Identification of European isolates of the lager yeast parent Saccharomyces eubayanus.

Lager brewing first occurred in Bavaria in the 15th century, associated with restrictions of brewing to colder months. The lager yeast, Saccharomyces pastorianus, is cold tolerant. It is a hybrid between Saccharomyces cerevisiae and Saccharomyces eubayanus, and has been found only in industrial settings. Natural isolates of S. eubayanus were first discovered in Patagonia 11 years ago. They have since been isolated from China, Tibet, New Zealand, and North America, but not from Europe. Here, we describe the first European strains UCD646 and UCD650, isolated from a wooded area on a university campus in Dublin, Ireland. We generated complete chromosome level assemblies of both genomes using long- and short-read sequencing. The UCD isolates belong to the Holarctic clade. Genome analysis shows that isolates similar to the Irish strains contributed to the S. eubayanus component of S. pastorianus, but isolates from Tibet made a larger contribution.

Evaluation of cryotolerant yeasts for the elaboration of a fermented pear beverage in Patagonia: Physicochemical and sensory attributes.

The production of pome fruits as pears and apples, as well as their derived industries, is of great economic importance in North Patagonia. The elaboration of fermented beverages as cider or perry has evidenced a substantial diversification during the last years, with the evaluation of different fruit varieties, yeast starters and technological changes. In this work, two cryotolerant yeasts belonging to the species Saccharomyces uvarum were evaluated at laboratory and pilot scale in sterile and no-sterile pear must. One of the strains was originally isolated from apple chicha (strain NPCC1314) and the other from apple cider (strain NPCC1420) in Patagonia. Both physicochemical and sensory features of the fermented products were evaluated. Both strains were able to successfully complete the fermentations, although strain NPCC1420 showed the better kinetic properties including a faster sugar consumption than the strain NPCC1314. Both strains showed excellent implantation capacity, but the fermented products showed different chemical profiles. The perry fermented with the strain NPCC1314 was characterized by better sensory attributes as assessed by trained panelists and a greater acceptance for untrained public than the same fermented with the strain NPCC1420. The two strains were able to consume sorbitol, both in pear must and in agar-plates supplemented with sorbitol as the sole carbon source. This ability is described for the first time in S. uvarum, at least for the two strains evaluated in this work.

Assessment of quorum sensing effects of tyrosol on fermentative performance by chief ethnic fermentative yeasts from northeast India.

AIM: Tyrosol, a quorum sensing molecule in yeasts, was reported to reduce lag phase and induces hyphae formation during cell proliferation. However, evidence of any enhancing effect of tyrosol in cellular proliferation within fermentative environment is unclear. In this investigation, selected yeast cells were assessed for their ability to synthesize tyrosol followed by examining the role of the molecule during fermentation. METHODS AND RESULTS: Tyrosols were characterized in four fermentative yeasts viz., Saccharomyces cerevisiae, Wickerhamomyces anomalus, Candida glabrata and Candida tropicalis isolated from traditional fermentative cakes of northeast India. All the isolates synthesized tyrosol while C. tropicalis exhibited filamentous growth in response to tyrosols retrieved from other isolates. Purified tyrosols showed protective behaviour in C. tropicalis and S. cerevisiae under ethanol mediated oxidative stress. During fermentation, tyrosol significantly enhanced growth of W. anomalus in starch medium while C. tropicalis exhibited growth enhancement in starch and glucose sources. The chief fermentative yeast S. cerevisiae showed notable enhancement in fermentative capacity in starch medium under the influence of tyrosol con-commitment of ethanol production. CONCLUSION: The study concludes that tyrosol exerts unusual effect in cellular growth and fermentative ability of both Saccharomyces and non-Saccharomyces yeasts. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of expression of tyrosol by non-conventional yeasts, where the molecule was found to exert enhancing effect during fermentation, thereby augmenting the process of metabolite production during traditional fermentation.

Elevated minimum inhibitory concentrations to antifungal drugs prevail in 14 rare species of candidemia-causing Saccharomycotina yeasts.

Antifungal susceptibility profiles of rare Saccharomycotina yeasts remain missing, even though an increase in prevalence of such rare Candida species was reported in candidemia. Majority of these rare yeast species carry intrinsic resistances against at least one antifungal compound. Some species are known to be cross-resistant (against multiple drugs of the same drug class) or even multi-drug resistant (against multiple drugs of different drug classes). We performed antifungal susceptibility testing (AFST) according to EUCAST broth microdilution for 14 rare species (Clavispora lusitaniae, Candida intermedia, Candida auris, Diutina rugosa, Wickerhamiella pararugosa, Yarrowia lipolytica, Pichia norvegensis, Candida nivariensis, Kluyveromyces marxianus, Wickerhamomyces anomalus, Candida palmioleophila, Meyerozyma guilliermondii, Meyerozyma caribbica, and Debaryomyces hansenii) known to cause candidemia. In total, 234 isolates were tested for amphotericin B, fluconazole, itraconazole, voriconazole, posaconazole, anidulafungin, micafungin, and caspofungin. Amphothericin B had the broadest efficiency against the 14 tested rare yeast species, while high minimum inhibitory concentrations (MICs) against azole drugs and echinocandins were common. Voriconazole was the most efficient azole drug. Multidrug resistance was observed for the species C. auris and K. marxianus. Multidrug resistant individual isolates were found for Y. lipolytica and M. caribbica. In conclusion, the observed high MIC values of the rare Saccharomycotina species tested limit antifungal treatment options, complicating the management of such infections.

Competition experiments in a soil microcosm reveal the impact of genetic and biotic factors on natural yeast populations.

The ability to measure microbial fitness directly in natural conditions and in interaction with other microbes is a challenge that needs to be overcome if we want to gain a better understanding of microbial fitness determinants in nature. Here we investigate the influence of the natural microbial community on the relative fitness of the North American populations SpB, SpC and SpC* of the wild yeast Saccharomyces paradoxus using DNA barcodes and a soil microcosm derived from soil associated with oak trees. We find that variation in fitness among these genetically distinct groups is influenced by the microbial community. Altering the microbial community load and diversity with an irradiation treatment significantly diminishes the magnitude of fitness differences among populations. Our findings suggest that microbial interactions could affect the evolution of yeast lineages in nature by modulating variation in fitness.

Probiotic potential of Saccharomyces strains isolated from Litchi chinensis (Lychee fruit).

Recently, probiotic yeasts have become an interesting topic of research all over the world. Saccharomyces cerevisiae is well proven probiotic yeast against several gastrointestinal diseases. Current study aimed to explore the probiotic potential and antibacterial properties of Saccharomyces strains isolated from fresh lychee fruits available in local markets of Karachi, Pakistan. Probiotic potential and antibacterial activity of locally isolated probiotic yeast strains (named as S. cerevisiae BEL 1 and S. cerevisiae BEL 9) was studied against gastrointestinal pathogens using standard in vitro screening methods. Comparative analysis was also carried out with commercially available S. boulardii probiotic preparations. Furthermore, for probiotic potential, all the studied yeast strains were exposed to various stress conditions inherent of gastrointestinal tract i.e., thermo tolerance, pH tolerance, bile salts survivability and osmo-tolerance. Isolated strains (BEL 1 and BEL 9) were able to tolerate at the temperatures (40oC and 45oC), moreover survived in the presence of gastric juices, extreme bile salt concentrations (range 0.5%-2%) and different osmotic stress conditions (1M and 1.5 M NaCl). Optimal growth was observed at 37oC. Similar growth pattern and viability of BEL 1 and 9 was found for most of the stress conditions, when compared with the commercially available strains of S. boulardii. Therefore, isolated yeast strains BEL 1 and 9 will be considered as a potential bio-therapeutic agent because of the promising probiotic potential.

Saccharomyces uvarum isolated from patagonian ciders shows excellent fermentative performance for low temperature cidermaking.

Saccharomyces uvarum has been recovered from natural habitats and traditionally fermented beverages (apple chicha) in Patagonia. However, this species has never been obtained from industrially relevant beverages like wine or cider in the same region. In this work, different strains belonging to the cryotolerant species S. uvarum were recovered from spontaneous cider fermentations carried out at low temperature in Red Delicious apple must. The strain S. uvarum NPCC1420 obtained from this cider and selected for its physiological and technological features, evidenced a better adaptation to the cidermaking process than a previously selected strain obtained from a less industrialized product called apple chicha. Some differences, like a higher ethanol and sulphite tolerance, seemed to be associated with differential domestication pressures suffered by each different strain. Moreover, the most important fermentative features of the strain NPCC1420 were a higher competition capacity than the strain NPCC1314 in non-sterile apple must, as well as significantly higher amounts of glycerol, 2-phenylethanol and 2-phenylethyl acetate than the strain isolated from apple chicha.

A new, rapid multiplex PCR method identifies frequent probiotic origin among clinical Saccharomyces isolates.

An increasing number of infections originating from probiotic use are reported worldwide, with the majority of such cases caused by the yeast Saccharomyces 'boulardii', a subtype of S. cerevisiae. Reliably linking infectious cases to probiotic products requires unequivocal genotyping data, however, these techniques are often time-consuming and difficult to implement in routine diagnostics. This leads to a widespread lack of genetic data regarding the origin of Saccharomyces infections. We propose a quick and reliable PCR-based protocol for the identification of S. 'boulardii' based on a combined analysis of interdelta fingerprinting and microsatellite typing. By applying various typing methods and our proposed method to the clinical yeast collection of a Hungarian hospital we show that probiotic origin is common among clinical Saccharomyces, and that the new multiplex method enables rapid and unequivocal identification of probiotic yeast infections. This method can be applied for the identification of yeast infection sources, helping decisions on probiotic use.

Quantifying the efficiency and biases of forest Saccharomyces sampling strategies.

Saccharomyces yeasts are emerging as model organisms for ecology and evolution, and researchers need environmental Saccharomyces isolates to test ecological and evolutionary hypotheses. However, methods for isolating Saccharomyces from nature have not been standardized, and isolation methods may influence the genotypes and phenotypes of studied strains. We compared the effectiveness and potential biases of an established enrichment culturing method against a newly developed direct plating method for isolating forest floor Saccharomyces spp. In a European forest, enrichment culturing was both less successful at isolating Saccharomyces paradoxus per sample collected and less labour intensive per isolated S. paradoxus colony than direct isolation. The two methods sampled similar S. paradoxus diversity: The number of unique genotypes sampled (i.e., genotypic diversity) per S. paradoxus isolate and average growth rates of S. paradoxus isolates did not differ between the two methods, and growth rate variances (i.e., phenotypic diversity) only differed in one of three tested environments. However, enrichment culturing did detect rare Saccharomyces cerevisiae in the forest habitat and also found two S. paradoxus isolates with outlier phenotypes. Our results validate the historically common method of using enrichment culturing to isolate representative collections of environmental Saccharomyces. We recommend that researchers choose a Saccharomyces sampling method based on resources available for sampling and isolate screening. Researchers interested in discovering new Saccharomyces phenotypes or rare Saccharomyces species from natural environments may also have more success using enrichment culturing. We include step-by-step sampling protocols in the supplemental materials.

Evolutionary journey and characterisation of a novel pan-gene associated with beer strains of Saccharomyces cerevisiae.

The sequencing of over a thousand Saccharomyces cerevisiae genomes revealed a complex pangenome. Over one third of the discovered genes are not present in the S. cerevisiae core genome but instead are often restricted to a subset of yeast isolates and thus may be important for adaptation to specific environmental niches. We refer to these genes as "pan-genes," being part of the pangenome but not the core genome. Here, we describe the evolutionary journey and characterisation of a novel pan-gene, originally named hypothetical (HYPO) open-reading frame. Phylogenetic analysis reveals that HYPO has been predominantly retained in S. cerevisiae strains associated with brewing but has been repeatedly lost in most other fungal species during evolution. There is also evidence that HYPO was horizontally transferred at least once, from S. cerevisiae to Saccharomyces paradoxus. The phylogenetic analysis of HYPO exemplifies the complexity and intricacy of evolutionary trajectories of genes within the S. cerevisiae pangenome. To examine possible functions for Hypo, we overexpressed a HYPO-GFP fusion protein in both S. cerevisiae and Saccharomyces pastorianus. The protein localised to the plasma membrane where it accumulated initially in distinct foci. Time-lapse fluorescent imaging revealed that when cells are grown in wort, Hypo-gfp fluorescence spreads throughout the membrane during cell growth. The overexpression of Hypo-gfp in S. cerevisiae or S. pastorianus strains did not significantly alter cell growth in medium-containing glucose, maltose, maltotriose, or wort at different concentrations.

Strain-level diversity of commercial probiotic isolates of Bacillus, Lactobacillus, and Saccharomyces species illustrated by molecular identification and phenotypic profiling.

Probiotic products are becoming more prevalent as awareness of the role of beneficial microbes in health increases. Ingredient labels of these products often omit identifications at the strain level, making it difficult to track down applicable published research. In this study, we investigated whether products labeled with the same species name contained different strains of those species. From 21 commercially available probiotic supplements and beverages, we cultured five main species: Bacillus coagulans, Bacillus subtilis, Lactobacillus plantarum, Lactobacillus rhamnosus, and the yeast Saccharomyces boulardii. To confirm the identity of each bacterial isolate, we applied standard molecular approaches: 16S rRNA gene sequencing and Matrix Assisted Laser Desorption Ionization Time-of-Flight mass spectrometry (MALDI-TOF MS). Phenotypic profiling and identification were performed with the Biolog Microbial Identification system. All of the bacterial isolates were correctly identified by at least one approach. Sequencing the 16S rRNA gene led to 82% of species identifications matching the product label, with 71% of isolates identified by MALDI-TOF MS and 60% identified correctly with the Biolog system. Analysis of the Biolog phenotypic profiles revealed different patterns of carbon source usage by each species, with sugars preferentially utilized by all except B. subtilis. To assess the strain-level differences, we compared strains of the same species and found variability in carbohydrate utilization and tolerance to environmental stressors (salt, acidity, antibiotics). By demonstrating that products listing the same species often contain strains with different 16S sequences and phenotypes, this study highlights that current labels of probiotic supplements do not sufficiently convey the strain diversity in these products.

Study of bacterial and fungal community structures in traditional koumiss from Inner Mongolia.

Koumiss is notable for its nutritional functions, and microorganisms in koumiss determine its versatility. In this study, the bacterial and fungal community structures in traditional koumiss from Inner Mongolia, China, were investigated. Our results demonstrated that 6 bacterial phyla represented by 126 genera and 49 species and 3 fungal phyla represented by 59 genera and 57 species were detected in 11 samples of artisanal koumiss. Among them, Lactobacillus was the predominant genus of bacterium, and Kluyveromyces and Saccharomyces dominated at the fungal genus level. In addition, there were no differences in the bacterial and fungal richness and diversity of koumiss from 3 neighboring administrative divisions in Inner Mongolia, and the bacterial and fungal community structures (the varieties and relative abundance of bacterial and fungal genera and species) were clearly distinct in individual samples. This study provides a comprehensive understanding of the bacterial and fungal population profiles and the predominant genus and species, which would be beneficial for screening, isolation, and culture of potential probiotics to simulate traditional fermentation of koumiss for industrial and standardized production in the future.

Bioprospecting for brewers: Exploiting natural diversity for naturally diverse beers.

The burgeoning interest in archaic, traditional, and novel beer styles has coincided with a growing appreciation of the role of yeasts in determining beer character as well as a better understanding of the ecology and biogeography of yeasts. Multiple studies in recent years have highlighted the potential of wild Saccharomyces and non-Saccharomyces yeasts for production of beers with novel flavour profiles and other desirable properties. Yeasts isolated from spontaneously fermented beers as well as from other food systems (wine, bread, and kombucha) have shown promise for brewing application, and there is evidence that such cross-system transfers have occurred naturally in the past. We review here the available literature pertaining to the use of nonconventional yeasts in brewing, with a focus on the origins of these yeasts, including methods of isolation. Practical aspects of utilizing nondomesticated yeasts are discussed, and modern methods to facilitate discovery of yeasts with brewing potential are highlighted.

Targeted metagenomics approach to capture the biodiversity of Saccharomyces genus in wild environments.

The species of the genus Saccharomyces are commonly inhabiting tree bark and the surrounding soil, but their abundance have likely been underestimated due to biases in culturing methods. Metagenomic studies have so far been unable to detect Saccharomyces species in wild environments. Here, we sequenced the mycobiome of soils surrounding different trees at various altitudes in the Italian Alps. To survey for yeasts species belonging to Saccharomyces genus rather than other fungal species, we performed a selectivity step involving the isolation of the internal transcribed spacer (ITS) region that is specific to this yeast group. Reads mapping to Saccharomyces species were detected in all soil samples, including reads for S. mikatae and for S. eubayanus. ITS1 alignment of the S. cerevisiae, S. paradoxus and S. kudriavzevii sequences showed up to three base pair polymorphisms with other known strains, indicating possible new lineages. Basidiomycetous fungi were still the dominant species, compared to the Ascomycota, but the selectivity step allowed for the first time the detection and study of the biodiversity of the Saccharomyces species in their natural environment.

Characterization of fungal dysbiosis in Japanese patients with inflammatory bowel disease.

BACKGROUND AND AIMS: There are no previous reports describing the fecal fungal microbiome of a Japanese population using advanced molecular techniques. In this study, we performed a molecular analysis on the fungal microbial community of a healthy Japanese population and patients with inflammatory bowel diseases (IBDs). PATIENTS AND METHODS: Fecal samples were obtained from 18 patients with inactive ulcerative colitis (UC, n = 18), Crohn's disease (CD, n = 20) and healthy volunteers (n = 20). Bacterial and fungal microbiome was analyzed by sequencing of 16S rRNA and the internal transcribed spacer (ITS) region, respectively. RESULTS: 16S rRNA sequencing of the bacterial microbiome revealed that the α-diversity indicated by the Chao-1 and Shannon indices was significantly lower in CD patients compared to healthy controls and/or UC patients. Principal coordinate (PCo) analysis of the bacterial community revealed significant structural differences in microbiome among healthy controls, UC and CD patients (PERMANOVA P = 0.0001). ITS sequencing of the fungal microbiome indicated no significant differences in α-diversity between healthy controls and IBD patients. However, the overall structure of the fungal microbial community of CD patients was significantly different from those of healthy controls and UC patients (PERMANOVA = 0.03). At the genus level, the genus Saccharomyces was dominant and this was followed by the genus Sarocladium in healthy controls. The abundance of the genus Candida was significantly higher in CD patients than healthy controls and/or UC patients. CONCLUSION: The fecal fungal microbiome of a Japanese population differed considerably from that of a Western population. We identified fungal dysbiosis in Japanese patients with IBD.

Fungal bio-sorption potential of chromium in Norkrans liquid medium by shake flask technique.

In this study, the myco-reduction potential of fungi isolated from soil was ascertained by Norkrans shake flask experiment contaminated with chromium(VI). Fungal tolerance assay and induced tolerance training of the fungi were also carried out. Aspergillus niger, Penicillium, and Saccharomyces strains were isolated from the soil samples using culture based technique. Norkrans samples were collected and analyzed for Cr(VI) concentration using diphenylcarbazide spectrophotometric method. Penicillium strain was observed to be most effect at Cr(VI) concentrations of 16.1 and 8.1 mg L-1 since it was able to reduce Cr(VI) more than Saccharomyces strain and A. niger on day 20. Bio-sorption kinetics for this study was better described by pseudo second order model while Langmuir isotherm model fitted better to the equilibrium data. There was virtually steady increase in fungal growth for all the treatments through-out the experimental period. Significant negative correlation (p < 0.05) was observed between fungal growth and Cr(VI) reduction rate. The results from the induced tolerance training showed that Penicillium had the highest tolerance index (TI) values at 18, 20, and 25 mg L-1 concentrations of Cr(VI) compared to A. niger and Saccharomyces strain. These results demonstrated that these fungi have the potential to bio-absorb Cr(VI) and if properly harnessed, could be used in place of conventional remediation technology to clean-up the Cr(VI) contaminant in the field.

Yeast distribution in Grignolino grapes growing in a new vineyard in Piedmont and the technological characterization of indigenous Saccharomyces spp. strains.

The aim of this study was to characterize the yeast consortium isolated from Grignolino grapes in a newly planted vineyard in Piedmont (Italy) via analysis of the intra-vineyard yeast distribution of grape samples from single rows. A two-phase approach allowed the identification of culturable yeasts present on grape skins and, through an enriching procedure via grape fermentation, the isolation of low frequency non-Saccharomyces and Saccharomyces spp. fermentative species, including S. paradoxus, which is highly unusual during grape fermentation, along with the intra-specific characterization of S. cerevisiae isolates. Culture-based molecular techniques revealed a grape yeast microbiota formed by (in order of abundance) Hanseniaspora uvarum, the yeast-like fungus Aerobasidium pullulans, Candida zemplinina, Pichia kluyveri, Candida californica, Curvibasidium cygneicollum, Meyerozima caribbica, Rhodotorula babjevae, Metschnikowia pulcherrima and Cryptococcus flavescens. Technological properties of isolated Saccharomyces spp. strains were analysed, identifying strains, including S. paradoxus, potentially suitable as an ecotypical starter for territorial wines.

Share of the Saccharomyces genus in the mycobiota of the gastrointestinal tract of oncology patients ­ potential effects of a fruit-based diet

This paper concerns the determination of the share of fungi of the Saccharomyces genus in the mycobiota of the gastrointestinal tract of patients with colorectal cancer. The biological material were fungi isolated from the mouth, oesophagus, duodenum, stomach, large intestine and anus of 92 patients with colorectal cancer. They were subjected to standard mycological diagnostic testing. The analysis was only carried out on isolates containing fungi of the Saccharomyces genus. In 140 isolates (24.5% of all positive results), the species S. cerevisiae (68.5%), S. fragilis (5.71%), S. italicus (11.43%), S. ludwigii (8.60%) and S. rouxii (5.76%) were found, which occurred individually or were accompanied species of the Candida, Trichosporon, and Rhodotorula genera. Most isolates were obtained from the large intestine and anus, and the least from the stomach. It follows from the comparison of the obtained results with those of previous studies into the occurrence of fungi of the Saccharomyces genus in various segments of the gastrointestinal tract, that their prevalence in the gastrointestinal tract is gradually increasing.

Phylogeography of the wild Lager-brewing ancestor (Saccharomyces eubayanus) in Patagonia.

Saccharomyces eubayanus is the close relative of the Lager-brewing yeast and was firstly found in North Patagonia associated with Nothofagus trees. In recent years additional strains were found in North America, Asia and New Zealand, and genomic analyses showed the existence of two main populations of this yeast, both of them present in Patagonia. Here, we performed the most comprehensive study of S. eubayanus in Patagonia natural environments (400 samples) and confirmed that this region has the highest isolation success rate for this species described worldwide (more than 10-fold). The genetic characterization of 200 isolates (COX2, DCR1, intFR) revealed five geographically structured subpopulations. We hypothesized that marine ingressions and glaciations, which shaped the Patagonian landscape, contributed on population differentiation. The first large screening of fermentation performance of 60 wild S. eubayanus strains indicated which subpopulations would be more suitable for beer production.