Vladimir Jiranek

Lactic Acid Bacteria as a Potential Source of Enzymes for Use in Vinification

Two key groups of organisms are involved in the production of red, white, and sparkling wine. The yeasts, typically strains of Saccharomyces cerevisiae , carry out the primary or alcoholic fermentation, in which sugars are converted to ethanol and CO2. Lactic acid bacteria (LAB), especially

Screening of Lactobacillus spp. and Pediococcus spp. for glycosidase activities that are important in oenology.

Aims:  To assess glycosidase activities from a range of Lactobacillus and Pediococcus species and characterize these activities under conditions pertinent to the wine industry.

High power ultrasonics as a novel tool offering new opportunities for managing wine microbiology

Industrial scale food and beverage processes that utilize microorganisms are typically faced with issues related to the exclusion, suppression or elimination of spoilage organisms. Yet the use of traditional anti-microbial treatments such as heat, chemical biocides or sterile filtration may themselves be restricted by regulations or else be undesirable due to their adverse sensory impacts on the product. High power ultrasound (HPU) is a technology whose application has been evaluated if not exploited in several food and beverage processes but has yet to be introduced into the wine industry. This review examines the research findings from related industries and highlights possible applications and likely benefits of the use of HPU in winemaking.

Potential Predictors of Timely Completion among Dissertation Research Students at an Australian Faculty of Sciences

Successful and timely completion by Dissertation Research (DR) students (i.e. research Masters and PhD) is increasingly important for students, supervisors, the university, and funding bodies alike. Previous studies highlight the importance of contributing factors, which can be classified according to their relating to the student, the supervisory arrangement, or the research infrastructure. However, the relative importance of specific factors for individual students can vary markedly across studies. In recognition of this fact, a survey of DR student completion rates and candidature duration within an Australian Faculty of Sciences (i.e. the author’s home institution and broad field of research activity) was conducted. Available information was extracted for a 10-year window (1996-2006) from faculty-level and university-level databases and included data relating to demographics, field of study, attendance mode, scholarships held, and duration of candidature for up to 1688 students. The results suggest an attrition rate of 33% with shorter completion times being seen within the School of Chemistry & Physics and among male and/or international students with scholarships. Possible predictors of HRD student success are considered, thereby beginning to provide a framework within which to develop practices to increase retention and completion rates in other areas of the faculty.

Cloning and Characterization of an Intracellular Esterase from the Wine-Associated Lactic Acid Bacterium Oenococcus oeni

ABSTRACT We report the cloning and characterization of EstB28, the first esterase to be so characterized from the wine-associated lactic acid bacterium, Oenococcus oeni. The published sequence for O. oeni strain PSU-1 was used to identify putative esterase genes and design PCR primers in order to amplify the corresponding region from strain Ooeni28, an isolate intended for inoculation of wines. In this way a 912-bp open reading frame (ORF) encoding a putative esterase of 34.5 kDa was obtained. The amino acid sequence indicated that EstB28 is a member of family IV of lipolytic enzymes and contains the GDSAG motif common to other lactic acid bacteria. This ORF was cloned into Escherichia coli using an appropriate expression system, and the recombinant esterase was purified. Characterization of EstB28 revealed that the optimum temperature, pH, and ethanol concentration were 40°C, pH 5.0, and 28% (vol/vol), respectively. EstB28 also retained marked activity under conditions relevant to winemaking (10 to 20°C, pH 3.5, 14% [vol/vol] ethanol). Kinetic constants were determined for EstB28 with p-nitrophenyl (pNP)-linked substrates ranging in chain length from C2 to C18. EstB28 exhibited greatest specificity for C2 to C4pNP-linked substrates.

Application of the reuseable, KanMX selectable marker to industrial yeast: construction and evaluation of heterothallic wine strains of Saccharomyces cerevisiae, possessing minimal foreign DNA sequences

The characterisation of wine yeasts and the complex metabolic processes influencing wine fermentation and the quality of wine might best be achieved by exploiting the standard classical and recombinant genetic techniques which have been successfully used with laboratory strains. However, application of these techniques to industrial strains has been restricted because such strains are typically prototrophic and often polyploid. To overcome this problem, we have identified commercial wine strains with good mating and sporulation properties from which heterothallic derivatives were constructed by disruption of the HO gene. Consequently, these haploids are amenable to genetic analysis, whilst retaining desirable wine-making properties. The approach used was an adaptation of a previously published gene disruption procedure for laboratory yeast and is based on the acquisition of geneticin resistance from a removable KanMX marker. The present work is the first report of the application of a construct of this type to the disruption of the HO gene in wine yeasts that are in common commercial use. Most of the 4.9-kb disruption construct was successfully removed from the genome of the haploid derivative strains by loop-out of the KanMX marker through meiotic recombination. Sequencing of the HO region confirmed the reduction of foreign sequences to a 582-bp fragment comprised largely of a single direct repeat at the target gene. The removal of the active foreign gene (conferring antibiotic resistance) allows the application of other constructs based on the KanMX module without the need to resort to other selectable marker systems. Laboratory-scale fermentation trials typically showed minimal differences between the HO disruptants and the parental wine strains in terms of fermentation kinetics and formation of key metabolites.

Survey of enzyme activity responsible for phenolic off-flavour production by Dekkera and Brettanomyces yeast

Volatile phenols are produced by Dekkera yeasts and are of organoleptic importance in alcoholic beverages. The key compound in this respect is 4-ethylphenol, responsible for the medicinal and phenolic aromas in spoiled wines. The microbial synthesis of volatile phenols is thought to occur in two steps, beginning with naturally occurring hydroxycinnamic acids (HCAs). The enzyme phenolic acid decarboxylase (PAD) converts HCAs to vinyl derivatives, which are the substrates of a second enzyme, postulated to be a vinylphenol reductase (VPR), whose activity results in the formation of ethylphenols. Here, both steps of the pathway are investigated, using cell extracts from a number of Dekkera and Brettanomyces species. Dekkera species catabolise ferulic, caffeic and p-coumaric acids and possess inducible enzymes with similar pH and temperature optima. Brettanomyces does not decarboxylate HCAs but does metabolise vinylphenols. Dekkera species form ethylphenols but the VPR enzyme appears to be highly unstable in cell extracts. A partial protein sequence for PAD was determined from Dekkera anomala and may indicate the presence of a novel enzyme in this genus.

Pleiotropic effects of the opi1 regulatory mutation of yeast: its effects on growth and on phospholipid and inositol metabolism.

Key factors which impact on the biosynthesis and subsequent fate of the phospholipid precursor inositol were studied as a function of growth phase in the yeast Saccharomyces cerevisiae. Both wild-type and strains disrupted for the OPI1 gene, the principal negative regulator of the phospholipid biosynthetic genes, were examined. Overexpression of the INO1 gene and overproduction of both inositol and the major inositol-containing phospholipid, phosphatidylinositol, varied as a function of growth phase. In opi1 cells, INO1 expression was constitutive at a high level throughout growth, although the level of transcript was reduced at stationary phase when the cells were grown in defined medium. In the wild-type strain, INO1 expression was limited to a peak in the exponential phase of growth in cells grown in the absence of inositol. Interestingly, the pattern of OPI1 expression in the wild-type strain resembled that of its putative target, INO1. Intracellular inositol contents of the opi1 strain were higher than those of the wild-type strain, with peak levels occurring in the stationary phase. Membrane phosphatidylinositol content paralleled intracellular inositol content, with opi1 strains having a higher phosphatidylinositol content in stationary phase. The proportion of the predominant phospholipid, phosphatidylcholine, exhibited a profile that was the inverse of the phosphatidylinositol content: phosphatidylcholine content was lowest in opi1 cells in stationary phase. The opi1 mutation was also found to have effects beyond phospholipid biosynthesis. opi1 cells were smaller, and opi1 cultures achieved a cell density twice as high as comparable wild-type cultures. opi1 cells were also more salt tolerant than wild-type cells: they were partly resistant to shrinking, more rapidly resumed growth, and attained a higher culture density after upshift to medium supplemented with 8% NaCl.

Ethanol production and maximum cell growth are highly correlated with membrane lipid composition during fermentation as determined by lipidomic analysis of 22 Saccharomyces cerevisiae strains.

ABSTRACT Optimizing ethanol yield during fermentation is important for efficient production of fuel alcohol, as well as wine and other alcoholic beverages. However, increasing ethanol concentrations during fermentation can create problems that result in arrested or sluggish sugar-to-ethanol conversion. The fundamental cellular basis for these problem fermentations, however, is not well understood. Small-scale fermentations were performed in a synthetic grape must using 22 industrial Saccharomyces cerevisiae strains (primarily wine strains) with various degrees of ethanol tolerance to assess the correlation between lipid composition and fermentation kinetic parameters. Lipids were extracted at several fermentation time points representing different growth phases of the yeast to quantitatively analyze phospholipids and ergosterol utilizing atmospheric pressure ionization-mass spectrometry methods. Lipid profiling of individual fermentations indicated that yeast lipid class profiles do not shift dramatically in composition over the course of fermentation. Multivariate statistical analysis of the data was performed using partial least-squares linear regression modeling to correlate lipid composition data with fermentation kinetic data. The results indicate a strong correlation (R 2 = 0.91) between the overall lipid composition and the final ethanol concentration (wt/wt), an indicator of strain ethanol tolerance. One potential component of ethanol tolerance, the maximum yeast cell concentration, was also found to be a strong function of lipid composition (R 2 = 0.97). Specifically, strains unable to complete fermentation were associated with high phosphatidylinositol levels early in fermentation. Yeast strains that achieved the highest cell densities and ethanol concentrations were positively correlated with phosphatidylcholine species similar to those known to decrease the perturbing effects of ethanol in model membrane systems.

Inhibitory effect of hydroxycinnamic acids on Dekkera spp.

Simple phenolic components of wine, hydroxycinnamic acids (HCAs) are known to have antimicrobial properties. This study sought to determine the potential of ferulic acid as an antifungal agent for the control of Dekkera. Growth was inhibited by all HCAs examined in this study, with ferulic acid being the most potent at all concentrations. In the presence of ethanol, the inhibitory effects of ferulic acid were amplified. Scanning electron microscopy images reveal cellular damage upon exposure to ferulic acid. Thus, manipulation of ferulic acid concentrations could be of industrial significance for control of Dekkera and may be the basis for differences in susceptibility of wines to Dekkera spoilage.