juice, and the headspace volumes were 700 ml; the 500-ml fermentors contained 500 ml of juice, and the headspace volumes were

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240 ml. The juices were saturated with air prior to inoculation. After inoculation, a constant flow of nitrogen gas at 10 ml/min was introduced through the headspace. A yeast hull preparation obtained from Fould Springer, Maisons Alfort, France, was used in the experiments. Either yeast hulls were directly added in the fermentor at a concentration of 1 g/liter or 1 g of hulls, was suspended in 20 ml of the juice and put inside a dialysis bag to physically separate the hulls from the fermenting yeast. Spectrapor (Spectrum Medical Industries, Inc., Los Angeles Calif.) dialysis membranes with a molecular weight cutoff of 6,000 to 8,000 (400 mm long, 23 mm wide) or 50,000 (800 mm long, 12 mm wide) were used for this purpose. Lipid extract from yeast hulls was prepared by extracting 1 g of yeast hulls with 30 ml of chloroform-methanol-5 M hydrochloric acid (5:6:1, vol/vol) for 20 min at room temperature (15). After addition of 6 ml of water and shaking, the organic layer was collected and dried under nitrogen gas. The extracts were redissolved in 5 ml of warm 95% ethanol and immediately added to the fermentor. A 5-ml portion of ethanol 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-ethanol mixture (12Brix, 5% ethanol). Yeast hulls (1 g) were suspended in 20 ml of the juice and put inside a dialysis bag (Spectrapor membrane; molecular weight cutoff, 50,000; 800 mm long, 12 mm in diameter). The sac was placed in a 250-ml Erlenmeyer flask containing 50 ml of the juice with 118 mg of decanoic 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 Scientific Co., Inc., Edison, N.J.) at 200 rpm and 14C for 24 h. After the experiment, the volumes of the juices inside and outside the dialysis bag were measured, and then the juices were analyzed for their decanoic acid contents. The yeast hulls were separated from the juice inside the bag by centrifugation (10 min at 3,000 x g and 4C), extracted with chloroform-methanol-5 M HCl (15), and then analyzed for their decanoic acid concentration. Experiments were run in duplicate. 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 were presented as specific gravity or converted to Brix by using prepared conversion tables (1). Viable yeast cells were counted in triplicate by the membrane filtration technique and expressed as millions of CFU per milliliter (14). Yeast cell mass was estimated gravimetrically after a buffer wash (14). The sterol contents of the yeast (collected, after fermentation, by centrifugation for 10 min at 3,000 x g and 4C and washed three times with 1/15 M potassium dihydrogen phosphate buffer [pH 4.5]) were analyzed by the LiebermanBuchard reaction (10). Ergosterol was used as a standard. Total fatty acids were extracted with chloroform-methanol-5 M HCI from 100 ml of final culture containing the yeast by the method of Kemp and White (15). The acids were methylated at 100 to 110C for 30 min with 2 ml of sulfuric acid-methanol-toluene (1:20:10, vol/vol/vol) reagent (13). After methylation, the esters were separated in a gas chromatograph (model 5750; Hewlett-Packard Co., Palo Alto, Calif.) with a stainless steel column (6 ft by 1/8 in. [1.8 m by 0.3 cm]) packed with GP 10% SP-2330 on 100/120 Chromosorb W/AW (Supelco, Inc., Bellefonte, Pa.). The temperature program used was as follows: 1 min at 130C, rising at

8C/min to 242C, followed by a 6-min hold at 242C. The carrier gas (helium) flow rate was 30 ml/min; the air flow rate was 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 4C) wine which had been passed through a 0.45-,um-pore-size membrane filter. The samples were saturated with NaCl and acidified to pH 1 with concentrated HCI. Nonanoic acid (0.1 mg/ml) was used as the internal standard. The juices were shaken for 15 min with an equal volume of chloroform-methanol (3:1). The samples were then centrifuged to separate the phases, and the organic phase was evaporated at room temperature in a rotary evaporator. After drying, the acids were refluxed for 1 h with 50 ml of 6% (wt/vol) potassium hydroxide in 95% ethanol. After the addition of 50 ml of water, the unsaponified material was removed by extraction three times with 40 ml of petroleum ether (bp 30 to 60C; BDH, Toronto, Canada). The aqueous phase was acidified to pH 1 with concentrated HCI in an ice bath, and the fatty acids were extracted 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 gas chromatograph (model 5750; Hewlett-Packard) on the column described above, with a temperature program of 6 min at 75C, a rise of 4C/min to 225C, and a 10-min hold at 2250C.
RESULTS Wine fermentation when yeast hulls are separated from the juice by a dialysis membrane. The 21.7Brix Chardonnay juices were air saturated before being inoculated with 2 x 106 CFU of yeast per ml. Fould-Springer yeast hulls were used in this fermentation. They were either freely suspended in the juice or physically separated from the fermenting yeast by being placed inside a dialysis sac. In both cases, yeast hulls were used at a concentration of 1 g/liter. Figure 1 presents the time course of a fermentation in which a dialysis membrane with a 50,000-molecular-weight cutoff was used. The enhancing effect of yeast hulls on fermentation rate and yeast growth, as reported earlier (25, 27), could be seen only when the hulls were in direct contact with the fermenting yeast. 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 a molecular weight cutoff of 6,000 to 8,000 was used (data not shown). Decanoic acid adsorbtion by the yeast hulls. If the increased growth and increased fermentation rate caused by yeast hulls in a sluggish fermentation were due to the adsorption of medium-chain fatty acids, the dialysis membrane in the experiment presented in Fig. 1 acted as a barrier, preventing these inhibitory substances from being adsorbed from the juice onto the hulls. This was considered unlikely, because of the low molecular weight of the fatty acids. To verify that medium-chain fatty acids can be adsorbed from the surrounding juice through the dialysis membrane onto yeast hulls, we conducted an experiment by using a sterile juiceethanol mixture containing 118 mg of decanoic acid per liter and 1 g of the hulls located inside a 50,000-molecularweight-cutoff dialysis sac as described in Materials and Methods. After 24 h at 14C, the contents of the sac and the juice outside the sac were reanalyzed for decanoic acid (Table 1). During the experiment, decanoic acid passed through the membrane, and 2.35 mg was adsorbed onto the hulls. The

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a

APPL. ENVIRON. MICROBIOL.

TABLE 2. Effect of lipid extracts or complete yeast hulls on fermentation rate and yeast growth in a 20.2Brix Chardonnay juice fermentation
Supplement

25 -

suspended
20

Maximum yeast growth Viable count Cell mass

Fermentationday) ratea (g/liter per

(106 CFU/ml)

(mg/ml)
4.8 5.5 4.7

.x 15
10

5
0

Lipid extract 1 g/liter 2 g/liter Yeast hulls (1 g/liter) None

80 91 72

14.6 15.2 13.9

46

3.1

10.3

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

5 10 15 20 25 30

Time(days)
50 E
U-

40

-'--3-*
C300

control suspended
sac

E 20

10

15

20

Time(days)
FIG. 1. Carbohydrate utilization (a) and cell viability (b) in 21.7Brix fermentations inoculated at 2 x 106 CFU/ml. Yeast hulls (1 g/liter) were either freely suspended (0) or put inside a 50,000molecular-weight-cutoff dialysis bag (U). The control experiment had no yeast hulls ([]).

concentration outside the sac dropped from 118 to 55.6 mg/liter (values are means of duplicate determinations). Mass balance shows that only 13% of the original acid in the juice outside the membrane was not detected in these two fractions. 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 through a dialysis membrane
Decanoic acid
location

Springer yeast hulls was used as a supplement in a series of Chardonnay juice fermentations. After air saturation, juices at 20.2Brix were inoculated with 4 x 106 to 5 x 106 CFU of the active dry yeast per ml. After inoculation, nitrogen gas at 10 ml/min was used for headspace flushing to prevent oxygen from entering into the fermentor headspace. Lipid extracts, corresponding to 1 or 2 g of yeast hull supplementation per liter, were prepared immediately before inoculation as described in Materials and Methods. The prepared extracts enhanced both fermentation rate and yeast growth as much as or more than the intact yeast hulls did when compared with the unsupplemented control. Data obtained from fermentations carried out with yeast hull and lipid extract supplementations, as well as without supplementation, are reported 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 a supplement instead of the extracts (12 and 9 g/liter, respectively). After day 2 of fermentation, when the fermentation rates became linear, lipid extracts increased growth and sugar consumption more than the hulls did. The larger amount 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 fatty acid composition and sterol contents. All the yeasts contained between 0.21 and 0.23% (wt/wt) sterol (measured as ergosterol). When the sterol contents were calculated per liter of culture, the differences between the control fermentation and the lipid extract-supplemented fermentations were considerable (Table 3). Table 3 also contains the data for yeast fatty acid analysis expressed as a ratio of the amounts 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 culture per 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 there was a limited oxygen supply for the yeast. Incorporation of
TABLE 3. Effect of yeast hull lipid extract on the amount of sterol and degree of unsaturation in yeast membrane fatty acids
Supplement
Amt of sterol

Recovery of decanoic acid

mg/liter

mg/sample
5.90 0.0

Before expt Juice Yeast hulls After expt Juice Adsorbed on hulls Juice inside bag
"

118.0

(mg/liter of culture)

Degree of unsaturation"
1.43 2.33 0.29

55.6
ND'

2.78 2.35 ND

Lipid extract 1 g/liter 2 g/liter

None

8.67 11.44 7.66

ND, Not detected.

" Ratio between unsaturated and saturated

Clf and Cl8 acids.

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TABLE 4. Effect of different inoculum levels on the enhancing effect of yeast hulls in a 20.7Brix juice fermentation
Inoculum level and supplement

enhancing effect of yeast hulls seen in a sluggish or a stuck

fermentation, however, cannot be attributed only to the

adsorption of medium-chain fatty acids from the juice onto the hulls. The hulls promoted vinification only when in close contact with the fermenting yeast. When a dialysis membrane formed a barrier between the yeast and the hulls, fermentation enhancement was not seen. Decanoic acid was shown above, however, to flow freely through the dialysis membrane. The decrease in the concentrations of medium-chain fatty acids seen in juices supplemented with Fould-Springer yeast hulls (20) can be explained by their content of unsaturated fatty acids. When unsaturated fatty acids are provided to the juice, increased amounts of both unsaturated fatty acids and longer-chain fatty acids are found in yeast membranes. A decrease in the concentration of cell-synthesized mediumchain acids follows (6). Incorporation of unsaturated fatty acids from the lipid extracts of Fould-Springer yeast hulls into the yeast during the fermentation was seen in the present work. The fatty acid adsorption theory presented for the action of yeast hulls is therefore in question. Enhancing effects shown by yeast hulls in sluggish wine fermentations are caused by their role as oxygen substitutes, under conditions where insufficient oxygen is present to allow sterol and unsaturated fatty acid synthesis to occur. The increase in the amounts of unsaturated fatty acids and sterol in the yeast when fermented with lipid extracts isolated from yeast hulls (Table 3) shows that unsaturated fatty acids and sterol have been incorporated into the yeast membranes. The control yeast harvested from the unsupplemented fermentation contained lower levels of unsaturated fatty acids, and the total amount of sterol present was smaller. Increasing the supply of sterol and unsaturated fatty acids in the fermentation medium by supplementation with yeast hulls enabled more yeast cells to be formed and enhanced the fermentation rate. Sterol levels regulate the growth of the yeast. When sterol levels have fallen to a certain concentration, growth will cease unless oxygen or external sterol is present (4). This level was determined to be 0.1% of the yeast dry weight for brewers' yeast (4) and 0.3%c of the dry weight for wine yeast (22). In the present work, the final sterol concentrations in the yeast were from 0.21 to 0.23%, which can be considered the lowest level for growth for the wine yeast used. In aerobically grown brewers' yeast, free sterol normally corresponds to only 0.3% of the dry weight; most of the sterol is present as sterol esters (5). The amount of free sterol present in yeast hulls is large enough to supply all the sterol accumulated in the yeast during the fermentation. In the present experiments, contact between the fermenting yeast and yeast hulls was essential to obtain improved growth and fermentation rate. The enzymes required for lipid hydrolysis and/or uptake were most probably membrane bound. The presence of membrane-bound lipases and phospholipases in bakers' yeast has been reported (26). Under microscopy, the yeast cells were seen to form aggregates around yeast hulls (cell debris). The lipid fraction of malt spent grain (29) and Aspergillius orvzcae proteolipids (11) have been reported to enhance growth, fermentation rate, and ethanol tolerance in brewery in sake fermentation. In both cases, as was seen in the present work, unsaturated fatty acids were incorporated into the yeast. In the beer fermentation, enough free sitosterol from spent grains was present to supply all the sterol incorporated in membranes (29). The presence of lipolytic activity in the yeast was

Fermentation rate" (g/liter per day)

Maximum growth

Cell count (106/ml)

Cell mass (mg/ml)

9 x 106 cells

Control Yeast hulls 4 x 106 cells Control Yeast hulls

12

12 10 14

83 96

3.4

3.8 3.1 4.7

46 73

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

the lipid extract from 2 g of yeast hulls into the juice increased the ratio almost 10-fold. A shift to longer-chain fatty acids was also seen with the lipid extract (data not shown). Lipid extract from Fould Springer yeast hulls was analyzed previously (25) and found to contain 1% sterol. It was high in palmitoleic and oleic acid content. The results in Table 3 show the incorporation of sterols and especially unsaturated fatty acids from the lipid extracts into the yeast membranes. Incorporation of unsaturated acids into yeast membranes 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 fermentation. The amount of oxygen or its substitutes (sterol and unsaturated fatty acids) needed in a particular fermentation depends on the size of the inoculum and the level of these compounds in the inoculum. Active dry yeast cells, as used in these experiments, are aerobically propagated and have close to maximum sterol content (1% of dry weight). They are also high in unsaturated fatty acids. An experiment was carried out in which air-saturated, yeast hull-supplemented juice (concentration, 1 g/liter) was fermented with two different yeast inocula. The effect of inoculum sizes of 4 x 106 to 5 x 106 and 9 x 106 CFU/ml on the enhancing effect of the hulls was studied. The results are presented in Table 4. When 9 x 106 cells per ml were used as the inoculum, the yeast hull preparation did not show a stimulatory effect on the fermentation rate. A slight increase in growth compared with that of the unsupplemented control was noticed. The high sterol content and amount of unsaturated fatty acids present in the inoculum yeast, in combination with the initial oxygen concentration of approximately 5 mg/liter (juice air saturation), were sufficient to permit yeast growth to a higher level than when the lower inoculation rate was used. Under these conditions, external sources of unsaturated acids or sterol as provided by the yeast hulls were not needed to promote higher yeast growth or fermentation rate.
DISCUSSION The theory concerning fermentation enhancement and the prevention of stuck fermentations by yeast hulls is based on two observations. The first is the removal of toxic decanoic, octanoic, and hexanoic acids and their esters by adsorption onto yeast hulls in a sterile (uninoculated) medium (17). The second is the actual decrease in the concentration of these acids in a wine fermented in the presence of hulls as compared with a control fermentation (20). The results of the present experiments support the claim that yeast hulls adsorb decanoic acid as previously shown (17, 18, 27). The

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speculated upon (29), or the aggregation of yeast cells around phospholipid fragments was seen (12). Stuck fermentations are a serious problem in the wine industry. The problem is more acute when white wines (in which the lipids of grape skins are not present) or juices with high sugar concentrations are fermented. To prevent stuck fermentations, a large number of yeast cells should be present in the fermentation tank. This can be achieved by using large inocula or by providing adequate yeast nutrition to allow the yeast to grow to desired high cell density. In
vinification, especially in Europe, where the natural flora is
often used without a pure-culture yeast, high inoculum levels are not possible. Under wine-making conditions, in which air is introduced to the juice only during the handling of juice before inoculation, oxygen easily becomes a limiting nutrient for subsequent yeast growth, and the lack of oxygen can cause 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 lack of oxygen. A more economical way would be to provide the fermentation with a higher concentration of oxygen by aeration or oxygenation initially; this practice is carried out in the brewing industry, and the technique has met with some success in California (23). Assimilable nitrogen (nitrogen usable by yeasts such as amino acids, ammonium salts, or the now banned urea) is the other essential nutrient required to increase cell yield. Some juices are very low in assimilable nitrogen (<40 mg/liter [14]), and increasing the level of this nutrient, together with provision of an adequate oxygen concentration, enhances both yeast growth and fermentation rate, thus preventing stuck fermentation (14). The quality of the resultant wine after increasing the nitrogen levels by vine fertilization (7) or after nitrogen supplementation during the fermentation (30) was reported to be

improved.
Provision of oxygen and extra assimilable nitrogen promotes yeast growth, increases the rate of fermentation, and eliminates stuck fermentations (14). It also allows white wine fermentation tankage to be used more than once per year.
ACKNOWLEDGMENT
We thank the Natural Science and Engineering Research Council of Canada for research support for this project.

8. Casey, G. P., and W. M. Ingledew. 1986. Ethanol tolerance in yeasts. Crit. Rev. Microbiol. 13:219-280. 9. Casey, G. P., C. A. Magnus, and W. M. Ingledew. 1984. High-gravity brewing: effects of nutrition on yeast composition, fermentative ability, and alcohol production. Appl. Environ. Microbiol. 48:639-646. 10. Giudici, P., and M. E. Guerzoni. 1982. Sterol content as a character for selecting yeast strains in enology. Vitis 21:5-14. 11. Hayashida, S., D. D. Feng, and M. Hongo. 1974. Function of the high concentration alcohol-producing factor. Agric. Biol. Chem. 38:2001-2006. 12. Hayashida, S., D. D. Feng, K. Ohta, S. Chaitiumvong, and M. Hongo. 1976. Composition and role of Aspergillus oryzae proteolipid as a high concentration alcohol-producing factor. Agric. Biol. Chem. 40:73-78. 13. Hitchcock, C., and E. W. Hammond. 1978. The determination of lipids in foods, p. 185-223. In R. D. King (ed.), Developments in food analysis techniques, vol. 2. Applied Science Publishers, London. 14. Ingledew, W. M., and R. E. Kunkee. 1985. Factors influencing sluggish fermentations of grape juice. Am. J. Enol. Vitic. 36: 65-75. 15. Kemp, P., and R. W. White. 1975. The hydrogenation of unsaturated fatty acids by five bacterial isolates from the sheep rumen including a new species. J. Gen. Microbiol. 90:100-114. 16. Lafon-Lafourcade, S. 1983. Wine and brandy, p. 81-163. In H.-J. Rehm and G. Reed (ed.), Biotechnology, vol. 5. Food and feed production and microorganisms. Verlag Chemie, Deerfield Beach, Fla. 17. Lafon-Lafourcade, S. 1984. Souches de levures. Bull. 0. I. V. 637:185-203. 18. Lafon-Lafourcade, S., C. Geneix, and P. Ribereau-Gayon. 1984. Les modalites de mise en oeuvre des ecorces de levure en vinification. Connaiss. Vigne Vin 18:111-125. 19. Lafon-Lafourcade, S., F. Larue, and P. Ribereau-Gayon. 1979. Evidence for the existence of "survival factors" as an explanation for some pecularities of yeast growth, especially in grape must of high sugar concentration. Appl. Environ. Microbiol.
38:1069-1073. 20. Larue, F., C. Geneix, S. Lafon-Lafourcade, A. Betrand, and P. Ribereau-Gayon. 1984. Premiers observations sur le mode d'ac-

tion des ecorces de levure. Connaiss. Vigne Vin 18:155-163.

21. Larue, F., and S. Lafon-Lafourcade. 1989. Survival factors in wine fermentation, p. 193-215. In N. van Uden (ed.), Alcohol tolerance in yeasts and bacteria. CRC Press, Inc., Boca Raton,

Fla.
22. Larue, F., S. Lafon-Lafourcade, and P. Ribereau-Gayon. 1980.

LITERATURE CITED
1. Amerine, M. A., and C. S. Ough. 1980. Methods of analysis of musts and wines, p. 18. John Wiley & Sons, Inc., New York. 2. Andreasen, A. A., and T. J. B. Stier. 1953. Anaerobic nutrition of Sacchacromyc es c erevisiace. Ergosterol requirement for

Relationship between the sterol content of yeast cells and their fermentation activity in grape must. Appl. Environ. Microbiol. 39:808-811.
23. Long, Z., and B. Lindblom. 1986. Juice oxidation experiments. Wines Vines 67(11):44-49. 24. Monk, P. R. 1982. Effect of nitrogen and vitamin supplements on yeast growth and rate of fermentation of Rhine Riesling grape juice. Food Technol. Aust. 34:328-332. 25. Munoz, E., and W. M. Ingledew. 1989. An additional explanation for the promotion of more rapid, complete fermentation by yeast hulls. Am. J. Enol. Vitic. 40:61-64. 26. Nurminen, T., and H. Suomalainen. 1970. The lipolytic activity of the isolated cell envelope fractions of baker's yeast. Biochem. J. 118:759-763. 27. Ribereau-Gayon, P. 1985. New developments in wine microbiology. Am. J. Enol. Vitic. 36:1-10. 28. Taylor, G. T., and B. H. Kirsop. 1977. The origin of the medium chain length fatty acids present in beer. J. Inst. Brew. 83:

growth in

defined medium. J. Cell. Comp. Physiol. 41:23-36.

3. Andreasen, A. A., and T. J. B. Stier. 1954. Anaerobic nutrition of Saccharomyces cereiisiae. Unsaturated fatty acid requirement for growth in a defined medium. J. Cell. Comp. Physiol.

43:271-281. Aries, V., and B. H. Kirsop. 1977. Sterol synthesis in relation to growth and fermentation by brewing yeasts inoculated at different concentrations. J. Inst. Brew. 83:220-223. 5. Aries, V., and B. H. Kirsop. 1978. Sterol biosynthesis by strains of Saccharomyces cerevisiae in the presence and absence of dissolved oxygen. J. Inst. Brew. 84:118-122. 6. Aries, V., B. H. Kirsop, and G. T. Taylor. 1977. Yeast lipids, p. 255-266. In Proceedings of the 16th Congress, Amsterdam. European Brewery Convention, Zoeterwonde, The Netherlands. 7. Bell, A. A., C. S. Ough, and W. M. Kliever. 1979. Effects on must and wine composition, rates of fermentation, and wine quality of nitrogen fertilization of Vitis i'nifera var. Thomson seedless grapes. Am. J. Enol. Vitic. 30:124-129.
4.

241-243.
29. Taylor, G. T., P. A. Thurston, and B. H. Kirsop. 1979. The influence of lipid derived from malt spent grains on yeast metabolism and fermentation. J. Inst. Brew. 85:219-227. 30. Vos, P. J. A., W. Zeeman, and H. Heyman. 1978. The effect on wine quality of diammonium phosphate additions to must. Proc. S. Afr. Enol. Soc. 1978:87-104.