Interactions between Brettanomyces bruxellensis and other yeast species during the initial stages of winemaking

AIMS: Wine is the product of complex interactions between yeasts and bacteria in grape must. Amongst yeast populations, two groups can be distinguished. The first, named non-Saccharomyces (NS), colonizes, with many other micro-organisms, the surface of grape berries. In the past, NS yeasts were primarily considered as spoilage micro-organisms. However, recent studies have established a positive contribution of certain NS yeasts to wine quality. Amongst the group of NS yeasts, Brettanomyces bruxellensis, which is not prevalent on wine grapes, plays an important part in the evolution of wine aroma. Some of their secondary metabolites, namely volatile phenols, are responsible for wine spoilage. The other group contributing to wine aroma, which is also the main agent of alcoholic fermentation (AF), is composed of Saccharomyces species. The fermenting must is a complex microbial ecosystem where numerous yeast strains grow and die according to their adaptation to the medium. Yeast-yeast interactions occur during winemaking right from the onset of AF. The aim of this study was to describe the interactions between B. bruxellensis, other NS and Saccharomyces cerevisiae during laboratory and practical scale winemaking. METHODS AND RESULTS: Molecular methods such as internal transcribed spacer-restriction fragment length polymorphism and polymerase chain reaction and denaturing gradient gel electrophoresis were used in laboratory scale experiments and cellar observations. The influence of different oenological practices, like the level of sulphiting at harvest time, cold maceration preceding AF, addition of commercial active dry yeasts on B. bruxellensis and other yeast interactions and their evolution during the initial stages of winemaking have been studied. Brettanomyces bruxellensis was the most adapted NS yeast at the beginning of AF, and towards the end of AF it appeared to be more resistant than S. cerevisiae to the conditions of increased alcohol and sugar limitation. CONCLUSIONS: Among all NS yeast species, B. bruxellensis is better adapted than other wild yeasts to resist in must and during AF. Moreover, B. bruxellensis appeared to be more tolerant to ethanol stress than S. cerevisiae and after AF B. bruxellensis was the main yeast species in wine. SIGNIFICANCE AND IMPACT OF THE STUDY: Brettanomyces bruxellensis interacts with other yeast species and adapts to the wine medium as the dominant yeast species at the end of AF. Contamination of B. bruxellensis might take place at the beginning of malolactic fermentation, which is a critical stage in winemaking.     

Chemical signaling and insect attraction is a conserved trait in yeasts

Yeast volatiles attract insects, which apparently is of mutual benefit, for both yeasts and insects. However, it is unknown whether biosynthesis of metabolites that attract insects is a basic and general trait, or if it is specific for yeasts that live in close association with insects. Our goal was to study chemical insect attractants produced by yeasts that span more than 250 million years of evolutionary history and vastly differ in their metabolism and lifestyle. We bioassayed attraction of the vinegar fly Drosophila melanogaster to odors of phylogenetically and ecologically distinct yeasts grown under controlled conditions. Baker's yeast Saccharomyces cerevisiae, the insect‐associated species Candida californica, Pichia kluyveri and Metschnikowia andauensis, wine yeast Dekkera bruxellensis, milk yeast Kluyveromyces lactis, the vertebrate pathogens Candida albicans and Candida glabrata, and oleophilic Yarrowia lipolytica were screened for fly attraction in a wind tunnel. Yeast headspace was chemically analyzed, and co‐occurrence of insect attractants in yeasts and flowering plants was investigated through a database search. In yeasts with known genomes, we investigated the occurrence of genes involved in the synthesis of key aroma compounds. Flies were attracted to all nine yeasts studied. The behavioral response to baker's yeast was independent of its growth stage. In addition to Drosophila, we tested the basal hexapod Folsomia candida (Collembola) in a Y‐tube assay to the most ancient yeast, Y. lipolytica, which proved that early yeast signals also function on clades older than neopteran insects. Behavioral and chemical data and a search for selected genes of volatile metabolites underline that biosynthesis of chemical signals is found throughout the yeast clade and has been conserved during the evolution of yeast lifestyles. Literature and database reviews corroborate that yeast signals mediate mutualistic interactions between insects and yeasts. Moreover, volatiles emitted by yeasts are commonly found also in flowers and attract many insect species. The collective evidence suggests that the release of volatile signals by yeasts is a widespread and phylogenetically ancient trait, and that insect–yeast communication evolved prior to the emergence of flowering plants. Co‐occurrence of the same attractant signals in yeast and flowers suggests that yeast‐insect communication may have contributed to the evolution of insect‐mediated pollination in flowers.     

Genomic characterization and selection of wine yeast to conduct industrial fermentations of a white wine produced in a SW Spain winery

Aims: To analyse the diversity of wild yeast in spontaneous fermentations of a white wine and to select the most suitable autochthonous starter yeasts. The selected yeasts would be used for inoculation of industrial fermentations in several years. Methods and Results: Yeasts were characterized by applying electrophoretic karyotyping. This technique was chosen because it can reveal the large‐scale mutations in the yeast genome induced by gross chromosomal rearrangements. This type of mutation is considered one of the main forces behind the rapid evolution of industrial yeasts. A heterogeneous population of yeast strains was observed in the spontaneous fermentations during two consecutive years. Four of the most abundant strains were isolated and tested for microbiological features of industrial importance. The selected autochthonous strains were used as starter yeasts for the following 7 years. In the majority of these experiences, we obtained homogeneous yeast populations, in which the karyotype of one of the inoculated strains – karyotype V – emerged as clearly dominant. Conclusions: The inoculation of the selected strain with karyotype V and a proper handling of the inoculum scaling‐up process led to the substitution of the spontaneous fermentations by controlled fermentations producing a highly satisfactory final product. Significance and Impact of the Study: We monitored the wine yeast population of an industrial system for a total of 9 years. Our work is one of the first examples made at industrial scale showing how molecular techniques can be successfully applied to improve the efficiency of the winemaking process.     

Remanence and survival of commercial yeast in different ecological niches of the vineyard

The use of commercial wine yeast strains as starters has been grown extensively over the past three decades. Wine yeasts are annually released in winery environments; however, little is known about the fate of these strains in the vineyard. To evaluate the industrial starter yeasts' ability to survive in nature and become part of the natural microbiota of musts, commercial yeast was disseminated voluntarily in an experimental vineyard in the Madrid region (Spain). A large sampling plan was devised over 3 years, including samples of grapes, leaves, bark and soil. The disseminated yeast was well represented in the vineyard during the first 8 months. After 2 years, the commercial yeast strain had not survived in the sprayed plants, but a residual population was found in plants situated 50 m east of the sprayed area. After 3 years, commercial yeast disseminated was not found in the sampled vineyard. Grapes and soil showed the highest number of yeasts isolated in the vegetative period, the bark being the main natural reservoir during the resting stages. The result of analysis of population variations from year to year indicated that permanent implantation of commercial strain (K1M) in the vineyard did not occur and its presence was limited in time.     

Isolation of Marine Yeasts Collected from the Pacific Ocean Showing a High Production of γ-Aminobutyric Acid

Marine yeasts were collected from coastal and deep sea areas in the Pacific Ocean and the Sea of Japan around central and northern Japan to prepare a novel type of natural seasoning. It was found that one of the marine yeasts collected from the Pacific Ocean off Hachinohe showed a high concentration of γ-aminobutyric acid (GABA) in its extract, about 7–10 times higher than those of commercially available bread yeast and other marine yeasts. The marine yeast isolated and named Hachinohe No. 6 catalyzed the reaction from monosodium glutamate to GABA only in the presence of glucose. Subsequently, several marine yeasts belonging to the genera Pichia and Candida were found to have such catalytic activities, but not those belonging to the genus Saccharomyces. Isolate Hachinohe No. 6 was found to have the highest catalytic activity among the yeasts examined in this study.     

Prevention of yeast spoilage in feed and food by the yeast mycocin HMK

The yeast Williopsis mrakii produces a mycocin or yeast killer toxin designated HMK; this toxin exhibits high thermal stability, high pH stability, and a broad spectrum of activity against other yeasts. We describe construction of a synthetic gene for mycocin HMK and heterologous expression of this toxin in Aspergillus niger. Mycocin HMK was fused to a glucoamylase protein carrier, which resulted in secretion of biologically active mycocin into the culture media. A partial purification protocol was developed, and a comparison with native W. mrakii mycocin showed that the heterologously expressed mycocin had similar physiological properties and an almost identical spectrum of biological activity against a number of yeasts isolated from silage and yoghurt. Two food and feed production systems prone to yeast spoilage were used as models to assess the ability of mycocin HMK to act as a biocontrol agent. The onset of aerobic spoilage in mature maize silage was delayed by application of A. niger mycocin HMK on opening because the toxin inhibited growth of the indigenous spoilage yeasts. This helped maintain both higher lactic acid levels and a lower pH. In yoghurt spiked with dairy spoilage yeasts, A. niger mycocin HMK was active at all of the storage temperatures tested at which yeast growth occurred, and there was no resurgence of resistant yeasts. The higher the yeast growth rate, the more effective the killing action of the mycocin. Thus, mycocin HMK has potential applications in controlling both silage spoilage and yoghurt spoilage caused by yeasts.     

Yeast killer toxins,molecular mechanisms of their action and their applications

Killer toxins secreted by some yeast strains are the proteins that kill sensitive cells of the same or related yeast genera. In recent years, many new yeast species have been found to be able to produce killer toxins against the pathogenic yeasts, especially Candida albicans. Some of the killer toxins have been purified and characterized, and the genes encoding the killer toxins have been cloned and characterized. Many new targets including different components of cell wall, plasma membrane, tRNA, DNA and others in the sensitive cells for the killer toxin action have been identified so that the new molecular mechanisms of action have been elucidated. However, it is still unknown how some of the newly discovered killer toxins kill the sensitive cells. Studies on the killer phenomenon in yeasts have provided valuable insights into a number of fundamental aspects of eukaryotic cell biology and interactions of different eukaryotic cells. Elucidation of the molecular mechanisms of their action will be helpful to develop the strategies to fight more and more harmful yeasts.     

Candida kazuoi sp. nov. and Candida hasegawae sp. nov., two new species of ascomycetous anamorphic yeasts isolated from insect frass in Thailand

Two strains of anamorphic yeasts isolated from insect frass collected in southern Thailand were assigned to the genus Candida based on the conventional taxonomic criteria used for yeast classification. In the phylogenetic tree based on the D1/D2 domain of the 26S rDNA, these strains are distant from the known species of yeasts and considered to represent two different new species. They are named Candida kazuoi sp. nov. and Candida hasegawae sp. nov.     

Prevention of Yeast Spoilage in Feed and Food by the Yeast Mycocin HMK

The yeast Williopsis mrakii produces a mycocin or yeast killer toxin designated HMK; this toxin exhibits high thermal stability, high pH stability, and a broad spectrum of activity against other yeasts. We describe construction of a synthetic gene for mycocin HMK and heterologous expression of this toxin in Aspergillus niger. Mycocin HMK was fused to a glucoamylase protein carrier, which resulted in secretion of biologically active mycocin into the culture media. A partial purification protocol was developed, and a comparison with native W. mrakii mycocin showed that the heterologously expressed mycocin had similar physiological properties and an almost identical spectrum of biological activity against a number of yeasts isolated from silage and yoghurt. Two food and feed production systems prone to yeast spoilage were used as models to assess the ability of mycocin HMK to act as a biocontrol agent. The onset of aerobic spoilage in mature maize silage was delayed by application of A. niger mycocin HMK on opening because the toxin inhibited growth of the indigenous spoilage yeasts. This helped maintain both higher lactic acid levels and a lower pH. In yoghurt spiked with dairy spoilage yeasts, A. niger mycocin HMK was active at all of the storage temperatures tested at which yeast growth occurred, and there was no resurgence of resistant yeasts. The higher the yeast growth rate, the more effective the killing action of the mycocin. Thus, mycocin HMK has potential applications in controlling both silage spoilage and yoghurt spoilage caused by yeasts.     

Effects of culture conditions on the in vitro infection of fibroblasts by Candida albicans.

The effects of yeast culture age, carbon source, growth temperature, and germ-tube inducers on adherence to primary fibroblast cultures was studied in conjunction with the determination of adherence-mediated mammalian cell damage by measuring chromium-51 release from fibroblast monolayers. The results indicated that yeast culture age affected adherence only when the yeasts were grown at 37 degrees C, not after growth at 28 degrees C. At 37 degrees C, quantitatively fewer exponential-phase, glucose- or galactose-grown yeasts adhered to fibroblasts than did yeasts that were in lag or stationary phases. The reduced adherence correlated with less chromium-51 release and reduced germ-tube formation. The addition of germ-tube inducers, such as N-acetyl-D-glucosamine or serum, to exponential-phase yeasts caused an increase in germ-tube formation with a concomitant increase in yeast adherence and release of chromium-51 from the monolayers. Exponential-phase galactose-grown yeasts were more responsive to serum-induced germ-tube formation, germ-tube elongation, and fibroblast adherence than were exponential-phase glucose-grown yeasts. In addition, exponential-phase galactose-grown yeasts caused more chromium-51 release from monolayers in the presence of serum than did glucose-grown yeasts. Overall, conditions that enhanced germ-tube formation and elongation resulted in greatest adherence-mediated damage to the monolayers.     

Alteration of cell wall structure in Saccharomyces cerevisiae and Saccharomyces bayanus during autolysis

Summary After culture in a synthetic and in a wine medium, the autolysis of Saccharomyces cerevisiae and Saccharomyces bayanus produced typical cell wall alterations depending on the yeast growth conditions. After growth in a wine medium, cell wall thickness did not change in either of the two yeasts even when there is an important loss of amino acids and glucans. This loss of wall material and especially of glucan involved a slackening of wall structures. The thickness of cell wall of yeast grown in a synthetic medium decreased by 50% after autolysis. This change was the consequence of a loss of amino acids and sugars which more specifically were constituents of the peripheral layer of the wall.     

Potentially pathogenic yeast isolated from the throat and cloaca of an Arctic colonial seabird: the little auk (Alle alle)

Yeasts are a distinctive group of microfungi, but compared to other microorganisms, their ecological function and biodiversity are poorly known. This is especially so where polar ecosystems are concerned. With climate changes and increasing pollution levels in the Arctic, it can be anticipated that there will be an increase in the prevalence and diversity of fungi colonizing live organisms. With these changes, it is crucial to investigate and monitor species diversity and prevalence of fungi in this fragile environment. In this study, yeasts were examined from throat and cloaca of a small colonial seabird, the little auk (Alle alle), a keystone species in the Arctic ecosystem. Samples were collected from 94 adults and 17 nestlings in breeding colony in Magdalenefjorden (NW Spitsbergen) in 2009. In total, twelve species of yeast from eight genera were found in 12 % of the samples, with the Dipodascus genus being the most prevalent. All yeast species were found in the adults, but only one species, Cryptococcus macerans, was found in a single nestling. In individuals where fungus was isolated, it was only isolated from either the throat or the cloaca, except for two cases, where fungus was found in both throat and cloaca. The presence of yeast was not related to sex but age of the birds, with adults being more prone to colonization by yeasts than the nestlings. The relatively low prevalence and diversity of yeast in little auks suggest that these birds are random carriers of fungi, with minor health impacts.     

Herman Jan Phaff: professor,mentor, friend and colleague

Herman Jan Phaff, the father of yeast ecology, was born in the Netherlands in 1913. In his early years, he spent much time in his family's winery, which sparked his interest in microbes. Trained in the famous Delft tradition, Phaff discovered many unrecognized ecological niches of yeast, such as shellfish, rabbit stomach, frass of bark beetles, tree exudates, cactus roots, Capri figs, sewage, Drosophila flies and shrimp. He is also remembered for his pioneering work on the coevolution of yeasts, insects and plants as well as for his work on yeast -glucanase, which resulted in major advances in the understanding of the nature of the yeast cell wall. Phaff's legacy includes research on pectin degradation by fungal enzymes and the application of molecular approaches to yeast systematics. He discovered and described many yeasts, such as the yeast named in his honor, Phaffia rhodozyma, which led to the establishment of a very important industrial fermentation process yielding high concentrations of the pigment astaxanthin, used throughout the world to provide a natural source of this important carotenoid.     

石河子不同树龄蟠桃土壤中可培养酵母多样性分析及功能酵母的筛选

为了探究新疆本土蟠桃园可培养酵母菌多样性,并挖掘功能酵母资源,本研究以新疆石河子蟠桃园3年、8年、15年树龄的根际和非根际土壤以及桃树叶片为材料,经过传统的分离培养方法获得可培养的酵母菌菌株,并进行形态学、生理生化以及26S r DNA的D1/D2区序列分析,共获得可培养酵母菌129株,从属于12个属17个种,其中子囊菌酵母为优势菌群,占分离属的88%,分布于威克汉姆酵母属(Wickerhamomyces),Vanrija属,Barnettozyma属和有孢圆酵母属(Torulaspora)等11个属的15个种。担子菌占分离属的12%,分布于隐球酵母属(Cryptococcus)的2个种。其中优势属威克汉姆酵母属,包括异常威克汉姆酵母(W. anomalus)和W. pijperi两个种,占总比例的33%,优势种异常威克汉姆酵母所占总株数比例为17%。从可培养酵母中共筛选出23株功能酵母,其中富硒酵母21株,优势种为白地霉(Galactomyces candidum),产蛋白酶酵母2株均属于隐球酵母属的Cryptococcus albidus。结果表明,新疆桃园中蕴含丰富的酵母菌资源,非根际土壤中的酵母多样性大于根际及叶片酵母多样性,且分离得到富硒酵母及产蛋白酶酵母。本研究挖掘了新疆本土可培养酵母菌资源,同时也为功能酵母的开发和利用提供理论指导。     

Psychrophilic yeasts in glacial environments of Alpine glaciers

The presence of psychrophilic yeasts in supra- and subglacial sediments, ice and meltwater collected from two glaciers of the Italian Alps (Forni and Sforzellina-Ortles-Cevedale group) was investigated. After incubation at 4 degrees C, subglacial sediments contained from 1.3 x 10(3) to 9.6 x 10(3) CFU of yeasts g(-1). The number of yeast cells in supraglacial sediments was c. 10-100-fold lower. A significant proportion of isolated yeasts exhibited one or more extracellular enzymatic activities (starch-degrading, lipolytic, esterolytic, proteolytic and pectinolytic activity) at 4 degrees C. Selected isolates were able to grow at 2 degrees C under laboratory-simulated in situ conditions. In all, 106 isolated yeasts were identified by MSP-PCR fingerprinting and 26S rRNA gene sequencing of the D1/D2 region as belonging to 10 species: Aureobasidium pullulans, Cryptococcus gilvescens (over 50% of the total), Cryptococcus terricolus, Mrakia gelida, Naganishia globosa, Rhodotorula glacialis, Rhodotorula psychrophenolica, Rhodotorula bacarum, Rhodotorula creatinivora and Rhodotorula laryngis. Four strains, all belonging to a new yeast species, yet to be described, were also isolated.     

High-Gravity Brewing: Effects of Nutrition on Yeast Composition, Fermentative Ability, and Alcohol Production

A number of economic and product quality advantages exist in brewing when high-gravity worts of 16 to 18% dissolved solids are fermented. Above this level, production problems such as slow or stuck fermentations and poor yeast viability occur. Ethanol toxicity has been cited as the main cause, as brewers' yeasts are reported to tolerate only 7 to 9% (vol/vol) ethanol. The inhibitory effect of high osmotic pressure has also been implicated. In this report, it is demonstrated that the factor limiting the production of high levels of ethanol by brewing yeasts is actually a nutritional deficiency. When a nitrogen source, ergosterol, and oleic acid are added to worts up to 31% dissolved solids, it is possible to produce beers up to 16.2% (vol/vol) ethanol. Yeast viability remains high, and the yeasts can be repitched at least five times. Supplementation does not increase the fermentative tolerance of the yeasts to ethanol but increases the length and level of new yeast cell mass synthesis over that seen in unsupplemented wort (and therefore the period of more rapid wort attenuation). Glycogen, protein, and sterol levels in yeasts were examined, as was the importance of pitching rate, temperature, and degree of anaerobiosis. The ethanol tolerance of brewers' yeast is suggested to be no different than that of sake or distillers' yeast.     

微紫青霉菌(Penicillium janthinellum)P-type ATPase及金属硫蛋白相关基因的克隆

本研究以高抗多种重金属盐的微紫青霉菌(Penicillium janthinellum)菌株GXCR为材料构建基因组fosmid文库。其插入片段集中在36～50kb,含13348个克隆,重组率为100%,大约覆盖了GXCR基因组的14.83倍。基于序列特异性和简并引物,利用PCR扩增分析了与酿酒酵母(Saccharomyces cerevisiae)重金属盐抗性相关的CRS5和CUP2基因;基于兼并引物和序列特异性引物,利用PCR扩增分析了GXCR的P-type ATPase基因。通过菌落原位杂交和Southern blot鉴定了一个含铜转运P-type ATPase基因的阳性fosmid克隆,经亚克隆测序分析表明该基因与棒曲霉(Aspergillus clavatus菌株)NRRL1的P-type copper ATPase相似性达97%。没有筛选到与CRS5和CUP2基因同源的克隆,说明GXCR中可能不存在与酿酒酵母CUP2和CRS5高度同源的MT基因,同时也暗示酵母与丝状真菌的重金属盐的抗性机制有本质上的差异或者独特性。     

Engineering of non-conventional yeasts for efficient synthesis of macromolecules: the methylotrophic genera

Methylotrophic yeasts, named after their ability to grow on methanol as the sole carbon source, have raised large interest as recombinant protein factories. In this review, we explain the basic mechanisms underlying this interest and describe the minimal requirements to transform the two genera recognized as methylotrophic, Pichia and Candida, into a powerful protein production tool. We present a comparison between this group of yeasts and the conventional yeasts used as expression system in view of productivity, level of secretion and quality of post-translational modifications. Selected examples of recombinant protein produced by methylotrophic yeast are also included.     

Ultrastructural investigation of acidocalcisomes and ATPase activity in yeast Candida guilliermondii NP-4 as ‘complementary’ stress-targets

As a result of electron microscopic studies of morphogenesis in yeast Candida guilliermondii NP-4, the formation of new structures of volutin acidocalcisomes has been established within the cell cytoplasm. Under influence of X-irradiation, the changes in morphometric and electron-dense properties of yeast cells were identified: in yeast cytoplasm, the electron-dense volutin granules were increased up to 400 nm in size. After 24-h post-irradiation incubation of yeasts, the large volutin pellets are fragmented into smaller number particles in size up to 25–150 nm. The ATPase activity in yeast mitochondria was changed under X-irradiation. In latent phase of growth, ATPase activity was decreased 1·35-fold in comparison with non-irradiated yeasts. In logarithmic phase of growth, ATPase activity was three times higher than in latent phase, and in stationary phase of growth it has a value similar to the latent phase. Probably, the cells receive the necessary energy from alternative energy sources, such as volutin. Electron microscopy of volutin granule changes might serve as convenient method for evaluation of damages and repair processes in cells under influence of different environmental stress-factors.     

Extracellular polymer of Candida albicans: isolation, analysis and role in adhesion

Extracellular polymeric material (EP) was isolated from culture supernatants of Candida albicans grown on carbon sources (50 mM-glucose, 500 mM-sucrose or 500 mM-galactose) known to promote yeast adhesion to different extents. Galactose-grown yeasts, which are the most adherent, produced more EP than sucrose-grown organisms, particularly after incubation for 5 d, while glucose-grown yeasts (the least adherent) gave the lowest yield. EP produced on all three carbon sources was of similar composition and contained carbohydrate (65 to 82%; mannose with some glucose), protein (7%), phosphorus (0.5%) and glucosamine (1.5%). Serological studies indicated that these EP preparations were immunologically identical but that galactose-grown yeasts had more antigenic determinants than sucrose-grown organisms while glucose-grown yeasts had the fewest determinants. Antigenic differences were apparent between EP preparations of some strains of C. albicans. Pretreatment of acrylic strips with EP to form a polymeric coating promoted yeast adhesion to the acrylic surface, but similar pretreatment of buccal epithelial cells with EP inhibited subsequent yeast adhesion. These results indicate that EP originates from the cell surface of C. albicans and that it contains the surface component(s), probably mannoprotein in nature, responsible for yeast adhesion.