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Hanseniaspora guilliermondii is a well-recognized producer of acetate esters associated with fruity and floral aromas. The molecular mechanisms underneath this production or the environmental factors modulating it remain unknown. Herein, we found that, unlike Saccharomyces cerevisiae, H. guilliermondii over-produces acetate esters and higher alcohols at low carbon-to-assimilable nitrogen (C:N) ratios, with the highest titers being obtained in the amino acid-enriched medium YPD. The evidences gathered support a model in which the strict preference of H. guilliermondii for amino acids as nitrogen sources results in a channeling of keto-acids obtained after transamination to higher alcohols and acetate esters. This higher production was accompanied by higher expression of the four HgAATs, genes, recently proposed to encode alcohol acetyl transferases. In silico analyses of these HgAat’s reveal that they harbor conserved AATs motifs, albeit radical substitutions were identified that might result in different kinetic properties. Close homologues of HgAat2, HgAat3, and HgAat4 were only found in members of Hanseniaspora genus and phylogenetic reconstruction shows that these constitute a distinct family of Aat’s. These results advance the exploration of H. guilliermondii as a bio-flavoring agent providing important insights to guide future strategies for strain engineering and media manipulation that can enhance production of aromatic volatiles.

This study aimed to explore communities and the ethanol-fermenting ability of yeasts in fresh coconut, palmyra, and nipa palm saps. From the 90 samples of coconut, palmyra, and nipa palm saps, 204 yeast isolates were isolated and identified as 15 species in the phylum Ascomycota and a species (one strain) in Basidiomycota. Saccharomyces cerevisiae, Hanseniaspora guilliermondii, and Lachancea thermotolerans were found in the saps of all three palm species. Candida tropicalis and Pichia kudriavzevii were obtained from the coconut and palmyra palm saps, Hanseniaspora vineae, Lachancea fermentati, and Pichia manshurica were present in the coconut and nipa palm saps, whereas Torulaspora delbrueckii was found in the palmyra and nipa palm saps. The species with the highest occurrence in the saps of coconut, palmyra, and nipa palms was S. cerevisiae with 76.67%, 86.70%, and 100% frequency of occurrence, respectively. Using principal coordinates analysis for ordination, no marked difference was observed in the yeast communities from the saps of the three palm species. A total of 199 isolates were found to possess ethanol-fermentation ability when cultivated using shake flask in 160 g/L of glucose medium at 28°C for 48 h. Lachancea fermentati YSP-383, isolated from nipa palm sap, produced the highest amount of ethanol (76.74 g/L). Twenty-six isolates of Candida sanyaensis (1), C. tropicalis (1), H. guilliermondii (7), L. fermentati (8), L. thermotolerans (1), Pichia kudriavzevii (2), and S. cerevisiae (6) produced high amounts of ethanol ranging from 69.57 to 76.74 g/L. The result demonstrated that yeasts in the palm saps could play roles in the natural fermentation of palm saps.

Wine production in Cyprus has strong cultural ties with the island’s tradition, influencing local and foreign consumers’ preferences and contributing significantly to Cyprus’ economy. A key contributor to wine quality and sensorial characteristics development is the microbiota that colonizes grapes and performs alcoholic fermentation. Still, the microbial patterns of wines produced in different geographic regions (terroir) in Cyprus remain unknown. The present study investigated the microbial diversity of five terroirs in Cyprus, two from the PGI Lemesos region [Kyperounta (PDO Pitsilia) and Koilani (PDO Krasochoria)], and three from the PGI Pafos region [Kathikas (PDO Laona Akamas), Panayia, and Statos (PDO Panayia)], of two grape varieties, Xynisteri and Maratheftiko, using high-throughput amplicon sequencing. Through a longitudinal analysis, we examined the evolution of the bacterial and fungal diversity during spontaneous alcoholic fermentation. Both varieties were characterized by a progressive reduction in their fungal alpha diversity (Shannon index) throughout the process of fermentation. Additionally, the study revealed a distinct separation among different terroirs in total fungal community composition (beta-diversity) for the variety Xynisteri. Also, Kyperounta terroir had a distinct total fungal beta-diversity from the other terroirs for Maratheftiko. Similarly, a significant distinction was demonstrated in total bacterial diversity between the PGI Lemesos region and the PGI Pafos terroirs for grape juice of the variety Xynisteri. Pre-fermentation, the fungal diversity for Xynisteri and Maratheftiko was dominated by the genera Hanseniaspora, Aureobasidium, Erysiphe, Aspergillus, Stemphylium, Penicillium, Alternaria, Cladosporium, and Mycosphaerella. During and post-fermentation, the species Hanseniaspora nectarophila, Saccharomyces cerevisiae, Hanseniaspora guilliermondii, and Aureobasidium pullulans, became the predominant in most must samples. Regarding the bacterial diversity, Lactobacillus and Streptococcus were the predominant genera for both grape varieties in all stages of fermentation. During fermentation, an increase was observed in the relative abundance of some bacteria, such as Acetobacter, Gluconobacter, and Oenococcus oeni. Finally, the study revealed microbial biomarkers with statistically significant higher relative representation, associated with each geographic region and each grape variety, during the different stages of fermentation. The present study’s findings provide an additional linkage between the grape microbial community and the wine terroir.

The phloem sap tapped from unopened inflorescence (spadix) of coconut palm using a novel collecting device, “coco-sap chiller,” has been branded Kalparasa® (henceforth as Kalparasa in the text) to distinguish its properties not found in sap harvested by traditional methods. To know its hitherto unidentified microbiome profile, we employed high-throughput sequencing to uncover the bacteriome and mycobiome in fresh and 12-h fermented samples. Fresh Kalparasa had a pH of 7.2, which dropped to 4.5 after 12 h, signifying fermentation of the sap. Diversity analysis indicated fresh Kalparasa having higher bacterial species than the fermented one. Contrary to this, fresh sap had lower fungal/yeast diversity than the fermented sample. Fresh Kalparasa had relatively higher abundance of probiotic-type Leuconostoc genus followed by equal proportions of Gluconobacter, Acetobacter, and Fructobacillus. The 12-h fermented Kalparasa showed a significant increase in Gluconobacter with a sharp decrease in Leuconostoc. Mycobiome data revealed fresh Kalparasa to be preponderant in Saccharomyces and Hanseniaspora genera of yeasts while the fermented sap had higher representation of Hanseniaspora and Cortinarius and lesser Saccharomyces. This suggested that the fermentation of Kalparasa was probably driven by symbiotic culture of bacteria and yeasts (SCOBY), particularly acetic acid bacteria and non-Saccharomyces yeasts. The bacteriome-function predictions highlighted the enrichment of glycerophospholipid, ABC transporters, purine, and pyrimidine metabolisms. Based on our findings, Kalparasa containing large population of Leuconostoc mesenteroides, Fructobacillus fructosus, Saccharomyces cerevisiae, and Hanseniaspora guilliermondii can be promoted as a healthy “unfermented” plant edible food containing live probiotic-type microbiome during its consumption.

Beer production is predominantly carried out by Saccharomyces species, such as S. cerevisiae and S. pastorianus. However, the introduction of non-Saccharomyces yeasts in the brewing process is now seen as a promising strategy to improve and differentiate the organoleptic profile of beer. In this study, 17 non-Saccharomyces strains of 12 distinct species were isolated and submitted to a preliminary sensory evaluation to determine their potential for beer bioflavouring. Hanseniaspora guilliermondii IST315 and H. opuntiae IST408 aroma profiles presented the highest acceptability and were described as having 'fruity' and 'toffee' notes, respectively. Their presence in mixed-culture fermentations with S. cerevisiae US-05 did not influence attenuation and ethanol concentration of beer but had a significant impact in its volatile composition. Notably, while both strains reduced the total amount of ethyl esters, H. guilliermondii IST315 greatly increased the concentration of acetate esters, especially when sequentially inoculated, leading to an 8.2-fold increase in phenylethyl acetate ('rose', 'honey' aroma) in the final beverage. These findings highlight the importance of non-Saccharomyces yeasts in shaping the aroma profile of beer and suggest a role for Hanseniaspora spp. in improving it.

In winemaking, non-Saccharomyces yeast species contribute important organoleptic complexity. Current interest focuses on abundant and dominant strains characteristically present in the early phase of spontaneous alcoholic fermentations. Non-Saccharomyces species are particularly relevant in Port wine production such that the fermentation is prematurely stopped, after the metabolism of only one half of the available sugar, through fortification with aguardente. This work aimed to isolate, identify and characterize non-Saccharomyces species present in spontaneously fermenting Port. To accomplish these goals, yeasts were isolated from a selection of frozen must samples (2012&ndash, 2016 harvests), using a pre-screening process choosing only the best candidates based on the organoleptic quality of the corresponding fortified wine. From five hundred non-Saccharomyces isolates, twelve species were identified. The three most abundant species, Hanseniaspora uvarum, Lachancea thermotolerans, and Metschnikowia pulcherrima, representing 89% of the isolates, exhibited particularly high diversity with high growth performance variability when exposed to typical stress conditions associated with common enological parameters. Less abundant species included Issatchenkia orientalis, Torulaspora delbrueckii, Hanseniaspora vineae, Hanseniaspora osmophila, Candida zemplinina, Rhodotorula mucilaginosa, Hanseniaspora guilliermondii, Issatchenkia occidentalis, and Zygosaccharomyces bisporus. This is the first study providing insights into the identification and characterization of non-Saccharomyces species responsible for spontaneous Port wine production.

Hanseniaspora, a predominant yeast genus of grape musts, includes sister species recently reported as fast evolving. The aim of this study was to investigate the genetic relationships between the four most closely related species, at the population level. A multi-locus sequence typing strategy based on five markers was applied on 107 strains, confirming the clear delineation of species H. uvarum, H. opuntiae, H. guilliermondii, and H. pseudoguilliermondii. Huge variations were observed in the level of intraspecific nucleotide diversity, and differences in heterozygosity between species indicate different life styles. No clear population structure was detected based on geographical or substrate origins. Instead, H. guilliermondii strains clustered into two distinct groups, which may reflect a recent step toward speciation. Interspecific hybrids were detected between H. opuntiae and H. pseudoguilliermondii. Their characterization using flow cytometry, karyotypes and genome sequencing showed different genome structures in different ploidy contexts: allodiploids, allotriploids, and allotetraploids. Subculturing of an allotriploid strain revealed chromosome loss equivalent to one chromosome set, followed by an auto-diploidization event, whereas another auto-diploidized tetraploid showed a segmental duplication. Altogether, these results suggest that Hanseniaspora genomes are not only fast evolving but also highly dynamic.

Hanseniaspora, a predominant yeast genus of grape musts, includes sister species recently reported as fast evolving. The aim of this study was to investigate the genetic relationships between the four most closely related species, at the population level. A multi-locus sequence typing strategy based on five markers was applied on 107 strains, confirming the clear delineation of species H. uvarum, H. opuntiae, H. guilliermondii, and H. pseudoguilliermondii. Huge variations were observed in the level of intraspecific nucleotide diversity, and differences in heterozygosity between species indicate different life styles. No clear population structure was detected based on geographical or substrate origins. Instead, H. guilliermondii strains clustered into two distinct groups, which may reflect a recent step toward speciation. Interspecific hybrids were detected between H. opuntiae and H. pseudoguilliermondii. Their characterization using flow cytometry, karyotypes and genome sequencing showed different genome structures in different ploidy contexts: allodiploids, allotriploids, and allotetraploids. Subculturing of an allotriploid strain revealed chromosome loss equivalent to one chromosome set, followed by an auto-diploidization event, whereas another auto-diploidized tetraploid showed a segmental duplication. Altogether, these results suggest that Hanseniaspora genomes are not only fast evolving but also highly dynamic.

Saccharomyces yeast species are currently the most important yeasts involved in industrial-scale food fermentations. However, there are hundreds of other yeast species poorly studied that are highly promising for flavour development, some of which have also been identified in traditional food fermentations. This work explores natural yeast biodiversity in terms of aroma formation, with a particular focus on aromas relevant for industrial fermentations such as wine and beer. Several non-Saccharomyces species produce important aroma compounds such as fusel alcohols derived from the Ehrlich pathway, acetate esters and ethyl esters in significantly higher quantities than the well-known Saccharomyces species. These species are Starmera caribaea, Hanseniaspora guilliermondii, Galactomyces geotrichum, Saccharomycopsis vini and Ambrosiozyma monospora. Certain species revealed a strain-dependent flavour profile while other species were very homogenous in their flavour profiles. Finally, characterization of a selected number of yeast species using valine or leucine as sole nitrogen sources indicates that the mechanisms of regulation of the expression of the Ehrlich pathway exist amongst non-conventional yeast species.

Saccharomyces cerevisiae produces antimicrobial peptides (AMPs) during alcoholic fermentation that are active against several wine-related yeasts (e.g. Hanseniaspora guilliermondii) and bacteria (e.g. Oenococcus oeni). In the present study, the physiological changes induced by those AMPs on sensitive H. guilliermondii cells were evaluated in terms of intracellular pH (pHi), membrane permeability and culturability. Membrane permeability was evaluated by staining cells with propidium iodide (PI), pHi was determined by a fluorescence ratio imaging microscopy (FRIM) technique and culturability by a classical plating method. Results showed that the average pHi of H. guilliermondii cells dropped from 6.5 (healthy cells) to 5.4 (damaged cells) after 20 min of exposure to inhibitory concentrations of AMPs, and after 24 h 77.0% of the cells completely lost their pH gradient (∆pH=pHi-pHext). After 24h of exposure to AMPs, PI-stained (dead) cells increased from 0% to 77.7% and the number of viable cells fell from 1×10(5) to 10 CFU/ml. This means that virtually all cells (99.99%) became unculturable but that a sub-population of 22.3% of the cells remained viable (as determined by PI staining). Besides, pHi results showed that after 24h, 23% of the AMP-treated cells were sub-lethally injured (with 0

This study aimed to enhance the bio-ethanol production from date molasses as a cheap and renewable resource by local species of non-Saccharomyces yeasts by optimization the production’s conditions. Hanseniaspora guilliermondii KKUY-0036 and H. uvarum KKUY-0078 were used based on their ability to ferment the date molasses efficiently. They were identified by the sequencing of D1/D2 domain of the 26S rRNA gene. Their identity was confirmed by comparing the obtained sequence with similar sequences allocated in the GenBank. To enhance the ethanol productivity, temperature, pH, fermentation period, molasses concentration and addition of some elements were optimized. Results revealed that the two yeast species exhibited their maximum productivity of ethanol at 30°C when they were grown on 20-25% of the date molasses after 96-120 h of incubation. The highest ethanol concentration was achieved in weak acidic medium (pH 4-6). Addition of zinc, magnesium and manganese induced the production of ethanol by the two yeasts and the optimum concentrations were 0.6, 0.2-0.3 and 0.03 g LG1, respectively. The study introduces both H. guilliermondii KKUY-0036 and H. uvarum KKUY-0078 as new ethanol-producers that ferment date molasses efficiently and will greatly reduce the biofuel production cost.

The increase of infections due to non-Candida albicans species made it very necessary to conduct adequate characterization to be able to identify the species of Candida isolated from traditional fermented foods. In this study, based on their hue on Candida Chromogenic Agar medium, a total of 136 yeast strains were isolated from tchapalo and bangui. Molecular identification based on PCR-RFLP of internal transcribed spacers of rDNA (ITS) and sequencing of the ITS and the D1/D2 regions allowed us to assign these isolates to seven species: Candida tropicalis, Candida inconspicua, Candida rugosa, Saccharomyces cerevisiae, Kluyveromyces marxianus, Hanseniaspora guilliermondii, Trichosporon asahii. With the respect to each beverage, six species were found among with four species are regarded as opportunistic pathogens. From these, C. tropicalis, C. inconspicua and K. marxianus were the most commonly encountered. The enzyme activities of the potential pathogens assessed using API ZYM system showed that almost strains had esterase, esterase lipase, valine and cystine arylamidase, alpha chymotrypsin, alkaline phosphatase and naphthol phosphohydrolase activities. The activity of α-glucosidase was found only in C. tropicalis and C. inconspicua strains isolated from tchapalo while β-glucosidase activity was found in all strains from tchapalo and only in C. inconspicua isolated from bangui.

International audience; Although differences in yeast biodiversity have often been found between vineyards subjected to organic protection or conventional protection, little is known about the effect of copper and sulfur fungicides (the only fungicides allowed in organic farming) on yeast populations. The sensitivity to copper and sulfur of 158 yeast isolates of seven different species (Aureobasidium pullulans, Hanseniaspora guilliermondii, Hanseniaspora uvarum, Metschnikowia sp., Pichia membranifaciens, Saccharomyces cerevisiae and Starmerella bacillaris) was evaluated. The species Aureobasidium pullulans and Starmerella bacillaris appeared to be more resistant to copper than the other species tested. The species Aureobasidium pullulans, Hanseniaspora guilliermondii and Metschnikowia sp. had the highest sulfur resistance. Thus, only isolates of the species Aureobasidium pullulans exhibited high resistance to both antifungal agents. These results may explain the lower diversity of yeasts present on berries and the strong dominance of the species Aureobasidium pullulans for vineyards protected by copper or sulfur-based fungicides compared to other vineyards reported by several studies.

Article History The aim of this study was to determine the intermediate chemical compounds during the fermentation of spoilage date juice (SDJ) by newly isolated yeast strain Hanseniaspora guilliermondii KKUY-0045. This yeast strain was identified based on sequence of the variable D1/D2 domain of the large subunit (26S) ribosomal DNA and comparing it with similar sequences in the Genbank database. Phylogenetic analysis using rDNA sequences indicated that strain KKUY-00045 shared one cluster with Hanseniaspora guilliermondii. In 7-L fermenetor, this strain produced 87.74 g/L of ethanol after 3 days of incubation. The chemical analysis of the cell free extract of the yeast strain grown on the SDJ by using GC-MS revealed the presence of some important chemical compound such as pregnane and nonacosane that could be used in the medical and pharmaceutical industries. The study recommends extensive studies to discover the chemicals associated with fermentation process and their industrial role.

Non- Saccharomyces yeasts include different species which comprise an ecologically and biochemically diverse group capable of altering fermentation dynamics and wine composition and flavour. In this study, single- and mixed-culture of Hanseniaspora guilliermondii and Saccharomyces cerevisiae were used to ferment natural grape-juice, under two nitrogen regimes. In single-culture the strain H. guilliermondii failed to complete total sugar breakdown even though the nitrogen available has not been a limiting factor of its growth or fermentative activity. In mixed-culture, that strain negatively interfered with the growth and fermentative performance of S. cerevisiae , resulting in lower fermentation rate and longer fermentation length, irrespective of the initial nitrogen concentration. The impact of co-inoculation on the volatile compounds profile was more evident in the wines obtained from DAP-supplemented musts, characterised by increased levels of ethyl and acetate esters, associated with fruity and floral character of wines. Moreover, the levels of fatty acids and sulphur compounds which are responsible for unpleasant odours that depreciate wine sensory quality were significantly lower. Accordingly, data obtained suggests that the strain H. guilliermondii has potential to be used as adjunct of S. cerevisiae in wine industry, although possible interactions with S. cerevisiae still need to be elucidated.

International audience; Different genera and/or species of yeasts present on grape berries, in musts and wines are widely described. Nevertheless, the community of non-Saccharomyces yeasts present in the cellar is still given little attention. Thus it is not known if the cellar is a real ecological niche for these yeasts or if it is merely a transient habitat for populations brought in by grape berries during the winemaking period. This study focused on three species of non-Saccharomyces yeasts commonly encountered during vinification: Starmerella bacillaris (synonymy with Candida zemplinina), Hanseniaspora guilliermondii and Hanseniaspora uvarum. More than 1200 isolates were identified at the strain level by FT-IR spectroscopy (207 different FTIR strain pattern). Only a small proportion of non-Saccharomyces yeasts present in musts came directly from grape berries for the three species studied. Some strains were found in the must in two consecutive years and some of them were also found in the cellar environment before the arrival of the harvest of second vintage. This study demonstrates for the first time the persistence of non-Saccharomyces yeast strains from year to year in the cellar. Sulfur dioxide can affect yeast populations in the must and therefore their persistence in the cellar environment.

In Georgia, one of the most ancient vine-growing environment, the homemade production of wine is still very popular in every rural family and spontaneous fermentation of must, without addition of chemical preservatives, is the norm. The present work investigated the yeast biodiversity in five Georgian areas (Guria, Imereti, Kakheti, Kartli, Ratcha-Lechkhumi) sampling grapes and wines from 22 different native cultivars, in 26 vineyards and 19 family cellars. One hundred and eighty-two isolates were ascribed to 15 different species by PCR-ITS and RFLP, and partial sequencing of D1/D2 domain 26S rDNA gene. Metschnikowia pulcherrima (F’ = 0.56, I’ = 0.32), Hanseniaspora guilliermondii (F’ = 0.49, I’ = 0.27), and Cryptococcus flavescens (F’ = 0.31, I’ = 0.11) were the dominant yeasts found on grapes, whereas Saccharomyces cerevisiae showed the highest prevalence into wine samples. Seventy four isolates with fermentative potential were screened for oenological traits such as ethanol production, resistance to SO2, and acetic acid, glycerol and H2S production. Three yeast strains (Kluyveromyces marxianus UMY207, S. cerevisiae UMY255, Torulaspora delbrueckii UMY196) were selected and separately inoculated in vinifications experiments at a Georgian cellar. Musts were prepared from healthy grapes of local varieties, Goruli Mtsvane (white berry cultivar) and Saperavi (black berry cultivar). Physical (°Brix) and microbial analyses (plate counts) were performed to monitor the fermentative process. The isolation of indigenous S. cerevisiae yeasts beyond the inoculated strains indicated that a co-presence occurred during the vinification tests. Results from quantitative GC-FID analysis of volatile compounds revealed that the highest amount of fermentation flavors, such as 4-ethoxy-4-oxobutanoic acid (monoethyl succinate), 2-methylpropan-1-ol, ethyl 2-hydroxypropanoate, and 2-phenylethanol, were significantly more produced in fermentation conducted in Saperavi variety inoculated with K. marxianus, whereas other aromatic compounds like 3-methylbutyl acetate, ethyl hexanoate and dihydrofuran-2(3H)-one (γ- butyrolactone) showed a higher content in Goruli Mtsvane variety samples fermented by S. cerevisiae. The selected yeast strains have proved to be promising for enhancing the flavor potential in low aromatic Georgian cultivars. This work intends to be a knowledge contribution for a precision oenology toward the strategic concept of “one grape variety-one yeast”.

The fermented cereal-based gruel togwa is used as weaning food for children in Tanzania. Togwa is rich in minerals but these are often not available for uptake in the human intestine due to natural inhibitors, such as phytate (IP(6)). The yeasts Pichia kudriavzevii TY13, Hanseniaspora guilliermondii TY14 and TY20, isolated from Tanzanian togwa, and selected for high phytase activity in complex yeast medium YPD, were now studied regarding their ability to degrade IP(6) in maize-based model togwa. A modified constitutively high-phytase producing Saccharomyces cerevisiae BY80 and commercial Aspergillus ficuum phytase were included for comparison. In addition, a strain of Lactobacillus plantarum was included in the model-togwa set-up. All yeasts in the study grew and reached final cell density 1.5-2 log units higher than the start value. S. cerevisiae BY80 degraded 85% of the IP(6) in 48 h; the same degradation level as with A. ficuum phytase (89%). Of the togwa-isolated yeasts, P. kudriavzevii TY13 and H. guilliermondii TY14 showed strong phytate degradation in the model-togwa; 95% or more of the initial IP(6) was degraded after 48 h. This corresponds to a remaining level of 0.4 and 0.3μmol IP(6)/g dw. Co-inoculation with L. plantarum did not increase IP(6) degradation. Moreover, fermentation with P. kudriavzevii TY13 yielded a successive increase in inorganic phosphate (P(i)), from 0.7 to 5.4 mM, suggesting a phytase production in TY13 which is fairly insensitive to P(i) repression. The study shows that phytate in a model togwa is available for yeast phytase enzymes, and addresses the importance of strain selection for effectively degrading the phytate. Certain yeasts originating from togwa seem to have developed a natural high phytase production, and P. kudriavzevii TY13 and H. guilliermondii TY14 seem particularly well adapted to phytate degradation in togwa, and is our choice for further studies and strain improvement.

Traditionally, it was assumed that non-Saccharomyces (NS) yeasts could only survive in the early stages of alcoholic fermentations. However, recent studies applying culture-independent methods have shown that NS populations persist throughout the fermentation process. The aim of the present work was to analyze and quantify Saccharomyces cerevisiae (Sc) and Hanseniaspora guilliermondii (Hg) populations during alcoholic fermentations by plating and culture-independent methods, such as fluorescence in situ hybridization (FISH) and quantitative PCR (QPCR). Species-specific FISH probes labeled with fluorescein (FITC) were used to directly hybridize Sc and Hg cells from single and mixed cultures that were enumerated by epifluorescence microscopy and flow cytometry. Static and agitated fermentations were performed in synthetic grape juice and cell density as well as sugar consumption and ethanol production were determined throughout fermentations. Cell density values obtained by FISH and QPCR revealed the presence of high populations (10⁷-10⁸ cells/ml) of Sc and Hg throughout fermentations. Plate counts of both species did not show significant differences with culture-independent results in pure cultures. However, during mixed fermentations Hg lost its culturability after 4-6 days, while Sc remained culturable (about 10⁸ cells/ml) throughout the entire fermentation (up to 10 days). The rRNA content of cells during mixed fermentations was also analyzed by flow cytometry in combination with FISH probes. The fluorescence intensity conferred by the species-specific FISH probes was considerably lower for Hg than for Sc. Moreover, the rRNA content of Hg cells, conversely to Sc cells, remained almost unchanged after boiling, which showed that rRNA stability is species-dependent.

Aims: To identify and compare microbiota in Chinese liquor Daqu, which were produced in the different regions using different production process. Methods and Results: The DNA exacted from Daqu samples was used as a template for PCR with universal primers of 16S rRNA, 26S rRNA and 18S rRNA, respectively. The amplicons were analysed using denaturing gradient gel electrophoresis (DGGE). It was observed that the bacterial DGGE profile indicated high diversity and predominance of lactic acid bacteria. The results showed that Saccharomycopsis fibuligera and Pichia anomal were dominant yeast species and that several non-Saccharomyces yeasts including Hanseniaspora guilliermondii, Debaryomyces hansenii, Issatchenkia orientalis and Trichosporon asahii were also detected. As for fungal DGGE, Aspergillus oryzae and Absidia blakesleeana were the most common species amongst different samples. Based on the DGGE analysis, a few differences in community structure were found between Daqu samples. Conclusions: A variety of bacteria, yeast and moulds were identified in Daqu samples, in addition to the present knowledge obtained mainly through the traditional culture-dependent methods. Moreover, production temperature played a more decisive role on the formation of micro-organism composition in Daqu than geographical region. Significance and Impact of the Study: PCR-DGGE technique was used in this study to fully observe and asses all microbial community (including bacteria, yeast and mould) in Chinese liquor Daqu for the first time and proved to be effective in profiling Daqu microbial diversity.

For the first time, an ecological survey of wine yeasts present in grapes growing in two vineyards located in the region of "Serranía de Ronda" (Málaga, southern Spain) has been carried out. During the 2006 and 2007 vintages, grapes from different varieties were aseptically collected and allowed to ferment spontaneously in the laboratory. From a total of 1586 colonies isolated from microvinifications, 1281 were identified according to ITS polymorphisms and their identity confirmed by sequencing of the D1/D2 region of 26S rDNA. Most of the isolates (84%) corresponded to thirteen different non-Saccharomyces species with Kluyveromyces thermotolerans, Hanseniaspora guilliermondii, Hanseniaspora uvarum and Issatchenkia orientalis accounting for 42.7% of the total. Mitochondrial DNA restriction analysis from the Saccharomyces cerevisiae isolates revealed a low diversity since only eleven different profiles were found, nine of them corresponding to local strains and two to commercial ones that had been used in different campaigns and that very likely were disseminated from the winery to the adjacent vineyard. A different distribution of strains was found in the three grape varieties studied.

The focus of the present investigation was on the Tanzanian fermented food togwa as a source for dietary iron and zinc, and the potential for mineral availability improvements using selected yeasts. To establish the content of target minerals and main inhibitor for intestinal uptake, iron and zinc as well as the mineral chelating phytic acid, (IP(6) or phytate) were determined in naturally fermented togwa. Yeasts were isolated from sorghum, maize and cassava based togwa, and identified by sequencing the D1/D2 region of the LSU rRNA gene. The isolated yeasts were subsequently screened for phytase activity. The total iron content in sorghum, maize and cassava based togwa were 41.5 (+/-7.2), 85.4 (+/-31.9) and 28.6 (+/-3.8) microg/g dw (dry weight) respectively. The zinc content was 12.3 (+/-3.1), 11.0 (+/-1.1) and 6.4 (+/-4.5) microg/g dw in sorghum, maize and cassava based togwa, and the phytate content in the three varieties were 2.6+/-1.2, 4.7+/-0.8 and 0.4+/-0.4 micromol/g dw respectively. The phytate levels in the sorghum and maize based togwa are expected to substantially reduce the availability of iron. The molar ratio phytate to iron for these two varieties were estimated to be 3.5:1 and 3.1:1 respectively. In general, a phytate to iron molar ratio below 1 is needed to increase the availability of iron. Among 26 isolates, 9 different species could be distinguished: Issatchenkia orientalis, Pichia anomala, Pichia norvegensis, Pichia burtonii, Pichia guilliermondii, Kluyveromyces marxianus, Saccharomyces cerevisiae, Hanseniaspora guilliermondii and Candida glabrata. The strains were screened for phytase activity in YPD supplemented with 0.5 mM IP(6). Of 26 screened strains, the phytase activity was most prominent in strains of I. orientalis and H. guilliermondii. The strains and data constitute a basis for further improvements of iron and zinc bioavailability in togwa.

The nature of the toxic compounds produced by Saccharomyces cerevisiae CCMI 885 that induce the early death of Hanseniaspora guilliermondii during mixed fermentations, as well as their ability to inhibit the growth of other non-Saccharomyces wine-related strains, was investigated. The killing effect of mixed supernatants towards H. guilliermondii was inactivated by protease treatments, thus revealing the proteinaceous nature of the toxic compounds. Analysis of the protein pattern of mixed supernatants on Tricine SDS-PAGE showed that this S. cerevisiae strain secretes peptides (

Thirty-eight yeast strains belonging to the genera Candida, Hanseniaspora, Pichia, Torulaspora and Zygosaccharomyces were screened for ester formation on synthetic microbiological medium. The genera Hanseniaspora and Pichia stood out as the best acetate ester producers. Based on the ester profile Hanseniaspora guilliermondii 11027 and 11102, Hanseniaspora osmophila 1471 and Pichia membranifaciens 10113 and 10550 were selected for further characterization of enological traits. When growing on must H. osmophila 1471, which displayed a glucophilic nature and was able to consume more than 90% of initial must sugars, produced levels of acetic acid, medium chain fatty acids and ethyl acetate, within the ranges described for wine. On the other hand, it was found to be a strong producer of 2-phenylethyl acetate. Our data suggest that H. osmophila 1471 is a good candidate for mixed starters, although the possible interactions with S. cerevisiae deserve further research.

The introduction of yeast starter cultures consisting in a blend of Saccharomyces cerevisiae and non-Saccharomyces yeast strains is emerging for production of wines with improved complexity of flavor. The rational use of this approach is, however, dependent on knowing the impact that co-inoculation has in the physiology of S. cerevisiae. In this work the transcriptome of S. cerevisiae was monitored throughout a wine fermentation, carried out in single culture or in a consortium with Hanseniaspora guilliermondii, this being the first time that this relevant yeast–yeast interaction is examined at a genomic scale. Co-inoculation with H. guilliermondii reduced the overall genome-wide transcriptional response of S. cerevisiae throughout the fermentation, which was attributable to a lower fermentative activity of S. cerevisiae while in the mixed-fermentation. Approximately 350 genes S. cerevisiae genes were found to be differently expressed (FDR

Fructophily has been described in yeasts as the ability to utilize fructose preferentially when fructose and glucose are available in the environment. In Zygosaccharomyces bailii and Zygosaccharomyces rouxii, fructophilic behaviour has been associated with the presence of a particular type of high-capacity and low-affinity fructose transporters designated Ffz. In this study, a PCR screening was performed in several yeasts using degenerate primers suitable to detect FFZ-like genes. In parallel, fructophilic character was evaluated in the same strains by comparing the relative consumption rate of fructose and glucose. For all the strains in which FFZ-like genes were detected, fructophilic behaviour was observed (25 strains). Results show that FFZ genes are ubiquitous in the Zygosaccharomyces and Starmerella clades. Strains of Lachancea fermentati, Torulaspora microellipsoides and Zygotorulaspora florentina were not fructophilic and did not harbour FFZ genes. It is of note that these new species were recently removed by taxonomists from the Zygosaccharomyces clade, supporting the view that the presence of FFZ-like genes is a main characteristic of Zygosaccharomyces. Among the strains tested, only Hanseniaspora guilliermondii NCYC2380 was an exception, having a preference for fructose in medium with high sugar concentrations, despite no FFZ-like genes being detected in the screening. Furthermore, this study supports the previous idea of the emergence of a new family of hexose transporters (Ffz facilitators) distinct from the Sugar Porter family.

Yeast, lactic acid bacteria (LAB) and acetic acid bacteria (AAB) populations, isolated from cocoa bean heap and box fermentations in West Africa, have been investigated. The fermentation dynamicswere determined by viable counts, and 106 yeasts, 105 LAB and 82 AAB isolateswere identified by means of rep-PCR grouping and sequencing of the rRNA genes. During the box fermentations, the most abundant species were Saccharomyces cerevisiae, Candida ethanolica, Lactobacillus fermentum, Lactobacillus plantarum, Acetobacter pasteurianus and Acetobacter syzygii, while S. cerevisiae, Schizosaccharomyces pombe, Hanseniaspora guilliermondii, Pichia manshurica, C. ethanolica, Hanseniaspora uvarum, Lb. fermentum, Lb. plantarum, A. pasteurianus and Acetobacter lovaniensis were identified in the heap fermentations. Furthermore, the most abundant species were molecularly characterized by analyzing the rep-PCR profiles. Strains grouped according to the type of fermentations and their progression during the transformation process were also highlighted. The yeast, LAB and AAB isolates were physiologically characterized to determine their ability to grow at different temperatures, as well as at different pH, and ethanol concentrations, tolerance to osmotic stress, and lactic acid and acetic acid inhibition. Temperatures of 45 °C, a pH of 2.5 to 3.5, 12% (v/v) ethanol and high concentrations of lactic and acetic acid have a significant influence on the growth of yeasts, LAB and AAB. Finally, the yeastswere screened for enzymatic activity, and the S. cerevisiae, H. guilliermondii, H. uvarumand C. ethanolica species were shown to possess several enzymes that may impact the quality of the final product.

A molecular approach is used for the identification of yeast isolated from table olives. Our results validate those obtained in the past by the classical biochemical methodology. Yeast were isolated from both aerobically and anaerobically processed black table olives and also from canned seasoned green table olives. Molecular identification methodology used included restriction pattern analysis of both PCRamplified 5.8S rRNA gene and internal transcribed spacers ITS1 and ITS2. For some species, sequence analysis of the 26S rRNA gene was necessary. These techniques allowed the identification of three yeast species (Issatchenkia occidentalis, Geotrichum candidum and Hanseniaspora guilliermondii) which had not been described previously in table olives. Saccharomyces cerevisiae and Candida boidinii were the most frequent species in green seasoned olives and processed black olives, respectively. The molecular study of total DNA variability among the S. cerevisiae strains isolated indicates a quite heterogeneous population, with at least four different restriction patterns.

The yeast populations associated with the fermentation of Ghanaian cocoa were investigated using denaturing gradient gel electrophoresis (DGGE). Samples were collected at 12-24 h intervals from heap and tray fermentations, at three different fermentation sites and different periods during the season. Eukaryotic universal primers were used to amplify a fragment of the 26S rRNA gene. The DGGE profiles were relatively complex, underlining that the fermentation of cocoa is a complex microbial process. The identities of selected fragments in the denaturing gels were revealed by sequencing. Hanseniaspora guilliermondii, Candida krusei and Pichia membranifaciens were detected from most fermentations, indicating their possible important role in the fermentation of Ghanaian cocoa. Saccharomyces cerevisiae and Candida zemplinina were almost exclusively detected during tray fermentations. The developed DGGE protocol was compared with traditional culture-based isolations. The results were comparable but slightly different, as one yeast species (C. zemplinina) was only detected using DGGE. On the other hand, Trichosporon asahii yielded only faint bands in the denaturing gels, despite the fact that it was detected using culture-based methods. Analysis of pure cultures showed that the targeted region of the 26S rRNA gene was poorly amplified in T. asahii, whereas all other investigated isolates were amplified efficiently using the chosen PCR approach. Cluster analysis revealed that the DGGE profiles clustered according to fermentation method and fermentation site. Furthermore, clustering according to progress in the fermentation was observed. The DGGE technique therefore seems to offer a relatively fast and reliable method for studying yeast population dynamics during cocoa fermentations.

Samples of cocoa beans were taken on two separate occasions during heap and tray fermentations in Ghana, West Africa. In total 496 yeast isolates were identified by conventional microbiological analyses and by amplification of their ITS1-5.8S rDNA-ITS2 regions. For important species the identifications were confirmed by sequencing of the D1/D2 domain of the 5' end of the large subunit (26S) rDNA. Assimilations of organic acids and other carbon compounds were conducted. For dominant yeasts intraspecies variations were examined by determination of chromosome length polymorphism (CLP) using pulsed-field gel electrophoresis. For the heap fermentations maximum yeast cell counts of 9.1 x 10(7) were reached, whereas maximum yeast counts of 6.0 x 10(6) were reached for the tray fermentations. Candida krusei was found to be the dominant species during heap fermentation, followed by P. membranifaciens, P. kluyveri, Hanseniaspora guilliermondii and Trichosporon asahii, whereas Saccharomyces cerevisiae and P. membranifaciens were found to be the dominant species during tray fermentation followed by low numbers of C. krusei, P. kluyveri, H. guilliermondii and some yeast species of minor importance. For isolates within all dominant species CLP was evident, indicating that several different strains are involved in the fermentations. Isolates of C. krusei, P. membranifaciens, H. guilliermondii, T. asahii and Rhodotorula glutinis could be found on the surface of the cocoa pods and in some cases on the production equipment, whereas the origin of e.g. S. cerevisiae was not indicated by the results obtained. In conclusion, the results obtained show that fermentation of cocoa beans is a very inhomogeneous process with great variations in both yeast counts and species composition. The variations seem to depend especially on the processing procedure, but also the season and the post-harvest storage are likely to influence the yeast counts and the species composition.

The kinetics of cell inactivation at high concentrations of ethanol (22.5% and 25% v v −1 ) was studied with particular reference to the influence of different cultivation conditions (aerobiosis, semi-aerobiosis, anaerobiosis, addition of survival factors) prior to ethanol challenge. The levels of fatty acids and sterols in cells grown under these different conditions were analysed in order to derive a potential relationship between the ethanol tolerance and the lipid composition of the different strains studied. Under the conditions tested, Hanseniaspora guilliermondii showed an ethanol tolerance very similar to that of Saccharomyces cerevisiae , and very different to that of the other apiculate yeast, H. uvarum . The survival of S. cerevisiae and H. guilliermondii at 25% (v v −1 ) ethanol was strongly influenced by the conditions of cultivation prior to the ethanol challenge. A small increase in survival was observed for H. uvarum and Torulaspora delbrueckii in the cultures grown in aerobiosis. Growth of these yeasts in anaerobiosis in the presence of added ergosterol and Tween 80 (as a source of oleic acid) did not induce a considerable tolerance to the ethanol challenge, in spite of the incorporation of these compounds by the cells. In these growth conditions Debaryomyces hansenii was not capable of incorporating the survival factors and did not increase the ethanol tolerance. It is shown in this work that, besides S. cerevisiae , the presence of oxygen and the addition of survival factors to the culture conditions and the subsequent increase in the proportion of cell sterols and unsaturated fatty acids may play an important role in the ethanol tolerance of some non- Saccharomyces yeasts. This effect was not, however, observed in all of the yeasts studied.

Fourteen apiculate yeast strains isolated from various sources in South Africa, North America and the Hawaiian islands were found to be genetically divergent from other Hanseniaspora-Kloeckera species by using randomly amplified polymorphic DNA (RAPD)-PCR. After cluster analysis of the RAPD-PCR fingerprints, five groups were recognized. DNA reassociation values among representatives of these groups and strains of Hanseniaspora-Kloeckera species revealed that the strains represent five novel species. Four are described here as novel species of HANSENIASPORA: Hanseniaspora meyeri sp. nov. (type CBS 8734(T)), Hanseniaspora clermontiae sp. nov. (type CBS 8821(T)), Hanseniaspora lachancei sp. nov. (type CBS 8818(T)) and Hanseniaspora opuntiae sp. nov. (type CBS 8733(T)). The fifth novel species, which is represented by only a single strain, CBS 8772, is not introduced as a new taxon. Phylogenetic analyses of the D1/D2 region of the 26S rDNA and internal transcribed spacer (ITS) regions with 5.8S rDNA sequences placed H. meyeri, H. clermontiae, H. lachancei, H. opuntiae and strain CBS 8772 close to Hanseniaspora uvarum and Hanseniaspora guilliermondii. The key characteristics for standard physiological identification of H. clermontiae and H. lachancei were respectively maximal growth temperature and assimilation of 2-keto-D-gluconate. However, physiological characteristics did not allow the distinction of H. opuntiae and strain CBS 8772 from H. guilliermondii or H. meyeri from H. uvarum. These three novel taxa can be identified by either ITS sequencing or PCR-RFLP of ITS regions using restriction enzymes MboII and HinfI.

Two non-Saccharomyces wine yeast strains, Hanseniaspora guilliermondii 11104 and Pichia anomala 10590, selected as good producers of acetate esters when grown on synthetic microbiological medium, have been tested in wine fermentations as mixed cultures together with Saccharomyces cerevisiae. Wines produced using mixed cultures showed levels of acetaldehyde, acetic acid, glycerol and total higher alcohols within the ranges described for wine, whereas an increase in acetate ester concentrations was found. Ethyl acetate was the main ester produced, and isoamyl acetate and 2-phenylethyl acetate made up the next largest group of ester compounds in the wines analysed. H. guilliermondii 11104 was found to be a strong producer of 2-phenylethyl acetate in both pure and mixed cultures whereas S. cerevisiae was the best producer of ethyl esters. Mixed cultures did not influence ethyl ester levels at all.

The physiology of Hanseniaspora guilliermondii was studied under aerobic glucose-limited conditions using the accelerostat procedure (continuous acceleration of dilution rate) and classical chemostat cultures. By both cultivation techniques this yeast was found to be Crabtree-positive. Up to a dilution rate of 0.25 h(-1), glucose was completely metabolised into biomass, glycerol and carbon dioxide. Above this value, an increase in the dilution rate was accompanied by the production of other metabolites like ethanol, acetic and malic acids. Biomass yield during the purely oxidative growth was 0.49 g g(-1) and decreased to 0.26 g g(-1) for D=0.34 h(-1). A maximal specific ethanol production rate of 1.36 mmol g(-1) h(-1) and a maximal ethanol yield of 0.05 g g(-1) were achieved at D=0.34 h(-1).

Phytate is known as a strong chelate of minerals causing their reduced uptake by the human intestine. Ninety-three yeast isolates from traditional African fermented food products, belonging to nine species (Pichia kudriavzevii, Saccharomyces cerevisiae, Clavispora lusitaniae, Kluyveromyces marxianus, Millerozyma farinosa, Candida glabrata, Wickerhamomyces anomalus, Hanseniaspora guilliermondii and Debaryomyces nepalensis) were screened for phytase production on solid and liquid media. 95% were able to grow in the presence of phytate as sole phosphate source, P. kudriavzevii being the best growing species. A phytase coding gene of P. kudriavzevii (PHYPk) was identified and its expression was studied during growth by RT-qPCR. The expression level of PHYPk was significantly higher in phytate-medium, compared to phosphate-medium. In phytate-medium expression was seen in the lag phase. Significant differences in gene expression were detected among the strains as well as between the media. A correlation was found between the PHYPk expression and phytase extracellular activity.

The sequences of the internal transcribed spacers (ITS regions) and the 5.8S rRNA gene, together with the electrophoretic karyotypes of 27 strains representative of the six species belonging to the genus Hanseniaspora, were examined. From the analysis of the 5.8S rRNA gene and the ITS regions, the genus Hanseniaspora is monophyletic and can be divided into two subgroups. This subdivision was supported by electrophoretic chromosome patterns. Hanseniaspora guilliermondii, H. uvarum and H. valbyensis show 6-7 bands (8 to 9 chromosomes), while the second group comprises the species H. occidentalis, H. osmophila and H. vineae which have only 5 chromosomes.

Yeasts were isolated by the enrichment technique from the phylloplane of 94 samples of sugarcane leaf collected from seven provinces in Thailand. All sugarcane leaf samples contained yeasts and 158 yeast strains were obtained. On the basis of the D1/D2 domain of the large subunit rRNA gene sequence analysis, 144 strains were identified to 24 known species in 14 genera belonging to the Ascomycota viz. Candida akabanensis, Candida dendronema, Candida mesorugosa, Candida michaelii, Candida nivariensis, Candida rugosa, Candida orthopsilosis, Candida quercitrusa, Candida tropicalis, Candida xylopsoci, Cyberlindnera fabianii, Cyberlindnera rhodanensis, Debaryomyces nepalensis, Hannaella aff. coprosmaensis, Hanseniaspora guilliermondii, Kluyveromyces marxianus, Lachancea thermotolerans, Lodderomyces elongisporus, Metschnikowia koreensis, Meyerozyma caribbica, Millerozyma koratensis, Pichia kudriavzevii, Torulaspora delbrueckii and Wickerhamomyces edaphicus, and 12 species in six genera of the Basidiomycota viz . Cryptococcus flavescens, Cryptococcus laurentii, Cryptococcus rajasthanensis, Kwoniella heveanensis, Rhodosporidium fluviale, Rhodosporidium paludigenum, Rhodotorula mucilaginosa, Rhodotorula sesimbrana, Rhodotorula taiwanensis, Sporidiobolus ruineniae, Sporobolomyces carnicolor and Sporobolomyces nylandii. Seven strains were identical or similar to four undescribed species. Another seven strains represented four novels species in the genus Metschnikowia, Nakazawaea, Wickerhamomyces and Yamadazyma. The results revealed 69 % of the isolated strains were ascomycete yeasts and 31 % were basidiomycete yeast. The most prevalent species was M. caribbica with a 23 % frequency of occurrence followed by Rh. taiwanensis (11 %) and C. tropicalis (10 %). All strains were assessed for indole-3-acetic acid (IAA) producing capability showing that 69 strains had the capability of producing IAA when cultivated in yeast extract peptone dextrose broth supplemented with 1 g/L L-tryptophan. The highest IAA concentration of 565.1 mg/L was produced by R. fluviale DMKU-RK253.

The aim of the present work was to investigate the microbiological, chemical, and sensory characteristics of red wine subjected to post-fermentation maceration that was extended to 90 days. For this purpose, the ‘Aglianico di Taurasi’ grape was used as a case study. The total yeast concentration increased until day 40 of maceration and decreased thereafter, whereas the concentration of lactic acid bacteria slightly increased. Dekkera/Brettanomyces spp. and acetic acid bacteria were not detected. The yeast community was composed of Saccharomyces cerevisiae, Zygosaccharomyces bisporus, Metschnikowia pulcherrima, Hanseniaspora guilliermondii, Hanseniaspora uvarum, Pichia guilliermondii, Aureobasidium pullulans and Debaryomyces carsonii. Nine S. cerevisiae strains were detected at high levels at different times of maceration. The results of all the conventional chemical analyses of the wines were in agreement with the regulations of commercial production and, interestingly, the changes in terms of concentration demonstrated the presence of yeast and LAB populations that were not only alive but also in a metabolically active state until day 90 of maceration. The alcohol and glycerol contents slightly increased until day 90. The concentrations of malic acid decreased, whereas those of lactic acid slightly increased throughout the maceration process. Furthermore, different durations of maceration resulted in significant differences in the total polyphenol content, which was higher at 40–50 days. The main phenolic compounds were benzoic and cinnamic acids and catechins. Interestingly, the highest ratio between (+)-catechin and (−)-epicatechin was found on day 40. In addition, the highest antioxidant activity was observed between days 40 and 50. The concentration of volatile organic compounds, which were mainly represented by alcohols, increased until the end of the maceration process. Sensory analysis revealed that samples that were subjected to maceration for a long period of time showed the highest odour and taste complexity and no off-odours and/or off-flavours were detected. These data confirmed that extending post-fermentation maceration to 90 days has no negative impact on the microbiological, chemical and sensory composition of wines, but affects the polyphenol content and potential health benefits of the resulting wine.

Populations and frequency of occurrence of yeasts in frozen apple, cherry, grape, orange, and pineapple juice concentrates were determined. Total yeast populations ranged from log10 < 1.00 to 5.41 CFU/ml of diluted (1:4) concentrate and increased to 2.42-7.34 CFU/ml after 24 h of incubation at 25°C. One hundred fifty-four isolates from 33 samples of fruit juice concentrates represented 21 species and 12 genera. The most frequently isolated species were Saccharomyces cerevisiae (24.7% of isolates), Candida stellata (22.1%), and Zygosaccharomyces rouxii (14.3%), followed by, in decreasing order of frequency, Torulaspora delbrueckii , Rhodotorula mucilaginosa , Issatchenkia orientalis , Hanseniaspora occidentalis , Lodderomyces elongisporus , Kluyveromyces thermotolerans , Hanseniaspora guilliermondii , Candida glabrata , and Pichia anomala , each representing 3-8% of isolates. Candida magnoliae , Candida maltosa , Candida parapsilosis , Candida tropicalis , Clavispora lusitaniae , Cryptococcus humicolus , C. laurentii , Pichia membranaefaciens , and Sporidiobolus salmonicolor were represented by single isolates. Populations in various samples consisted of 24-100% S. cerevisiae , 52-100% C. stellata , and 3-56% for Z. rouxii . S. cerevisiae and Z. rouxii were isolated, respectively, from five and four types of fruit concentrates investigated. This may be the first observation of this high frequency of C. stellata in juice concentrates.

Having more information on the yeast ecology of grapes is important for wine-makers to produce wine with high quality and typical attributes. China is a significant wine-consuming country and is becoming a serious wine-producer, but little has been reported about the yeast ecology of local ecosystems. This study provides the first step towards the exploitation of the yeast wealth in China's vine-growing regions. The aim of this study was to investigate the yeast population density and diversity on three grape varieties cultivated in four representative vine-growing regions of China. Yeast species diversity was evaluated by using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and sequence analysis of the 5.8S internal transcribed spacer (ITS) ribosomal DNA (rDNA) region of cultivable yeasts. The grapes harbored yeast populations at 10(2)-10(6)CFU/mL, consisting mostly of non-Saccharomyces species. Seventeen different yeast species belonging to eight genera were detected on the grape samples tested, including Hanseniaspora uvarum, Cryptococcus flavescens, Pichia fermentans, Candida zemplinina, Cryptococcus carnescens, Candida inconpicua, Zygosaccharomyces fermentati, Issatchenkia terricola, Candida quercitrusa, Hanseniaspora guilliermondii, Candida bombi, Zygosaccharomyces bailii, Sporidiobolus pararoseus, Cryptococcus magnus, Metschnikowia pulcherrima, Issatchenkia orientalis and Pichia guilliermondii. H. uvarum and C. flavescens were the dominant species present on the grapes. For the first time Sporidiobolus pararoseus was discovered as an inhabitant of the grape ecosystem. The yeast community on grape berries was influenced by the grape chemical composition, vine-variety and vine-growing region. This study is the first to identify the yeast communities associated with grapes in China using molecular methods. The results enrich our knowledge of wine-related microorganisms, and can be used to promote the development of the local wine industry.

The natural Saccharomyces and non-Saccharomyces yeast flora present on the grape berries significantly affect wine production. Six grape varieties, Bangalore blue, Zinfandel, Cabernet, Chenin Blanc, Sauvignon Blanc and Shiraz are being used in India for wine making. The yeast diversity was studied on the basis of morphological, colony, physiological characteristics and 5.8S-ITS sequencing of rDNA of the isolates. Eleven different species belonging to seven genera were identified as: Candida azyma, Candida quercitrusa, Debaryomyces hansenii, Hanseniaspora guilliermondii, Hanseniaspora viniae, Hanseniaspora uvarum, Issatchenkia orientalis, Issatchenkia terricola, Pichia membranifaciens, Saccharomyces cerevisiae and Zygoascus steatolyticus. H. guilliermondii was the predominant species while S. cerevisiae was observed occasionally in the six vine varieties. For the first time, C. azyma was isolated from Bangalore blue and Cabernet varieties grown in different localities. This association may be attributed to the change in cropping pattern from sugarcane to viticulture in the vine growing regions and the known association of C. azyma with sugarcane phylloplane. Further analysis of the indigenous strains and the qualitative and quantitative changes in the flora during fermentation will be useful to understand wine quality and to design preservation strategies to control wine spoilage.

The physiological and kinetic capabilities of 233 Saccharomyces cerevisiae isolates, originating from traditional Brazilian cachaca fermentation, were evaluated under laboratory conditions to select flocculent and non-H2S producing strains to be employed in beverage production. Three flocculent S. cerevisiae strains were selected, two non-H2S producing and one H2S producing, and their kinetic performances were analysed during two large-scale fermentation experiments in a traditional cachaca distillery. One non-flocculent H2S-producing S. cerevisiae strain was also used for comparison with the flocculent strains. The results of mitochondrial DNA restriction analysis showed that the three flocculent starter S. cerevisiae strains, as well as the non-flocculent strain, remained in the process during the whole fermentation period, with cells numbering around 10(7) cfu/ml. All selected strains produced ethanol yields that were typically higher in the distillery than in the laboratory conditions, except for strain UFMGA-1240. The greatest diversity of non-Saccharomyces yeasts was observed prior to day 21 of cachaca fermentation; Pichia membranifaciens and Hanseniaspora guilliermondii were the most frequently isolated species. These yeasts were present in lower densities throughout the whole process. The cachaca produced by the selected strains contained concentrations of chemical compounds in accordance with current Brazilian legislation, and all cachacas scored well in sensory effective tests. In addition to the advantage of being flocculent, the strain UFMGA-1031 is non-H2S producing and also produces cachaca with good sensory acceptance. Therefore, this flocculent and non-H2S producing S. cerevisiae strain is highly suitable as a starter for production of high quality traditional cachaca.

Hanseniaspora guilliermondii and Hanseniaspora uvarum were tested in grape must fermentations as pure and mixed starter cultures with Saccharomyces cerevisiae. In pure cultures, the specific growth rates found were 0.29 h- 1 for H. uvarum, 0.23 h- 1 for H. guilliermondii and 0.18 h- 1 for S. cerevisiae. No significant differences were observed between these values and those obtained in mixed cultures. Results presented in this work show that growth of apiculate yeasts during the first days of fermentation enhances the production of desirable compounds, such as esters, and may not have a negative influence on the production of higher alcohols and undesirable heavy sulphur compounds. Growth of apiculate yeasts reduced the total content of higher alcohols in wines, when compared to those produced by a pure culture of S. cerevisiae. Furthermore, the highest levels of 2-phenylethyl acetate were obtained when H. guilliermondii was inoculated in grape musts, whereas H. uvarum increased the isoamyl acetate content of wines. Apiculate yeasts produced high amounts of ethyl acetate; however, the level of this compound decreased in mixed cultures of apiculate yeasts and S. cerevisiae. When S. cerevisiae was used as a starter culture, wines showed higher concentrations of glycerol, 2-phenylethanol and ethyl hexanoate. In mixed cultures of apiculate yeasts and S. cerevisiae, wines presented amounts of methionol, acetic acid-3-(methylthio)propyl ester, 4-(methylthio)-1-butanol, 2-mercaptoethanol and cis-2-methyltetrahydro-thiophen-3-ol similar to those produced by a pure culture of S. cerevisiae. An increase in the amounts of 3-(ethylthio)-1-propanol, trans-2-methyltetrahydro-thiophen-3-ol and 3-mercapto-1-propanol was obtained in wines produced from mixed cultures with H. guilliermondii.

The early death of two non-Saccharomyces wine strains (H. guilliermondii and H. uvarum) during mixed fermentations with S. cerevisiae was studied under enological growth conditions. Several microvinifications were performed in synthetic grape juice, either with single non-Saccharomyces or with mixed S. cerevisiae/non-Saccharomyces inocula. In all mixed cultures, non-Saccharomyces yeasts grew together with S. cerevisiae during the first 1-3 days (depending on the initial inoculum concentration) and then, suddenly, non-Saccharomyces cells began to die off, regardless of the ethanol concentrations present. Conversely, in both non-Saccharomyces single cultures the number of viable cells remained high (ranging 107-108 CFU ml-1) even when cultures reached significant ethanol concentrations (up to 60-70 g l-1). Thus, at least for these yeast strains, it seems that ethanol is not the main death-inducing factor. Furthermore, mixed cultures performed with different S. cerevisiae/ H. guilliermondii inoculum ratios (3 : 1; 1 : 2; 1 : 10; 1 : 100) revealed that H. guilliermondii death increases for higher inoculum ratios. In order to investigate if the nature of the yeast-yeast interaction was related or not with a cell-cell contact-mediated mechanism, cell-free supernatants obtained from 3 and 6 day-old mixed cultures were inoculated with H. guilliermondii pure cultures. Under these conditions, cells still died and much higher death rates were found for the 6 days than for the 3 day-old supernatants. This strongly indicates that one or more toxic compounds produced by S. cerevisiae triggers the early death of the H. guilliermondii cells in mixed cultures with S. cerevisiae. Finally, although it has not been yet possible to identify the nature of the toxic compounds involved in this phenomenon we must emphasise that the S. cerevisiae strain used in the present work is killer sensitive with respect to the classical killer toxins, K1, K2 and K28, whereas the H. guilliermondii and H. uvarum strains are killer neutral.

Export of cocoa beans is of great economic importance in Ghana and several other tropical countries. Raw cocoa has an astringent unpleasant taste and a spontaneous fermentation is the first step in a process leading to cocoa beans with the characteristic cocoa flavour and taste. The microbiology of Ghanaian cocoa fermentations was investigated using culture-dependent and culture-independent methods. Samples were collected at 12 hour intervals during 96-144 hour tray and traditional heap fermentations. Yeast, Lactic Acid Bacteria (LAB), Acetic Acid Bacteria (AAB) and Bacillus spp. were enumerated on suitable substrates and identified using phenotypic and molecular methods. The yeast and bacterial micro-populations involved in the cocoa fermentation were further investigated using the culture-independent method Denaturing Gradient Gel Electrophopresis (DGGE). A microbiological succession was observed during the fermentations. At the onset of fermentation yeasts were the dominating microorganisms. Lactic Acid Bacteria became dominant after 12-24 h of fermentation and remained predominant throughout the fermentations with AAB reaching high counts in the mid phase of fermentation. Bacillus spp. were only detected during heap fermentations where they reached high numbers during the later stages of fermentation. Hanseniaspora guilliermondii was the predominant yeast during the initial phase and Pichia membranifaciens during the later phases of fermentation. A number of other yeast species including three putatively undescribed species were isolated during the fermentations. Lactobacillus fermentum was the dominant LAB in most samples. Several other LAB including Lactobacillus plantarum, Leuconostoc pseudomesenteroides, Leuconostoc pseudoficulneum, Pediocococcus acidilactici and a putatively undescribed LAB species were detected during the fermentations. Acetobacter syzygii, Acetobacter pasteurianus and Acetobacter tropicalis were the predominant AAB in all investigated fermentations. During the later stages of heap fermentation Bacillus licheniformis and occasionally other Bacillus spp. were detected in high numbers. In general the culture-based findings were confirmed using DGGE. However, DGGE indicated that Lc. pseudoficulneum plays a more important role during the fermentation of cocoa than expected from the culture-based findings as it yielded a strong band in most DGGE fingerprints. Cluster analysis of the DGGE fingerprints revealed that the DGGE fingerprints clustered according to fermentation site. Within each fermentation site the profiles clustered according to fermentation time. The DGGE method seems to offer a relatively fast and reliable tool for studying yeast and bacterial dynamics during cocoa fermentations.

Multistarter fermentations of Hanseniaspora uvarum, Torulaspora delbrueckii and Kluyveromyces thermotolerans together with Saccharomyces cerevisiae were studied. In grape musts with a high sugar content, mixed trials showed a fermentation behaviour and analytical profiles of wines comparable to or better than those exhibited by a pure culture of S. cerevisiae. Sequential trials of T. delbrueckii and K. thermotolerans revealed a sluggish fermentation, while those of H. uvarum exhibited an unacceptable increase in ethyl acetate content (175 ml l(-1)). A low fermentation temperature (15 degrees C) of multistarter trials of H. uvarum resulted in a stuck fermentation that was not due to a deficiency of assimilable nitrogenous compounds since lower amounts of these compounds were used. Sequential fermentation carried out by H. uvarum at 15 degrees C confirmed the high production of ethyl acetate. The persistence and level of non-Saccharomyces yeasts during multistarter fermentations under stress conditions (high ethanol content and/or low temperature) can cause stuck fermentations.

Strains of Hanseniaspora uvarum, Hanseniaspora guilliermondii and Saccharomyces cerevisiae were used as pure or mixed starter cultures in commercial medium, in order to compare their kinetic parameters and fermentation patterns. In pure and mixed cultures, yeasts presented similar ethanol yield and productivity. Pure cultures of H. uvarum and S. cerevisiae showed a specific growth rate of 0.38 h-1; however, this value decreased when these yeasts were grown in mixed cultures with H. guilliermondii. The specific growth rate of pure cultures of H. guilliermondii was 0.41 h-1 and was not affected by growth of other yeasts. H. guilliermondii was found to be the best producer of 2-phenylethyl acetate and 2-phenylethanol in both pure and mixed cultures. In pure cultures, H. uvarum led to the highest contents of heavy sulphur compounds, but H. guilliermondii and S. cerevisiae produced similar levels of methionol and 2-methyltetrahydrothiophen-3-one. Growth of apiculate yeasts in mixed cultures with S. cerevisiae led to amounts of 3-methylthiopropionic acid, acetic acid-3-(methylthio)propyl ester and 2-methyltetrahydrothiophen-3-one similar to those obtained in a pure culture of S. cerevisiae; however, growth of apiculate yeasts increased methionol contents of fermented media.

A double coupling bioreactor system was used to fast screen yeast strains for the production of acetate esters. Eleven yeast strains were used belonging to the genera Candida, Hanseniaspora, Metschnikowia, Pichia, Schizosaccharomyces and Zygosacharomyces, mainly isolated from grapes and wine, and two wine Saccharomyces cerevisiae strains. The acetate ester forming activities of yeast strains belonging to the genera Hanseniaspora (Hanseniaspora guilliermondii and H. uvarum) and Pichia (Pichia anomala) showed different substrate specificities and were able to produce ethyl acetate, geranyl acetate, isoamyl acetate and 2-phenylethyl acetate. The influence of aeration culture conditions on the formation of acetate esters by non-Saccharomyces wine yeast and S. cerevisiae was examined by growing the yeasts on synthetic microbiological medium. S. cerevisiae produced low levels of acetate esters when the cells were cultured under highly aeration conditions, while, under the same conditions, H. guilliermondii 11104 and P. anomala 10590 were found to be strong producers of 2-phenylethyl acetate and isoamyl acetate, respectively.

P. ROMANO, G. SUZZI, G. COMI AND R. ZIRONI. 1992. Ninety-six strains of apiculate wine yeasts were investigated for their ability to produce higher alcohols and acetic acid in synthetic medium. Less isoamyl alcohol and more n-propanol and isobutanol were formed by Hanseniaspora guilliermondii than by Kloeckera apiculata. The latter produced twice as much acetic acid as H. guilliermondii. The production of higher alcohols and acetic acid was found to be a characteristic of individual strains and was statistically significant. In a multivariate analysis of higher alcohol production two main groupings were formed at 86%S, corresponding to the taxa H. guilliermondii and K. apiculata. Strains that produced low amounts (50 mg/1) of acetic acid, comparable with that of Saccharomyces cerevisiae, were found in both species of apiculate yeasts.