Background. With growing interest in skin-fermented white wines, more winemakers in North America are producing this style of wine with cold-tolerant, interspecific hybrid grape varieties rather than only Vitis vinifera species. Objective. In this study we sought to characterize the level of phenolic extraction using extended skin contact post-fermentation using two hybrid white grape cultivars. Alcoholic fermentations were conducted with Cayuga White and Vidal Blanc separately, followed by five months of extended skin contact post-fermentation. Phenolic compounds and color quantification were monitored over the course of post-fermentative aging on grape skins. The parameters were analyzed using standard UV/Visible spectroscopy and HPLC-MS-based methods. Conclusions. For both hybrid cultivars, there were no significant changes in phenolic content or in brown or yellow color over five months of post-fermentation skin contact. Under the winemaking conditions used in this study, for the interspecific hybrid grape cultivars Cayuga White and Vidal Blanc, we found that extended skin contact did not increase phenolic extraction beyond the level achieved by completion of alcoholic fermentation, providing useful guidance for winemakers to make production decisions regarding potential benefits (increased compound extraction) and risks (increased spoilage or oxidation potential due to longer periods of atmospheric exposure) of extended skin contact post-fermentation.

Hard apple cider is considered to be a low-risk product for food spoilage and mycotoxin contamination due to its alcoholic nature and associated food sanitation measures. However, the thermotolerant mycotoxin-producing fungus Paecilomyces niveus may pose a significant threat to hard cider producers. P. niveus is known to infect apples (Malus xdomestica), and previous research indicates that it can survive thermal processing and contaminate finished apple juice with the mycotoxin patulin. To determine if hard apple cider is susceptible to a similar spoilage phenomenon, cider apples were infected with P. niveus or one of three patulin-producing Penicillium species and the infected fruits underwent benchtop fermentation. Cider was made with lab inoculated Dabinett and Medaille d'Or apple cultivars, and patulin was quantified before and after fermentation. Results show that all four fungi can infect cider apples and produce patulin, some of which is lost during fermentation. Only P. niveus was able to actively grow throughout the fermentation process. To determine if apple cider can be treated to hinder P. niveus growth, selected industry-grade sanitation measures were tested, including chemical preservatives and pasteurization. High concentrations of preservatives inhibited P. niveus growth, but apple cider flash pasteurization was not found to significantly impact spore germination. This study confirms that hard apple cider is susceptible to fungal-mediated spoilage and patulin contamination. P. niveus is an important concern for hard apple cider producers due to its demonstrated thermotolerance, survival in fermentative environments, and resistance to sanitation measures.

Plasmid shuttle vectors are a common tool used to study yeast physiology. The majority of yeast plasmids have been optimized for Saccharomyces cerevisiae lab strain compatibility, relying on auxotrophic complementation as their selective property. We sought to construct a series of plasmid shuttle vectors to extend functionality beyond strains with auxotrophic requirements, and test compatibility across a diverse panel of yeasts. We constructed 18 plasmids which were successfully maintained by yeasts from several genera. From a panel of 24 yeast strains, these plasmids were maintained by 18 yeasts, spanning 11 species within the genera Lachancea, Metschnikowia, Pichia, Saccharomyces, and Torulaspora. Additionally, an integrated gene expression reporter was assayed for functional compatibility with the 18 strains. Plasmid-derived gene expression was observed for 13 strains, spanning five species within the Saccharomyces genus, in addition to Torulaspora delbrueckii. These results indicate that this plasmid series is broadly useful for advancements and applications within academia, biotechnology, and the food and fermentation industries for research utilizing diverse Saccharomyces and non-Saccharomyces yeasts.

With the sustained popularity of hazy IPAs, brewers have explored multiple approaches to maximizing stable haze that will remain in suspension throughout the shelf life of the beer. Our recent investigations into yeast-dependent haze have uncovered specific brewing yeast strains that promote the formation of haze in heavily dry-hopped beer styles. These brewing strains have been termed “haze-positive” and furthermore, the timing of dry hop additions has been found to be another key factor in producing this stable haze. Classical genetics have identified YIL169C (herein referred to as HZY1 ) as both necessary and sufficient for the haze-positive phenotype in the yeast strain most widely used for Hazy IPAs. HZY1 encodes a candidate glycoprotein and our recent findings suggest it is localized to the cell wall through a GPI anchor. Surprisingly, using long-read sequencing data we uncovered extensive genetic variation in HZY1 across brewing strains. The haze-positive phenotype correlates with an expansion in the N-terminal serine-rich region. We propose that the Hzy1 glycoprotein is a critical component to yeast-dependent colloidal haze and the genetic variation in this locus contributes the range of haze phenotypes observed across industrial brewing strains.

In the yeast Saccharomyces cerevisiae, 2 types of trehalase activities have been described. Neutral trehalases (Nth1 and Nth2) are considered to be the main proteins that catalyze intracellular trehalose mobilization. In addition to Nth1 and Nth2, studies have shown that acid trehalase Ath1 is required for extracellular trehalose degradation. Although both neutral and acid-type trehalases have been predominantly investigated in laboratory strains of S. cerevisiae, we sought to examine the phenotypic consequences of disrupting these genes in wild strains. In this study, we constructed mutants of the trehalose degradation pathway (NTH1, NTH2, and ATH1) in 5 diverse S. cerevisiae strains to examine whether published lab strain phenotypes are also exhibited by wild strains. For each mutant, we assessed a number of phenotypes for comparison to trehalose biosynthesis mutants, including trehalose production, glycogen production, cell size, acute thermotolerance, high-temperature growth, sporulation efficiency, and growth on a variety of carbon sources in rich and minimal medium. We found that all trehalase mutants including single deletion nth1Δ, nth2Δ, and ath1Δ, as well as double deletion nth1nth2Δ, accumulated higher intracellular trehalose levels compared to their isogenic wild-type cells. Also, nth1Δ and nth1Δnth2Δ mutants exhibited mild thermal sensitivity, suggesting a potential minor role for trehalose mobilization when cells recover from stress. In addition, we evaluated phenotypes more directly relevant to trehalose degradation, including both extracellular and intracellular trehalose utilization. We discovered that intracellular trehalose hydrolysis is critical for typical spore germination progression, highlighting a role for trehalose in cell cycle regulation, likely as a storage carbohydrate providing glycolytic fuel. Additionally, our work provides further evidence suggesting Ath1 is indispensable for extracellular trehalose utilization as a carbon source, even in the presence of AGT1.

Chemical preservatives are ubiquitously used to suppress the growth of or kill microorganisms across numerous industries, including the food industry. Utilizing yeast functional genomic techniques, genes and their functions can be observed at a genomic scale to elucidate how environmental stressors (e.g., chemical preservatives) impact microbial survival. These types of chemical genomics approaches can reveal genetic mutations that result in preservative resistance or sensitivity, assist in identification of preservative mechanism of action, and can be used to compare different preservatives for rational design of preservative mixtures. In this proof-of-concept study, we performed deletion and high-copy genetic expression screens to identify mutants that confer drug resistance to sodium benzoate, potassium sorbate, rosemary extract, and Natamax. By observing overlapping mutant genes between genetic screens, we were able to identify functional overlap between chemical preservatives and begin to explain mechanisms of action for these compounds.

The fission yeast Schizosaccharomyces pombe and the budding yeast Saccharomyces cerevisiae are highly diverged (530 mya), single- celled, and model eukaryotic organisms. Scientists employ mating, meiosis, and the plating of ascospores and cells to generate strains with novel genotypes and to discover biological processes. Our 3 laboratories encountered independently sudden-onset, major impediments to such research. Spore suspensions and vegetative cells no longer plated effectively on minimal media. By systematically analyzing multiple different media components from multiple different suppliers, we identified the source of the problem. Specific lots of agar were toxic. We report that this sporadic toxicity affects independently the agar stocks of multiple vendors, has occurred repeatedly over at least 3 decades, and extends to species in highly diverged taxa. Interestingly, the inhibitory effects displayed variable penetrance and were attenuated on rich media. Consequently, quality control checks that use only rich media can provide false assurances on the quality of the agar. Lastly, we describe likely sources of the toxicity and we provide specific guidance for quality control measures that should be applied by all vendors as preconditions for their sale of agar.

Overconsumption of added sugars is associated with higher incidences of obesity, type II diabetes, and cardiovascular disease. Alternative sweeteners have long been relied on as a strategy to reduce consumption of added sugars; however these alternatives differ notably from sucrose in their sensory properties. Recently, consumers have also been looking to reduce consumption of ingredients deemed "artificial," seeking natural alternatives despite poor definitions of these terms on the whole. Wider knowledge of the sensory properties of natural sweeteners would greatly aid in this goal. Descriptive analysis and time-intensity scaling were used to characterize the temporal profile and off-flavors of allulose, erythritol, rebaudioside (Reb) A, Reb D, Reb M, monk fruit, and thaumatin, as compared to sucrose. Further, each was blended to reduce sucrose by 50% and 75% in binary mixtures. Where significant off-flavors were frequently reported with sweeteners, when delivered in binary mixtures for partial sugar reduction, these were much mitigated, while also bringing temporal profiles closer to that of sucrose. This suggests that partial sugar reduction may be an effective way of reducing caloric intake while not compromising sensory experience. Practical ApplicationThe use of both descriptive analysis and time-intensity scaling allows for a more complete understanding of the sensory properties of natural alternative sweeteners. Understanding their sensory deficits and ways to improve these sweeteners plays a critical role in creating healthier products that will be accepted by consumers.

Auxotrophic strains starving for their cognate nutrient, termed auxotrophic starvation, are characterized by a shorter lifespan, higher glucose wasting phenotype, and inability to accomplish cell cycle arrest when compared to a "natural starvation," where a cell is starving for natural environmental growth-limiting nutrients such as phosphate. Since evidence of this physiological response is limited to only a subset of auxotrophs, we evaluated a panel of auxotrophic mutants to determine whether these responses are characteristic of a broader range of amino acid auxotrophs. Based on the starvation survival kinetics, the panel of strains was grouped into three categories-short-lived strains, strains with survival similar to a prototrophic wild type strain, and long-lived strains. Among the short-lived strains, we observed that the tyrosine, asparagine, threonine, and aspartic acid auxotrophs rapidly decline in viability, with all strains unable to arrest cell cycle progression. The three basic amino acid auxotrophs had a survival similar to a prototrophic strain starving in minimal media. The leucine, tryptophan, methionine, and cysteine auxotrophs displayed the longest lifespan. We also demonstrate how the phenomenon of glucose wasting is limited to only a subset of the tested auxotrophs, namely the asparagine, leucine, and lysine auxotrophs. Furthermore, we observed pleiotropic phenotypes associated with a subgroup of auxotrophs, highlighting the importance of considering unintended phenotypic effects when using auxotrophic strains especially in chronological aging experiments.

Fermentation is a critical step in the production of coffee when following standard wet processing, one of the most common methods used to remove the mucilage layer from coffee cherries. During this step, the de-pulped coffee cherries undergo fermentation with native yeast that modifies the flavor profile of the resultant coffee. This study aimed to ferment green coffee beans using commercial yeast strains from beer and wine prized for their ability to produce specific flavors, and subsequently evaluate the aroma and flavor of the coffee using coffee consumers. Four Saccharomyces cerevisiae strains were used: Belgian Ale, Sourvisiae, 71 B, and Tropical IPA, along with one non-Saccharomyces, Toluraspora delbrueckii (Biodiva), and a non-inoculated control sample. The green coffee beans underwent a controlled wet fermentation for 72 h, followed by roasting, grinding, and brewing. Results showed that flavor profiles varied broadly by yeast strain, suggesting that producing novel flavors in coffee through fermentation is feasible and that these flavors survive the roasting process; however, higher liking scores were still reported for the control sample compared to the fermented samples. Biodiva, a strain used in wine to produce esters and fruity flavors, resulted in coffee with highly fruity notes, and all strains were rated more floral than the control, while the sample fermented with Sourvisiae yeast used in the brewing of sour ales resulted in coffee that was both perceived as more sour and had the lowest pH, likely due to the degree of lactic acid this strain is engineered to produce. Further, there were significant color differences between the samples. In conclusion, fermenting green coffee beans with brewing and winemaking yeast strains strongly impacted the flavor and aroma of the resultant coffee; however, evaluating larger panels of strains or optimizing strain performance may yield flavor profiles more suitable for coffee.

Determining the minimal concentration of a substance-whether a compound used to inhibit cell growth or a growth-limiting nutrient-can be an arduous process. Carrying out the experiment in flasks or tubes and estimating cell density in a single-sample spectrophotometer has been a routine method of choice due to the ease of measurement and standardized conversions from optical density measurements to cell concentrations. However, when dealing with dozens or more samples, several challenges arise, including increased processing time, increased risk of contamination through repeated sampling, and an increased risk of confounding one sample for another. The protocol described here details a rapid method to estimate cell concentrations for such experiments using a microplate spectrophotometer. Using a microplate spectrophotometer to measure optical density of many cultures can be automated for high throughput and benefits from a reduced risk of contamination. Since one of the caveats of a microplate spectrophotometer is its low saturation limit, we further describe how to convert optical density readings of the microplate spectrophotometer into its single-sample spectrophotometric equivalent. To further illustrate the applicability of this protocol, we compare OD600 readings generated using a microplate spectrophotometer and a single-sample spectrophotometer for different nutrient starvations and show that the results are comparable. Overall, this method reduces the required resources, reduces the risk of contamination, and allows for faster processing of samples. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Generating an equation to convert microplate spectrophotometer readings to single-sample spectrophotometric values Basic Protocol 2: Evaluating growth-limiting nutrient concentrations using a microplate spectrophotometer.

In Saccharomyces cerevisiae , trehalose-6-phosphate synthase (Tps1) catalyzes the formation of trehalose-6-phophate in trehalose synthesis. Deletion of the TPS1 gene is associated with phenotypes including inability to grow on fermentable carbon sources, survive at elevated temperatures, or sporulate. To further understand these pleiotropic phenotypes, we conducted a genetic suppressor screen and identified a novel suppressor, grr1 Δ, able to restore tps1 Δ growth on rapidly fermentable sugars. However, disruption of GRR1 did not rescue tps1 Δ thermosensitivity. These results support the model that trehalose metabolism has important roles in regulating glucose sensing and signaling in addition to regulating stress resistance.

Trehalose is a naturally occurring, non-reducing disaccharide comprising two covalently-linked glucose molecules. It possesses unique physiochemical properties, which account for multiple biological roles in a variety of prokaryotic and eukaryotic organisms. In the past few decades, intensive research on trehalose has uncovered its functions, and extended its uses as a sweetener and stabilizer in the food, medical, pharmaceutical, and cosmetic industries. Further, increased dietary trehalose consumption has sparked research on how trehalose affects the gut microbiome. In addition to its role as a dietary sugar, trehalose has gained attention for its ability to modulate glucose homeostasis, and potentially as a therapeutic agent for diabetes. This review discusses the bioactive effects of dietary trehalose, highlighting its promise in future industrial and scientific contributions.

While sequentially inoculating non-Saccharomyces yeasts with Saccharomyces cerevisiae can lower the alcohol contents of wine, the abilities of these yeasts to utilize/produce ethanol or generate other byproducts remained unclear. Metschnikowia pulcherrima or Meyerozyma guilliermondii were inoculated into media with or without S. cerevisiae to assess byproduct formation. Both species metabolized ethanol in a yeast-nitrogen-base medium but produced the alcohol in a synthetic grape juice medium. In fact, Mt. pulcherrima and My. guilliermondii generated less ethanol per gram of metabolized sugar (0.372 and 0.301 g/g, respectively) compared to S. cerevisiae (0.422 g/g). Sequentially inoculating each non-Saccharomyces species with S. cerevisiae into grape juice media achieved up to 3.0% v/v alcohol reduction compared to S. cerevisiae alone while producing variable glycerol, succinic acid, and acetic acid concentrations. However, neither non-Saccharomyces yeasts released appreciable CO2 under fermentative conditions regardless of incubation temperature. Despite equivalent peak populations, S. cerevisiae produced more biomass (2.98 g/L) than the non-Saccharomyces yeasts while sequential inoculations yielded higher biomass with Mt. pulcherrima (3.97 g/L) but not My. guilliermondii (3.03 g/L). To reduce ethanol concentrations, these non-Saccharomyces species may metabolize ethanol and/or produce less from metabolized sugars compared to S. cerevisiae but also divert carbon towards glycerol, succinic acid, and/or biomass.

This study is to investigate the use of a constitutively expressed trehalose transport protein to directly control intracellular trehalose levels and protect baker's yeast (Saccharomyces cerevisiae) cells against freeze–thaw stress in vivo.

In the yeast Saccharomyces cerevisiae, trehalose-6-phospahte synthase (Tps1) and trehalose-6-phosphate phosphatase (Tps2) are the main proteins catalyzing intracellular trehalose production. In addition to Tps1 and Tps2, 2 putative regulatory proteins with less clearly defined roles also appear to be involved with trehalose production, Tps3 and Tsl1. While this pathway has been extensively studied in laboratory strains of S. cerevisiae, we sought to examine the phenotypic consequences of disrupting these genes in wild strains. Here we deleted the TPS1, TPS2, TPS3, and TSL1 genes in 4 wild strains and 1 laboratory strain for comparison. Although some tested phenotypes were not shared between all strains, deletion of TPS1 abolished intracellular trehalose, caused inability to grow on fermentable carbon sources and resulted in severe sporulation deficiency for all 5 strains. After examining tps1 mutant strains expressing catalytically inactive variants of Tps1, our results indicate that Tps1, independent of trehalose production, is a key component for yeast survival in response to heat stress, for regulating sporulation, and growth on fermentable sugars. All tps2Δ mutants exhibited growth impairment on nonfermentable carbon sources, whereas variations were observed in trehalose synthesis, thermosensitivity and sporulation efficiency. tps3Δ and tsl1Δ mutants exhibited mild or no phenotypic disparity from their isogenic wild type although double mutants tps3Δ tsl1Δ decreased the amount of intracellular trehalose production in all 5 strains by 17-45%. Altogether, we evaluated, confirmed, and expanded the phenotypic characteristics associated trehalose biosynthesis mutants. We also identified natural phenotypic variants in multiple strains that could be used to genetically dissect the basis of these traits and then develop mechanistic models connecting trehalose metabolism to diverse cellular processes.

Acid whey from Greek-style yogurt (YAW) is an underutilized byproduct and a challenge for the dairy industry. One alternative is the fermentation of YAW by yeasts such as Saccharomyces, Brettanomyces, and Kluyveromyces spp., to produce new styles of fermented beverages. Previous research in our group suggested that the sugar profiles of the dairy coproducts impacted the fermentation profiles produced by B. claussenii. The present work aims to describe the fermentation of dairy sugars by S. cerevisiae, K. marxianus, and B. claussenii, under conditions comparable to those of YAW. For this purpose, four preparations of yeast nitrogen base, each containing 40 g/L of either lactose (LAC), glucose (GLU), galactose (GAL), or a 1:1 mixture of glucose and galactose (GLU:GAL), all at pH 4.20, were used as fermentation media. The fermentation was performed independently by each organism at 25 °C under anoxic conditions, while density, pH, cell count, ethanol, and organic acids were monitored. Non-linear modeling was used to characterize density curves, and Analysis of Variance and Tukey’s Honest Significant Difference tests were used to compare fermentation products. K. marxianus and S. cerevisiae displayed rapid sugar consumption with consistent ethanol yields in all media, as opposed to B. claussenii, which showed more variable results. The latter organism exhibited what appears to be a selective glucose fermentation in GLU:GAL, which will be explored in the future. These results provide a deeper understanding of dairy sugar utilization by relevant yeasts, allowing for future work to optimize fermentations to improve value-added beverage and ingredient production from YAW.

Trehalose is a nonreducing disaccharide composed of two glucose molecules linked by α, α-1,1-glycosidic bond. It is present in a wide variety of organisms, including bacteria, fungi, insects, plants, and invertebrate animals. Trehalose has distinct physical and chemical properties that have been investigated for their biological importance in a range of prokaryotic and eukaryotic species. Emerging research on trehalose has identified untapped opportunities for its application in the food, medical, pharmaceutical, and cosmetics industries. This review summarizes the chemical and biological properties of trehalose, its occurrence and metabolism in living organisms, its protective role in molecule stabilization, and natural and commercial production methods. Utilization of trehalose in the food industry, in particular how it stabilizes protein, fat, carbohydrate, and volatile compounds, is also discussed in depth. Challenges and opportunities of its application in specific applications (e.g., diagnostics, bioprocessing, ingredient technology) are described. We conclude with a discussion on the potential of leveraging the unique molecular properties of trehalose in molecular stabilization for improving the safety, quality, and sustainability of our food systems.

Diastatic yeasts are a major contamination risk to packaged beer. Brewery quality control programs rely on selective microbial growth media and molecular detection of the STA1 gene; however, there is a wide functional range of diastatic activity that remains unresolved by these current methods. Herein, a comprehensive analysis of diastatic yeast selection media using our collection of STA1+ strains is provided. For the preparation of microbial selection media, several factors to control including plate age, autoclave cycling conditions, cupric sulfate, and dipotassium phosphate concentrations are highlighted. Further several functional assays that confirm the variation in diastatic activity across STA1+ strains and show a clear correlation with earlier onset and increasing strength in strains that contain an intact STA1 promoter are presented. Furthermore, a robust and simple plate-based assay to confirm diastatic activity of potential contaminants has been developed. Collectively, these functional assays provide improved risk assessment of re-fermentation in packaged product with the potential for determining contaminant thresholds for different STA1+ strains and weighing in on the decision to destroy beer or issue a product recall.

Wine and alcoholic apple cider are commonly back-sweetened with unpasteurized juice to produce fresh, natural, and palatable sweetened alcoholic beverages. Foodborne pathogens may be introduced from unpasteurized juice into alcoholic beverages through this back-sweetening process. Although foodborne pathogens generally do not survive under low pH conditions or a high alcohol environment, the die-off of these pathogens has not been established to ensure the microbiological safety of the products. To establish the holding conditions that would provide the required 5-log pathogen reduction requirements for these back-sweetened beverages, we evaluated the survival of three common foodborne pathogens, E. coli O157:H7, Salmonella enterica, and Listeria monocytogenes, in modified white grape juice and apple juice models. White grape juice and apple juice were modified with hydrochloric acid and sodium hydroxide and with ethanol to achieve conditions that are similar to back-sweetened white wine and alcoholic apple cider in regard to pH and ethanol content. Foodborne pathogen cocktails were inoculated separately into modified juice models, and their survival in the juice models was recorded over a 96-h period. Our results show that a combination of low pH and high ethanol content resulted in faster pathogen die-off compared with higher pH and lower ethanol conditions. The holding times required for different combinations of pH and ethanol concentration for each juice model to achieve a 5-log reduction were reported. This research provides data to validate pathogen die-off to comply with juice hazard analysis and critical control point 5-log pathogen inactivation requirements for back-sweetened wine and alcoholic apple cider.