Directed evolution of Saccharomyces cerevisiae for low volatile acidity during winemaking under aerobic conditions.

The use of yeast respiratory metabolism has been proposed as a promising approach to solve the problem of increasing ethanol content in wine, which is largely due to climate change. The use of S. cerevisiae for this purpose is mostly hampered by acetic acid overproduction generated under the necessary aerobic conditions. However, it was previously shown that a reg1 mutant, alleviated for carbon catabolite repression (CCR), showed low acetic acid production under aerobic conditions. In this work directed evolution of three wine yeast strains was performed to recover CCR-alleviated strains, expecting they will also be improved concerning volatile acidity. This was done by subculturing strains on galactose, in the presence of 2-deoxyglucose for around 140 generations. As expected, all evolved yeast populations released less acetic acid than their parental strains in grape juice, under aerobic conditions. Single clones were isolated from the evolved populations, either directly or after one cycle of aerobic fermentation. Only some clones from one of three original strains showed lower acetic acid production than their parental strain. Most clones isolated from EC1118 showed slower growth. However, even the most promising clones failed to reduce acetic acid production under aerobic conditions in bioreactors. Therefore, despite the concept of selecting low acetic acid producers by using 2-deoxyglucose as selective agent was found to be correct, especially at the population level, the recovery of strains with potential industrial utility by this experimental approach remains a challenge.

Production and characterization of anthocyanin-rich beer from black wheat by an efficient isolate Saccharomyces cerevisiae CMS12.

Beer is the world's third most popular fermented beverage. It is typically made from malted barley. Tropical countries must import barley from temperate countries for brewing, which is an expensive process. Therefore, it is critical to investigate alternative possible substrates for beer production in order to meet the growing demand for high-nutritional-quality beer. The current study involves the creation of a fermented beverage from anthocyanin-rich black wheat with the help of yeast, Saccharomyces cerevisiae CMS12, isolated from fruit waste. Characterization (UV, HPLC, NMR, FTIR, and ICPMS) was then performed, as well as a comparative study with white (amber) wheat beer. Further, process parameters optimization included initial sugar concentration, inoculum size, and pH. Black wheat wort contained 568 mg GAE/L total phenolic content, 4.67 mg/L anthocyanin concentration, 6.8% (v/v) alcohol content, and a pH of 4.04. The sensory analysis revealed that black wheat beer was more acceptable than white wheat beer. The developed fermented beverage has enormous commercialization potential.

Morphometric quantification of a pseudohyphae forming Saccharomyces cerevisiae strain using in situ microscopy and image analysis.

Yeast morphology and counting are highly important in fermentation as they are often associated with productivity and can be influenced by process conditions. At present, time-consuming and offline methods are utilized for routine analysis of yeast morphology and cell counting using a haemocytometer. In this study, we demonstrate the application of an in situ microscope to obtain a fast stream of pseudohyphae images from agitated sample suspensions of a Saccharomyces cerevisiae strain, whose morphology in cell clusters is frequently found in the bioethanol fermentation industry. The large statistics of microscopic images allow for online determination of the principal morphological characteristics of the pseudohyphae, including the number of constituent cells, cell-size, number of branches, and length of branches. The distributions of these feature values are calculated online, constituting morphometric monitoring of the pseudohyphae population. By providing representative data, the proposed system can improve the effectiveness of morphological characterization, which in turn can help to improve the understanding and control of bioprocesses in which pseudohyphal-like morphologies are found.

Saccharomyces cerevisiae fungemia: Risk factors, outcome and links with S. boulardii-containing probiotic administration.

Saccharomyces cerevisiae fungemia: risk factors, outcome and links with S. boulardii-containing probiotic administration. OBJECTIVE: The aim of our study was to review cases of S. cerevisiae fungemia along with the corresponding risk factors (including S. boulardii probiotic intake), treatment and outcomes. PATIENTS AND METHODS: Retrospective study (2005-2017) of S. cerevisiae fungemia. All the data were extracted from medical files. RESULTS: We identified 10 patients with S. cerevisiae fungemia. Mean age was 59.4 years (range 21-88). Four fifths (80%) were on total parenteral or enteral nutrition, 70% had a central venous line, and 30% were admitted in an Intensive Care Unit (ICU). S. boulardii-containing probiotic prescription was identified in 6 subjects. Three patients with no risk factors such as ICU or central venous catheter were 80 years old or more. Mortality rate was 50%. CONCLUSION: S. cerevisiae fungemia is a rare but life-threatening infection, associated with intake of probiotics containing S. boulardii. Besides classical risk factors, older age should be a contraindication for these probiotics.

Presence of Saccharomyces cerevisiae subsp. diastaticus in industry and nature and spoilage capacity of its vegetative cells and ascospores.

Saccharomyces cerevisiae sub-species diastaticus (S. diastaticus) is the main fungal cause of spoilage of carbonated fermented beverages in the brewing industry. Here, prevalence of S. diastaticus in nature and breweries was assessed as well as the spoilage capacity of its vegetative cells and spores. S. diastaticus could only be enriched from 1 out of 136 bark and soil samples from the Netherlands, being the first described natural isolate of this yeast outside South America. On the other hand, it was identified by PCR and selective enrichment in 25 and 21 out of 54 biofilm samples from beer filling halls in Asia, Africa, Europe and North America. ITS sequencing revealed that S. cerevisiae (including S. diastaticus) represented <0.05% of fungal DNA in 17 out of 20 samples, while it represented 0.1, 2 and 32% in samples VH6, VH1 and VH3 respectively. Next, vegetative cells and ascospores of the natural S. diastaticus isolate MB523 were inoculated in a variety of beer products containing 0.0-5.0% alcohol (v/v). Ascospores spoiled all beer products, while vegetative cells did not grow in Radler lemon 0.0, Radler lime mint 0.0 and Radler lemon lime 0.0. Notably, vegetative cells could spoil these Radlers when they first had been grown in alcohol free beer either or not mixed with Radler lemon lime 0.0. Conversely, vegetative cells that had been grown in Radler lemon lime lost their spoilage potential of this beer product when they had grown in YPD medium for more than 24 h. In addition, it was shown that cells grown in alcohol free beer were more heat resistant than cells grown in YPD (D52 40 min and ≤ 10.3 min, respectively). Together, these data show that S. diastaticus is a less prevalent variant of S. cerevisiae in nature, while it accumulates in breweries in mixed biofilms. Data also show that both vegetative cells and spores can spoil all tested beer products, the latter cell type irrespective of its environmental history.

An update on water kefir: Microbiology, composition and production.

Water kefir is a sparkling, slightly acidic fermented beverage produced by fermenting a solution of sucrose, to which dried fruits have been added, with water kefir grains. These gelatinous grains are a symbiotic culture of bacteria and yeast embedded in a polysaccharide matrix. Lactic acid bacteria, yeast and acetic acid bacteria are the primary microbial members of the sugary kefir grain. Amongst other contributions, species of lactic acid bacteria produce the exopolysaccharide matrix from which the kefir grain is formed, while yeast assists the bacteria by a nitrogen source that can be assimilated. Exactly which species predominate within the grain microbiota, however, appears to be dependent on the geographical origin of the grains and the fermentation substrate and conditions. These factors ultimately affect the characteristics of the beverage produced in terms of aroma, flavour, and acidity, for example, but can also be controlled and exploited in the production of a beverage of desired characteristics. The production of water kefir has traditionally occurred on a small scale and the use of defined starter cultures is not commonly practiced. However, as water kefir increases in popularity as a beverage - in part because of consumer lifestyle trends and in part due to water kefir being viewed as a health drink with its purported health benefits - the need for a thorough understanding of the biology and dynamics of water kefir, and for defined and controlled production processes, will ultimately increase. The aim of this review is to provide an update into the current knowledge of water kefir.

Rapid isolation method of Saccharomyces cerevisiae based on optically induced dielectrophoresis technique for fungal infection diagnosis.

Saccharomyces cerevisiae(S. cerevisiae) has been classically used as a treatment for diarrhea and diarrhea-related diseases. However, cases of the fungal infections caused by S. cerevisiae have been increasing in the last two decades among immunocompromised patients, while a long time was spent on S. cerevisiae isolation clinically so it was difficult to achieve timely diagnosis the diseases. Here, a novel approach for isolation and selection of S. cerevisiae is proposed by designing a microfluidic chip with an optically induced dielectrophoresis (ODEP) system. S. cerevisiae was isolated from the surroundings by ODEP due to different dielectrophoretic forces. Two special light images were designed and used to block and separate S. cerevisiae, respectively, and several manipulation parameters of ODEP were experimentally optimized to acquire the maximum isolation efficiency of S. cerevisiae. The results on the S. cerevisiae isolation declared that the purity of the S. cerevisiae selected by the method was up to 99.5%±0.05, and the capture efficiency was up to 65.0%±2.5 within 10 min. This work provides a general method to isolate S. cerevisiae as well as other microbial cells with high accuracy and efficiency and paves a road for biological research in which the isolation of high-purity cells is required.

Phenotypic characterization of indigenous Saccharomyces cerevisiae strains associated with sorghum beer and palm wines.

In order to phenotypically characterized Saccharomyces cerevisiae strains isolated from sorghum beer and palm wines for a possible selection of a starter culture, 30 strains were tested for killer activity, temperature resistance, ethanol tolerance, carbohydrate fermentation, enzyme profile and sorghum wort fermentation. Of the tested strains, three showed a killer profile, while four showed a neutral profile and 23 were found to be sensitive to K2 toxin. Temperatures of 40 °C and 44 °C allowed to distinguish strains into four thermal groups with only three strains may grow at 44 °C. Almost tested strains were tolerant to 5% ethanol with viability rates up to 73%. But at 10% and 15% ethanol, respectively 18 and 7 strains were tolerant. Carbohydrate fermentation revealed 13 fermentation profiles, including one typical and 12 atypical profiles. The typical profile strains (16.13% of the strains) fermented glucose, galactose, fructose, sucrose, maltose, trehalose and raffinose. Most of the strains secreted lipases (mainly esterase and esterase-lipase), proteases (mainly valine and cysteine arylamidase, chrymotrypsin) and phosphatases (mainly acid phosphatase and naphthol phosphohydrolase). On contrary, only five strains isolated from sorghum beer exhibited glucosidase activity, mainly α-glucosidase. The analyse of fermented sorghum wort revealed that fermentative performance is strain dependent. Furthermore, the Hierarchical Cluster Analysis showed that the strains were separated in three distinct clusters with the strains from sorghum beer clustered separately.

Distribution and function of dominant yeast species in the fermentation of strong-flavor baijiu.

The role of the yeast community in Chinese strong-flavor baijiu fermentation was investigated by culture-independent and culture-dependent methods based on 26S rDNA sequence analysis. 92 yeast species belonging to 54 genera were found by Illumina sequencing during the fermentation of strong-flavor baijiu "Wuliangye" and 306 strains belonging to 28 species, which covered all species with more than 1% of relative abundance, were isolated and identified. Kazachstania exigua, Geotrichum silvicola, Pichia kudriavzevii, Saccharomyces cerevisiae, Zyosaccharomyces bailii, and K. humilis were found to be the first six dominant species, and their proportion varied with different fermentation stages. K. exigua was the most important yeast species responsible for the production of ethanol with the assistance of P. kudriavzevii and Z. bailii in the vigorous stage and P. kudriavzevii and G. silvicola in the continuous fermentation stage. Higher alcohols were mainly produced by K. exigua, P. kudriavzevii, S. cerevisiae, and Z. bailii in the vigorous fermentation stage, which was stimulated by more oxygen in the grains of the upper strata. K. exigua, P. kudrizevii, S. cerevisiae, Z. bailii, G. silvicola, and Trichosporon ovoides promoted the formation of ethyl alcohol. The results revealed the key roles of K. exigua, G. silvicola, and P. kudriavzevii in the fermentation of strong-flavor baijiu. The functions of these species should be confirmed by a further study based on the fermentation characteristics of isolated yeast strains gathered in this study. Distribution and function of dominant yeast species in the fermentation of strong flavor baijiu.

Phenotypic and genomic differences among S. cerevisiae strains in nitrogen requirements during wine fermentations.

Nitrogen requirements by S. cerevisiae during wine fermentation are highly strain-dependent. Different approaches were applied to explore the nitrogen requirements of 28 wine yeast strains. Based on the growth and fermentation behaviour displayed at different nitrogen concentrations, high and low nitrogen-demanding strains were selected and further verified by competition fermentation. Biomass production with increasing nitrogen concentrations in the exponential fermentation phase was analysed by chemostat cultures. Low nitrogen-demanding (LND) strains produced a larger amount of biomass in nitrogen-limited synthetic grape musts, whereas high nitrogen-demanding (HND) strains achieved a bigger biomass yield when the YAN concentration was above 100 mg/L. Constant rate fermentation was carried out with both strains to determine the amount of nitrogen required to maintain the highest fermentation rate. Large differences appeared in the analysis of the genomes of low and high-nitrogen demanding strains showed for heterozygosity and the amino acid substitutions between orthologous proteins, with nitrogen recycling system genes showing the widest amino acid divergences. The CRISPR/Cas9-mediated genome modification method was used to validate the involvement of GCN1 in the yeast strain nitrogen needs. However, the allele swapping of gene GCN1 from low nitrogen-demanding strains to high nitrogen-demanding strains did not significantly influence the fermentation rate.

In Vitro Assessment of Probiotic Potential of Saccharomyces Cerevisiae DABRP5 Isolated from Bollo Batter, a Traditional Goan Fermented Food.

Bollo is a traditional Goan fermented food in which coarse wheat/wheat and finger millet is leavened with toddy. We here isolated 42 yeast strains from Bollo batter. Initial screening of the isolates with probiotic properties yielded four yeast isolates (DABRP1, DABRP2, DABRP5 and DABRP12). These isolates exhibited tolerance to high bile salt concentration and acidic pH and resistance to various antibiotics, which indicated their probiotic nature. All these yeast isolates were identified as Saccharomyces cerevisiae through D1D2-LSU-rDNA sequencing. These yeast isolates also showed higher percent hydrophobicity towards chloroform followed by n-hexadecane and o-xylene indicating their mucosal surface-adhesive property. To evaluate the safety of the isolates for them to be called as generally recognized as safe, the pathogenic behavior of the isolates determined through the in vitro hemolysis assay and evaluation of DNase and gelatinase activities. None of the isolates exhibited hemolysis or produced DNase or gelatinase and thus were considered potentially safe. In terms of beneficial effects, the most potent isolate S. cerevisiae DABRP5 showed antibacterial activity against the test pathogens. It also showed excellent antioxidant activity with DPPH free radical scavenging activity of 68.85 ± 0.69%, anti-inflammatory activity with 60.39 ± 0.34% inhibition of protein denaturation, and antidiabetic activity with 71.75 ± 0.45% inhibition of α-amylase activity. The isolate produced α-amylase, lipase, and ß-galactosidase. The probiotic potential of the isolate S. cerevisiae DABRP5 was similar to that of the reference strain (Saccharomyces boulardii CNCM I-745) used in this study. The results thus indicate that yeast isolates from Bollo batter have probiotic potential.

Separation and Enrichment of Yeast Saccharomyces cerevisiae by Shape Using Viscoelastic Microfluidics.

Yeast Saccharomyces cerevisiae (S. Cerevisiae) is one of the most attractive microbial species used for industrial production of value-added products and is an important model organism to understand the biology of the eukaryotic cells and humans. S. Cerevisiae has different shapes, such as spherical singlets, budded doublets, and clusters, corresponding to phases of the cell cycle, genetic, and environmental factors. The ability to obtain high-purity populations of uniform-shaped S. Cerevisiae cells is of significant importance for a wide range of applications in basic biological research and industrial processes. In this work, we demonstrate shape-based separation and enrichment of S. Cerevisiae using a coflow of viscoelastic and Newtonian fluids in a straight rectangular microchannel. Due to the combined effects of lift inertial and elastic forces, this label-free and continuous separation arises from shape-dependent migration of cells from the Newtonian to the non-Newtonian viscoelastic fluid. The lateral position of S. Cerevisiae cells with varying morphologies is found to be dependent on cell major axis. We also investigate the effects of sheath and sample flow rate, poly(ethylene oxide) (PEO) concentration and channel length on the performance of the viscoelastic microfluidic device for S. Cerevisiae enrichment and separation by shape. Moreover, the separation efficiency, cell extraction yield, and cell viability after sorting operations are studied.

Impact of stress-response related transcription factor overexpression on lignocellulosic inhibitor tolerance of Saccharomyces cerevisiae environmental isolates.

Numerous transcription factor genes associated with stress response are upregulated in Saccharomyces cerevisiae grown in the presence of inhibitors that result from pretreatment processes to unlock simple sugars from biomass. To determine if overexpression of transcription factors could improve inhibitor tolerance in robust S. cerevisiae environmental isolates as has been demonstrated in S. cerevisiae haploid laboratory strains, transcription factors were overexpressed at three different expression levels in three S. cerevisiae environmental isolates. Overexpression of the YAP1 transcription factor in these isolates did not lead to increased growth rate or reduced lag in growth, and in some cases was detrimental, when grown in the presence of either lignocellulosic hydrolysates or furfural and 5-hydroxymethyl furfural individually. The expressed Yap1p localized correctly and the expression construct improved inhibitor tolerance of a laboratory strain as previously reported, indicating that lack of improvement in the environmental isolates was due to factors other than nonfunctional expression constructs or mis-folded protein. Additional stress-related transcription factors, MSN2, MSN4, HSF1, PDR1, and RPN4, were also overexpressed at three different expression levels and all failed to improve inhibitor tolerance. Transcription factor overexpression alone is unlikely to be a viable route toward increased inhibitor tolerance of robust environmental S. cerevisiae strains.

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.

Dysbiosis of Gut Fungal Microbiota in Children with Autism Spectrum Disorders.

In this study, we tested the feces of children with ASD and those of healthy children, and the overall changing of the gut fungal community was observed in ASD children compared with controls. However, there were no abundant fungi populations showed significant variations between the ASD and Control group both at phylum and class level. Among the 507 genera identified, Saccharomyces and Aspergillus showed significant differences between ASD (59.07%) and Control (40.36%), indicating that they may be involved in the abnormal gut fungal community structure of ASD. When analyzed at the species level, a decreased abundance in Aspergillus versicolor was observed while Saccharomyces cerevisiae was increased in children with ASD relative to controls. Overall, this study characterized the fungal microbiota profile of children with ASD and identified potential diagnostic species closely related to the immune response in ASD.

Systematic Analysis of Phosphatidylinositol-5-phosphate-Interacting Proteins Using Yeast Proteome Microarrays.

We used yeast proteome microarrays (∼5800 purified proteins) to conduct a high-throughput and systematic screening of PI5P-interacting proteins with PI5P-tagged fluorescent liposomal nanovesicles. Lissamine rhodamine B-dipalmitoyl phosphatidylethanol was incorporated into the liposome bilayer to provide the nanovesicles with fluorescence without any encapsulants, which not only made the liposome fabrication much easier without the need for purification but also improved the chip-probing quality. A special chip assay was washed very gently without the traditional spin-dry step. Forty-five PI5P-interacting proteins were identified in triplicate with this special chip assay. Subsequently, we used flow cytometry to validate these interactions, and a total of 41 PI5P-interacting proteins were confirmed. Enrichment analysis revealed that these proteins have significant functions associated with ribosome biogenesis, rRNA processing, ribosome binding, GTP binding, and hydrolase activity. Their component enrichment is located in the nucleolus. The InterPro domain analysis indicated that PI5P-interacting proteins are enriched in the P-loop containing nucleoside triphosphate hydrolases domain (P-loop). Additionally, using the MEME program, we identified a consensus motif (IVGPAGTGKSTLF) that contains the Walker A sequence, a well-known nucleotide-binding motif. Furthermore, using a quartz crystal microbalance, both the consensus motif and Walker A motif showed strong affinities to PI5P-containing liposomes but not to PI5P-deprived liposomes or PI-containing liposomes. Additionally, the glycine (G6) and lysine (K7) residues of the Walker A motif (-GPAGTG6K7S-) were found to be critical to the PI5P-binding ability. This study not only identified an additional set of PI5P-interacting proteins but also revealed the strong PI5P-binding affinity (Kd = 1.81 × 10-7 M) of the Walker A motif beyond the motif's nucleotide-binding characteristic.

Wine produced from date palm (Phoenix dactylifera L.) fruits using Saccharomyces cerevisiae X01 isolated from Nigerian locally fermented beverages.

The study focused on the production of wine from date palm fruits (Phoenix dactylifera L.) using a strain of yeast isolated from selected Nigerian locally fermented beverages ('pito', 'brukutu' and palm wine). Seven (7) distinct yeasts were isolated and identified using cultural, microscopy and biochemical tests (temperature tolerance, flocculation, ethanol tolerance, H2S production and killer toxin production and the ability to assimilate and ferment sugars. The yeast isolates were screened using refractometric and spectrophotometric methods to select the isolate with the best ability for wine production. This isolate was molecularly characterized, grown in 1 L of potatoes dextrose broth, freeze dried and used for wine production. The population of yeast, bacteria and the physicochemical analysis of the must were monitored during fermentation. Populations of bacteria in the wine were assessed by standard pour plate count. The proximate content and physicochemical properties of the produced wine before and after ageing, as well as the sensorial quality of the produced wine was determined. All the yeast isolates possessed the ability to flocculate, tolerate ethanol concentration of between 5 and 20% and temperature range of 30-45 °C, produced low concentration of H2S and does not produce killer toxins. A palm wine isolate, identified as Saccharomyces cerevisiae X01 was selected as the best isolate with the most ability for wine production. The total yeast count increases as the period of fermentation progressed while the total viable bacterial count reduced as the fermentation period progressed. There was a significant difference (P < 0.05) in the physicochemical properties of the must during fermentation. At the end of the fermentation, the produced wine had 5.22% and 4.86% ethanol content for S. cerevisiae QA23 and S. cerevisiae X01 respectively. There was no significant difference (P > 0.05) between the proximate and physicochemical compositions of the produced wine before and after ageing using Saccharomyces cerevisiae X01 when compared to the control S. cerevisiae QA23. This study revealed that the Nigerian locally sourced S. cerevisiae X01 can be used as an alternative substrate for industrial scale production of wine with a mild alcoholic content.

Quantifying the effect of human practices on S. cerevisiae vineyard metapopulation diversity.

Saccharomyces cerevisiae is the main actor of wine fermentation but at present, still little is known about the factors impacting its distribution in the vineyards. In this study, 23 vineyards and 7 cellars were sampled over 2 consecutive years in the Bordeaux and Bergerac regions. The impact of geography and farming system and the relation between grape and vat populations were evaluated using a collection of 1374 S. cerevisiae merlot grape isolates and 289 vat isolates analyzed at 17 microsatellites loci. A very high genetic diversity of S. cerevisiae strains was obtained from grape samples, higher in conventional farming system than in organic one. The geographic appellation and the wine estate significantly impact the S. cerevisiae population structure, whereas the type of farming system has a weak global effect. When comparing cellar and vineyard populations, we evidenced the tight connection between the two compartments, based on the high proportion of grape isolates (25%) related to the commercial starters used in the cellar and on the estimation of bidirectional geneflows between the vineyard and the cellar compartments.