Modeling Yeast Spoilage in Cold-Filled Ready-To-Drink Beverages with Saccharomyces cerevisiae, Zygosaccharomyces bailii, and Candida lipolytica

Mathematical models were developed to predict the probability of yeast spoilage of cold-filled ready-to-drink beverages as a function of beverage formulation. A Box-Behnken experimental design included five variables, each at three levels: pH (2.8, 3.3, and 3.8), titratable acidity (0.20, 0.40, and 0.60%), sugar content (8.0, 12.0, and 16.0 °Brix), sodium benzoate concentration (100, 225, and 350 ppm), and potassium sorbate concentration (100, 225, and 350 ppm). Duplicate samples were inoculated with a yeast cocktail (100 μl/50 ml) consisting of equal proportions of Saccharomyces cerevisiae, Zygosaccharomyces bailii, and Candida lipolytica (~5.0 × 10⁴ CFU/ml each). The inoculated samples were plated on malt extract agar after 0, 1, 2, 4, 6, and 8 weeks. Logistic regression was used to create the predictive models. The pH and sodium benzoate and potassium sorbate concentrations were found to be significant factors controlling the probability of yeast growth. Interaction terms for pH and each preservative were also significant in the predictive model. Neither the titratable acidity nor the sugar content of the model beverages was a significant predictor of yeast growth in the ranges tested.     

Aerobic sugar metabolism in the spoilage yeast Zygosaccharomyces bailii

Despite the importance of some Zygosaccharomyces species as agents causing spoilage of food, the carbon and energy metabolism of most of them is yet largely unknown. This is the case with Zygosaccharomyces bailii. In this study the occurrence of the Crabtree effect in the petite-negative yeast Z. bailii ATCC 36947 was investigated. In this yeast the aerobic ethanol production is strictly dependent on the carbon source utilised. In glucose-limited continuous cultures a very low level of ethanol was produced. In fructose-limited continuous cultures ethanol was produced at a higher level and its production increased with the dilution rate. As a consequence, on fructose the onset of respiro-fermentative metabolism caused a reduction in biomass yield. An immediate aerobic alcoholic fermentation in Z. bailii was observed during the transition from sugar limitation to sugar excess, both on glucose and on fructose. The analysis of some key enzymes of the fermentative metabolism showed a high level of acetyl-CoA synthetase in Z. bailii growing on fructose. At high dilution rates, the activities of glucose- and fructose-phosphorylating enzymes, as well as of pyruvate decarboxylase and alcohol dehydrogenase, were higher in cells during growth on fructose than on glucose.     

Reactions of Saccharomyces cerevisiae and Zygosaccharomyces bailii to sulphite

Sulphite inhibited growth of all four yeasts studied, Zygosaccharomyces bailii NCYC 563 being most sensitive and Saccharomyces cerevisiae NCYC 431 the least. Vertical Woolf-Eadie plots were obtained for initial velocities of 35S accumulation by all four yeasts suspended in high concentrations of sulphite. Equilibrium levels of 35S accumulation were reached somewhat faster with strains of S. cerevisiae than with those of Z. bailii. With all four yeasts, the greater the extent of 35S accumulation, the larger was the decline in internal pH value. Growth of S. cerevisiae TC8 and Z. bailii NCYC 563, but to a lesser extent of S. cerevisiae NCYC 431 and Z. bailii NCYC 1427, was inhibited when mid exponential-phase cultures were supplemented with 1.0 or 2.0 mM-sulphite, the decrease in growth being accompanied by a decline in ethanol production. Unless growth was completely inhibited, the sulphite-induced decline in growth was accompanied by production of acetaldehyde and additional glycerol.     

Oxygen requirements of the food spoilage yeast Zygosaccharomyces bailii in synthetic and complex media

Most yeast species can ferment sugars to ethanol, but only a few can grow in the complete absence of oxygen. Oxygen availability might, therefore, be a key parameter in spoilage of food caused by fermentative yeasts. In this study, the oxygen requirement and regulation of alcoholic fermentation were studied in batch cultures of the spoilage yeast Zygosaccharomyces bailii at a constant pH, pH 3.0. In aerobic, glucose-grown cultures, Z. bailii exhibited aerobic alcoholic fermentation similar to that of Saccharomyces cerevisiae and other Crabtree-positive yeasts. In anaerobic fermentor cultures grown on a synthetic medium supplemented with glucose, Tween 80, and ergosterol, S. cerevisiae exhibited rapid exponential growth. Growth of Z. bailii under these conditions was extremely slow and linear. These linear growth kinetics indicate that cell proliferation of Z. bailii in the anaerobic fermentors was limited by a constant, low rate of oxygen leakage into the system. Similar results were obtained with the facultatively fermentative yeast Candida utilis. When the same experimental setup was used for anaerobic cultivation, in complex YPD medium, Z. bailii exhibited exponential growth and vigorous fermentation, indicating that a nutritional requirement for anaerobic growth was met by complex-medium components. Our results demonstrate that restriction of oxygen entry into foods and beverages, which are rich in nutrients, is not a promising strategy for preventing growth and gas formation by Z. bailii. In contrast to the growth of Z. bailii, anaerobic growth of S. cerevisiae on complex YPD medium was much slower than growth in synthetic medium, which probably reflected the superior tolerance of the former yeast to organic acids at low pH.     

Production of L-ascorbic acid by metabolically engineered Saccharomyces cerevisiae and Zygosaccharomyces bailii

Yeasts do not possess an endogenous biochemical pathway for the synthesis of vitamin C. However, incubated with l-galactose, L-galactono-1,4-lactone, or L-gulono-1,4-lactone intermediates from the plant or animal pathway leading to l-ascorbic acid, Saccharomyces cerevisiae and Zygosaccharomyces bailii cells accumulate the vitamin intracellularly. Overexpression of the S. cerevisiae enzymes d-arabinose dehydrogenase and D-arabinono-1,4-lactone oxidase enhances this ability significantly. In fact, the respective recombinant yeast strains even gain the capability to accumulate the vitamin in the culture medium. An even better result is obtainable by expression of the plant enzyme L-galactose dehydrogenase from Arabidopsis thaliana. Budding yeast cells overexpressing the endogenous D-arabinono-1,4-lactone oxidase as well as L-galactose dehydrogenase are capable of producing about 100 mg of L-ascorbic acid liter(-1), converting 40% (wt/vol) of the starting compound L-galactose.     

Acetic acid induces a programmed cell death process in the food spoilage yeast Zygosaccharomyces bailii

Here we show that 320-800 mM acetic acid induces in Zygosaccharomyces bailii a programmed cell death (PCD) process that is inhibited by cycloheximide, is accompanied by structural and biochemical alterations typical of apoptosis, and occurs in cells with preserved mitochondrial and plasma membrane integrity (as revealed by rhodamine 123 (Rh123) and propidium iodide (PI) staining, respectively). Mitochondrial ultrastructural changes, namely decrease of the cristae number, formation of myelinic bodies and swelling were also seen. Exposure to acetic acid above 800 mM resulted in killing by necrosis. The occurrence of an acetic acid-induced active cell death process in Z. bailii reinforces the concept of a physiological role of the PCD in the normal yeast life cycle.     

The spoilage yeast Zygosaccharomyces bailii forms mitotic spores: a screening method for haploidization

Zygosaccharomyces bailii ISA 1307 and the type strain of this spoilage yeast show a diploid DNA content. Together with a rather peculiar life cycle in which mitotic but no meiotic spores appear to be formed, the diploid DNA content explains the observed difficulties in obtaining auxotrophic mutants. Mitotic chromosome loss induced by benomyl and selection on canavanine media resulted in three haploid strains of Z. bailii. This new set of Z. bailii strains allows the easy isolation of recessive mutants and is suitable for further molecular genetic studies.     

Zygosaccharomyces kombuchaensis: the physiology of a new species related to the spoilage yeasts Zygosaccharomyces lentus and Zygosaccharomyces bailii

Zygosaccharomyces kombuchaensis was recently discovered in the 'tea fungus' used to make fermented tea. Z. kombuchaensis was shown by ribosomal DNA sequencing to be a novel species, and a close relative of Zygosaccharomyces lentus, from which it could not be distinguished by conventional physiological tests. Z. lentus was originally established as a new taxon by growth at 4 degrees C, sensitivity for heat and oxidative stress, and lack of growth in aerobic shaken culture at temperatures above 25 degrees C. Subsequent analysis of Z. kombuchaensis reveals that this species shares these unusual characteristics, confirming its close genealogical relationship to Z. lentus. Detailed physiological data from a number of Z. kombuchaensis and Z. lentus strains clearly demonstrate that these two species can in fact be distinguished from one another based on their differing resistance/sensitivity to the food preservatives benzoic acid and sorbic acid. The spoilage yeasts Zygosaccharomyces bailii and Z. lentus are resistant to both acetic acid and sorbic acid, whereas Z. kombuchaensis is resistant to acetic acid but sensitive to sorbic acid. This would indicate that Z. kombuchaensis strains lack the mechanism for resistance to sorbic acid, but possess the means of resistance to acetic acid. This observation would therefore suggest that these two resistance mechanisms are different, and that in all probability acetic and sorbic acids inhibit yeast growth by different modes of action. Z. kombuchaensis strains were also sensitive to benzoic acid, again suggesting inhibition dissimilar from that to acetic acid.     

A peculiar behaviour for cell death induced by weak carboxylic acids in the wine spoilage yeast Zygosaccharomyces bailii

In glucose-grown cells of Zygosaccharomyces bailii , ISA 1307 acetic acid and other carboxylic acids enhanced death. The effects were much lower than those described for Saccharomyces cerevisiae , being only detectable at higher acid concentrations. In Z. bailii, acetic acid and other weak acids also induced intracellular acidification, but this effect was less pronounced than that of death and no relationship was found with death enhancement. The results suggested that in Z. bailii , unlike S. cerevisiae , intracellular acidification induced by weak acids is less pronounced and appears not to have a significant role in death at the temperature range used.     

Differetiation of the spoilage yeast Zygosaccharomyces bailii from other Zygosaccharomyces species using 18S rDNA as target for a non-radioactive ligase detection reaction

A non-radioactive PCR coupled ligase detection reaction was developed to discriminate the food spoilage yeasts Zygosaccharomyces bailii and Z. bisporus from each other and from other members of the genus. A short region of the 18S rRNA gene was amplified from boiled cell lysates and polymerase chain reaction (PCR) products used as target in the template directed ligation of two adjacent oligonucleotides. Ligated products were captured using biotin-streptavidin chemistry and detected using digoxigenin
immuno-chemiluminescence. The ligase detection reaction was able to discriminate to the species level, targeting a single base deletion. The specificity of the reaction was assessed using seven species of the genus Zygosaccharomyces . Only strains of Z. bailii and Z. bisporus gave positive results with their respective primer sets. The lower detection limit of the strategy was 10pg (3 times 10⁷ targets) of amplified product.     

Individual cells of Saccharomyces cerevisiae and Zygosaccharomyces bailii exhibit different short-term intracellular pH responses to acetic acid

The effects of perfusion with 2.7 and 26 mM undissociated acetic acid in the absence or presence of glucose on short-term intracellular pH (pH(i)) changes in individual Saccharormyces cerevisiae and Zygosaccharomyces bailii cells were studied using fluorescence-ratio-imaging microscopy and a perfusion system. In the S. cerevisiae cells, perfusion with acetic acid induced strong short-term pH(i) responses, which were dependent on the undissociated acetic acid concentration and the presence of glucose in the perfusion solutions. In the Z. bailii cells, perfusion with acetic acid induced only very weak short-term pH(i) responses, which were neither dependent on the undissociated acetic acid concentration nor on the presence of glucose in the perfusion solutions. These results clearly show that Z. bailii is more resistant than S. cerevisiae to short-term pH(i) changes caused by acetic acid.     

Accumulation of sulphite by Saccharomyces cerevisiae and Zygosaccharomyces bailii as affected by phospholipid fatty-acyl unsaturation and chain length

Analyses were made of the fatty-acyl composition of phospholipids from each of two strains of Saccharomyces cerevisiae and Zygosaccharomyces bailii grown aerobically. Residues of C16:0, C16:1 and C18:1 predominated in phospholipids from strains of the first yeast, while phospholipids from Z. bailii contained mainly C16:0, C18:1 and C18:2 residues. S. cerevisiae NCYC 431, grown anaerobically in media supplemented with ergosterol and C14:1, C16:1, C18:1, C18:2, C18:3 or C20:1 fatty acids, contained phospholipids enriched with residues of the exogenously provided acid, to a greater extent with shorter chain than longer chain acids. A plot of the permeability coefficient for sulphite, derived from Woolf-Eadie plots, against the degree of unsaturation in phospholipids (expressed as delta mol-1 value) showed that the coefficient was greater the lower the degree of unsaturation in the phospholipids. A plot of the permeability coefficient against values for the mean fatty-acyl chain length divided by the delta mol-1 value, which is an approximation of the cross-section surface area of a phospholipid molecule, showed that the permeability coefficient tended to increase the greater the surface-area value.     

Glucose respiration and fermentation in Zygosaccharomyces bailii and Saccharomyces cerevisiae express different sensitivity patterns to ethanol and acetic acid

In the yeast Zygosaccharomyces bailii ISA 1307, respiration and fermentation ofglucose were exponentially inhibited by ethanol, both processes displaying similar sensitivity tothe alcohol. Moreover, the degree of inhibition on fermentation was of the same magnitude as thatreported for Saccharomyces cerevisiae. Acetic acid also inhibited these two metabolicprocesses in Z. bailii , with the kinetics of inhibition again being exponential. However,inhibition of fermentation was much less pronounced than in S. cerevisiae. The valuesestimated with Z. bailii for the minimum inhibitory concentration of acetic acid rangedfrom 100 to 240 mmol l⁻¹ total acetic acid compared with values of near zeroreported for S. cerevisiae. The inhibitory effects of acetic acid on Z. bailii were notsignificantly potentiated by ethanol.     

Modelling bacterial spoilage in cold-filled ready to drink beverages by Acinetobacter calcoaceticus and Gluconobacter oxydans

AIMS: Characterization of a bacterial isolate (strain MAE2) from intertidal beach sediment capable of degrading linear and branched alkanes. METHODS AND RESULTS: A Gram-positive, aerobic, heterotrophic bacterium (strain MAE2), that was capable of extensive degradation of alkanes in crude oil but had a limited capacity for the utilization of other organic compounds, was isolated from intertidal beach sediment. MAE2 had an obligate requirement for NaCl but could not tolerate high salt concentrations. It was capable of degrading branched and n-alkanes in crude oil from C11 to C33, but was unable to degrade aromatic hydrocarbons. Comparative 16S rRNA sequence analysis placed the isolate with members of the genus Planococcus. That finding was corroborated by chemotaxonomic and physiological data. The fatty acid composition of strain MAE2 was very similar to the type species of the genus Planococcus, P. citreus (NCIMB 1493T) and P. kocurii (NCIMB 629T), and was dominated by branched acids, mainly a15:0. However, the 16S rRNA of strain MAE2 had less than 97% sequence identity with the type strains of P. citreus (NCIMB 1439T), P. kocurii (NCIMB 629T) and two Planococcus spp. (strain MB6-16 and strain ICO24) isolated from Antarctic sea ice. This indicated that strain MAE2 represented a separate species from these planococci. Morphologically, the isolate resembled P. okeanokoites (NCIMB 561T) and P. mcmeekinii S23F2 (ATCC 700539T). The cellular fatty acid composition of P. okeanokoites and P. mcmeekinii was considerably different from strain MAE2, and the mol % G + C content of P. mcmeekinii was far lower than that of MAE2. CONCLUSION: On the basis of phenotypic and genotypic data, it is proposed that strain MAE2 is a new species of Planococcus, Planococcus alkanoclasticus sp. nov., for which the type strain is P. alkanoclasticus MAE2 (NCIMB 13489T). SIGNIFICANCE AND IMPACT OF THE STUDY: Planococcus species are abundant members of the bacterial community in a variety of marine environments, including some in sensitive Antarctic ecosystems. The occurrence of hydrocarbon-degrading Planococcus spp. is potentially of importance in controlling the impact of hydrocarbon contamination in sensitive marine environments.     

The ZbYME2 gene from the food spoilage yeast Zygosaccharomyces bailii confers not only YME2 functions in Saccharomyces cerevisiae, but also the capacity for catabolism of sorbate and benzoate, two major weak organic acid preservatives

A factor influencing resistances of food spoilage microbes to sorbate and benzoate is whether these organisms are able to catalyse the degradation of these preservative compounds. Several fungi metabolize benzoic acid by the beta-ketoadipate pathway, involving the hydroxylation of benzoate to 4-hydroxybenzoate. Saccharomyces cerevisiae is unable to use benzoate as a sole carbon source, apparently through the lack of benzoate-4-hydroxylase activity. However a single gene from the food spoilage yeast Zygosaccharomyces bailii, heterologously expressed in S. cerevisiae cells, can enable growth of the latter on benzoate, sorbate and phenylalanine. Although this ZbYME2 gene is essential for benzoate utilization by Z. bailii, its ZbYme2p product has little homology to other fungal benzoate-4-hydroxylases studied to date, all of which appear to be microsomal cytochrome P450s. Instead, ZbYme2p has strong similarity to the matrix domain of the S. cerevisiae mitochondrial protein Yme2p/Rna12p/Prp12p and, when expressed as a functional fusion to green fluorescent protein in S. cerevisiae growing on benzoate, is largely localized to mitochondria. The phenotypes associated with loss of the native Yme2p from S. cerevisiae, mostly apparent in yme1,yme2 cells, may relate to increased detrimental effects of endogenous oxidative stress. Heterologous expression of ZbYME2 complements these phenotypes, yet it also confers a potential for weak acid preservative catabolism that the native S. cerevisiae Yme2p is unable to provide. Benzoate utilization by S. cerevisiae expressing ZbYME2 requires a functional mitochondrial respiratory chain, but not the native Yme1p and Yme2p of the mitochondrion.     

Raman Spectroscopy and Chemometrics for Identification and Strain Discrimination of the Wine Spoilage Yeasts Saccharomyces cerevisiae,Zygosaccharomyces bailii,and Brettanomyces bruxellensis

The yeasts Zygosaccharomyces bailii, Dekkera bruxellensis (anamorph, Brettanomyces bruxellensis), and Saccharomyces cerevisiae are the major spoilage agents of finished wine. A novel method using Raman spectroscopy in combination with a chemometric classification tool has been developed for the identification of these yeast species and for strain discrimination of these yeasts. Raman spectra were collected for six strains of each of the yeasts Z. bailii, B. bruxellensis, and S. cerevisiae. The yeasts were classified with high sensitivity at the species level: 93.8% for Z. bailii, 92.3% for B. bruxellensis, and 98.6% for S. cerevisiae. Furthermore, we have demonstrated that it is possible to discriminate between strains of these species. These yeasts were classified at the strain level with an overall accuracy of 81.8%.     

A new strategy for inhibition of the spoilage yeasts Saccharomyces cerevisiae and Zygosaccharomyces bailii based on combination of a membrane-active peptide with an oligosaccharide that leads to an impaired glycosylphosphatidylinositol (GPI)-dependent yeast wall protein layer

Glycosylphosphatidylinositol (GPI)-dependent cell wall proteins in yeast are connected to the beta-1,3-glucan network via a beta-1,6-glucan moiety. Addition of gentiobiose or beta-1,6-glucan oligomers to growing cells affected the construction of a normal layer of GPI-dependent cell wall proteins at the outer rim of the Saccharomyces cerevisiae cell wall. Treated S. cerevisiae cells secreted significant amounts of cell wall protein 2, were much more sensitive to the lytic action of zymolyase 20T and displayed a marked increase in sensitivity to the small amphipathic antimicrobial peptide MB-21. Similar results in terms of sensitization of yeast cells to the antimicrobial peptide were obtained with the notorious food spoilage yeast Zygosaccharomyces bailii. Our results indicate that treating cells with a membrane-perturbing compound together with compounds that lead to an impaired construction of a normal GPI-dependent yeast wall protein layer represents an effective strategy to prevent the growth of major food spoilage yeasts.     

Construction of a genomic library of the food spoilage yeast Zygosaccharomyces bailii and isolation of the beta-isopropylmalate dehydrogenase gene (ZbLEU2)

A genomic library of the yeast Zygosaccharomyces bailii ISA 1307 was constructed in pRS316, a shuttle vector for Saccharomyces cerevisiae and Escherichia coli. The library has an average insert size of 6 kb and covers the genome more than 20 times assuming a genome size similar to that of S. cerevisiae. This new tool has been successfully used, by us and others, to isolate Z. bailii genes. One example is the beta-isopropylmalate dehydrogenase gene (ZbLEU2) of Z. bailii, which was cloned by complementation of a leu2 mutation in S. cerevisiae. An open reading frame encoding a protein with a molecular mass of 38.7 kDa was found. The nucleotide sequence of ZbLEU2 and the deduced amino acid sequence showed a significant degree of identity to those of beta-isopropylmalate dehydrogenases from several other yeast species. The sequence of ZbLEU2 has been deposited in the EMBL data library under accession number AJ292544.     

Influence of ethanol and temperature on the cellular fatty acid composition of Zygosaccharomyces bailii spoilage yeasts

Changes in the fatty acid profile of Zygosaccharomyces bailii strains, isolated from different sources, after growth at increasing concentrations of ethanol and/or decreasing temperatures were determined. Differences in fatty acid composition between Zygosaccharomyces bailii strains at standard conditions (25°C, 0% initial ethanol) were observed and could be related to ethanol tolerance. Zygosaccharomyces bailii strain isolated from wine showed the highest ethanol tolerance in relation to growth rate. Surprisingly, an increase in ethanol concentration or a decrease in growth temperature caused a decrease in the degree of unsaturation of total cellular fatty acids. On the other hand, the mean chain length increased (high ethanol concentration) or decreased (low temperature) depending on the stress factor. When both stress situations (high ethanol concentration and low temperature) were present at the same time, the degree of unsaturation remained approximately constant. With decreasing temperatures, the C16/C18 ratio increased in studies of initial ethanol content below 5%, and above 5% ethanol, decreased.