Biology subjects

Vrhovsek, U.

Publications and source records attributed to Vrhovsek, U..

8 recordsLinked to original sources

bioRxiv2026

Characterisation of Saccharomyces cerevisiae and Saccharomyces jurei hybrids for oenological applications

The Saccharomyces genus present as a genetically diverse set of microorganisms capable of tolerating a range of environmental stressors, demonstrating high fermentation capacity and producing interesting flavour compounds. Such diversity can be exploited in a single organism by producing interspecies hybrids, overcoming the limitations of a single species. Here, as proof of principle, we exploited a library of F12 interspecies diploid hybrids between Saccharomyces cerevisiae and Saccharomyces jurei (Sc/Sj), to identify new strains harbouring traits of oenological relevance, such as ethanol and glycerol production, superior tolerance to acetic acid, limited production of H2S, and optimal growth on fructose. Moreover, we calculated robustness across strains tested on solid media comparing the trade-off between performance on a single conditions and overall robustness. Progeny from a range of robustness values with favourable H2S production profiles were isolated for further analysis via micro and small-scale fermentations. Micro-scale fermentations in synthetic grape must containing individual wine-associated stressors were performed, and the robustness of the selected strains was confirmed. The wine characteristics of the hybrids were tested in small-scale fermentations in synthetic grape media. We found that both ethanol and glycerol production across our selected progeny matched or exceeding that from the commercial wine strain, and most hybrids produced less acetic acid and more fatty-acids, including higher quantities of associated esters. Overall, we were able to identify suitable Sc/Sj hybrids with interesting aroma profiles to develop further for wine fermentation.

microbiology↗

bioRxiv2026

Colon-delivered multivitamin supplementation enhances working memory-related fMRI responses in older adults: a randomized, placebo-controlled trial

BackgroundVitamins are important modulators of intestinal health and may affect the gut-brain axis through microbial metabolites such as short-chain fatty acids (SCFAs). However, the neurocognitive effects of colon-delivered vitamins in older adults remain unexplored - a critical gap given the gut-brain axiss emerging role in cognitive aging. ObjectiveWe investigated the effect of a colon-delivered multivitamin (CDMV) supplement on intestinal health and neurocognitive outcomes in older adults at risk of cognitive decline. MethodsWithin the double-blind randomized placebo-controlled trial COMBI (ClinicalTrials.govID: NCT05675007), we included 75 older adults (60-75 years) at risk of cognitive decline based on lifestyle-related factors. Participants consumed a colon-delivered capsule with vitamins B2, B3, B6, B9, C and D3, or a placebo, daily for six weeks. Pre- and post-intervention, we employed neuroimaging, feces- and blood collection. Primary outcomes were fecal SCFA concentrations, working memory (WM)-related fMRI responses, and WM performance measured with the n-back task. ResultsAfter adjusting for baseline values, we found no significant between-group differences in total fecal SCFA levels (p=0.30) and WM performance (p=0.50). Post-intervention WM-related fMRI responses in the hippocampus (p=0.01; p{superscript 2}=0.09) and dorsolateral prefrontal cortex (dlPFC) (p=0.06; p{superscript 2}=0.04), driven by the right dlPFC (p=0.02), were higher in the CDMV group compared to placebo. Independent of intervention group, post-pre increases in fecal SCFA levels were significantly correlated to increases in dlPFC fMRI responses ({rho}=0.31; p=0.02) and WM performance ({rho}=0.43; p=0.001). ConclusionsOur findings suggest that CDMV supplementation increases WM-related responses of the dlPFC and hippocampus in older adults, but this effect was not accompanied by changes in fecal SCFA levels or WM performance. The positive correlation of within-subject changes in fecal SCFAs with changes in WM dlPFC responses and performance across intervention groups provides human evidence for gut-brain communication in cognitive aging beyond cross-sectional associations.

neuroscience↗

bioRxiv2025

Multi-omics approach combined with functional characterization of genes enlightens the formation of certain aromatic compounds in Vitis vinifera cv. Assyrtiko grape berries

Assyrtiko, a grape variety with considerable historical importance in Greece, has recently garnered global interest due to its unique citrus and floral fragrances, elevated alcohol content, and its wines created on the volcanic island of Thira (Santorini). However, grown in various regions, such as Central Greece, its aromatic characteristics may vary, prompting an investigation into the molecular mechanisms accounting for these differences. This research focused on two Assyrtiko clones-- A16 and E11--grown in the Nemea, Peloponnese, which displayed distinct aromatic traits. Chemical analysis during mid-ripening indicated that clone E11 yielded significantly greater amounts of floral terpenoids, including linalool, geraniol, nerol, and -terpineol, while A16 was rich in C compounds like (Z)-3-hexenol and (Z)-3-hexenal that are linked to green and earthy scents. Transcriptomic analysis corroborated these results: clone E11 demonstrated an upregulation of genes related to pathogen resistance and to the terpene and phenylpropanoid biosynthetic pathways, such as DXS, MECPS, CCD1, COMT, AADC, and vanillin synthase, whereas clone A16 showed increased expression of genes related to volatile thiol production and flavonoid metabolism, including GST3 and GST4. Further, five key genes (RZS1, AAT, EGS1, AADC, and CYP76F14) from A16 were functionally characterized through heterologous expression in Saccharomyces cerevisiae and in vitro enzymatic assays. These findings offer new insights into the biochemical pathways determining aroma diversity in Vitis vinifera cv. Assyrtiko. HighlightAn integrated analysis of chemical and transcriptomic profiles from two clonal variants of Vitis vinifera var. Assyrtiko elucidates the molecular basis of aroma compound biosynthesis during grape berry ripening. This study includes the cloning and functional characterization of key aroma-related enzymes: raspberry ketone synthase (RZS1), eugenol synthase (EGS1), acetyltransferase of coniferyl alcohol (AAT), 8-hydroxylinalool synthase (CYP76F14) and phenylacetaldehyde synthase (AADC) from Assyrtiko berries.

plant biology↗

bioRxiv2024

Aroma compounds profile is affected by the initial yeast ratio in wort co-fermentations

In recent years, the boom of the craft beer industry refocused the biotech interest from ethanol production to diversification of beer aroma profiles. This study analyses the fermentative phenotype of a collection of non-conventional yeasts and examines their role in creating new flavours, particularly through co-fermentation with industrial Saccharomyces cerevisiae. High-throughput solid and liquid media fitness screening compared the ability of eight Saccharomyces and four non-Saccharomyces yeast strains to grow in wort. We determined the volatile profile of these yeast strains and found that Hanseniaspora vineae displayed a particularly high production of the desirable aroma compounds ethyl acetate and 2-phenethyl acetate. Given that H. vineae on its own was a poor fermenter, we carried out mixed wort co-fermentations with a S. cerevisiae brewing strain at different ratios. The two yeast strains were able to co-exist throughout the experiment, regardless of their initial inoculum, and the increase in the production of the esters observed in the H. vineae monoculture was maintained, alongside with a high ethanol production. Moreover, different inoculum ratios yielded different aroma profiles: the 50/50 S. cerevisiae/H. vineae ratio produced a more balanced profile, while the 10/90 ratio generated stronger floral aromas. Our findings show the potential of using different yeasts and different inoculum combinations to tailor the final aroma, thus offering new possibilities for a broader range of beer flavours and styles. IMPORTANCECraft brewing underwent an unprecedented growth in the last years due to customer demand for more unique and complex beverages. Brewers started to explore innovative fermentation methods using new ingredients, different brewing conditions, and new yeasts to explore a larger flavour landscape. The use of non-Saccharomyces yeasts has emerged as an effective strategy to produce novel distinct flavour profiles, however, knowledge regarding their fermentation performance and volatiles production is still limited, which hinders their industrial application. In this study, we expand on the knowledge of several non-Saccharomyces yeasts in terms of their brewing application and highlight the potential of H. vineae in co-fermentation with S. cerevisiae for producing unique fruity beers with a standard ethanol content. Our findings provide the craft beer industry with a new strategy to produce distinctive fruity beers.

microbiology↗

bioRxiv2024

Circulating (poly)phenol metabolites in the brain: unveiling in vitro and in vivo blood-brain barrier transport

Circulating metabolites resulting from colonic metabolism of dietary (poly)phenols are highly abundant in the bloodstream, though still marginally explored, particularly concerning their brain accessibility. Our goal is to disclose (poly)phenol metabolites blood-brain barrier (BBB) transport, in vivo and in vitro, as well as their role at BBB level. For three selected metabolites, benzene-1,2-diol-3-sulfate/benzene-1,3-diol-2-sulfate (pyrogallol-sulfate - Pyr-sulf), benzene-1,3-diol-6-sulfate (phloroglucinol-sulfate - Phlosulf), and phenol-3-sulfate (resorcinol-sulfate - Res-sulf), BBB transport was assessed in human brain microvascular endothelial cells (HBMEC). Their potential in modulating in vitro BBB properties at circulating concentrations was also studied. Metabolites fate towards the brain, liver, kidney, urine, and blood was disclosed in Wistar rats upon injection. Transport kinetics in HBMEC highlighted different BBB permeability rates, where Pyr-sulf emerged as the most in vitro BBB permeable metabolite. Pyr-sulf was also the most potent regarding BBB properties improvement, namely increased beta({beta})-catenin membrane expression and reduction of zonula occludens-1 membrane gaps. Whereas no differences were observed for transferrin, increased expression of caveolin-1 upon Pyr-sulf and Res-sulf treatments was found. Pyrsulf was also capable of modulating gene and protein expression of some solute carrier transporters. Notably, each of the injected metabolites exhibited a unique tissue distribution in vivo, with the remarkable ability to almost immediately reach the brain.

biochemistry↗

bioRxiv2022

Volatile aroma compound production is affected by growth rate in S. cerevisiae

The initial growth rate of a yeast strain is a key parameter in the production of fermented beverages. Fast growth is linked with higher fermentative capacity and results in less slow and stuck fermentations unable to reach the expected final gravity. As concentrations of metabolites are in constant state of flux, quantitative data on how growth rate affects the production of aromatic compounds becomes an important factor for brewers. Chemostats allow to set and keep a specific dilution rate throughout the fermentation and are ideal system to study the effect of growth on aroma production. In this study, we run chemostats alongside batch and fed-batch cultures, compared volatile profiles detected at different growth rates, and identified those affected by the different feeding profiles. Specifically, we quantified six abundant aroma compounds produced in anaerobic glucose-limited continuous cultivations of S. cerevisiae at different dilution rates. We found that volatile production was affected by the growth rate in four out of six compounds assayed, with higher alcohols and esters following opposite trends. Batch and fed-batch fermentations were devised to study the extent by which the final concentration of volatile compounds is influenced by glucose availability. When compared to the batch system, fed-batch fermentations, where the yeast growth was artificially limited by a slow constant release of nutrients in the media, resulted in a significant increase in concentration of higher alcohols, mirroring the results obtained in continuous fermentations. This study paves the way to further process development optimization for the production of fermented beverages. ImportanceThe production of fermentation beverages will need to quickly adapt to changes in both the climate and in customer demands, requiring the development of new strains and processes. Breakthroughs in the field are hindered by the limited knowledge on the physiological role of aroma compounds production in yeast. No quantitative data on how growth rate affects aroma profile is available in the literature to guide optimisation of the complex flavours in fermented beverages. In this study, we exploited the chemostat system, alongside with batch and fed-batch cultures to compare volatile profiles at different growth rates. We identified the aromatic compounds affected by the different feeding profiles and nutrient limitations. Moreover, we uncovered the correlation between yeast growth, esters and higher alcohols production. This study showcases the potential of the application of feeding profiles for the manipulation of aroma in the craft beverage industry.

microbiology↗

bioRxiv2022

Enhanced flavour profiles through radicicol induced genomic variation in S. pastorianus lager yeast

The yeasts, Saccharomyces pastorianus, are hybrids of Saccharomyces cerevisiae and Saccharomyces eubayanus and have acquired traits from the combined parental genomes such as ability to ferment a range of sugars at low temperatures and to produce aromatic flavour compounds, allowing for the production of lager beers with crisp, clean flavours. The polyploid strains are sterile and have reached an evolutionary bottleneck for genetic variation. Here we describe an accelerated evolution approach to obtain lager yeasts with enhanced flavour profiles. As the relative expression of orthologous alleles is a significant contributor to the transcriptome during fermentation, we aimed to induce genetic variation by altering the S. cerevisiae to S. eubayanus chromosome ratio. Aneuploidy was induced through the temporary inhibition of the cells stress response and strains with increased production of aromatic amino acids via the Shikimate pathway were selected by resistance to amino acid analogues. Genomic changes such as gross chromosomal rearrangements, chromosome loss and chromosome gain were detected in the characterised mutants, as were Single Nucleotide Polymorphisms in ARO4, encoding for DAHP synthase, the catalytic enzyme in the first step of the Shikimate pathway. Transcriptome analysis confirmed the upregulation of genes encoding enzymes in the Ehrlich pathway and the concomitant increase in the production of higher alcohols and esters such as 2-phenylethanol, 2-phenylethyl acetate, tryptophol, and tyrosol. We propose that the plasticity of polyploid S. pastorianus genomes is an advantageous trait supporting opportunities for genetic diversity in otherwise sterile strains. Significance StatementLager beer is the product of fermentations conducted with Saccharomyces pastorianus, which are hybrids of Saccharomyces cerevisiae and Saccharomyces eubayanus. A quintessential property of lager beers is the distinctive flavours produced during fermentation. Hybrids are sterile and have reached an evolutionary bottleneck. Finding ways to introduce genetic variation as a means of enhancing the flavour profiles is a challenge. Here, we describe an approach to introduce genetic variation by inducing aneuploidy through the temporary inhibition of the cells stress response. Strains with an enhanced flavour production were selected by resistance to amino acid analogues. We identified genomic changes and transcriptome analysis confirmed the upregulation of genes in the Ehrlich pathway which is responsible for the production of flavour compounds.

microbiology↗

bioRxiv2021

Biotechnological exploitation of Saccharomyces jurei and its hybrids in craft beer fermentation uncovers new aroma combinations.

Hybridization is an important evolutionary mechanism to bring about novel phenotypes and may produce new hybrids with advantageous combinations of traits of industrial importance. Within the Saccharomyces group, Saccharomyces jurei is a newly discovered species and its biotechnological potential has not yet been fully explored. This yeast was found to be able to grow well in synthetic wort and at low temperatures, qualities necessary in good candidates for fermentated bevarages. Here, we analysed its fermentation and aroma profile and created novel non-GMO hybrids between S. jurei and S. cerevisiae ale yeasts to develop new starter strains with interesting flavours for the craft beer industry. Pilot beer fermentations with specific hybrids showed a good fermentation performance, similar to the ale parent strain, while presenting a better sugar attenuation and a more complex flavour profile. This study exploits the genetic diversity of yeasts and show how inter-specific hybridisation and clone selection can be effectively used in brewing to create new products and to eliminate or increase specific traits.

microbiology↗