Yeast Extract Peptone Dextrose Medium Research Articles

Cost-effective production of ergothioneine using Rhodotorula mucilaginosa DL-X01 from molasses and fish bone meal enzymatic hydrolysate

Ergothioneine (EGT) is a high-value natural antioxidant that cannot be synthesized by the human body. This study showed that Rhodotorula mucilaginosa DL-X01 can use untreated molasses and fish bone meal enzymatic hydrolysate as the substrates to synthesize EGT. By optimizing the growth conditions, the EGT yield reached 29.39 mg/L when molasses and fish bone meal (FBM) were added at 60 g/L and 400 g/L respectively. Finally, the EGT yield was increased to 216.25 mg/L by fed-batch fermentation in a 5 L bioreactor. Compared with the fermentation by yeast extract peptone dextrose medium, the feedstock cost of EGT production was reduced by 330.91 % by using molasses and FBM as substrates. These results showed that R. mucilaginosa DL-X01 can produce high-value EGT using two cheap processing by-products, molasses and FBM, which is of great significance for environmental protection and sustainable development.

Optimization of xylanase production by Pichia kudriavzevii and Candida tropicalis isolated from the wood product workshop.

Enzymatic compounds can be found abundantly and provide numerous advantages in microbial organisms. Xylanases are used in various pharmaceutical, food, livestock, poultry, and paper industries. This study aimed to investigate xylanase-producing yeasts, xylose concentration curve and their enzymatic activity under various factors including carbon and nitrogen sources, temperature, and pH. Enzyme activity was evaluated under different conditions before, during, and after purification. The yeast strains were obtained from the wood product workshop and were subsequently cultivated on YPD (yeast extract peptone dextrose) medium. Additionally, the growth curve of the yeast and its molecular identification were conducted. The optimization and design process of xylan isolated from corn wood involved the use of Taguchi software to test different parameters like carbon and nitrogen sources, temperature, and pH, with the goal of determining the most optimal conditions for enzyme production. In addition, the Taguchi method was utilized to conduct a multifactorial optimization of xylanase enzyme activity. The isolated species were partially purified using ammonium sulfate precipitation and dialysis bag techniques. The results indicated that 3 species (8S, 18S, and 16W) after molecular identification based on 18S rRNA gene sequencing were identified as Candida tropicalis SBN-IAUF-1, Candida tropicalis SBN-IAUF-3, and Pichia kudriavzevii SBN-IAUF-2, respectively. The optimal parameters for wheat carbon source and peptone nitrogen source were found at 50°C and pH 9.0 through single-factor optimization. By using the Taguchi approach, the best combination for highest activity was rice-derived carbon source and peptone nitrogen source at 50°C and pH 6.0. The best conditions for xylanase enzyme production in single-factor optimization of wheat bran were 2135.6 U/mL, peptone 4475.25 U/mL, temperature 50°C 1868 U/mL, and pH 9.0 2002.4 U/mL. Among the tested yeast, Candida tropicalis strain SBN-IAUF-1 to the access number MZ816946.1 in NCBI was found to be the best xylanase product. The highest ratio of enzyme production at the end of the delayed phase and the beginning of the logarithmic phase was concluded by comparing the growth ratio of 8S, 16W, and 18S yeasts with the level of enzymatic activity. This is the first report on the production of xylan polymer with a relative purity of 80% in Iran. The extracellular xylanases purified from the yeast species of C. tropicalis were introduced as a desirable biocatalyst due to their high enzymatic activity for the degradation of xylan polymers.

Cultivation of Microalgae Chlorella vulgaris in Open Reactor for Bioethanol Production

Microalgae have a high growth rate, high CO2 absorption capacity, and high content of chlorophyll, proteins, vitamins, mineral salts, carbohydrates, antioxidant substances, and fatty acids. In recent years, Chlorella vulgaris has been widely used as a feedstock for producing third-generation biofuels, such as bioethanol. Thus, this work aims to develop a strategy to increase the production scale of the microalgae Chlorella vulgaris grown in distilled reused water, supplemented with a modified BG-11 medium, to use biomass in the production of bioethanol. The total cultivation of 72 L presented a concentration of 0.415 g·L−1·d−1, with 61.32 g of final biomass. To improve carbohydrate extraction, the biomass was pre-treated with sulfuric acid at different concentrations (1.5% and 3% v/v). The hydrolyzed solution was supplemented with YPD (yeast extract peptone dextrose) medium and inoculated with Saccharomyces cerevisae yeast, initiating fermentation. In each sample, the Brix degree, cell concentration, reducing sugar concentration, and alcohol content were analyzed. The sample pre-treated with sulfuric acid 1.5% v/v was the one that presented the best result, with alcohol content after distillation of 68 °GL (Gay-Lussac). It appears that the cultivation of the microalgae Chlorella vulgaris in scale-up, with reused water, has high potential in the production of third-generation biofuel.

Efficient biosynthesis of resveratrol via combining phenylalanine and tyrosine pathways in Saccharomyces cerevisiae

BackgroundResveratrol is a commercially available stilbenoid widely used as dietary supplements, functional food ingredients, and cosmetic ingredients due to its diverse physiological activities. The production of resveratrol in microorganisms provides an ideal source that reduces the cost of resveratrol, but the titer in Saccharomyces cerevisiae was still much lower than that in other hosts.ResultsTo achieve enhanced production of resveratrol in S. cerevisiae, we constructed a biosynthetic pathway via combining phenylalanine and tyrosine pathways by introducing a bi-functional phenylalanine/tyrosine ammonia lyase from Rhodotorula toruloides. The combination of phenylalanine pathway with tyrosine pathway led to a 462% improvement of resveratrol production in yeast extract peptone dextrose (YPD) medium with 4% glucose, suggesting an alternative strategy for producing p-coumaric acid-derived compounds. Then the strains were further modified by integrating multi-copy biosynthetic pathway genes, improving metabolic flux to aromatic amino acids and malonyl-CoA, and deleting by-pathway genes, which resulted in 1155.0 mg/L resveratrol in shake flasks when cultured in YPD medium. Finally, a non-auxotrophic strain was tailored for resveratrol production in minimal medium without exogenous amino acid addition, and the highest resveratrol titer (4.1 g/L) ever reported was achieved in S. cerevisiae to our knowledge.ConclusionsThis study demonstrates the advantage of employing a bi-functional phenylalanine/tyrosine ammonia lyase in the biosynthetic pathway of resveratrol, suggesting an effective alternative in the production of p-coumaric acid-derived compounds. Moreover, the enhanced production of resveratrol in S. cerevisiae lays a foundation for constructing cell factories for various stilbenoids.Graphical

Examining How the Fermentation Medium Influences Thiol Expression and Its Perceived Aroma in Commercial Brewing Yeast Strains.

In Saccharomyces, the IRC7 gene encodes for a cysteine S-conjugate β-lyase enzyme which can release polyfunctional thiols from their cysteinylated precursor forms, thereby promoting thiol aroma in beer. This study examined the thiol production of 10 commercial yeast strains in two different media, a hopped yeast extract-peptone-dextrose (YPD) medium and a 100% barley malt wort to explore how differences in yeast strain and medium conditions influence the release of polyfunctional thiols. 3-Sulfanylhexan-1-ol was most affected by medium conditions, and its concentrations were highest in wort fermentations. The higher nitrogen content and pH of the YPD medium relative to the wort fermentations were notable differences, and significant correlations between these variables and the extent of free thiol production were observed. A strong association existed between polyfunctional thiol concentrations and the fermentation-derived, malt, and hop-derived compounds 2-phenylethanol, β-damascenone, and β-ionone. The sensory impressions of thiol character in beer were influenced by the presence of other aromatic compounds such as esters and terpene alcohols, and aroma attributes such as "tropical" were not the most suitable for describing beers brewed with yeasts that fully express homozygous IRC7F. Sensory attributes "sweaty", "vegetal", and "overripe fruit" were more strongly associated with these strains.

Quorum sensing molecules in yeast wastewater treatment and their regulation of yeast cell morphology

Yeast cells can transform from yeast form into mycelium form under some circumstances, resulting in poor cell settleability and seriously deteriorating the stability of system operation. The feasibility of mediating cell morphology by quorum sensing was carried out in this study. The results showed that the predominant species in the system, Candida tropicalis, could secrete quorum sensing molecules(QSMs) in some media. The supernatant of yeast extract-peptone-dextrose medium(YPD) or yeast filamentation-induced medium(YCBP) after cultivating Candida tropicalis could inhibit mycelial transformation in YCBP, implying that this strain could secrete QSMs. Further, QSMs-(E, E)-farnesol, 2-phenylethanol(2-PE) and a cell filamentation inhibitor 2,4-di-tert-butylphenol(2,4-di-TBP) were detected in YPD supernatant and a lab-scale SBR system in treating oil-containing wastewater. 2-PE and 2,4-di-TBP were detected in the supernatant of YCBP, indicating that the kinds of QSMs depended on substrate components. Finally, the detected compounds were added to a filamentation-induced media to investigate the optimal supplement time to antagonize cell morphological transformation. The results showed that the addition of (E, E)-farnesol, 2-PE, or 2,4-di-TBP could inhibit the morphological transformation from yeast form into mycelium form within the first 3 h of induction, with the maximum inhibition rates of mycelia number and length of 78.1 ± 3.5 % and 59.4 ± 12.7 % for 300 μM (E, E)-farnesol, respectively. For 100 μM 2,4-di-TBP, the highest mycelia number and length inhibition rates were achieved at 98.5 ± 2.3 % and 86.8 ± 5 %. Supplements of QSMs or filamentation inhibitors to the system are expected to inhibit yeast filamentation and establish an efficient and stable yeast wastewater treatment system.

Enhancing biofuels production by engineering the actin cytoskeleton in Saccharomyces cerevisiae

Saccharomyces cerevisiae is widely employed as a cell factory for the production of biofuels. However, product toxicity has hindered improvements in biofuel production. Here, we engineer the actin cytoskeleton in S. cerevisiae to increase both the cell growth and production of n-butanol and medium-chain fatty acids. Actin cable tortuosity is regulated using an n-butanol responsive promoter-based autonomous bidirectional signal conditioner in S. cerevisiae. The budding index is increased by 14.0%, resulting in the highest n-butanol titer of 1674.3 mg L−1. Moreover, actin patch density is fine-tuned using a medium-chain fatty acid responsive promoter-based autonomous bidirectional signal conditioner. The intracellular pH is stabilized at 6.4, yielding the highest medium-chain fatty acids titer of 692.3 mg L−1 in yeast extract peptone dextrose medium. Engineering the actin cytoskeleton in S. cerevisiae can efficiently alleviate biofuels toxicity and enhance biofuels production.

Methods for Oxygenation of Continuous Cultures of Brewer’s Yeast, Saccharomyces cerevisiae

Maintaining steady-state, aerobic cultures of yeast in a bioreactor depends on the configuration of the bioreactor system as well as the growth medium used. In this paper, we compare several conventional aeration methods with newer filter methods using a novel optical sensor array to monitor dissolved oxygen, pH, and biomass. With conventional methods, only a continuously stirred tank reactor configuration gave high aeration rates for cultures in yeast extract peptone dextrose (YPD) medium. For filters technologies, only a polydimethylsiloxan filter provided sufficient aeration of yeast cultures. Further, using the polydimethylsiloxan filter, the YPD medium gave inferior oxygenation rates of yeast compared to superior results with Synthetic Complete medium. It was found that the YPD medium itself, not the yeast cells, interfered with the filter giving the low oxygen transfer rates based on the volumetric transfer coefficient (KLa). The results are discussed for implications of miniaturized bioreactors in low-gravity environments.

Metabolic Engineering of Non-carotenoid-Producing Yeast Yarrowia lipolytica for the Biosynthesis of Zeaxanthin.

Zeaxanthin is vital to human health; thus, its production has received much attention, and it is also an essential precursor for the biosynthesis of other critical carotenoids such as astaxanthin and crocetin. Yarrowia lipolytica is one of the most intensively studied non-conventional yeasts and has been genetically engineered as a cell factory to produce carotenoids such as lycopene and β-carotene. However, zeaxanthin production by Y. lipolytica has not been well investigated. To fill this gap, β-carotene biosynthesis pathway has been first constructed in this study by the expression of genes, including crtE, crtB, crtI, and carRP. Three crtZ genes encoding β-carotene hydroxylase from different organisms were individually introduced into β-carotene-producing Y. lipolytica to evaluate their performance for producing zeaxanthin. The expression of crtZ from the bacterium Pantoea ananatis (formerly Erwinia uredovora, Eu-crtZ) resulted in the highest zeaxanthin titer and content on the basis of dry cell weight (DCW). After verifying the function of Eu-crtZ for producing zeaxanthin, the high-copy-number integration into the ribosomal DNA of Y. lipolytica led to a 4.02-fold increase in the titer of zeaxanthin and a 721% increase in the content of zeaxanthin. The highest zeaxanthin titer achieved 21.98 ± 1.80 mg/L by the strain grown on a yeast extract peptone dextrose (YPD)–rich medium. In contrast, the highest content of DCW reached 3.20 ± 0.11 mg/g using a synthetic yeast nitrogen base (YNB) medium to culture the cells. Over 18.0 g/L of citric acid was detected in the supernatant of the YPD medium at the end of cultivation. Furthermore, the zeaxanthin-producing strains still accumulated a large amount of lycopene and β-carotene. The results demonstrated the potential of a cell factory for zeaxanthin biosynthesis and opened up an avenue to engineer this host for the overproduction of carotenoids.

Two-Stage Fermentation of Lipomyces starkeyi for Production of Microbial Lipids and Biodiesel.

The high operating cost is currently a limitation to industrialize microbial lipids production by the yeast Lipomyces starkeyi. To explore economic fermentation technology, the two-stage fermentation of Lipomyces starkeyi using yeast extract peptone dextrose (YPD) medium, orange peel (OP) hydrolysate medium, and their mixed medium were investigated for seven days by monitoring OD600 values, pH values, cell growth status, C/N ratios, total carbon concentration, total nitrogen concentration, residual sugar concentration, lipid content, lipid titer, and fatty acids profiles of lipids. The results showed that two-stage fermentation with YPD and 50% YPD + 50% OP medium contributed to lipid accumulation, leading to larger internal lipid droplets in the yeast cells. However, the cells in pure OP hydrolysate grew abnormally, showing skinny and angular shapes. Compared to the one-stage fermentation, the two-stage fermentation enhanced lipid contents by 18.5%, 27.1%, and 21.4% in the flasks with YPD medium, OP medium, and 50%YPD + 50%OP medium, and enhanced the lipid titer by 77.8%, 13.6%, and 63.0%, respectively. The microbial lipids obtained from both one-stage and two-stage fermentation showed no significant difference in fatty acid compositions, which were mainly dominated by palmitic acid (33.36–38.43%) and oleic acid (46.6–48.12%). Hence, a mixture of commercial medium and lignocellulosic biomass hydrolysate could be a promising option to balance the operating cost and lipid production.

Genetically Engineered Oleaginous Yeast Lipomyces starkeyi for Sesquiterpene α-Zingiberene Production.

Oleaginous yeast, such as Lipomyces starkeyi, are logical organisms for production of higher energy density molecules like lipids and terpenes. We demonstrate that transgenic L.starkeyi strains expressing an α-zingiberene synthase gene from lemon basil or Hall's panicgrass can produce up to 17 mg/L α-zingiberene in yeast extract peptone dextrose (YPD) medium containing 4% glucose. The transgenic strain was further examined in 8% glucose media with C/N ratios of 20 or 100, and YPD. YPD medium resulted in 59 mg/L α-zingiberene accumulation. Overexpression of selected genes from the mevalonate pathway achieved 145% improvement in α-zingiberene synthesis. Optimization of the growth medium for α-zingiberene production led to 15% higher titer than YPD medium. The final transgenic strain produced 700 mg/L α-zingiberene in fed-batch bioreactor culture. This study opens a new synthetic route to produce α-zingiberene or other terpenoids in L.starkeyi and establishes this yeast as a platform for jet fuel biosynthesis.

Isolation, Identification and Function of Pichia anomala AR2016 and Its Effects on the Growth and Health of Weaned Pigs.

Simple SummaryPichia anomala is a simple pseudomycelium with a round cell shape, product killer factors, β-1,3-glucanase, and phytase. P. anomala had the functions of anticorrosion, improving the nutritive value of feed, increasing the protein content, and reducing the concentration of nutritive substances. In this study, P. anomala AR2016 is isolated and identified from solid wine koji, and its culture conditions are optimized. Heat tolerance, bile salt tolerance, and artificial gastric and intestinal juice tolerance are evaluated. In our methodology, thirty weaned pigs were randomly divided into three groups with 10 barrows in each, and fed a maize-soybean meal diet and orally administered 0.85% saline (CK), 1 mL 1 × 109 cfu/mL Candida utilis (C. utilis), and 1 mL 1 × 109 cfu/mL P. anomala once daily for 28 days (P. anomala). Our results show that P. anomala AR2016 grew best in yeast extract peptone dextrose medium with pH 5.0 at 28 °C, 180 r/min and could tolerate 45 °C for 0.5 h, 0.2% pig bile salts, simulated gastric fluid, and 1.0% simulated intestinal fluid. Our study indicates that P. anomala AR2016 can tolerate high acidity and high bile salts, and has high survivability in the artificial gastric intestinal juice environment. In conclusion, oral administration of P. anomala AR2016 improves the growth performance, reduces the incidence of diarrhea, enhances intestinal barrier function, and improves microflora in weaned pigs.This study isolates and identifies Pichia anomala (P. anomala) AR2016, and studies its effect on the growth and health of weaned pigs. A P. anomala strain from solid wine koji is isolated and identified using 26S rDNA analysis, and its culture conditions are optimized. Heat tolerance, bile salt tolerance, artificial gastric, and intestinal juice tolerance are evaluated. In our methodology, thirty 28 d Large White × Landrace × Rongchang weaned pigs were randomly divided into three groups with 10 barrows in each, and fed a maize-soybean meal diet and orally administered 0.85% saline (CK), 1 mL 1 × 109 cfu/mL Candida utilis (C. utilis), and 1 mL 1 × 109 cfu/mL P. anomala once daily for 28 days. A P. anomala strain was identified and named P. anomala AR2016. P. anomala AR2016 grew best in yeast extract peptone dextrose medium with pH 5.0 at 28 °C, 180 r/min and could tolerate 45 °C for 0.5 h, 0.2% pig bile salts, simulated gastric fluid, and 1.0% simulated intestinal fluid. Our results show that compared with the CK group, orally administered P. anomala AR2016 increases average daily gain, the ileal villus height, the ileal mucosal concentrations of occludin and zonula occluens-1, the serum glucose and total protein concentration, total superoxide dismutase, glutathione peroxidase, and total antioxidative capacity activity, the trypsin and lipase activity in jejunal and ileal contents, the jejunal and ileal mucosa mRNA levels of ALP, TNF-α, and TLR-2, and the relative abundance of Bacteroidetes, Actinobacteria, Succinivibrionaceae, Lachnospiraceae, and Prevotellaceae (p < 0.05). Compared with the CK group, oral administration of P. anomala AR2016 decreased the incidence of diarrhea, aspartate aminotransferase activity, alanine amino-transferase-activity, malondialdehyde, D-lactic acid and endotoxin content in serum, the mRNA level of aminopeptidase N of ileum mucosa, and the relative abundance of Proteobacteria, Clostridiaceae, Campylobacteraceae, Vibrionaceae, Bacillus, and Pseudon (p < 0.05). Collectively, the study indicates that P. anomala AR2016 can tolerate high acidity and high bile salts, and has high survivability in the artificial gastric intestinal juice environment. Oral administration of P. anomala AR2016 improves the growth performance, reduces the incidence of diarrhea, enhances intestinal barrier function, and improves microflora in weaned pigs.

Microbial Culture in Minimal Medium With Oil Favors Enrichment of Biosurfactant Producing Genes.

The waste produced by petrochemical industries has a significant environmental impact. Biotechnological approaches offer promising alternatives for waste treatment in a sustainable and environment-friendly manner. Microbial consortia potentially clean up the wastes through degradation of hydrocarbons using biosurfactants as adjuvants. In this work, microbial consortia were obtained from a production water (PW) sample from a Brazilian oil reservoir using enrichment and selection approaches in the presence of oil as carbon source. A consortium was obtained using Bushnell-Haas (BH) mineral medium with petroleum. In parallel, another consortium was obtained in yeast extract peptone dextrose (YPD)-rich medium and was subsequently compared to the BH mineral medium with petroleum. Metagenomic sequencing of these microbial communities showed that the BH consortium was less diverse and predominantly composed of Brevibacillus genus members, while the YPD consortium was taxonomically more diverse. Functional annotation revealed that the BH consortium was enriched with genes involved in biosurfactant synthesis, while the YPD consortium presented higher abundance of hydrocarbon degradation genes. The comparison of these two consortia against consortia available in public databases confirmed the enrichment of biosurfactant genes in the BH consortium. Functional assays showed that the BH consortium exhibits high cellular hydrophobicity and formation of stable emulsions, suggesting that oil uptake by microorganisms might be favored by biosurfactants. In contrast, the YPD consortium was more efficient than the BH consortium in reducing interfacial tension. Despite the genetic differences between the consortia, analysis by a gas chromatography-flame ionization detector showed few significant differences regarding the hydrocarbon degradation rates. Specifically, the YPD consortium presented higher degradation rates of C12 to C14 alkanes, while the BH consortium showed a significant increase in the degradation of some polycyclic aromatic hydrocarbons (PAHs). These data suggest that the enrichment of biosurfactant genes in the BH consortium could promote efficient hydrocarbon degradation, despite its lower taxonomical diversity compared to the consortium enriched in YPD medium. Together, these results showed that cultivation in a minimal medium supplemented with oil was an efficient strategy in selecting biosurfactant-producing microorganisms and highlighted the biotechnological potential of these bacterial consortia in waste treatment and bioremediation of impacted areas.

Effect of Optisol Supplementation With 0.255 μg/mL Amphotericin B on Elimination of Yeast at 5°C.

Fungal infections in lamellar keratoplasty are a growing concern. Optisol-GS does not contain an antifungal agent and supplementation with 0.255 μg/mL Amphotericin B (AmpB) has been considered. This study tested the ability of 0.255 μg/mL AmpB in Optisol-GS to eliminate yeast contamination of corneal tissue. Three isolates of Candida albicans, 1 of Candida parapsilosis, and 1 of Candida glabrata were tested in Optisol with and without AmpB. Corneoscleral rims stored at -80°C were thawed and placed in 10 multiwell plates (4 per plate). The rims were inoculated with 4 respective loads of yeast: 0, 10, 10, and 10 colony-forming units in 2 sets of 5 for 5 yeasts. One set was filled with Optisol plus AmpB and the other with Optisol only. All 10 plates were incubated at cold storage (2°C-8°C) for 48 hours. After 48 hours, all corneal rims were placed into 10 mL of yeast extract peptone dextrose medium; a swab culture of each well was plated onto Sabouraud plates; and all plates with the remaining Optisol were incubated at 30°C. Yeast growth was monitored for 10 days. Minimum inhibitory concentration and minimum fungicidal concentration were determined. All corneoscleral specimens were positive regardless of fungal load or presence of AmpB. All controls remained negative. Minimum inhibitory concentrations and minimum fungicidal concentrations were equivalent and ranged between 0.5 and 2.0 μg/mL. AmpB at a concentration of 0.255 μg/mL in Optisol-GS at cold storage (2°C-8°C) over 48 hours did not eliminate yeast from corneal tissue.

The production of yeast cell wall using an agroindustrial waste influences the wall thickness and is implicated on the aflatoxin B1 adsorption process

The objectives of this study were: to evaluate the use of dry distillery grain soluble extract - DDGse to produce yeast biomass and to obtain cell wall (CW), to use the CW as an aflatoxin B1 (AFB1) adsorbent, to study the variation in the composition and thickness of the CW under the influence of DDGse to evaluate their implication on the adsorption process using transmission electron microscopy (TEM) and fourier-transform infrared spectroscopy (FITR). The production of biomass and CW were variable. The CW thickness values showed that S. boulardii strain grown in yeast extract peptone dextrose (YPD) or DDGse medium, with no significant differences observed. The thickness of the CW for S. cerevisiae (RC012 and VM014) were increased when the cells were grown in DDGse medium, the thickness was almost double compared to the values obtained in YPD medium. The spectra IR of each CW in the two culture media shown regions corresponding to polysaccharides, proteins and lipids. Cells grown in DDGse medium adsorbed more AFB1 than those grown in YPD. The CW adsorbed more AFB1 than the same amount of whole cell. Future studies should be done to determine the type of carbohydrates and the relationship between chitin - beta glucans responsible for mycotoxin adsorption.

Enhanced antifungal activity of voriconazole-loaded nanostructured lipid carriers against Candida albicans with a dimorphic switching model.

Candida commonly adheres to implanted medical devices and forms biofilms. Due to the minimal activity of current antifungals against biofilms, new drugs or drug-delivery systems to treat these persistent infections are urgently needed. In the present investigation, voriconazole-loaded nanostructured lipid carriers (Vrc-NLCs) were formulated for enhanced drug-delivery efficiency to C. albicans to increase the antifungal activity of Vrc and to improve the treatment of infectious Candida diseases. Vrc-NLCs were prepared by a hot-melt, high-pressure homogenization method, and size distribution, ζ-potential, morphology, drug-encapsulation efficiency, drug loading, and physical stability were characterized. The antifungal activity of Vrc-NLCs in vitro was tested during planktonic and biofilm growth in C. albicans. The mean particle size of the Vrc-NLCs was 45.62±0.53 nm, and they exhibited spheroid-like morphology, smooth surfaces, and ζ-potential of −0.69±0.03 mV. Encapsulation efficiency and drug loading of Vrc-NLCs were 75.37%±2.65% and 3.77%±0.13%, respectively. Physical stability results revealed that despite the low measured ζ-potential, the dispersion of the Vrc-NLCs was stable during their 3-week storage at 4°C. The minimum inhibitory concentration of Vrc-NLCs was identical to that of Vrc. However, the inhibition rate of Vrc-NLCs at lower concentrations was significantly higher than that of Vrc during planktonic growth in C. albicans in yeast-extract peptone dextrose medium. Surprisingly, Vrc-NLCs treatment reduced cell density in biofilm growth in C. albicans and induced more switches form hyphal cells to yeast cells compared with Vrc treatment. In conclusion, Vrc-NLCs maintain antifungal activity of Vrc and increase antifungal drug-delivery efficiency to C. albicans. Therefore, Vrc-NLCs will greatly contribute to the treatment of infectious diseases caused by C. albicans.

Screening of Anti-inflammatory Compound-producing Wild Yeasts and Their Microbiological Characteristics

비병원성 야생효모들로부터 새로운 항염물질을 생산하고자 우리나라 주요 섬과 산에서 분리, 동정한 야생 효모들 중 비병원성 효모들의 무세포 추출물들을 제조하여 이들의 nitric oxide (NO) 생성 저해활성을 측정하였다. 182 균주 중 Metschnikowia reukaufii SY20-7, Rhodotorula ingeniosa SY1-1, Yarrowia lipolytica SY51-1, 51-3, Meyerozyma guilliermondii YJ34-2, Rhodotorula graminis YJ36-1, Rhodotorula nothofagi YJ1-1, YJ22-2, Metschnikowia koreensis UL32-1, Sporobolomyces carnicolor UL32-3 등 10 균주들의 무세포 추출물들이 30% 이상의 NO 생성 저해활성을 보였다. 이들 중 유포자효모들에서는 Rhodotorula graminis YJ36-1가 가장 높은 57.4%의 NO 생성 저해활성을 보였고 무포자효모들에서는 Meyerozyma guilliermondii YJ34-2가 47.0%로 가장높아 항염물질생산 우수효모로 최종 선발하였다. 선발된 야생효모들의 미생물학적 특성을 조사한 결과 Rhodotorula graminis YJ36-1 균주는 자낭포자와 위균사를 형성하였고 <TEX>$25^{\circ}C$</TEX>에서 잘 생육하였으며 Meyerozyma guilliermondii YJ34-2는 자낭포자와 위균사를 형성하지 않았고 <TEX>$30^{\circ}C$</TEX>, 5%의 NaCl 함유 YPD 배지에서도 잘 생육하였다. 또한, 두 효모 모두 pH 4.0~8.0에서 생육하였고 YM, PD, YPD, 비타민 무함유 배지 등에서 잘 생육하였다. To screen for potent anti-inflammatory compound-producing yeasts, we evaluated nitric oxide production inhibitory activities in RAW 264.7 macrophage cells using cell-free extracts from 182 non-pathogenic yeasts. Rhodotorula graminis YJ36-1 and Meyerozyma guilliermondii YJ34-2 showed high inhibitory activities of 57.4% and 47.0%, respectively. The microbiological characteristics of these yeasts were investigated. Rhodotorula graminis YJ36-1 formed ascospores and pseudomycelium. This species grew well at <TEX>$25^{\circ}C$</TEX> in yeast extract-peptone-dextrose (YPD) medium, vitamin-free medium, and 5% NaCl-containing YPD medium. Meyerozyma guilliermondii YJ34-2 was an asporogenous yeast and did not form pseudomycelium. This strain also grew well at <TEX>$30^{\circ}C$</TEX> in YPD medium, vitamin-free medium, and 5% NaCl-containing YPD medium.

Microtubules and Actin Cytoskeleton of Cryptococcus neoformans as Targets for Anticancer Agents to Potentiate a Novel Approach for New Antifungals

Background: We investigated the targeting of microtubules (MT) and F-actin cytoskeleton (AC) of the human pathogenic yeast Cryptococcus neoformans with agents for cancer therapy, in order to examine whether this yeast cytoskeleton could become a new antifungal target for the inhibition of cell division. Methods: Cells treated with 10 cytoskeleton inhibitors in yeast extract peptone dextrose medium were investigated by phase-contrast and fluorescence microscopy, and growth inhibition was estimated by cell counts using a Bürker chamber and measuring absorbance for 6 days. Results: Docetaxel, paclitaxel, vinblastine sulfate salt, cytochalasin D and chlorpropham [isopropyl N-(3-chlorophenyl) carbamate] did not inhibit proliferation. The MT inhibitors methyl benzimidazole-2-ylcarbamate (BCM), nocodazole, thiabendazole (TBZ) and vincristine (VINC) disrupted MT and inhibited mitoses, but anucleated buds emerged on cells that increased in size, vacuolated and seemed to die after 2 days. The response of the cells to the presence of the actin inhibitor latrunculin A (LA) included the disappearance of actin patches, actin cables and actin rings; this arrested budding and cell division. However, in 3-4 days, resistant budding cells appeared in all 5 inhibitors. Disruption of the MT and AC and inhibition of cell division and budding persisted only when the MT and AC inhibitors were combined, i.e. VINC + LA, BCM + LA or TBZ + LA. Conclusion: The MT and AC of C. neoformans are new antifungal targets for the persistent inhibition of cell division by combined F-actin and MT inhibitors.

Effects of Selenium on Morphological Changes in Candida utilis ATCC 9950 Yeast Cells.

This paper presents the results of microscopic examinations of the yeast cells cultured in yeast extract–peptone–dextrose (YPD) media supplemented with sodium selenite(IV). The analysis of the morphological changes in yeast cells aimed to determine whether the selected selenium doses and culturing time may affect this element accumulation in yeast cell structures in a form of inorganic or organic compounds, as a result of detoxification processes. The range of characteristic morphological changes in yeasts cultivated in experimental media with sodium selenite(IV) was observed, including cell shrinkage and cytoplasm thickening of the changes within vacuole structure. The processes of vacuole disintegration were observed in aging yeast cells in culturing medium, which may indicate the presence of so-called ghost cells lacking intracellular organelles The changes occurring in the morphology of yeasts cultured in media supplemented with sodium selenite were typical for stationary phase of yeast growth. From detailed microscopic observations, larger surface area of the cell (6.03 μm2) and yeast vacuole (2.17 μm2) were noticed after 24-h culturing in the medium with selenium of 20 mg Se4+/L. The coefficient of shape of the yeast cells cultured in media enriched with sodium selenite as well as in the control YPD medium ranged from 1.02 to 1.22. Elongation of cultivation time (up to 48 and 72 h) in the media supplemented with sodium selenite caused a reduction in the surface area of the yeast cell and vacuole due to detoxification processes.

Nanofiltration concentration of extracellular glutathione produced by engineered Saccharomyces cerevisiae

This study aimed to optimize extracellular glutathione production by a Saccharomyces cerevisiae engineered strain and to concentrate the extracellular glutathione by membrane separation processes, including ultrafiltration (UF) and nanofiltration (NF). Synthetic defined (SD) medium containing 20gL(-1) glucose was fermented for 48h; the fermentation liquid was passed through an UF membrane to remove macromolecules. Glutathione in this permeate was concentrated for 48h to 545.1±33.6mgL(-1) using the NF membrane; this was a significantly higher concentration than that obtained with yeast extract peptone dextrose (YPD) medium following 96h NF concentration (217.9±57.4mgL(-1)). This higher glutathione concentration results from lower cellular growth in SD medium (final OD600=6.9±0.1) than in YPD medium (final OD600=11.0±0.6) and thus higher production of extracellular glutathione (16.0±1.3 compared to 9.2±2.1mgL(-1) in YPD medium, respectively). Similar fermentation and membrane processing of sweet sorghum juice containing 20gL(-1) total sugars provided 240.3±60.6mgL(-1) glutathione. Increased extracellular production of glutathione by this engineered strain in SD medium and subsequent UF permeation and NF concentration in shortend time may help realize industrial recovery of extracellular glutathione.