Yeast Populations Research Articles

Multi-omics framework to reveal the molecular determinants of fermentation performance in wine yeast populations

BackgroundConnecting the composition and function of industrial microbiomes is a major aspiration in microbial biotechnology. Here, we address this question in wine fermentation, a model system where the diversity and functioning of fermenting yeast species are determinant of the flavor and quality of the resulting wines.ResultsFirst, we surveyed yeast communities associated with grape musts collected across wine appellations, revealing the importance of environmental (i.e., biogeography) and anthropic factors (i.e., farming system) in shaping community composition and structure. Then, we assayed the fermenting yeast communities in synthetic grape must under common winemaking conditions. The dominating yeast species defines the fermentation performance and metabolite profile of the resulting wines, and it is determined by the initial fungal community composition rather than the imposed fermentation conditions. Yeast dominance also had a more pronounced impact on wine meta-transcriptome than fermentation conditions. We unveiled yeast-specific transcriptomic profiles, leveraging different molecular functioning strategies in wine fermentation environments. We further studied the orthologs responsible for metabolite production, revealing modules associated with the dominance of specific yeast species. This emphasizes the unique contributions of yeast species to wine flavor, here summarized in an array of orthologs that defines the individual contribution of yeast species to wine ecosystem functioning.ConclusionsOur study bridges the gap between yeast community composition and wine metabolite production, providing insights to harness diverse yeast functionalities with the final aim to producing tailored high-quality wines.74dL8hDE5Rym1TqBfqxSSDVideo

Assessing variability among culturable phylloplane basidiomycetous yeasts from Italian agroecosystems

This study analysed basidiomycetous yeasts isolated from the phylloplane of crops and spontaneous plants in Italian agroecosystems. A total of 25 species belonging to 17 genera were recognized by analysing 83 isolates from vineyards and orchards, that are not treated with synthetic fungicides, and adjacent natural areas. Rhodotorula graminis and Filobasidium magnum were the most frequent species but 13 others were represented by a single isolate (e.g., Buckleyzyma salicina, Pseudozyma prolifica, and Moniliella megachiliensis). Preliminary analysis of (GTG)5-PCR fingerprinting revealed high genetic intraspecific heterogeneity. All isolates were characterized by their production of extracellular hydrolytic enzymes and their sensitivity to six commercial fungicides used in Italy. The isolates displayed great variability in these phenotypic traits, which play an important role in the survival of yeast populations in agroecosystems. Most of them exhibited lipolytic, proteolytic, β-glucosidase and pectinolytic activities, but only three (F. magnum, Kwoniella mangroviensis and Ps. prolifica) also had cellulolytic and amylolytic activity. Most isolates were sensitive to four fungicides, and one R. graminis isolate was resistant to all six. This heterogeneity was not related to the geographical origin of the isolates. The lack of selective factors (i.e. pesticide treatments) in the sampling fields and the presence of adjacent natural areas may have favored the maintenance of an elevated level of strain diversity. This study provides new information on phylloplane basidiomycetous yeasts in agroecosystems and opens the way to further investigations into the impact of agricultural practices on the microbial diversity of these natural habitats.

Dynamic Changes in Yeast Populations and Aroma Composition of ‘Beimei’ (Vitis vinifera × Vitis amurensis) during Spontaneous Fermentation

‘Beimei’ is a hybrid cultivar of Vitis vinifera and wild Vitis amurensis with fungus- and cold-resistant characteristics that is widely grown in many regions of China. However, studies on the yeast diversity and aroma compound composition of this cultivar remain scarce. Here, indigenous yeast diversity, aroma compound composition, and their dynamic changes during the spontaneous fermentation of ‘Beimei’ were investigated and monitored using high-throughput sequencing and gas chromatography-mass spectrometry. The result indicated that non-Saccharomyces yeasts were predominant at the initial stages of ‘Beimei’ spontaneous fermentation and then sharply decreased. Cladosporium herbarum and Hanseniaspora uvarum were identified as the dominant species of filamentous fungi and yeast, respectively, at the beginning and on the first day of fermentation, whereas unclassified_f_Saccharomycetacease was dominant throughout fermentation, starting from the third day until the end of fermentation. The main volatile aroma chemicals were esters and alcohols, which accumulated abundantly at the late stage of fermentation. Taken together, this study represents the first step in exploring untapped yeast species and aroma compounds, which is helpful for the screening of native yeasts from ‘Beimei’ and producing distinctive aroma wines.

Tunable cell differentiation via reprogrammed mating-type switching

This study introduces a synthetic biology approach that reprograms the yeast mating-type switching mechanism for tunable cell differentiation, facilitating synthetic microbial consortia formation and cooperativity. The underlying mechanism was engineered into a genetic logic gate capable of inducing asymmetric sexual differentiation within a haploid yeast population, resulting in a consortium characterized by mating-type heterogeneity and tunable population composition. The utility of this approach in microbial consortia cooperativity was demonstrated through the sequential conversion of xylan into xylose, employing haploids of opposite mating types each expressing a different enzyme of the xylanolytic pathway. This strategy provides a versatile framework for producing and fine-tuning functionally heterogeneous yet isogenic yeast consortia, furthering the advancement of microbial consortia cooperativity and offering additional avenues for biotechnological applications.

PSXIII-26 Effects of Lentilactobacillus lactis and Lentilactobacillus buchneri on fermentation and aerobic stability in corn grain silages

Abstract The inclusion of corn grain silages (early-harvest and reconstitute grain) in the diets has gained wide acceptance in the last decade among Brazilian farms. Thus, reducing losses in these silages is one of the main goals. The objective of this study was to evaluate the effects of an inoculant containing Lentilactobacillus lactis O224 and Lentilactobacillus buchneri LB1819 on fermentation, microbial counts, and aerobic stability in reconstituted corn grain (RCG) and high-moisture corn (HMC). Two experiments were carried out, which tested the inclusion of no additive (CON) and the addition of L. lactis and L. buchneri. For both trials, the inoculant was added at a theoretical rate of 1.5 x 105 CFU/g of feed. For HMC, corn kernels (30.1% of moisture) were processed and ensiled in 5-L jars for 44 or 64 d. For RCG, corn kernels were purchased at 14% moisture, ground, and water was added to reach 35% moisture. Afterwards, ground kernels were ensiled in 5-L jars for 21 or 42 d. Measurements included fermentation end-products (acids, alcohols, and NH3-N), microbial counts (LAB, yeasts, and molds), and aerobic stability. Both trials were set up as a completely randomized design using a 2 x 2 factorial arrangement. Data were analyzed using MIXED procedure of SAS, followed by a Tukey test at P ≤ 0.05. In both experiments, inoculated silages had reduced concentrations of lactic acid and greater concentrations of acetic acid and 1,2-propanediol. These three fermentation end products increased over time for both treatments. In HMC, there was an interaction between inoculation and time of conservation for propionic acid concentrations. Also, the concentration of NH3-N was greater in treated silages. In both experiments, the population of LAB was greater in inoculated silages. For RCG, yeast population was greater in CON treatment. During aerobic exposure, treated silages remained more stable in both trials. In HMC, the aerobic stability was > 240 h for treated silages, whereas CON silages had 94 h of stability. Still in HMC, the peaks of temperature were 39.5 and 28.2 oC and the pH values (after 10 d of aerobic exposure) were 6.81 and 4.38 for CON and inoculated silages, respectively. In RCG, the aerobic stability was more pronounced at 42 d of conservation (35 vs 66 h for CON and inoculated silages, respectively). Overall, the combination of L. lactis and L. buchneri modulates corn grain silages fermentation and improves aerobic stability.

Experimental evolution of Saccharomyces cerevisiae for caffeine tolerance alters multidrug resistance and target of rapamycin signaling pathways.

Caffeine is a natural compound that inhibits the major cellular signaling regulator target of rapamycin (TOR), leading to widespread effects including growth inhibition. Saccharomyces cerevisiae yeast can adapt to tolerate high concentrations of caffeine in coffee and cacao fermentations and in experimental systems. While many factors affecting caffeine tolerance and TOR signaling have been identified, further characterization of their interactions and regulation remain to be studied. We used experimental evolution of S. cerevisiae to study the genetic contributions to caffeine tolerance in yeast, through a collaboration between high school students evolving yeast populations coupled with further research exploration in university labs. We identified multiple evolved yeast populations with mutations in PDR1 and PDR5, which contribute to multidrug resistance, and showed that gain-of-function mutations in multidrug resistance family transcription factors Pdr1, Pdr3, and Yrr1 differentially contribute to caffeine tolerance. We also identified loss-of-function mutations in TOR effectors Sit4, Sky1, and Tip41 and showed that these mutations contribute to caffeine tolerance. These findings support the importance of both the multidrug resistance family and TOR signaling in caffeine tolerance and can inform future exploration of networks affected by caffeine and other TOR inhibitors in model systems and industrial applications.

Survival of the probiotic strain Lacticaseibacillus paracasei subsp. paracasei F19 in high-hopped beers

This study aims to enhance understanding of probiotic lactic acid bacteria (LAB) survival in high-hopped beer formulations and their interactions with different yeasts and highlights the fermentation processes, microbial metabolism, and production of distinctive beer flavors. For this, this research used Lacticaseibacillus paracasei F19 (F19), Saccharomycodes ludwigii, and Saccharomyces cerevisiae strains US-05 (US-05) and Kveik (Kveik) for brewing. Bacterial and yeast cultures were prepared, fermented in wort, and analyzed in different hop concentrations (International Bitterness Units − IBU 0, 20, 40). Methods included physicochemical analysis, yeast and bacterial counts, RT-qPCR for gene expression, statistical analysis, and sensory evaluation by sommeliers following BJCP guidelines. Physicochemical analysis showed efficient fermentation across all hop concentrations (IBU 0, 20, 40), with decreasing SG and pH over time due to lactic acid bacteria and yeast metabolism. Higher hop levels (IBU 20 and 40) resulted in less acidic beer, indicating hop interference with bacterial activity. Yeast populations remained stable regardless of hop content, with Saccharomyces cerevisiae and Saccharomycodes ludwigii performing well. Probiotic strain F19 exhibited robust viability in all formulations. Sensory analysis favored higher hop content beers, suggesting consumer acceptance and potential health benefits of probiotic, high-hop beers. Higher hop content hindered sour beer production as only hop-free beers reached low pH levels. Probiotic strain F19 remained viable under high IBU formulations (20 and 40), with these being preferred by sommeliers using BJCP methodology. All yeast strains supported F19 survival. Further studies are needed on gastrointestinal resistance and clinical benefits.

Yeasts, arthropods, and environmental matrix: a triad to disentangle the multi-level definition of biodiversity

Our understanding of the spread of yeasts in natural ecosystems remains somewhat limited. The recent momentum of yeast ecology research has unveiled novel habitats and vectors that, alongside human activities, impact yeast communities in their natural environments. Yeasts, as non-airborne microorganisms, rely on animal vectors, predominantly insects. However, the overlooked actor in this interplay is the environmental matrix, a player potentially influencing yeast populations and their vectors. This study aims to delve deeper into the intricate, multi-layered connections between yeast populations and ecosystems, focusing on the interactions between the attributes of the environmental matrix, arthropod diversity, and the mycobiota within a renowned yeast-inhabited framework: the vineyard. To investigate these relationships, we sampled both invertebrate and yeast diversity in six organic and conventional vineyards described in terms of management and landscape composition. We identified 80 different invertebrate taxa and isolated 170 yeast strains belonging to 18 species. Notably, new species-specific yeast-insect associations were observed, including the exclusive association between Candida orthopsilosis and Hymenoptera and between Metschnikowia pulcherrima and Coleoptera. These newly identified potential associations provide valuable insights into insect and yeast physiology, hence holding the promise of enhancing our understanding of yeast and arthropod ecology and their collective impact on overall ecosystem health.

Improving wine fermentation efficiency of Torulaspora delbrueckii by increasing the ploidy of yeast inocula

The life cycle of most non-conventional yeasts, such as Torulaspora delbrueckii (Td), is not as well-understood as that of Saccharomyces cerevisiae (Sc). Td is generally assumed to be haploid, which detracts from some biotechnological properties compared to diploid Sc strains. We analyzed the life cycle of several Td wine strains and found that they were mainly diploid during exponential growth in rich medium. However, most cells became haploid in stationary phase, as observed for Sc haploid heterothallic strains. When transferred and incubated in nutrient-deficient media, these haploid cells became polymorphic, enlarged, and transitioned to diploid or polyploid states. The increased ploidy, that mainly results from supernumerary mitosis without cytokinesis, was followed by sporulation. A similar response was observed in yeasts that remained alive during the second fermentation of base wine for sparkling wine making, or during growth in ethanol-supplemented medium. This response was not observed in the Sc yeast populations under any of the experimental conditions assayed, which suggests that it is a specific adaptation of Td to the stressful fermentation conditions. This response allows Td yeasts to remain alive and metabolically active longer during wine fermentation. Consequently, we designed procedures to increase the cell size and ploidy of haploid Td strains. Td inocula with increased ploidy showed enhanced fermentation efficiency compared to haploid inocula of the same strains.

Highly parallelized laboratory evolution of wine yeasts for enhanced metabolic phenotypes

Adaptive Laboratory Evolution (ALE) of microorganisms can improve the efficiency of sustainable industrial processes important to the global economy. However, stochasticity and genetic background effects often lead to suboptimal outcomes during laboratory evolution. Here we report an ALE platform to circumvent these shortcomings through parallelized clonal evolution at an unprecedented scale. Using this platform, we evolved 104 yeast populations in parallel from many strains for eight desired wine fermentation-related traits. Expansions of both ALE replicates and lineage numbers broadened the evolutionary search spectrum leading to improved wine yeasts unencumbered by unwanted side effects. At the genomic level, evolutionary gains in metabolic characteristics often coincided with distinct chromosome amplifications and the emergence of side-effect syndromes that were characteristic of each selection niche. Several high-performing ALE strains exhibited desired wine fermentation kinetics when tested in larger liquid cultures, supporting their suitability for application. More broadly, our high-throughput ALE platform opens opportunities for rapid optimization of microbes which otherwise could take many years to accomplish.

Real-time assessment of mitochondrial DNA heteroplasmy dynamics at the single-cell level.

Mitochondrial DNA (mtDNA) is present in multiple copies within cells and is required for mitochondrial ATP generation. Even within individual cells, mtDNA copies can differ in their sequence, a state known as heteroplasmy. The principles underlying dynamic changes in the degree of heteroplasmy remain incompletely understood, due to the inability to monitor this phenomenon in real time. Here, we employ mtDNA-based fluorescent markers, microfluidics, and automated cell tracking, to follow mtDNA variants in live heteroplasmic yeast populations at the single-cell level. This approach, in combination with direct mtDNA tracking and data-driven mathematical modeling reveals asymmetric partitioning of mtDNA copies during cell division, as well as limited mitochondrial fusion and fission frequencies, as critical driving forces for mtDNA variant segregation. Given that our approach also facilitates assessment of segregation between intact and mutant mtDNA, we anticipate that it will be instrumental in elucidating the mechanisms underlying the purifying selection of mtDNA.

Psychrotrophic Bacteria and Yeast Populations in Turkish White and Tulum Cheeses

In this study, pH and microbiological (psychrotrophic bacteria and yeast) properties of White and Tulum cheeses were investigated. According to the results of our study, the lowest pH value identified was 4.42, and the highest pH was 5.84 in the cheese samples. Psychrotrophic bacteria counts ranged from 4.15 to 6.21 log CFU/g for White cheese and 4.02 to 6.15 log CFU/g for Tulum cheese. Psychrotrophic yeast counts ranged from 2.98 to 7.58 log CFU/g for White cheese and 2.99 to 6.77 log CFU/g for Tulum cheese. The highest values of psychrotrophic yeast were detected in the Tulum cheese samples. As a result, it was determined that the numbers of psychrotrophic bacteria and yeast were high in both Tulum and White cheese samples. These high numbers of psychrotrophic microorganisms during cheese storage in the refrigerator may increase even further and reduce shelf life. To extend the shelf life of these cheeses, it is recommended to ensure hygienic handling practices and explore the use of natural preservatives or modified atmosphere packaging.

Characteristics of whole wheat steamed bread production by two-stage fermentation procedure with Jiaozi as starter

Given the beneficial role of biological acidification in improving the quality of cereal-based products, this study evaluated the applicability of Jiaozi as a starter for whole wheat dough fermentation and steamed bread quality improvement. During the first 12 h of dough fermentation, yeast and lactic acid bacteria (LAB) populations increased significantly, resulting in rapid carbohydrate consumption and acidification of the dough. The lactic/acetic acid molar ratio was approximately 3.3, which was within the recommended fermentation quotient (FQ). Compared with the unfermented dough and the dough fermented for 12 h, the dough fermented for 6 h had an improved protein network structure, and the steamed bread made from it also showed the best quality. At a low leavening temperature of 28 °C, satisfactory specific volume and height/diameter ratio were obtained. The dough with an inoculation amount of 50% of the fully fermented dough showed the highest CO2 production capacity. Compared with yeast, the whole wheat steamed bread produced by Jiaozi using the two-stage fermentation process showed higher specific volume, resistant starch (RS) content and protein digestibility. The results showed that the use of Jiaozi as a starter has great potential for application in improving the quality of whole wheat steamed bread.

YEAST POPULATION OF SACCHAROMYCES CEREVISIAE IN THE FECES OF YOUNG AND ADULT ETAWA CROSSBREED GOATS IN SUKAWATI DISTRICT, GIANYAR REGENCY

Sacharomyces cerevisiae is a yeast that has great potential to increase the nutritional value of feed ingredients and increase the activity of rumen microbial performance, by utilizing oxygen so that rumen conditions are anaerobic, which makes cellulolytic bacteria develop well. This study aims to determine the population of S. cerevisiae yeast in young and adult etawa crossbreed goat feces. The samples used were 27 of etawa crossbreed goat feces from etawa crossbreed goat farms in Sukawati Sub-District, Gianyar Regency. There are three treatments given, namely etawa crossbreed goat feces from young goats aged 0-2 months, 3-5 months and adult goats aged over 6 months. This study used a Randomized Group Design by comparing the yeast population of S. cerevisiae in the feces of young and adult etawa crossbreed goats, which was done microscopically using a hemocytometer. The results showed that the average yeast content of S. cerevisiae in the feces of young etawa crossbreed goats aged 0-2 months was 25.56 x 104 ± 16,666.667 cells/ml, 3-5 months 42.22 x 104 ± 26,352.314 cells/ml, and adult goats aged 6 months to above 94.44 x 104 ± 30,459.445 cells/ml. There is a very significant difference, the burden of the yeast S. cerevisiae in the feces of adult Etawa crossbreed goats is higher compared to young goats. Further testing needs to be done on the type of yeast found in the feces and stomach of Etawa crossbreed goats.

Influence of different stress factors during the elaboration of grape must's pieddecuve on the dynamics of yeast populations during alcoholic fermentation

The pieddecuve (PdC) technique involves using a portion of grape must to undergo spontaneous fermentation, which is then used to inoculate a larger volume of must. This allows for promoting autochthonous yeasts present in the must, which can respect the typicality of the resulting wine. However, the real impact of this practice on the yeast population has not been properly evaluated. In this study, we examined the effects of sulphur dioxide (SO2), temperature, ethanol supplementation, and time on the dynamics and selection of yeasts during spontaneous fermentation to be used as PdC. The experimentation was conducted in a synthetic medium and sterile must using a multi-species yeast consortium and in un-inoculated natural grape must. Saccharomyces cerevisiae dominated both the PdC and fermentations inoculated with commercial wine yeast, displaying similar population growth regardless of the tested conditions. However, using 40 mg/L of SO2 and 1% (v/v) ethanol during spontaneous fermentation of Muscat of Alexandria must allowed the non-Saccharomyces to be dominant during the first stages, regardless of the temperature tested. These findings suggest that it is possible to apply the studied parameters to modulate the yeast population during spontaneous fermentation while confirming the effectiveness of the PdC methodology in controlling alcoholic fermentation.

Tuning interdomain conjugation to enable in situ population modification in yeasts.

The ability to modify and control natural and engineered microbiomes is essential for biotechnology and biomedicine. Fungi are critical members of most microbiomes, yet technology for modifying the fungal members of a microbiome has lagged far behind that for bacteria. Interdomain conjugation (IDC) is a promising approach, as DNA transfer from bacterial cells to yeast enables in situ modification. While such genetic transfers have been known to naturally occur in a wide range of eukaryotes and are thought to contribute to their evolution, IDC has been understudied as a technique to control fungal or fungal-bacterial consortia. One major obstacle to the widespread use of IDC is its limited efficiency. In this work, we manipulated metabolic and physical interactions between genetically tractable Escherichia coli and Saccharomyces cerevisiae to control the incidence of IDC. We test the landscape of population interactions between the bacterial donors and yeast recipients to find that bacterial commensalism leads to maximized IDC, both in culture and in mixed colonies. We demonstrate the capacity of cell-to-cell binding via mannoproteins to assist both IDC incidence and bacterial commensalism in culture and model how these tunable controls can predictably yield a range of IDC outcomes. Furthermore, we demonstrate that these controls can be utilized to irreversibly alter a recipient yeast population, by both "rescuing" a poor-growing recipient population and collapsing a stable population via a novel IDC-mediated CRISPR/Cas9 system.IMPORTANCEFungi are important but often unaddressed members of most natural and synthetic microbial communities. This work highlights opportunities for modifying yeast microbiome populations through bacterial conjugation. While conjugation has been recognized for its capacity to deliver engineerable DNA to a range of cells, its dependence on cell contact has limited its efficiency. Here, we find "knobs" to control DNA transfer, by engineering the metabolic dependence between bacterial donors and yeast recipients and by changing their ability to physically adhere to each other. Importantly, we functionally validate these "knobs" by irreversibly altering yeast populations. We use these controls to "rescue" a failing yeast population, demonstrate the capacity of conjugated CRISPR/Cas9 to depress or collapse populations, and show that conjugation can be easily interrupted by disrupting cell-to-cell binding. These results offer building blocks toward in situ mycobiome editing, with significant implications for clinical treatments of fungal pathogens and other fungal system engineering.

Optimal trade-off between boosted tolerance and growth fitness during adaptive evolution of yeast to ethanol shocks

BackgroundThe selection of Saccharomyces cerevisiae strains with higher alcohol tolerance can potentially increase the industrial production of ethanol fuel. However, the design of selection protocols to obtain bioethanol yeasts with higher alcohol tolerance poses the challenge of improving industrial strains that are already robust to high ethanol levels. Furthermore, yeasts subjected to mutagenesis and selection, or laboratory evolution, often present adaptation trade-offs wherein higher stress tolerance is attained at the expense of growth and fermentation performance. Although these undesirable side effects are often associated with acute selection regimes, the utility of using harsh ethanol treatments to obtain robust ethanologenic yeasts still has not been fully investigated.ResultsWe conducted an adaptive laboratory evolution by challenging four populations (P1–P4) of the Brazilian bioethanol yeast, Saccharomyces cerevisiae PE-2_H4, through 68–82 cycles of 2-h ethanol shocks (19–30% v/v) and outgrowths. Colonies isolated from the final evolved populations (P1c–P4c) were subjected to whole-genome sequencing, revealing mutations in genes enriched for the cAMP/PKA and trehalose degradation pathways. Fitness analyses of the isolated clones P1c–P3c and reverse-engineered strains demonstrated that mutations were primarily selected for cell viability under ethanol stress, at the cost of decreased growth rates in cultures with or without ethanol. Under this selection regime for stress survival, the population P4 evolved a protective snowflake phenotype resulting from BUD3 disruption. Despite marked adaptation trade-offs, the combination of reverse-engineered mutations cyr1A1474T/usv1Δ conferred 5.46% higher fitness than the parental PE-2_H4 for propagation in 8% (v/v) ethanol, with only a 1.07% fitness cost in a culture medium without alcohol. The cyr1A1474T/usv1Δ strain and evolved P1c displayed robust fermentations of sugarcane molasses using cell recycling and sulfuric acid treatments, mimicking Brazilian bioethanol production.ConclusionsOur study combined genomic, mutational, and fitness analyses to understand the genetic underpinnings of yeast evolution to ethanol shocks. Although fitness analyses revealed that most evolved mutations impose a cost for cell propagation, combination of key mutations cyr1A1474T/usv1Δ endowed yeasts with higher tolerance for growth in the presence of ethanol. Moreover, alleles selected for acute stress survival comprising the P1c genotype conferred stress tolerance and optimal performance under conditions simulating the Brazilian industrial ethanol production.

Current and novel approaches in yeast cell death research.

The study of cell death mechanisms in fungi, particularly yeasts, has gained substantial interest in recent decades driven by the potential for biotechnological advancements and therapeutic interventions. Examples include the development of robust yeast strains for industrial fermentations and high-value compound production, novel food preservation strategies against spoilage yeasts, and the identification of targets for treating fungal infections in the clinic. In this review, we discuss a wide range of methods to characterize cellular alterations associated with yeast cell death, noting the advantages and limitations. We describe assays to monitor reversible events versus those that mark a commitment to cell death (point-of-no-return), as these distinctions are important to decipher the underlying regulatory mechanisms. Several well-known challenges remain, including the varied susceptibilities to death within a cell population and the delineation of detailed cell death mechanisms. The identification and characterization of morphologically distinct subsets of dying yeast cells within dynamic yeast populations provides opportunities to reveal novel vulnerabilities and survival mechanisms. Elucidating the intricacies of yeast regulated cell death (yRCD) will contribute to the advancement of scientific knowledge and foster breakthrough discoveries with broad-ranging implications.

Strain prevalence and killer factor only partially influence the fermentation activity of pairwise Saccharomyces cerevisiae wine strains inoculation.

Commercial Saccharomyces cerevisiae starters are single-strain cultures widely used in winemaking to optimise the fermentation process and improve the organoleptic quality of wine. Unfortunately, the worldwide extensive use of a limited number of industrial strains led to the standardisation of the sensory properties, reducing the identity of wines. Therefore, the use of multi-strain S. cerevisiae starters can be an alternative tool to alter the sensory profile of wines, increasing the diversity of wine styles. However, this strategy may be interesting only if the overall fermentation kinetics is not affected. To date, there is a lack of information regarding the influence of multi-strain starters on the overall fermentation process in wine. In this context, killer toxins, affecting the viability of sensitive strains, can play a significant role. This study aimed to evaluate the effects of pairing eight wine strains of S. cerevisiae (two sensitive, three neutral and three killer) in co-fermentations compared to single-strain fermentations. Results evidenced that, among co-fermentations where the strain prevalence was significant, the killer strains constituted 79% to 100% of the total yeast population when co-inoculated with a sensitive one. However, in most of the cases, co-fermentations kinetics were similar to those of sensitive strains or worse than both strains. Thus, the presence of a killer strain alone is not sufficient to predict the overall fermentation progress, which is an essential information in winemaking. Interestingly, the neutral strain P304.4 was always prevalent, regardless of the second strain and, in most of the co-fermentations, the overall fermentation trend was similar to the P304.4 single-strain fermentation. Regardless of killer activity, our results suggest that the effect of strains on fermentative kinetics is still unpredictable, and further studies are needed to thoroughly explore strain to strain interactions in winemaking.

A comparison of free amino nitrogen and yeast-assimilable nitrogen measurement methods for use in alcoholic fermentation of whey

The continued efficient use of whey as a fermentable substrate to produce alcoholic beverages will benefit from improving the measurement of key components relevant to the fermentability of whey. One component of fermentation that is not well studied in whey is the concentration of fermentable nitrogen, either as free amino nitrogen (FAN) or yeast-assimilable nitrogen (YAN). Fermentable nitrogen in media is essential for yeast cells to replicate. Insufficient concentrations of FAN or YAN to support the growing yeast population can result in sluggish or "stuck" fermentations. We evaluated 3 common methods of fermentable nitrogen determination and compared them for use in whey fermentation systems, based on ninhydrin, nitrogen by o-phthalaldehyde (NOPA), and formaldehyde pH (Sørensen titration) values. Each measurement method was evaluated independently using standard addition curves and compared for overall accuracy using paired t-tests (α = 0.05). Although the formaldehyde pH method showed high precision, it did not measure nitrogen accurately, as it overestimated YAN in whey by up to 6 times relative to other tests. The ninhydrin and NOPA methods both showed accuracy for fermentable nitrogen determination in whey based on analysis of standardized nitrogen sources. We concluded that either the ninhydrin FAN or NOPA YAN method may be used to determine the fermentable nitrogen content in whey. This is expected to improve the ability of producers to use whey as a fermentation medium.