High Levels Of Ethanol Research Articles

Stress-Driven Production of γ-Aminobutyric Acid Using Non-Conventional Yeast Strains Kluyveromyces marxianus JMY140K and Metschnikowia reukaufii JMY075.

γ-Aminobutyric acid (GABA) is a valuable amino acid widely used in food, healthcare, and agriculture. GABA bioproduction by budding yeasts has been commonly reported, but related studies using non-conventional yeasts remain limited. In this study, two non-conventional natural yeast strains, namely, Kluyveromyces marxianus JMY140K and Metschnikowia reukaufii JMY075, were identified as promising GABA producers, and M. reukaufii JMY075 was discovered to be a GABA producer. Enhanced GABA production was observed in the two yeast strains under stress conditions, including high temperature and high ethanol and acetic acid levels. In particular, K. marxianus JMY140K showed 7.93 times higher GABA titers under thermal stress than that of the control. External stress conditions significantly influenced the GABA production of these two yeast strains. The culture filtrate of K. marxianus JMY140K also showed promising activities in human skin cells. In addition, K. marxianus JMY140K could also produce GABA using rice straw hydrolysate, which indicated that it has the potential to produce GABA using renewable biomass. Our studies provide insight for further enhancing the GABA production of natural yeasts and promoting its biotechnology applications.

Saccharomyces cerevisiae for lignocellulosic ethanol production: a look at key attributes and genome shuffling.

These days, bioethanol research is looking at using non-edible plant materials, called lignocellulosic feedstocks, because they are cheap, plentiful, and renewable. However, these materials are complex and require pretreatment to release fermentable sugars. Saccharomyces cerevisiae, the industrial workhorse for bioethanol production, thrives in sugary environments and can handle high levels of ethanol. However, during lignocellulose fermentation, S. cerevisiae faces challenges like high sugar and ethanol concentrations, elevated temperatures, and even some toxic substances present in the pretreated feedstocks. Also, S. cerevisiae struggles to efficiently convert all the sugars (hexose and pentose) present in lignocellulosic hydrolysates. That's why scientists are exploring the natural variations within Saccharomyces strains and even figuring out ways to improve them. This review highlights why Saccharomyces cerevisiae remains a crucial player for large-scale bioethanol production from lignocellulose and discusses the potential of genome shuffling to create even more efficient yeast strains.

The relevant and complex role of ethanol in the sensory properties of model wines

In a context of increasing interest in the production of wine-based beverages with lower ethanol content, the present work explored the role of ethanol in the sensory properties of model wines (MW). Four sets of MWs (red and white) with alcohol contents ranging from 0.5 to 15 % (1st study) and from 12 to 15 % (2nd study), the non-volatile fraction of real wines and fixed aroma compositions were all prepared and characterised by sensory descriptive analysis. The results indicate that sensory effects were stronger in red MWs than in white MWs, and that alcohol levels below 10 % in reds and 7.5 % in whites cause the models to become unbalanced with excessive sourness. In whites, low-alcohol MWs also showed reduced levels of perceived bitterness, and only at high ethanol levels were body and alcoholic aroma more intense. In reds, the low alcohol MWs showed less positive aroma persistence (perceived retronasally) and less body; at 0.5 % ethanol the positive odour intensity (perceived orthonasally) was minimal and at higher ethanol levels, the models became sweeter and more alcoholic. Overall, although samples with similar alcoholic contents maintained some sensory similarity, there were relevant discontinuities caused a slight increase in ethanol (0,5 %) to have a dramatic sensory impact, with some sensory descriptors showing significant maxima or minima at these alcohol levels. In reds, sourness, alcohol and astringency perceptions were maximum at 13.5 % and 15 % and body at 13 % and 15 %; meanwhile, red fruit was minimum at 13 % and 15 %, peaking at 14.5 %. In whites, alcohol perception was also maximum at 13.5 % and 15 %, while sweetness was maximum at 13.5 % and 14.5 %. These results demonstrate that ethanol exerts profound effects on sensory properties, probably due to strong perceptual interactions with odour, taste and tactile properties.

Investigating the Influence of Vessel Shape on Spontaneous Fermentation in Winemaking

The earliest archaeological evidence of wine came from ceramic vessels of the Transcaucasian ‘Shulaveri-Shomutepe’ or ‘Aratashen-Shulaveri-Shomutepe culture’ (SSC/AShSh: c. 6000–5200 BC). Western European ‘Bell Beaker culture’ (BB: c. 2500–2000 BC) is characterized by bell-shaped pottery vessels but has so far not been found with residues consistent with wine. Knowing that wild grapes populated both habitats, the absence of wine during the Bell Beaker period remains to be explained. The main goal of this work was to investigate whether the shape of the vessels could influence the performance of spontaneous fermentation, specifically regarding the production of volatile acidity. Crushed grapes or juices from various grape cultivars were fermented in two types of vessels: (i) borosilicate glass beakers (4–5 L) to imitate bell beakers and (ii) Erlenmeyer flasks (5 L) to imitate SSC/AShSh vessels. Fermentations occurred spontaneously, and the wines were analyzed for their conventional physical–chemical parameters (e.g., ethanol content, total acidity, volatile acidity, pH), chromatic characteristics (e.g., wine color intensity, wine hue), and volatile composition by gas-chromatography-flame ionization detection (GC-FID). At the end of fermentation, the yeast species were identified by molecular methods. In addition, wine yields and phenolic composition (e.g., total phenols, anthocyanins, total pigments) were determined for wild grapes in comparison with six red varieties Vitis vinifera L. subsp. sativa (Vinhão, Marufo, Branjo, Melhorio, Castelão and Tempranillo Tinto), chosen as a function of their genetic relatedness with the wild counterpart. Wines produced from V. sylvestris grapes showed higher total acidity and color intensity when compared to the cultivated varieties. Saccharomyces cerevisiae dominated at the end of all spontaneous fermentations in all types of vessels and conditions. Wines fermented in Erlenmeyers showed ethanol concentrations as high as 14.30% (v/v), while the highest ethanol level was 12.30% (v/v) in beakers. Volatile acidity increased to a maximum of 4.33 g/L (acetic acid) in Erlenmeyers and 8.89 g/L in beakers. Therefore, the shape of the vessels influenced the performance of fermentation, probably due to the different exposures to air, leading to vinegary ferments more frequently in open mouths than in conical-shaped flasks. These results provide a hypothesis based on fermentation performance for the absence of wine produced in the Iberian Peninsula until the arrival of Phoenician settlers.

Natural variation in yeast reveals multiple paths for acquiring higher stress resistance

BackgroundOrganisms frequently experience environmental stresses that occur in predictable patterns and combinations. For wild Saccharomyces cerevisiae yeast growing in natural environments, cells may experience high osmotic stress when they first enter broken fruit, followed by high ethanol levels during fermentation, and then finally high levels of oxidative stress resulting from respiration of ethanol. Yeast have adapted to these patterns by evolving sophisticated “cross protection” mechanisms, where mild ‘primary’ doses of one stress can enhance tolerance to severe doses of a different ‘secondary’ stress. For example, in many yeast strains, mild osmotic or mild ethanol stresses cross protect against severe oxidative stress, which likely reflects an anticipatory response important for high fitness in nature.ResultsDuring the course of genetic mapping studies aimed at understanding the mechanisms underlying natural variation in ethanol-induced cross protection against H2O2, we found that a key H2O2 scavenging enzyme, cytosolic catalase T (Ctt1p), was absolutely essential for cross protection in a wild oak strain. This suggested the absence of other compensatory mechanisms for acquiring H2O2 resistance in that strain background under those conditions. In this study, we found surprising heterogeneity across diverse yeast strains in whether CTT1 function was fully necessary for acquired H2O2 resistance. Some strains exhibited partial dispensability of CTT1 when ethanol and/or salt were used as mild stressors, suggesting that compensatory peroxidases may play a role in acquired stress resistance in certain genetic backgrounds. We leveraged global transcriptional responses to ethanol and salt stresses in strains with different levels of CTT1 dispensability, allowing us to identify possible regulators of these alternative peroxidases and acquired stress resistance in general.ConclusionsUltimately, this study highlights how superficially similar traits can have different underlying molecular foundations and provides a framework for understanding the diversity and regulation of stress defense mechanisms.

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.

Sensory-directed approach to explore cider typicity: the case of ciders from the Canary Islands (Spain)

PurposeThe main aim of this study is to characterise and identify specific chemo-sensory profiles of ciders from the Canary Islands (Spain).Design/methodology/approachCommercial samples of Canary ciders were compared to ciders from the Basque Country and Asturias. In total, 18 samples were studied, six for each region. The analysis comprised their sensory profiling and chemical characterisation of their polyphenolic profile, volatile composition, conventional chemical parameters and CIELAB colour coordinates. In parallel, the sensory profile of the samples from the Canary Islands was first compared with their Basque and Asturian counterparts by labelled sorting task. Then, their specific aroma profile was characterised by flash profile. Further quantification of sensory-active compounds was performed by GC–MS and GC-FID to identify the volatile compounds involved in their aroma profile.FindingsResults show that Canary ciders present a specific chemical profile characterised by higher levels of ethanol, and hydroxycinnamic acids, mainly t-ferulic, t-coumaric and neochologenic acids, and lower levels of volatile and total acidity than their Asturian and Basque counterparts. They also present a specific aroma profile characterised by fruity aroma, mainly fruit in syrup and confectionary, and sweet flavours related to their highest levels of vinylphenols formed by transformation of hydroxycinnamic acids.Originality/valueAn integrated strategy to explore the typicity of the currently existing Canary ciders in the market was developed. The results are important in that they will help other regions to identify specific typical chemo-sensory profiles and to promote the creation of certifications supporting regional typicity.

Effects of long-term metformin administration associated with high-intensity interval training on physical performance, glycogen concentration, GLUT-4 content, and NMR-based metabolomics in healthy rats.

The aim was to investigate the long-term effects of metformin ingestion on high-intensity interval training on performance, glycogen concentration (GC), GLUT-4 content, and metabolomics outcomes in rats. Fifty male Wistar rats were randomly divided into baseline, metformin (500 mg daily), and control groups. Training consisted of 4 sets of 10 jumps with 30 s of passive recovery per day, 5 days/week for 8 weeks. The intensity equivalent was 50% of body mass (BM) in the first four weeks and 70% of BM in the last four weeks. The animals were submitted to a weekly jump test until exhaustion at 50% of BM. Serum and tissues were collected at baseline and after 4 and 8 weeks for biochemical and metabolomics analysis. The number of jumps increased in the Control group without a significant difference between groups at 4 and 8 weeks. GLUT4 was lower in the gastrocnemius muscle in the Metformin at the fourth week compared to Control (P=0.03) and compared to Metformin (P=0.02) and Control (P=0.01) at eight weeks. Hepatic and soleus GC were not altered by metformin. Gastrocnemius GC was lower after 8 weeks in the Metformin group compared to Control (P=0.01). Significantly lower levels of pyruvate and phenylalanine and higher levels of ethanol, formate, betaine, very low-density lipoprotein, low-density lipoprotein, and creatine were found in the Metformin compared to the Control. Although chronic administration of metformin decreased food intake and negatively influenced the synthesis of muscle glycogen, it did not significantly change physical performance compared to the Control.

Formulation and evaluation of tenoxicam ethosomes as a novel drug carrier

The aim of this study is to create an alcohol-based tenoxicam gel for transversal delivery. Tenoxicam is a non-steroidal anti-inflammatory BCSII drug with low solubility and high permeability. It is prepared thermally using alcohol, phospholipids and ethanol. Alcosomes are phospholipid- based elastic nanoparticles containing high levels of ethanol (20-45%), which is known to be highly accessible. Ethanol systems are more effective at delivering the speed and depth of medication to the skin than liposomes or hydroalcoholic solutions. FTIR studies show that there is no interaction between the drug and the additive. Formulation F8 (ethanol 30% v/v and lecithin 1% w/w) was selected as the best formulation due to its small size, encapsulation efficiency, low turbidity, and highest in vitro release. A 3-month stability study was carried out on the F8 formulation using Carbopol 934 base (1,1.5, 2% w/w) at two different temperatures, 25°±2°C and 4°±2°C. The maximum in vitro release rate of carbomer concentration in rat skin at 1.5% w/w is 95.06 ± 0.15%, and the in vitro release rate is 86.65 ± 0.38%.

Seed dispersal syndrome predicts ethanol concentration of fruits in a tropical dry forest.

Studying fruit traits and their interactions with seed dispersers can improve how we interpret patterns of biodiversity, ecosystem function and evolution. Mounting evidence suggests that fruit ethanol is common and variable, and may exert selective pressures on seed dispersers. To test this, we comprehensively assess fruit ethanol content in a wild ecosystem and explore sources of variation. We hypothesize that both phylogeny and seed dispersal syndrome explain variation in ethanol levels, and we predict that fruits with mammalian dispersal traits will contain higher levels of ethanol than those with bird dispersal traits. We measured ripe fruit ethanol content in species with mammal- (n = 16), bird- (n = 14) or mixed-dispersal (n = 7) syndromes in a Costa Rican tropical dry forest. Seventy-eight per cent of fruit species yielded measurable ethanol concentrations. We detected a phylogenetic signal in maximum ethanol levels (Pagel's λ = 0.82). Controlling for phylogeny, we observed greater ethanol concentrations in mammal-dispersed fruits, indicating that dispersal syndrome helps explain variation in ethanol content, and that mammals may be more exposed to ethanol in their diets than birds. Our findings further our understanding of wild fruit ethanol and its potential role as a selective pressure on frugivore sensory systems and metabolism.

Alcohol and vascular endothelial function: Biphasic effect highlights the importance of dose.

Alcohol (ethanol) consumption has different influences on arterial disease, being protective or harmful depending on the amount and pattern of consumption. The mechanisms mediating these biphasic effects are unknown. Whereas endothelial cells play a critical role in maintaining arterial health, this study compared the effects of moderate and high alcohol concentrations on endothelial cell function. Human coronary artery endothelial cells (HCAEC) were treated with levels of ethanol associated with either low-risk/moderate drinking (i.e., 25 mM) or high-risk/heavy drinking (i.e., 50 mM) after which endothelial function was assessed. The effect of ethanol's primary metabolite acetaldehyde (10 and 25 μM) was also determined. Moderate ethanol exposure (25 mM) improved HCAEC barrier integrity as determined by increased transendothelial electrical resistance (TEER), inhibited cell adhesion molecule (CAM) mRNA expression, decreased inflammatory cytokine (interferon-γ and interleukin 6) production, inhibited monocyte chemotactic protein-1 (MCP-1) expression and monocyte adhesion, and increased homeostatic Notch signaling. In contrast, exposure to high-level ethanol (50 mM) decreased TEER, increased CAM expression and inflammatory cytokine production, and stimulated MCP-1 and monocyte adhesion, with no effect on Notch signaling. Reactive oxygen species (ROS) generation and endothelial nitric oxide synthase activity were increased by both alcohol treatments, and to a greater extent in the 50 mM ethanol group. Acetaldehyde-elicited responses were generally the same as those of the high-level ethanol group. Ethanol has biphasic effects on several endothelial functions such that a moderate level maintains the endothelium in a nonactivated state, whereas high-level ethanol causes endothelial dysfunction, as does acetaldehyde. These data show the importance of dose when considering ethanol's effects on arterial endothelium, and could explain, in part, the J-shaped relationship between alcohol concentration and atherosclerosis reported in some epidemiologic studies.

Structural and functional insights of a cold-adaptive β-glucosidase with very high glucose tolerance from Microbacterium sp. CIAB417

A gene glu1 (WP_243232135.1) coding for β-glucosidase from the genome of Microbacterium sp. CIAB417 was characterized for its cold adaptive nature and tolerance to high levels of glucose and ethanol. The phylogenetic analysis suggested the close association of glu1 with a similar gene from a mesophilic bacterium Microbacterium indicum. The purified recombinant GLU1 displayed its optimal activity and stability at pH 5 and temperature 30ᴼC. Additionally, the presence of L3 loop in GLU1 suggested its cold adaptive nature. The glucose tolerant Gate keeper residues (Leu 174 & Trp 169) with a distance of ∼ 6.953 Å between them was also predicted in GLU1. The GLU1 enzyme showed ≥ 95% and ≥ 40% relative activity in the presence of 5 M glucose and 20% ethanol. The Vmax, Km, and Kcat values of GLU1 for cellobiose substrate were observed to be 45.22 U/mg, 3.5 mM, and 41.0157 s-1, respectively. The GLU1 was found to be highly efficient in hydrolysis of celloologosaccharides (C2-C5), lactose and safranal picrocrocin into glucose. Hence, cold adaptive GLU1 with very high glucose and ethanol tolerance could be very useful in bio-refinery, dairy, and flavor industries.

Repeated-Batch Ethanol Fermentation from Sweet Sorghum Stem Juice under a Very High Gravity Condition Using a Stirred Tank Bioreactor Coupled with a Column Bioreactor by Immobilized Saccharomyces cerevisiae

The ethanol fermentation efficiency of sweet sorghum stem juice (SSJ) under a very high gravity (VHG) condition (250 g/L of sugar) was improved by immobilized Saccharomyces cerevisiae SSJKKU01, using a stirred tank bioreactor (STR) coupled with a column bioreactor (CR). Dried rattan pieces (as carriers for cell immobilization) at 50% of the working volume of the CR were suitable for use in a batch ethanol fermentation. The average ethanol concentration (PE) and ethanol productivity (QP) of repeated-batch fermentation in the CR for eight successive cycles were 109.85 g/L and 1.88 g/L⋅h, respectively. Then an STR coupled with a CR was applied for repeated-batch ethanol fermentation in two systems. System I was an STR (1.8 L working volume), and System II was an STR (1 L) coupled with a CR, referred to as a CR-F (0.8 L). Both systems were connected to a new CR, called CR-I, containing sterile dried rattan pieces at 50% of its working volume. Active yeast cells were inoculated only into the STR, and the medium circulation rate between bioreactors was 5.2 mL/min. The results showed that at least eight successive cycles could be operated with an average PE of 108.51 g/L for System I and 109.44 g/L for System II. The average QP and SC values of both systems were also similar, with values of 1.87 to 1.88 g/L⋅h and 93 to 94%, respectively. The morphology of the carriers with and without immobilized cells before and after the fermentation was investigated. The obtained results demonstrated that a repeated-batch fermentation by immobilized cells on rattan pieces, using an STR coupled with a CR, was successfully used to produce high levels of ethanol from SSJ under a VHG condition.

Induction of a Consumption Pattern for Ethanol and Gasoline in Brazil

Historically, carbon dioxide emissions from transport have been a globally discussed and analyzed problem. The adoption of flex fuel vehicles designed to run ethanol–gasoline blends is important to mitigate these emissions. The main purpose of this paper is to analyze the impact of the ethanol–gasoline price ratio on different vehicle models, and discuss the opportunities to increase ethanol consumption from this perspective. Our analysis shows that the use of a unique fuel economy ratio for all flex–fuel vehicles in the country significantly reduces the opportunity of some customers to purchase hydrous ethanol. The paper also discusses possible actions to provide adequate information that may increase the possibility of fuelling vehicles with a high-level ethanol blend.

Petechial hemorrhages, ethanol, and opioids in victims from intoxication

Petechial hemorrhages are of interest to forensic pathologists because of their association with pressure on the neck. This study shows the associations between ethanol, opioids in blood and the risk of petechiae in conjunctivae and eye lids of 865 medico-legally examined victims from intoxication, 112 (12.9 %) with petechiae. Livor mortis on the front, face down body position, higher body weight, and younger age of the victims were independently associated with higher risk of petechiae. These variables were used for adjustment in the logistic regression analyzes. We found associations between ethanol, opioids, and the risk of petechiae when analyzed simultaneously. The association between ethanol and the risk of petechiae differed in opioid negative and positive victims (interaction, p = 0.028). In the opioid negative group, the association was J-formed, victims with low to medium level ethanol having lower risk (OR = 0.77) than those without ethanol or opioids, whereas high ethanol level gave a 4-fold higher risk (OR = 3.97). In the opioid positive group, the J-formed pattern was reversed. Victims with low to medium level ethanol had more than 4 times higher risk (OR = 4.65), whereas high level ethanol gave a slightly elevated risk (OR = 1.34) only compared to no ethanol or opioids. The results suggest that ethanol and opioids have a complex association with the risk of petechiae independent of livor mortis, initial body position, body weight, and age in victims from intoxication. Of practical value for the post-mortem examination is that the pathologist must consider both the ethanol level and the presence of opioids when judging the significance of petechiae in the eye regions.

Beer ethanol and iso-α-acid level affect microbial community establishment and beer chemistry throughout wood maturation of beer

Sour beers produced by barrel-aging of conventionally fermented beers are becoming increasingly popular. However, as the intricate interactions between the wood, the microbes and the beer are still unclear, wood maturation often leads to inconsistent end products with undesired sensory properties. Previous research on industrial barrel-aging of beer suggests that beer parameters like the ethanol content and bitterness play an important role in the microbial community composition and beer chemistry, but their exact impact still remains to be investigated. In this study, an experimentally tractable lab-scale system based on an in-vitro community of four key bacteria (Acetobacter malorum, Gluconobacter oxydans, Lactobacillus brevis and Pediococcus damnosus) and four key yeasts (Brettanomyces bruxellensis, Candida friedrichii, Pichia membranifaciens and Saccharomyces cerevisiae) that are consistently associated with barrel-aging of beer, was used to test the hypotheses that beer ethanol and bitterness impact microbial community composition and beer chemistry. Experiments were performed using different levels of ethanol (5.2 v/v%, 8 v/v% and 11 v/v%) and bitterness (13 ppm, 35 ppm and 170 ppm iso-α-acids), and beers were matured for 60 days. Samples were taken after 0, 10, 20, 30 and 60 days to monitor population densities and beer chemistry. Results revealed that all treatments and the maturation time significantly affected the microbial community composition and beer chemistry. More specifically, the ethanol treatments obstructed growth of L. brevis and G. oxydans and delayed fungal growth. The iso-α-acid treatments hindered growth of L. brevis and stimulated growth of P. membranifaciens, while the other strains remained unaffected. Beer chemistry was found to be affected by higher ethanol levels, which led to an increased extraction of wood-derived compounds. Furthermore, the distinct microbial communities also induced changes in the chemical composition of the beer samples, leading to concentration differences in beer- and wood-derived compounds like 4-ethyl guaiacol, 4-ethyl phenol, cis-oak lactone, vanillin, furfural and 5-hydroxymethyl furfural. Altogether, our results indicate that wood-aging of beer is affected by biotic and abiotic parameters, influencing the quality of the final product. Additionally, this work provides a new, cost-effective approach to study the production of barrel-aged beers based on a simplified microbial community model.

Influence of Kazachstania spp. on the chemical and sensory profile of red wines

We report the fermentative traits of two Kazachstania species (K. aerobia and K. servazzii) in non-sterile red wine and the resulting chemical and sensory properties. This builds on our previous work which revealed that Kazachstania spp. increased acetate esters in sterilised white wine. In this study Kazachstania spp. were initially evaluated in laboratory-scale fermentations (500 mL) in Merlot must to assess whether similar increases in chemical/volatile compounds would occur. The impact of malolactic fermentation (MLF) by Oenococcus oeni (VP41) on aroma composition was considered and found to reduce ester profiles in Merlot wines. The sensory implications of sequential inoculation with Kazachstania spp., followed by Saccharomyces cerevisiae, were then evaluated in small-lot fermentations (7 kg) of Shiraz must. Fungal diversity was monitored during early fermentation stages and was influenced by the early implantation of Kazachstania spp., followed by the dominance of S. cerevisiae. The effect of MLF in Shiraz wines was inconclusive due to high ethanol levels providing an inhospitable environment for lactic acid bacteria. When compared to S. cerevisiae alone, Kazachstania spp. significantly increased acetate esters, particularly phenylethyl acetate and isoamyl acetate, in both Merlot and Shiraz. The Shiraz wines fermented with Kazachstania spp. had higher jammy and red fruit aroma/flavour compared to S. cerevisiae (monoculture) wines. No influence was observed on colour one-year post-bottling. Results from this study show the contribution of Kazachstania spp. to the aroma profile of red wines and demonstrate their potential as starter cultures for improving the aromatic complexity of wines.

A novel murine model of multi-day moderate ethanol exposure reveals increased intestinal dysfunction and liver inflammation with age

BackgroundThere are currently > 600 million people over the age of 65 globally and this number is expected to double by the year 2050. Alcohol use among this population is on the rise, which is concerning as aging is associated with increased risk for a number of chronic illnesses. As most studies investigating the effects of alcohol have focused on young/middle-aged populations, there is a dearth of information regarding the consequences of alcohol use in older consumers. In addition, most murine ethanol models have concentrated on exposure to very high levels of ethanol, while the vast majority of elderly drinkers do not consume alcohol in excess; instead, they drink on average 2 alcoholic beverages a day, 3–4 days a week.MethodsWe designed a murine model of aging and moderate ethanol consumption to determine if the deleterious effects of alcohol on the gut-liver axis are exacerbated in aged, relative to younger, animals. Aged and young mice were exposed to a multi-day moderate exposure ethanol regimen for 4 weeks and changes in gut permeability along with intestinal tight junction protein and antimicrobial peptide gene expression were measured. In addition, hepatic inflammation was assessed by histological analysis, inflammatory gene expression and flow cytometric analysis of inflammatory infiltrate.ResultsOur results reveal that in aged, but not young mice, moderate ethanol exposure yielded significantly worsened intestinal permeability, including increased bacterial translocation from the gut, elevated serum iFABP and leakage of FITC-dextran from the gut. Interestingly, moderate ethanol exposure in young animals led to gut protective transcriptional changes in the ileum while this protective response was blunted in aged mice. Finally, moderate ethanol exposure in aged mice also resulted in marked inflammatory changes in the liver.ConclusionsThese results demonstrate that aged mice are more susceptible to ethanol-induced gut barrier dysfunction and liver inflammation, even at moderate doses of ethanol. This increased vulnerability to ethanol’s gastrointestinal effects has important implications for alcohol use in the aging population. Future studies will explore whether improving intestinal barrier function can reverse these age-related changes.

Bioprospecting thermotolerant yeasts from distillery effluent and molasses for high-temperature ethanol production.

Isolation, characterization and assessment of inhibitor tolerance of thermotolerant yeasts associated with distillery effluent and molasses, and their use in high-temperature ethanol production from alkali-treated rice straw. A total of 92 thermotolerant yeasts were isolated from seven different distillery effluent and molasses samples. Based on MSP-PCR, 34 yeasts were selected and identified by sequencing the D1/D2 domain of LSU rDNA. These yeasts belonged to eight genera and nine different species. We assessed the inhibitor tolerance of these 34 well-characterized yeasts against various pre-treatment-generated inhibitors (furfural, 5-hydroxymethyl furfural and acetic acid) and also evaluated their ethanol yields at 40, 45 and 50℃. Among selected strains, Pichia kudriavzevii DSA3.2 exhibited the highest ethanol production (24.5gl-1 ) with an efficiency of 95.7% at 40℃ using 5% glucose. At 45℃, P. kudriavzevii DSA3.2 and Kluyveromyces marxianus MSS6.3 yielded maximum ethanol titres; 22.3 and 23gl-1 with 87.4% and 90% efficiency, respectively. While using alkali-treated RS at 45℃, K. marxianus MSS6.3 produced 10.5gl-1 of ethanol with 84.5% fermentation efficiency via separate hydrolysis and fermentation, and 10.9gl-1 of ethanol with 85% efficiency via simultaneous saccharification and fermentation. Pichia kudriavzevii DSA3.2, DSA3.1 and K. marxianus MSS6.3 also exhibited significant tolerance against multiple inhibitors. Yeast isolates P. kudriavzevii DSA3.2 and K. marxianus MSS6.3 exhibited significant inhibitor tolerance and proved to be suitable for high-temperature ethanol fermentation. After additional optimization and scale-up experiments, these isolates can be exemplary candidates for industrial-scale ethanol production from lignocellulosic biomass. Our study recognizes distillery effluents and molasses as specialized niches for yeasts with a broad substrate range, capable of tolerating multiple inhibitors and yielding high levels of ethanol at elevated temperatures. These yeasts can further be exploited for bioethanol production through SSF/SHF at a larger scale.

Integrated Multi-Omics Analysis of Mechanisms Underlying Yeast Ethanol Tolerance.

For yeast cells, tolerance to high levels of ethanol is vital both in their natural environment and in industrially relevant conditions. We recently genotyped experimentally evolved yeast strains adapted to high levels of ethanol and identified mutations linked to ethanol tolerance. In this study, by integrating genomic sequencing data with quantitative proteomics profiles from six evolved strains (data set identifier PXD006631) and construction of protein interaction networks, we elucidate exactly how the genotype and phenotype are related at the molecular level. Our multi-omics approach points to the rewiring of numerous metabolic pathways affected by genomic and proteomic level changes, from energy-producing and lipid pathways to differential regulation of transposons and proteins involved in cell cycle progression. One of the key differences is found in the energy-producing metabolism, where the ancestral yeast strain responds to ethanol by switching to respiration and employing the mitochondrial electron transport chain. In contrast, the ethanol-adapted strains appear to have returned back to energy production mainly via glycolysis and ethanol fermentation, as supported by genomic and proteomic level changes. This work is relevant for synthetic biology where systems need to function under stressful conditions, as well as for industry and in cancer biology, where it is important to understand how the genotype relates to the phenotype.