Zygosaccharomyces bailii is a species in the genus Zygosaccharomyces. It was initially described as Saccharomyces bailii by Lindner in 1895, but in 1983 it was reclassified as Zygosaccharomyces bailii in the work by Barnett et al.
Spoilage resulting from growth of the yeast Zygosaccharomyces is widespread, which has caused significant economic losses to the food industry. Within this genus, Z. bailii is one of the most troublesome species due to its exceptional tolerance to various stressful conditions. A wide range of acidic and/or high-sugar products such as fruit concentrates, wine, soft drinks, syrups, ketchup, mayonnaise, pickles, salad dressing, etc., are normally considered to be shelf-stable, i.e. they readily inactivate a broad range of food-borne microorganisms. However, these products are still susceptible to spoilage by Z. bailii.
Morphology and modes of reproduction Zygosaccharomyces bailii vegetative cells are usually ellipsoid, non-motile and reproduced asexually by multilateral budding, i.e. the buds can arise from various sites on the cells.Sutton, B.C., 1999. Overview of classification of the fungi. In: Robinson, R.K., Batt, C.A., Patel, P.D. (Eds.), Encyclopedia of Food Microbiology, vol. 2. Academic Press, London, pp. 860-871. During the budding process, a parent cell produces a bud on its outer surface. As the bud elongates, the parent cell's nucleus divides and one nucleus migrates into the bud. Cell wall material is filled in the gap between the bud and the parent cell; eventually the bud is separated to form a daughter cell of unequal size.Ketchum, P.A., 1988. Microbiology: Concepts and applications. John Wiley and Sons Inc., New York, pp. 379-400.Tortora, G.J., Funke, B.R., Case, C.L., 1992. Microbiology - An introduction, 4th ed. The Benjamin/Cummings Publishing Company, Redwood, pp. 296-331. Z. bailii cell size varies within a range of (3.5 - 6.5) x (4.5 - 11.5) μm and the cells exist singly or in pair, rarely in short chain.Pitt, J.I., Hocking, A.D., 1997. Fungi and food spoilage, 2nd ed. Blackie Academic and Professional, Cambridge. It has been observed that the doubling time of this yeast is approximately 3 hours at 23 °C in yeast nitrogen base broth containing 20% (w/v) fructose (pH 4.0). In more stressful conditions, this generation time is significantly extended.Cole, M.B., Keenan, M.H.J., 1987. A quantitative method for predicting shelf life of soft drinks using a model system. Journal of Industrial Microbiology 2, 59-62.
Besides the asexual reproduction mode, under certain conditions (e.g. nutritional stress) Z. bailii produces sexual spores (ascospores) in a sac called ascus (plural: asci). Normally, each ascus contains one to four ascospores, which are generally smooth, thin-walled, spherical or ellipsoidal.Mossel, D.A.A., Corry, J.E.L., Struijk, C.B., Baird, R.M., 1995. Essentials of the microbiology of foods - A textbook for advanced studies. John Wiley & Sons, Chichester, pp. 44-47. It should be mentioned that the ascospores are rarely observed as it is difficult and may take a long time to induce their formation; besides many yeast strains lose the ability to produce ascospores on repeated sub-cultures in the laboratory. On various nutrient agars, Z. bailii colonies are smooth, round, convex and white to cream coloured, with a diameter of 2 – 3 mm at 3 – 7 days. As the morphology properties of Zygosaccharomyces are identical to other yeast genera such as Saccharomyces, Candida and Pichia, it is impossible to differentiate Zygosaccharomyces from other yeasts or individual species within the genus based on macroscopic and microscopic morphology observations. Therefore, the yeast identification to species level is more dependent on physiological and genetic characteristics than on morphological criteria.
Culture conditions In general, any glucose-containing medium is suitable for the culture and counting of yeasts, e.g. Sabouraud medium, malt extract agar (MEA), tryptone glucose yeast extract agar (TGY), yeast glucose chloramphenicol agar (YGC).Bouix, M. and Leveau, J.Y., 1995. The yeasts. In: Bourgeois, C.M. and Leveau, J.Y. (Eds), Microbiological control for Foods and Agricultural products. VCH Publishers, New York, pp. 249-275. For the detection of acid-resistant yeasts like Z. bailii, acidified media are recommended, such as MEA or TGY with 0.5% (v/v) acetic acid added.Deak, T., 2003. Detection, enumeration and isolation of yeasts. In: Boekhout, T. and Robert, V. (Eds), Yeasts in food - Beneficial and detrimental aspects. Woodhead Publishing Ltd. and CRC Press, Hamburg, pp. 39-67. Plating with agar media is often used for counting of yeasts, with surface spreading technique is preferable to pour plate method because the former technique gives a better recovery of cells with lower dilution errors.Seiler, D.A.L., 1992. Report on a collaborative study on the effect of presoaking and mixing time on the recovery of fungi from foods. In: Samson, R.A., Hocking, A.D., Pitt, J.I., King, A.D. (Eds.), Modern methods in food mycology. Elsevier, Amsterdam, pp. 79-88. The common incubation conditions are aerobic atmosphere, temperature 25 °C for a period of 5 days. Nevertheless, a higher incubation temperature (30 °C) and shorter incubation time (3 days) can be applied for Z. bailii, as the yeast grows faster at this elevated temperature.
Physiological properties Among the Zygosaccharomyces spoilage species, Z. bailii possesses the most pronounced and diversified resistance characteristics, enabling it to survive and proliferate in very stressful conditions. It appears that Z. bailii prefers ecological environments characterized by high osmotic conditions. The most frequently described natural habitats are dried or fermented fruits, tree exudates (in vineyards and orchards), and at various stages of sugar refining and syrup production. Besides, it is seldom to encounter Z. bailii as a major spoilage agent in unprocessed foods; usually the yeast only attains importance in processed products when the competition with bacteria and moulds is reduced by intrinsic factors such as pH, water activity (aw), preservatives, etc.Dennis, C., Buhagiar, R.W.M., 1980. Yeast spoilage of fresh and processed fruits and vegetables. In: Skinner, F.A., Passmore, S.M., Davenport, R.R. (Eds.), Biology and activities of yeasts. The Society for Applied Bacteriology Symposium, series No. 9, Academic Press, London, pp. 123-133.
Resistance characteristics An outstanding feature of Z. bailii is its exceptional resistance to weak acid preservatives commonly used in foods and beverages, such as acetic, lactic, propionic, benzoic, sorbic acids and sulfur dioxide. In addition, it is reported that the yeast is able to tolerate high ethanol concentrations (≥ 15% (v/v)). The ranges of pH and aw for growth are wide, 2.0 - 7.0 and 0.80 - 0.99, respectively. Besides being preservative resistant, other features that contribute to the spoilage capacity of Z. bailii are: (i) its ability to vigorously ferment hexose sugars (e.g. glucose and fructose), (ii) ability to cause spoilage from an extremely low inoculum (e.g. one viable cell per package of any size), (iii) moderate osmotolerance (in comparison to Zygosaccharomyces rouxii). Therefore, foods at particular risk to spoilage by this yeast usually have low pH (2.5 to 5.0), low aw and contain sufficient amounts of fermentable sugars.
The extreme acid resistance of Z. bailii has been reported by many authors. On several occasions, growth of the yeast has been observed in fruit-based alcohols (pH 2.8 - 3.0, 40 - 45% (w/v) sucrose) preserved with 0.08% (w/v) benzoic acid, and in beverages (pH 3.2) containing either 0.06% (w/v) sorbic acid, 0.07% (w/v) benzoic acid, or 2% (w/v) acetic acid.Berry, J.M., 1979. Yeast problems in the food and beverage industry. In: Rhodes, M.E. (Ed), Food mycology. G.K. Hall and Co., Boston, pp. 82-90.Pitt, J.E., Richardson, K.C., 1973. Spoilage by preservative-resistant yeasts. CSIRO Food Research Quart 33, 80-85. Notably, individual cells in any Z. bailii population differ considerably in their resistance to sorbic acid, with a small fraction able to grow in preservative levels double that of the average population.Steels, H., James, S.A., Roberts, I.N., Stratford, M., 2000. Sorbic acid resistance: the inoculum effect. Yeast 16, 1173-1183. In some types of food, the yeast is even able to grow in the presence of benzoic and sorbic acids at concentrations higher than those legally permitted and at pH values below the pKa of the acids. For example, according to the European Union (EU) legislation, sorbic acid is limited to 0.03% (w/v) in soft drinks (pH 2.5 - 3.2);EU, 1995. European Parliament and Council Directive 95/2/EC of 20 February 1995 on food additives other than colours and sweeteners. however Z. bailii can grow in soft drinks containing 0.05% (w/v) of this acid (pKa 4.8).Neves, L., Pampulha, M.E., Loureiro-Dias, M.C., 1994. Resistance of food spoilage yeasts to sorbic acids. Letters in Applied Microbiology 19, 8-11. Particularly, there is strong evidence that the resistance of Z. bailii is stimulated by the presence of multiple preservatives. Hence, the yeast can survive and defeat synergistic preservative combinations that normally provide microbiological stability to processed foods. It has been observed that the cellular acetic acid uptake was inhibited when sorbic or benzoic acid was incorporated into the culture medium. Similarly, ethanol levels up to 10% (v/v) did not adversely influence sorbic and benzoic acid resistance of the yeast at pH 4.0 - 5.0. Moreover, Sousa et al. (1996) Sousa, M.J., Miranda, L., Corte-Real, M., Leao, C., 1996. Transport of acetic acid in Zygosaccharomyces bailii: Effects of ethanol and their implications on the resistance of the yeast to acidic environments. Applied and Environmental Microbiology 62, 3152-3157. have proved that in Z. bailii, ethanol plays a protective role against the negative effect of acetic acid by inhibiting the transport and accumulation of this acid intracellularly.
Like other microorganisms, Z. bailii has the ability to adapt to sub-inhibitory levels of a preservative, which enables the yeast to survive and grow in much higher concentrations of the preservative than before adaptation. In addition, it seems that Z. bailii resistance to acetic, benzoic and propionic acid is strongly correlated, as the cells which were adapted to benzoic acid also showed enhanced tolerances to the other preservatives.Warth, A.D., 1989. Relationships between the resistance of yeasts to acetic, propanoic and benzoic acids and to methyl paraben and pH. International Journal of Food Microbiology 8, 343-349.