Crabtree/Warburg-like aerobic xylose fermentation by engineered Saccharomyces cerevisiae.

Bottlenecks in the efficient conversion of xylose into cost-effective biofuels have limited the widespread use of plant lignocellulose as a renewable feedstock. The yeast Saccharomyces cerevisiae ferments glucose into ethanol with such high metabolic flux that it ferments high concentrations of glucose aerobically, a trait called the Crabtree/Warburg Effect. In contrast to glucose, most engineered S. cerevisiae strains do not ferment xylose at economically viable rates and yields, and they require respiration to achieve sufficient xylose metabolic flux and energy return for growth aerobically. Here, we evolved respiration-deficient S. cerevisiae strains that can grow on and ferment xylose to ethanol aerobically, a trait analogous to the Crabtree/Warburg Effect for glucose. Through genome sequence comparisons and directed engineering, we determined that duplications of genes encoding engineered xylose metabolism enzymes, as well as TKL1, a gene encoding a transketolase in the pentose phosphate pathway, were the causative genetic changes for the evolved phenotype. Reengineered duplications of these enzymes, in combination with deletion mutations in HOG1, ISU1, GRE3, and IRA2, increased the rates of aerobic and anaerobic xylose fermentation. Importantly, we found that these genetic modifications function in another genetic background and increase the rate and yield of xylose-to-ethanol conversion in industrially relevant switchgrass hydrolysate, indicating that these specific genetic modifications may enable the sustainable production of industrial biofuels from yeast. We propose a model for how key regulatory mutations prime yeast for aerobic xylose fermentation by lowering the threshold for overflow metabolism, allowing mutations to increase xylose flux and to redirect it into fermentation products.

Effect of air-blast drying and the presence of protectants on the viability of yeast entrapped in calcium alginate beads with an aim to improve the survival rate.

Five yeast strains, Saccharomyces cerevisiae D8, M12, and S13; Hanseniaspora uvarum S6; and Issatchenkia orientalis KMBL5774, isolated from Korean grapes, were entrapped in Ca-alginate beads, which are non-toxic, simple to use, and economical. Ca-alginate beads containing yeast cells were soaked in protective solutions, such as skim milk, saccharides, polyols, and nitrogen compounds, before air-blast drying to improve the yeast survival rate and storage ability. The results showed that both entrapment in Ca-alginate beads and soaking in protective agents favorably affected the survival of all strains. The microenvironment formed by the beads and protective agents can protect the yeast cells from harsh environmental conditions, such as low water (below 10 %). All the yeast strains entrapped in Ca-alginate beads showed greater than 80 % survival and less than 11 % water content after air-blast drying at 37 °C for 5 h. In addition, air-blast dried cells of S. cerevisiae D8, M12, S13; H. uvarum S6; and I. orientalis KMBL5774 entrapped in 2 % Ca-alginate beads and soaked in protective agents (10 % skim milk containing 10 % sucrose, 10 % raffinose, 10 % trehalose, 10 % trehalose, and 10 % glucose, respectively) after air-blast drying at 37 °C for 5 h showed 90, 87, 92, 90, and 87 % viability, respectively. All dried entrapped yeast cells showed survival rates of at least 51 % after storage at 4 °C for 3 months.

Effects of protectant and rehydration conditions on the survival rate and malolactic fermentation efficiency of freeze-dried Lactobacillus plantarum JH287.

In this study, Lactobacillus plantarum JH287 was used as a malolactic fermentation starter in Campbell Early wine production. L. plantarum JH287 was first lyophilized, and the malolactic fermentation potential of freeze-dried L. plantarum JH287 was investigated. Different protective media and rehydration conditions were tested to improve the survival rate of freeze-dried L. plantarum JH287. Optimal protective medium contained 10 % sorbitol and 10 % skim milk. The optimal rehydration condition was a 1-h rehydration time conducted in the same protective media, and the combination of these two methods produced a survival rate of 86.37 %. In addition, a 77.71 % survival rate was achieved using freeze-dried samples that were stored at 4 °C for 2 months. Freeze-dried L. plantarum JH287 and Saccharomyces cerevisiae Fermivin were used to inoculate the Campbell Early grape must to decrease its malic acid content. Using this mixed-fermentation method, wine showed a decrease in malic acid content after 9 days of fermentation. GC-MS analysis detected 15 volatile ester compounds in the wine. A sensory evaluation showed that the taste and aroma of mix-fermented wine were better than those of the control that had not been inoculated with L. plantarum JH287.

Improvement of volatile aromatic compound levels and sensory quality of distilled soju derived from Saccharomyces cerevisiae and Wickerhamomyces anomalus co-fermentation.

Distilled soju, a Korean traditional alcoholic beverage, is produced by fermenting rice with a variety of microorganisms including molds, yeasts, and lactic acid-producing bacteria, followed by distillation. Our study sought to improve the quality of distilled soju through co-fermentation with Saccharomyces cerevisiae and Wickerhamomyces anomalus, known for producing volatile aromatic compounds during the early stages of fermentation. Analysis of volatile aromatic compounds in co-fermented distilled soju revealed a substantial increase in compounds with boiling points below 200 °C. Among them, ethyl hexanoate, isobutanol, and isoamyl alcohol were identified as the major volatile aromatic compounds based on Log2 fold change analyses of the volatile aromatic compound pattern. In sensory evaluation, co-fermented distilled soju received higher scores in terms of odor and overall preference. Therefore, incorporating W. anomalus may improve the quality of distilled soju.

Inhibitory Effect of Phenolic Compounds on Vascular Endothelial Growth Factor-Induced Retinal Endothelial Permeability and Angiogenesis.

Age-related macular degeneration (AMD), often triggered by endothelial barrier disruption through vascular endothelial growth factor (VEGF), is a leading cause of blindness. This study investigated the inhibitory effects of phenolic compounds on VEGF-induced endothelial cell proliferation, migration, angiogenesis, and permeability using human retinal microvascular endothelial cells (hRECs). Thirty-seven polyphenolic compounds were selected from various databases based on their antioxidant properties, abundance in food, and solubility. These compounds significantly reduced migration, tube formation, and endothelial permeability in VEGF-stimulated hRECs. Notably, formononetin, eriodictyol, biochanin A, and p-coumaric acid were more effective in suppressing VEGF-induced angiogenesis and endothelial permeability than lutein. Molecular docking simulations revealed that formononetin, eriodictyol, and biochanin A had relatively lower binding energies with VEGF receptor 2 (VEGFR2) than lutein and sorafenib. These findings highlight the potential of phenolic compounds to be used as VEGFR2 inhibitors and an alternative strategy for preventing AMD.

Fermentation Characteristics of Unripe Citrus unshiu Vinegar Production Using Acetic Acid Bacteria Isolated from Traditional Fermented Vinegars.

Here, we aimed to isolate an acetic acid bacterium that is suitable for the production of unripe Citrus unshiu vinegar from traditional fermented vinegars. We compared the halo sizes of isolates to select a strain with superior acetic acid production capabilities and selected Komagataeibacter kakiaceti P6 (P6) as the final strain. Using Acetobacter pasteurianus CY (CY) and A. pasteurianus KACC 17058 (KACC 17058) as controls, we analyzed the total phenolic compounds, total flavonoid content, antioxidant activities, and organic acids of the selected strain to verify its suitability for acetic acid fermentation. On the 30th day of the fermentation period, P6 showed a total acidity of 4.86%, which was higher than that of control groups (CY, 4.16%; KACC 17058, 4.01%). The total phenolic compounds, total flavonoid content, 1,1-diphenyl-2-picrylhydrazyl scavenging activity, and ferric ion reducing antioxidant power values significantly increased during fermentation with P6 compared with the initial C. unshiu wine, and no significant differences were observed from the vinegars produced by CY and KACC 17058. Moreover, organic acid analysis revealed that the unripe C. unshiu vinegar produced with P6 had an acetic acid content of 26.15 mg/mL, which was significantly higher than those produced with CY and KACC 17058, indicating that the P6 strain effectively produces acetic acid without adversely affecting other quality aspects during fermentation. In conclusion, the novel P6 strain is expected to be used as a starter for fermenting unripe C. unshiu vinegar, and its excellent acetic acid production capabilities suggest potential applications for other vinegars.

Quality Characteristics and Antioxidant Activities of Six Types of Korean White Wine.

The cultivation of European grape cultivars suitable for winemaking in Korea presents challenges due to factors such as climate, soil conditions, precipitation, and sunlight. Consequently, Korea has traditionally resorted to adding sugar to its wine production to counteract the low sugar content in Korean grapes, yielding lower-quality wines. However, recent success in the cultivation of five European grape cultivars and the development of the domestic grape cultivar Cheongsoo have increased the possibility of achieving high-quality Korean wines. This study aimed to explore the potential of European grape cultivars and Cheongsoo as wine grapes in Korea. This study also conducted sensory evaluation and analyzed the physicochemical properties of the grapes and wines, including antioxidant capacity and color. Despite originating from the same vineyard, the composition of grapes and wines, including volatile aromatic compounds, significantly differed among the grape cultivars. In particular, Vidal wine exhibited superior antioxidant capacity compared with other wines. Moreover, Cheongsoo wine showed higher levels of essential volatile aromatic compounds, such as monoterpenes, than other wines. Sensory evaluation of these two wines also revealed excellent results. In conclusion, these findings hold promise for enhancing the diversity of Korean white wine and fostering growth in the wine industry.

The Antimutagenic and Antioxidant Activity of Fermented Milk Supplemented with Cudrania tricuspidata Powder.

In this study, Cudrania tricuspidata (CT) containing abundant phytochemicals, such as xanthones and flavonoids, was evaluated as an additive to fortify the functionality and organoleptic quality of fermented milk. The physicochemical, functional, and sensory properties of fermented milk supplemented with different concentrations of CT powder were investigated. Increasing amounts of CT powder elevated the malic acid concentration, increasing the total acidity and decreasing the pH of fermented milk supplemented with CT powder. The viable cell count and free sugar contents of fermented milk indicated that supplementing with CT powder improved lactic acid fermentation slightly. The color of fermented milk supplemented with CT powder was darker, redder, yellower, and more pleasing than the control fermented milk. The total phenolic and flavonoid contents of fermented milk supplemented with CT powder rose as the concentration of supplemented CT powder increased, resulting in enhanced antioxidant and antimutagenic activities. The CT powder improved the functionality of the fermented milk; still, at 2% or more, it had some unfavorable sensory properties, such as sourness, taste, and texture, which reduced the overall consumer preference. Therefore, a CT powder concentration of 0.5% or 1% may be acceptable to consumers.

Isolation and Investigation of Potential Non-Saccharomyces Yeasts to Improve the Volatile Terpene Compounds in Korean Muscat Bailey A Wine.

The Muscat Bailey A (MBA) grape, one of the most prominent grape cultivars in Korea, contains considerable amounts of monoterpene alcohols that have very low odor thresholds and significantly affect the perception of wine aroma. To develop a potential wine starter for Korean MBA wine, nine types of non-Saccharomyces yeasts were isolated from various Korean food materials, including nuruk, Sémillon grapes, persimmons, and Muscat Bailey A grapes, and their physiological, biochemical, and enzymatic properties were investigated and compared to the conventional wine fermentation strain, Saccharomyces cerevisiae W-3. Through API ZYM analysis, Wickerhamomyces anomalus JK04, Hanseniaspora vineae S7, Hanseniaspora uvarum S8, Candida railenensis S18, and Metschnikowia pulcherrima S36 were revealed to have ß-glucosidase activity. Their activities were quantified by culturing in growth medium composed of different carbon sources: 2% glucose, 1% glucose + 1% cellobiose, and 2% cellobiose. W. anomalus JK04 and M. pulcherrima S36 showed the highest ß-glucosidase activities in all growth media; thus, they were selected and utilized for MBA wine fermentation. MBA wines co-fermented with non-Saccharomyces yeasts (W. anomalus JK04 or M. pulcherrima S36) and S. cerevisiae W-3 showed significantly increased levels of linalool, citronellol, and geraniol compared to MBA wine fermented with S. cerevisiae W-3 (control). In a sensory evaluation, the flavor, taste, and overall preference scores of the co-fermented wines were higher than those for the control wine, suggesting that W. anomalus JK04 and M. pulcherrima S36 are favorable wine starters for improving Korean MBA wine quality.

Development of air-blast dried non-Saccharomyces yeast starter for improving quality of Korean persimmon wine and apple cider.

A total of 512 yeasts, including 422 non-Saccharomyces yeasts, were isolated from various fruits including apple, aronia, Muscat Bailey A grapes, and persimmon. These were used to prepare persimmon wine and apple cider starters that produced high levels of aromatic compounds, which contribute to high-quality fermented products. Environmental tolerance testing with 20% glucose and 8% EtOH, alongside a sniffing test, led to the selection of Wickerhamomyces anomalus (Synonym Pichia anomala) SJ20, Meyerozyma caribbica (Synonym Pichia caribbica) YP1, Pichia kluyveri CD34, Hanseniaspora uvarum SJ69 (for persimmon wine), W. anomalus CS7-16 (for apple cider), and Starmerella bacillaris (Synonym Candida zemplinina) CD80 (for both wines) as wine starters. These strains had high environmental stress tolerance and the highest sniffing test scores. Persimmon wine and apple cider were fermented using these strains in single- or mixed-culture with S. cerevisiae W-3 to determine the improved effect on wine aroma. In accordance with the results of volatile ester compounds and sensory evaluation, W. anomalus SJ20, H. uvarum SJ69, and W. anomalus CS7-16 had an excellent potential as persimmon wine and apple cider starters. Moreover, other strains also showed a good potential for a distinctive persimmon wine and apple cider because of the different compositions of the various volatile ester compounds. Six types of sugars (fructose, glucose, maltose, sucrose, raffinose, sucrose, and trehalose), four types of rehydration solutions (distilled water, 1× phosphate buffered saline, 0.85% NaCl, and 1% peptone water), and two types of antioxidants (l-ascorbic acid and glutathione) were examined to improve the survival rate of air-blast dried non-Saccharomyces yeast cells. Optimal sugar and rehydration conditions for each strain were validated, and scanning electron microscopy showed that each cell was surrounded by protectants, including sugar, skim milk, and lactomil. Storability assessment of air-blast dried yeast cells maintained at 4 °C for two months indicated that at least one condition in each strain had a higher survival rate than the control, regardless of the concentration or type of antioxidant treatment, except for M. caribbica YP1. These results suggest that antioxidant treatment contributes to maintaining the viability of air-blast dried cells in hostile environments.

Optimization of air-blast drying process for manufacturing Saccharomyces cerevisiae and non-Saccharomyces yeast as industrial wine starters.

Wine yeast (Saccharomyces cerevisiae D8) and non-Saccharomyces wine yeasts (Hanseniaspora uvarum S6 and Issatchenkia orientalis KMBL5774) were studied using air-blast drying instead of the conventional drying methods (such as freeze and spray drying). Skim milk-a widely used protective agent-was used and in all strains, the highest viabilities following air-blast drying were obtained using 10% skim milk. Four excipients (wheat flour, nuruk, artichoke powder, and lactomil) were evaluated as protective agents for yeast strains during air-blast drying. Our results showed that 7 g lactomil was the best excipient in terms of drying time, powder form, and the survival rate of the yeast in the final product. Finally, 7 types of sugars were investigated to improve the survival rate of air-blast dried yeast cells: 10% trehalose, 10% sucrose, and 10% glucose had the highest survival rate of 97.54, 92.59, and 79.49% for S. cerevisiae D8, H. uvarum S6, and I. orientalis KMBL5774, respectively. After 3 months of storage, S. cerevisiae D8 and H. uvarum S6 demonstrated good survival rates (making them suitable for use as starters), whereas the survival rate of I. orientalis KMBL5774 decreased considerably compared to the other strains. Air-blast dried S. cerevisiae D8 and H. uvarum S6 showed metabolic activities similar to those of non-dried yeast cells, regardless of the storage period. Air-blast dried I. orientalis KMBL5774 showed a noticeable decrease in its ability to decompose malic acid after 3 months of storage at 4 °C.