- Research Notes -
Zygosaccharomyces, A Spoilage Yeast Isolated from Wine
by
K.C. Fugelsang
CATI publication #960804
© copyright August 1996, all rights reserved

INTRODUCTION

Spoilage resulting from growth of the yeast Zygosaccharomyces is widespread in the food industry. Zygosaccharomyces has been identified as a spoilage agent in fruit concentrates and juices, jams, jellys and preserves, as well as in ketchup, salad dressings, relishes and pickles (Thomas and Davenport, 1985). Although the yeast has a long history of spoilage in the food industry, it has only recently received attention among winemakers. In part, this stems from increased production, transport and utilization of potentially contaminated grape and fruit concentrates.

The currently recognized species, Zygosaccharomyces bailii, was first described in 1985 by Linder as Saccharomyces bailii. Its affinity with other Saccharomyces (S. acidifacciens, S. elegans) was maintained until current genus Zygosaccharomyces, incorporating eight species, was adopted (Barnett, et. al., 1983). Of those recognized species of Zygosaccharomyces, Z. bailii, Z. bisporous, Z. rouxii and Z. Florentinus have been isolated from grape must and wine.

Zygosaccharomyces is frequently described as being osmophilic, suggesting a habitat restricted to high solute (in this case, sugar) environments. The Aw of typical concentrated syrups (> 65 percent soluable solids) ranges from 0.82-0.94 (Kaplow, 1970). Depending upon the solute and its relative concentration (salt, sugar, or sugar/glycerol), Z. rouxii has been reported to grow at Aw ranging from 0.62 (in fructose) to 0.65 (sucrose/glycerol) to 0.86 (in NaCl) (Corry, 1978).

Since the yeast grows over a wide range of sugar levels, including those typical of fruit concentrates as well as sweetened wines, it technically may be regarded as osmotolerant or osmoduric. When growing in high sugar environments, it likely grows in the thin film of water at the surface. As growth continues, it "creates" more water, thereby extending the contamination.

Aside from its ability to grow in the restrictive habitat of high osmotic pressure, Zygosaccharomyces is also extraordinarily resistant to common preservatives used in juice, concentrate and wine industries: 
  
      Sulfur Dioxide:   >3mg/L molecular  (at pH 3.4)1
      Sorbic Acid:      600-800 mg/L2,3   
      Benzoic Acid:     600->1,000 mg/L2,3   
      Acetic Acid:      2.0->2.5% (vol/vol)2,3   
      Alcohol:          >18%  (vol/vol)3  

  1 Thomas (1981)   
  2 Pitt  (1974)  
  3 Thomas and Davenport (1985)
Thomas and Davenport (1985) speculate that SO2 resistance is partially the result of formation of extracellular compounds (such as acetaldehyde) that bind SO2 and thereby reduce the concentrations of molecular form. Muller and Fugelsang (1994) have shown that carbon monoxide, at levels of 420 mg/L, is effective in control of Zygosaccharomyces. Saccharomyces, by comparison, was not affected by levels of CO >1,000 mg/L.

In nature, Zygosaccharomyces is isolated from dehydrated or mummified fruit as well as from fruit tree exudates. Although present on fruit, population increases are noted during and after processing when competitive microrganissms have been either eliminated or their numbers greatly reduced. Thus, raw materials serve to initially introduce the yeast. In the case of sweetened wines, spoilage usually results from contaminated concentrate (Rankinee and Pilone 1973).

Once introduced into the winery, difficult-to-sanitize places in processing lines serve as the principle reservoirs from which continued product contamination may occur. In this regard, Zygosaccharomyces sp. have also been isolated from atypical sites in the winery. For example, Rankine and Pilone (1973) reported issolation of established populations of the yeast from a pressure gauge on the post-filtration side of a sterile filter. In this case, the gauge's construction not only allowed the organism to survive steam sterilization, but to continually reenter sterile-filtered wine enroute to the bottling line. Other unusual sites of isolation include lubricating oils (Beech and Davenport, 1983). Aerosols created by interation of these contaminated oils and high speed machinery served to contaminate the previously sanitzed area.

Incidences of Zygosaccharomyces spoilage are frequently linked to sub-lethal doses of chemical sterilizing agents or steam/hot water protocol that doesn't meet the time x temperature requirements for cell death. In one study, Van Esch (1992) notes that aside from contaminated raw material (i.e., grape concentrate), 95 percent of the contamination occured because production demands of the bottling facility lead to shortcutting sanitation.

Overt growth in stored concentrate may go unnnotice at low temperature storage. However, warming the product during shipment and or changes in chemistry upon blend formulation may stimulate previously repressed populations resulting in spoilage.

Strains of Zygosaccharomyces are known to be extraordinary tolerant to alcohol. Thomas and Davenport (1985) report growth in wines at 18 percent (vol/vol) alcohol, explaining, in part, the high frequency of isolation from refermenting bottled table wine discussed by Peynaud and Domercq (1959).

Thomas (1981) reported that phenolics and anthocyanins in red wined may be inhibitiory. This supports the previos report of Peynaud and Domercq (1959) indicating that white wines were at greatest risk of spoilage. By comparison, our recent survey found that concentrate- sweetened rose and red wines experience greater incidences of refermentation (Fugelsang and Muller, 1994 unpublished results). Further, our survey suggests that cork or capsule-finished bottled wines seldom presented a problem whereas the same wine packaged bag-in-box may support growth. The two differ in the area of post-bottling oxygen incursion. Bag-in-box liners are somewhat oxygen permeable whereas incursion of oxygen in cork-finished bottled wine is limited. Further, the greater incidences of spoilage in bag-in-box packaged sweetened red and rose wines suggests the role of flavonoid phenols (and their polymers) as oxygen reservoirs.


MATERIALS AND METHODS

I. Recovery Studies: Over a two year period, samples were collected from wineries and/or concentrate facilities throughout central California. These represented juice, concentrate, sweetened packaged wines as well as swab samples from tanks, lines, etc. Appropriate dilutions of liquid samples were plated on Zygosaccharomyces-selective agar media as described in Zoecklein, et. al., (1995). Swab samples were immersed in sterile peptone broth, shaken for 5 minutes, and membrane (0.45 um) filtered. After filtration, membranes were also placed on selective agar. Plates were incubated 7-10 days at 80°F. Where colony development was noted, confirmation was obtained by plating on Wort agar or sporulation. On this medium, Zygosaccharomyces forms characteristic club-shaped asci.

II. Growth Studies: Zinfandel and Grenache blush style wines were adjusted from baseline 7.5 percent (v/v) to 8.0 percent, 8.5 percent, 9.0 percent, 9.5 percent and 10 percent alchol respectively, using NSFG. Each lot was sparged with nitrogen gas to reduce oxygen levels to 0.5 ppm (determined using a YSI Model 57 dissolved oxygen meter) and sterile (0.45 um) filtered into previously sterilized and nitrogen gas sparged five-gallon carboys. One-liter volumes of each of the above-described lots were transferred to (duplicate) sterile mylar bags and inoculated with stationary phase Zygosaccharomyces at a level of 70 cells/L. Initial cell titer and viability was determined by visual examination (Neubauer counting slide and methylene blue dye reduction) as decribed in Zoecklin, et. al., (1995). Verification of visual counts was carried out by direct plating on Wort agar and examination of cultures after 7 days. Duplicate lots representing each alcohol level were then incubated for a 90-day period at 60, 70, 80, and 90°F. Bags were examined daily for signs of fermentative activity (detected as expansion).


RESULTS

I. Growth Studies: As can be seen in Tables I and II, the only lots that did not show biological activity were those held at 90°F. At lower temperature, refermentation followed expected time and temperature relationships. It has been reported that osmophilic/osmoduric yeasts exhibit little heat resistance. This combined with our results suggest the potential for HTST thermal processing.
fig1

II. Recovery Studies: As of this report, more than 80 samples representing all stages of processing of grape juice concentrated have been screened for presence of Zygosaccharomyces. In that this is a continuing survey, detailed results will not be presented at this time. We can report that the majority of positive isolations occur in the proximity of concentrate storage tanks and associated transfer lines, pumps and filters.


ACKNOWLEDGMENTS

The author wishes to thank the California Agricultural Technology Institute (CATI) for its support of this project.


AUTHOR'S NOTE

This interim report presents preliminary results of ongoing research and has not been subject to peer review.


REFERENCES

Barnett, J.A., Payne, R.W., and Yarrow, D. Yeasts: Characterization and Identification. Cambridge University Press. 1983.

Beech, F.W., and Davenport, R.R. 1983. New prospects and problems in the beverage industry. In: Food Microbiology: Advances and Prospects (T.A. Roberts and F.A. Skinner, Eds.). The Soc. for Appl. Bacteriology Symp. Series No. 11. London Academic Press. pp. 241-56.

Corry, J.E.L. 1978. Relationships of Water Activity to Fungal Growth. In: Food and Beverage Mycology. L.R. Beuchat, ed. Avi Publishing Co., Westport, Conn. pp. 45-83.

Kaplow, M. 1970. Commercial development of intermediate moisture foods. Food Technol. 24:889-93.

Muller, C.J. and Fugelsang, K.C. 1994. Microbiological Stabilization of Juice and Wine: Effect of Carbon Monoxide on Spoilage Yeasts. Proceedings of the Office International de la Vigne et du Vin. Paris, France. June, 4-8.

Peynaud, E., and Domerq, S. 1959. A review of microbiological problems in winemaking in France. Am. J. Enol. Vitic. 10:69-77.

Pitt, J.I. 1974. Resistance of some food spoilage yeasts to preservatives. Food Tech. Australia 26:238-41.

Rankine, B.C., and Pilone, D.A. 1973. Saccharomyces bailii, a resistant yeast causing serious spoilage of bottled table wine. Am. J. Enol. Vitic. 24(2):55-58.

Thomas, D.S. 1981. Microbial spoilage in wine. Long Ashton Res. Station Report.

Thomas, S., and Davenport, R.R. 1985. Zygosaccharomyces bailii, a profile of characteristics and spoilage activities. Food Microbiology 2:157-169.

Van Esch, F. 1992. Yeast in soft drinks and concentrated fruit juices. Brygmesteren - NR 4:9-20.

Zoeklein, B.W., Fugelsang, K.C., Gump, B.H., and Nury, F.S. 1995. Wine Analysis and Production. Chapman & Hall Publ. Co. New York, NY.

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Copyright © 1996. All rights reserved.
CALIFORNIA AGRICULTURAL TECHNOLOGY INSTITUTE - CATI
College of Agricultural Sciences and Technology
California State University, Fresno