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Some yeast strains have been proposed as probiotics to improve the health of cultured fish. Cobia is a tropical benthopelagic fish species with potential for marine aquaculture; however, one of the main limitations to its large-scale production is the high mortality of fish larvae. In this study, we evaluated the probiotic potential of autochthonous yeasts from the intestines of cobia. Thirty-nine yeast isolates were recovered from the intestinal mucosa of 37 adult healthy cobia by culture methods. Yeasts were identified by sequencing of the ITS and D1/D2 regions of the 28S rRNA gene and typed by RAPD-PCR using the M13 primer. Yeast strains with unique RAPD patterns were characterized in terms of their cell biomass production ability; anti-Vibrio, enzymatic, and hemolytic activity; biofilm production; hydrophobicity; autoaggregation; polyamine production; safety; and protection of cobia larvae against saline stress. Candida haemuloni C27 and Debaryomyces hansenii C10 and C28 were selected as potential probiotics. They did not affect the survival of larvae and showed biomass production >1 g L-1, hydrophobicity >41.47%, hemolytic activity γ, and activity in more than 8 hydrolytic enzymes. The results suggest that the selected yeast strains could be considered as potential probiotic candidates and should be evaluated in cobia larvae.

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Pollutants, such as polycyclic aromatic hydrocarbons (PAHs), e.g., benzo(a)pyrene (BaP), are common components of contaminating mixtures. Such compounds are ubiquitous, extremely toxic, and they pollute soils and aquatic niches. The need for new microorganism-based remediation strategies prompted researchers to identify the most suitable organisms to eliminate pollutants without interfering with the ecosystem. We analyzed the effect caused by BaP on the growth properties of Candida albicans, Debaryomyces hansenii, Rhodotorula mucilaginosa, and Saccharomyces cerevisiae. Their ability to metabolize BaP was also evaluated. The aim was to identify an optimal candidate to be used as the central component of a mycoremediation strategy. The results show that all four yeast species metabolized BaP by more than 70%, whereas their viability was not affected. The best results were observed for D. hansenii. When an incubation was performed in the presence of a cytochrome P450 (CYP) inhibitor, no BaP degradation was observed. Thus, the initial oxidation step is mediated by a CYP enzyme. Additionally, this study identified the D. hansenii DhDIT2 gene as essential to perform the initial degradation of BaP. Hence, we propose that D. hansenii and a S. cerevisiae expressing the DhDIT2 gene are suitable candidates to degrade BaP in contaminated environments.

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This work aimed to evaluate the performance of co-cultivation of potential probiotic yeast and lactic acid bacteria (LAB) in producing plant-based fermented beverages. The co-culture comprised LAB Lactiplantibacillus plantarum CCMA0743 with the yeasts Pichia kluyveri CCMA 0615, Pichia guilliermondii CCMA 1753 and Debaryomyces hansenii CCMA 1761 separately. The plant substrate was 75 g oat, 175 g sunflower seeds, and 75 g almonds. The viability of microorganisms in the plant-based matrix was evaluated during fermentation, storage at 4 °C, and under simulated gastrointestinal tract (GIT) conditions. Chemical analysis, antioxidant activity, and sensory profile of the beverages were also determined. The three yeasts and the LAB showed counts greater than 6.0 log CFU/mL after fermentation, and the plant-based matrix protected the yeasts during simulated digestion. P. kluyveri and D. hansenii showed higher survival than P. guilliermondii and L. plantarum after exposure to simulated GIT conditions. The pH of the plant-based matrix reduced from approximately 7 to 3.8. Lactic acid was the main organic acid produced during fermentation. In addition, 113 volatile compounds were detected by gas chromatography-mass spectrometry (GC-MS), including alcohols, aldehydes, alkanes, alkenes, acids, ester, ether, ketones, phenol, and amides. The beverage sensory profile varied with the co-culture. The co-culture D. hansenii and L. plantarum showed higher antioxidant activity than the other co-culture tested, and the homogeneous texture attribute characterized the beverage produced with this combination. Results show the suitability of tested co-cultures to produce a plant-based fermented beverage and indicate more significant potential for D. hansenii and L. plantarum co-culture as a starter for its functionalization.

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The host microbiome plays an essential role in health and disease. Microbiome modification by pathogens or probiotics has been poorly explored especially in the case of probiotic yeasts. Next-generation sequencing currently provides the best tools for their characterization. Debaryomyces hansenii 97 (D. hansenii 97) and Yarrowia lipolytica 242 (Y. lipolytica 242) are yeasts that protect wildtype zebrafish (Danio rerio) larvae against a Vibrio anguillarum (V. anguillarum) infection, increasing their survival rate. We investigate the effect of these microorganisms on the microbiome and neutrophil response (inflammation) in zebrafish larvae line Tg(Bacmpx:GFP) i114. We postulated that preinoculation of larvae with yeasts would attenuate the intestinal neutrophil response and prevent modification of the larval microbiome induced by the pathogen. Microbiome study was performed by sequencing the V3-V4 region of the 16S rRNA gene and prediction of metabolic pathways by Piphillin in conventionally raised larvae. Survival and the neutrophil response were both evaluated in conventional and germ-free conditions. V. anguillarum infection resulted in higher neutrophil number in the intestinal area compared to non-infected larvae in both conditions. In germ-free conditions, infected larvae pre-inoculated with yeasts showed fewer neutrophil numbers than infected larvae. In both conditions, only D. hansenii 97 increased the survival of infected larvae. Beta diversity of the microbiota was modified by V. anguillarum and both yeasts, compared to non-inoculated larvae. At 3 days post-infection, V. anguillarum modified the relative abundance of 10 genera, and pre-inoculation with D. hansenii 97 and Y. lipolytica 242 prevented the modification of 5 and 6 of these genera, respectively. Both yeasts prevent the increase of Ensifer and Vogesella identified as negative predictors for larval survival (accounting for 40 and 27 of the variance, respectively). In addition, yeast pre-inoculation prevents changes in some metabolic pathways altered by V. anguillarum's infection. These results suggest that both yeasts and V. anguillarum can shape the larval microbiota configuration in the early developmental stage of D. rerio. Moreover, modulation of key taxa or metabolic pathways of the larval microbiome by yeasts can be associated with the survival of infected larvae. This study contributes to the understanding of yeast-pathogen-microbiome interactions, although further studies are needed to elucidate the mechanisms involved.

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The oxidation of tyrosine to 3-nitrotyrosine is irreversible, and due to this characteristic, 3-nitrotyrosine is used as a marker for oxidative stress in a range of diverse chronic and degenerative diseases. It has been established that the yeast Debaryomyces hansenii (D. hansenii) can assimilate free 3-nitrotyrosine as unique source of nitrogen, and during saline stress, has a high denitrase activity to detoxify this compound in a reaction that involves the liberation of nitrogen dioxide from 3-nitrotyrosine. However, until now it has not been determined whether D. hansenii can detoxify protein-bound 3-nitrotyrosine such as nitrated proteins present in different chronic illnesses. TThe aim of the present study was to evaluate the denitrase activity of D. hansenii to reduce 3-nitrotyrosine from liver proteins of mice with colitis. Firstly, the levels of reactive oxygen species of liver tissue of colitic and control mice were measured by the reaction with the 2'7'-dichlorofluorescein diacetate. Denitrase activity of D. hansenii was evaluated by incubating cell extracts of the yeast with protein extracts from livers of mice with colitis. Following incubation, 3-nitrotyrosine was measured, and to corroborate that denitrase reaction had occurred, the production of nitrites was measured. In samples of liver tissue from mice with colitis, the maximum levels of reactive oxygen species were up to two times higher compared with the control livers. Following the incubation of colitic liver samples with cell extracts of D. hansenii, it was observed that 3-nitrotyrosine decreased to the basal concentration of control liver samples, and that the concentration of nitrites was increased. These results indicate that denitrase of D. hansenii extracts can effectively detoxify 3-nitrotyrosine bound to proteins and that the extracts could be used to decrease protein oxidation damage in chronic degenerative diseases.

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Debaryomyces hansenii is a halotolerant and Na+-includer yeast that can be isolated from different food and low-water activity products. It has also been defined as a marine-occurring yeast but key aspects for this salt tolerant behavior are far from being understood. Here, we searched for clues helping to elucidate the basis of this ability. Our results on growth, Rb+ transport, total K+ and Na+ content and vacuolar fragmentation are compatible with a yeast species adapted to cope with salt stress. On the other hand, we confirmed the existence of D. hansenii strategies that are generally observed in sensitive organisms, such as the production of glycerol as a compatible solute and the efficient vacuolar sequestration of Na+. We propose a striking role of D. hansenii vacuoles in the maintenance of constant cytosolic K+ values, even in the presence of extracellular Na+ concentration values more than two orders of magnitude higher than extracellular K+. Finally, the ability to deal with cytosolic Na+ levels significantly higher than those found in S. cerevisiae, shows the existence of important and specific salt tolerance mechanisms and determinants in D. hansenii.

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Application of yeast is increasing to improve welfare and promotes growth in aquaculture. The halotolerant yeast Debaryomyces hansenii is normally a non-pathogenic yeast with probiotic properties and potential source of antioxidant enzymes as superoxide dismutase. Here, first, we characterized the sequence features of MnSOD and icCu/ZnSOD from Pacific red snapper, and second, we evaluated the potential antioxidant immune responses of the marine yeast Debaryomyces hansenii strain CBS004 in leukocytes which were then subjected to Vibrio parahaemolyticus infection. In silico analysis revealed that LpMnSOD consisted of 1186 bp, with an ORF of 678 bp encoding a 225 amino acid protein and LpicCu/ZnSOD consisted of 1090 bp in length with an ORF of 465 bp encoding a 154 amino acid protein. Multiple alignment analyzes revealed many conserved regions and active sites among its orthologs. In vitro assays using head-kidney and spleen leukocytes immunostimulated with D. hansenii and zymosan in response to V. parahaemolyticus infection reveled that D. hansenii strain CBS004 significantly increased transcriptions of MnSOD and icCu/ZnSOD genes. Flow cytometry assay showed that D. hansenii was able to inhibit apoptosis caused by V. parahaemolyticus in the Pacific red snapper leukocytes and enhanced the phagocytic capacity in head-kidney leukocytes. Immunological assays reveled an increased in superoxide dismutase and peroxidase activities, as well as, in nitric oxide production and reactive oxygen species production (respiratory burst) in fish stimulated with D. hansenii. Finally, our results. These results strongly support the idea that marine yeast Debaryomyces hansenii strain CBS004 can stimulate the antioxidant immune mechanism in head-kidney and spleen leukocytes.

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The yeast Debaryomyces hansenii overproduces riboflavin upon exposure to subtoxic levels of cobalt (Co(+2)). However, mechanisms for survival have yet to be studied and have been hindered by D. hansenii's high genetic heterogeneity among strains. In this study, we used transcriptomic analyses and RNA-seq in order to identify differentially expressed genes in D. hansenii in response to cobalt exposure. Highly upregulated genes under this condition were identified to primarily comprise DNA damage and repair genes, oxidative stress response genes, and genes for cell wall integrity and growth. The main response of D. hansenii to heavy metal stress is the activation of non-enzymatic oxidative stress response mechanisms and control of biological production of reactive oxygen species. Our results indicate that D. hansenii does not seem to be pre-adapted to survive high concentrations of heavy metals. These organisms appear to possess genetic survival and detoxification mechanisms that enable the cells to recover from heavy metal stress.

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Physiological uncoupling of mitochondrial oxidative phosphorylation (OxPhos) was studied in Debaryomyces hansenii. In other species, such as Yarrowia lipolytica and Saccharomyces cerevisiae, OxPhos can be uncoupled through differential expression of branched respiratory chain enzymes or by opening of a mitochondrial unspecific channel (ScMUC), respectively. However D. hansenii mitochondria, which contain both a branched respiratory chain and a mitochondrial unspecific channel (DhMUC), selectively uncouple complex I-dependent rate of oxygen consumption in the stationary growth phase. The uncoupled complex I-dependent respiration was only 20% of the original activity. Inhibition was not due to inactivation of complex I, lack of protein expression or to differential expression of alternative oxidoreductases. Furthermore, all other respiratory chain activities were normal. Decrease of complex I-dependent respiration was due to NAD(+) loss from the matrix, probably through an open of DhMUC. When NAD(+) was added back, coupled complex I-activity was recovered. NAD(+) re-uptake was independent of DhMUC opening and seemed to be catalyzed by a NAD(+)-specific transporter, which was sensitive to bathophenanthroline, bromocresol purple or pyridoxal-5'-phosphate as described for S. cerevisiae mitochondrial NAD(+) transporters. Loss of NAD(+) from the matrix through an open MUC is proposed as an additional mechanism to uncouple OxPhos.

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An intracellular ß-glucosidase from Debaryomyceshansenii UFV-1 was produced in an YP medium with cellobiose as the carbon source. This enzyme was purified, characterised and presented a Mr of 65.15kDa. Yeast cells containing the intracellular ß-glucosidase were immobilised in calcium alginate. The free ß-glucosidase and immobilised cells containing the enzyme presented optima values of pH and temperature of 6.0 and 45°C and 5.5 and 50°C, respectively. The free enzyme maintained 62% and 47% of its original activity after 90days at 4°C and after 15days at room temperature, respectively. The immobilisation process resulted in higher enzyme thermostability at 45 and 50°C. Soy molasses treatment with the free enzyme and the immobilised cells containing ß-glucosidase, for 2h at 40°C, promoted efficient hydrolysis of isoflavone glicosides to their aglycon forms. The results suggest that this enzyme could be used in the food industry, in the free or immobilised forms, for a safe and efficient process to hydrolyse isoflavone glycosides in soy molasses.

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The branched respiratory chain in mitochondria from the halotolerant yeast Debaryomyces hansenii contains the classical complexes I, II, III and IV plus a cyanide-insensitive, AMP-activated, alternative-oxidase (AOX). Two additional alternative oxidoreductases were found in this organism: an alternative NADH dehydrogenase (NDH2e) and a mitochondrial isoform of glycerol-phosphate dehydrogenase (MitGPDH). These monomeric enzymes lack proton pump activity. They are located on the outer face of the inner mitochondrial membrane. NDH2e oxidizes exogenous NADH in a rotenone-insensitive, flavone-sensitive, process. AOX seems to be constitutive; nonetheless, most electrons are transferred to the cytochromic pathway. Respiratory supercomplexes containing complexes I, III and IV in different stoichiometries were detected. Dimeric complex V was also detected. In-gel activity of NADH dehydrogenase, mass spectrometry, and cytochrome c oxidase and ATPase activities led to determine the composition of the putative supercomplexes. Molecular weights were estimated by comparison with those from the yeast Y. lipolytica and they were IV2, I-IV, III2-IV4, V2, I-III2, I-III2-IV, I-III2-IV2, I-III2-IV3 and I-III2-IV4. Binding of the alternative enzymes to supercomplexes was not detected. This is the first report on the structure and organization of the mitochondrial respiratory chain from D. hansenii.

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Traditional phenotypic methods and commercial kits based on carbohydrate assimilation patterns are unable to consistently distinguish among isolates of Pichia guilliermondii, Debaryomyces hansenii and Candida palmioleophila. As result, these species are often misidentified. In this work, we established a reliable method for the identification/differentiation of these species. Our assay was validated by DNA sequencing of the polymorphic region used in a real-time PCR assay driven by species-specific probes targeted to the fungal ITS 1 region. This assay provides a new tool for pathogen identification and for epidemiological, drug resistance and virulence studies of these organisms.

RESUMO

Objetivo. Determinar la respuesta antioxidante [actividad de superóxido dismutasa (SOD) y catalasa (CAT)] así como la cuenta total de hemocitos (CTH) y el contenido de proteínas (CP) en camarones (Litopenaeus vannamei) expuestos a diferentes dosis y cepas de la levadura Debaryomyces hansenii (DH5, DH6, LL1), y un inmunoestimulante comercial (LAM). Materiales y métodos. Las levaduras fueron cultivadas y suministradas diariamente en concentraciones diferentes (104 – 106 UFC/mL) directamente a los tanques de cultivo de los camarones (8 ± 0.2 g) mientras que LAM fue aplicado una vez a la semana (0.5 mg/L). Los organismos fueron mantenidos bajo condiciones de laboratorio (28°C, 35%, 80% de recambio diario de agua, dieta comercial para camarón ad libitum). Los tratamientos fueron distribuidos por duplicado y los resultados evaluados a los 15 días con un análisis de varianza y una prueba de Tukey. Resultados. Se registró un CTH significativo (p<0.05) en los tratamientos con DH6 y LL1 (106 UFC/mL) comparada con el control, mientras que las cepas DH5 y DH6 revelaron un incremento significativo (p<0.05) de CP con la dosis de 104 UFC/mL. Los camarones tratados con LAM incrementaron significativamente (p<0.05) los valores de SOD y CAT. Conclusiones. Los resultados obtenidos demuestran que D. hansenii incrementa la respuesta antioxidante y CTH en camarones.

Assuntos

RESUMO

Traditional phenotypic methods and commercial kits based on carbohydrate assimilation patterns are unable to consistently distinguish among isolates of Pichia guilliermondii, Debaryomyces hansenii and Candida palmioleophila. As result, these species are often misidentified. In this work, we established a reliable method for the identification/differentiation of these species. Our assay was validated by DNA sequencing of the polymorphic region used in a real-time PCR assay driven by species-specific probes targeted to the fungal ITS 1 region. This assay provides a new tool for pathogen identification and for epidemiological, drug resistance and virulence studies of these organisms.

Assuntos