Production and characterization of a β-glucosidase from Issatchenkia terricola and its use for hydrolysis of aromatic precursors in Cabernet Sauvignon wine

New enzymes isolated from the biodiversity of native wine ecosystems could contribute to increase the varietal character of regional wines. This study reports on the production and characterization of Issatchenkia terricola, beta-glucosidase and its potential to release red-wine aromatic compounds. The enzyme, a monomer of 48 kDa with an isoelectric point of 3.5 is tolerant to glucose and ethanol, properties compatible with enological use. Although fed-batch is usually the most suitable system for enzyme production in submerged culture, in this case the yield was practically the same as in batch culture. Enzyme productivity was increased 2-fold in synthetic medium with glucose with respect to the YPG and 3 to 8-fold with respect to other media assayed. After enzymatic treatment, GC-MS analysis of the released aglycones demonstrated significant increases in the concentration of phenols (83%) and norisoprenoids (65%). According to the judges of the sensory panel, the treatment resulted in a wine with dried fruits and raisins notes, as compared to the control, which was found more sweet and fruity. This, together with the lack of activity on anthocyanin glycosides, highlights the potential of this enzyme in enology, since its high selectivity allowed the development of aroma without compromising wine color.Centro de Investigación y Desarrollo en Fermentaciones Industriale

Production and characterization of a β-glucosidase from Issatchenkia terricola and its use for hydrolysis of aromatic precursors in Cabernet Sauvignon wine

New enzymes isolated from the biodiversity of native wine ecosystems could contribute to increase the varietal character of regional wines. This study reports on the production and characterization of Issatchenkia terricola, beta-glucosidase and its potential to release red-wine aromatic compounds. The enzyme, a monomer of 48 kDa with an isoelectric point of 3.5 is tolerant to glucose and ethanol, properties compatible with enological use. Although fed-batch is usually the most suitable system for enzyme production in submerged culture, in this case the yield was practically the same as in batch culture. Enzyme productivity was increased 2-fold in synthetic medium with glucose with respect to the YPG and 3 to 8-fold with respect to other media assayed. After enzymatic treatment, GC-MS analysis of the released aglycones demonstrated significant increases in the concentration of phenols (83%) and norisoprenoids (65%). According to the judges of the sensory panel, the treatment resulted in a wine with dried fruits and raisins notes, as compared to the control, which was found more sweet and fruity. This, together with the lack of activity on anthocyanin glycosides, highlights the potential of this enzyme in enology, since its high selectivity allowed the development of aroma without compromising wine color.Centro de Investigación y Desarrollo en Fermentaciones Industriale

Production and characterization of a β-glucosidase from Issatchenkia terricola and its use for hydrolysis of aromatic precursors in Cabernet Sauvignon wine

New enzymes isolated from the biodiversity of native wine ecosystems could contribute to increase the varietal character of regional wines. This study reports on the production and characterization of Issatchenkia terricola, beta-glucosidase and its potential to release red-wine aromatic compounds. The enzyme, a monomer of 48 kDa with an isoelectric point of 3.5 is tolerant to glucose and ethanol, properties compatible with enological use. Although fed-batch is usually the most suitable system for enzyme production in submerged culture, in this case the yield was practically the same as in batch culture. Enzyme productivity was increased 2-fold in synthetic medium with glucose with respect to the YPG and 3 to 8-fold with respect to other media assayed. After enzymatic treatment, GC-MS analysis of the released aglycones demonstrated significant increases in the concentration of phenols (83%) and norisoprenoids (65%). According to the judges of the sensory panel, the treatment resulted in a wine with dried fruits and raisins notes, as compared to the control, which was found more sweet and fruity. This, together with the lack of activity on anthocyanin glycosides, highlights the potential of this enzyme in enology, since its high selectivity allowed the development of aroma without compromising wine color.Centro de Investigación y Desarrollo en Fermentaciones Industriale

Cellular transport of microcystin-LR in rainbow trout (Oncorhynchus mykiss) across the intestinal wall: possible involvement of multidrug resistance-associated proteins

We studied Abcc mediated-transport in middle and posterior intestine of the rainbow trout, Oncorhynchus mykiss. Luminal and serosal transport were evaluated in everted and non-everted intestinal sacs, respectively, incubated with 1-chloro-2,4-dinitrobenzene (CDNB; 200 μM). CDNB enters the cells and is conjugated with glutathione via glutathione S-transferase (GST) to form 2,4-dinitrophenyl-S-glutathione (DNP-SG), a known Abcc substrate. DNP-SG concentration in the bath was recorded every 10 min, in order to calculate the mass-specific transport rate. For evaluating the possible involvement of Abcc proteins in microcystin-LR (MCLR) transport, 1.135 μM MCLR was added to the bath or inside the sacs, in everted or non-everted preparations, respectively. Both luminal and serosal DNP-SG efflux were significantly inhibited by MCLR. A concentration–response curve obtained using strips from middle intestine yielded an IC50 value of 1.33 μM MCLR. The Abcc inhibitor, MK571 produced concentration-dependent inhibition of DNP-SG similar to that produced by MCLR. Since competition of MCLR and CDNB as GST substrates could bias the DNP-SG transport results, we evaluated the effects of MCLR on calcein efflux, which does not depend on GST activity. We applied the non-fluorescent, cell-permeant compound calcein-AM (0.25 μM) to middle intestinal strips and recorded the efflux of its hydrolysis product, the fluorescent Abcc substrate calcein. 2.27 μM MCLR and 3 μM MK571 inhibited calcein efflux (17.39 and 20.2%, respectively). Finally, MCLR interaction with Abcc transporters was evaluated by measuring its toxic intracellular effects. Middle intestinal segments were incubated in saline solution with 1.135 μM MCLR (MC1), 2.27 μM MCLR (MC2), 3 μM MK571 (MK) or 1.135 μM MCLR + 3 μM MK571 (MC1/MK). After 1 h, GSH concentration, protein phosphatase 1 and 2A (PP1, PP2A) and GST activities were measured in each segment. MC1did not produce significant effect while MC1/MK and MC2 significantly inhibited PP1and PP2A in similar proportions (34–49%). MK alone significantly increased PP2A activity (40%) with no effect in any other variable. GST activity and GSH concentration were not affected by any treatment. Concentration–response curves for MCLR (1.135 to 13.62 μM) alone or plus 3 or 6 μM MK571 were obtained using PP1 activity as response variable. The IC50 values were 1.0, 0.52, and 0.37 μM, respectively. Our results suggest that O. mykiss enterocytes are capable of eliminating MCLR by GST-mediated conjugation and luminal excretion through an Abcc-like apical transporter. This mechanism would prevent toxic effects and reduce the toxin uptake into the blood, which is likely mediated by basolateral Abccs.Fil: Bieczynski, Flavia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Patagonia Norte. Instituto de Investigación En Biodiversidad y Medioambiente; Argentina. Universidad Nacional del Comahue; ArgentinaFil: de Anna, Julieta Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Patagonia Norte. Instituto de Investigación En Biodiversidad y Medioambiente; Argentina. Universidad Nacional del Comahue; ArgentinaFil: Pirez, Macarena. Universidad de la Republica. Facultad de Quimica; UruguayFil: Brena, Beatriz M.. Universidad de la Republica. Facultad de Quimica; UruguayFil: Villanueva, Silvina Stella Maris. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Fisiología Experimental (i); ArgentinaFil: Luquet, Carlos Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Patagonia Norte. Instituto de Investigación En Biodiversidad y Medioambiente; Argentina. Universidad Nacional del Comahue; Argentin

Nuevas metodologías para el análisis de microcistinas en peces. Estudio de Astraloheros facetus expuestos in vitro = New methodology in the determination of microcystins in fish. Study of Astraloheros facetus exposed in vitro

La alta incidencia de floraciones de cianobacterias productoras de microcistinas en el país y la región representa un riesgo muy elevado para humanos y animales. A fin de estudiar el impacto y la presencia de las microcistinas (MCs) en ani-males, es importante disponer de métodos simples de bajo costo. Como primera aproximación a estos objetivos en peces, se estudiaron Astraloheros facetus (Cas-tañetas) expuestas a una floración de Microcystis spp (MCs 60 y 600 &#956;gMCs/L) en un bioensayo subcrónico (18 días). Si bien no hubo mortalidad, la histopato-logía mostró infiltración grasa en el hígado, más relevante en los peces expuestos a la mayor concentración. Para analizar MCs en pescados se optimizaron dos mé-todos inmunoquímicos sensibles basados en un anticuerpo recombinante de llama (nanobody) de alta especificidad: ELISA y MALDI-TOF cuantitativo, utilizando partículas magnéticas funcionalizadas. Los métodos fueron recientemente desa-rrollados localmente. La excelente correlación ELISA/MALDI-TOF (rSpearman = 0,988, p< 10-7) resalta el potencial de este ELISA como herramienta simple y costo-efectiva para minimizar las muestras a analizar por métodos de referencia. Las concentraciones de MCs en las Castañetas fueron relevantes, acordes con bioensayos en otras especies y peces de la naturaleza. Esto destaca la importan-cia de analizar MCs en pescado para consumo

Las floraciones de cianobacterias tóxicas comprometen el uso del agua del Río Negro, Uruguay

Las floraciones de cianobacterias tóxicas generan impactos negativos a nivel ambiental, económico, y en la salud humana y animal. Se realizó un análisis de datos históricos (1989-2020, n = 423) y un experimento de enriquecimiento de nutrientes para estudiar las cianobacterias del Río Negro, principal río interno del país. En base a indicadores cuantitativos (biovolumen, abundancia de células de cianobacterias y observación visual), se definieron cuatro niveles de peligrosidad de exposición a cianobacterias tóxicas. Las cianobacterias más frecuentes (Microcystis sp. y Dolichospermum sp.) son productoras potenciales de diversas toxinas, incluyendo algunas que no han sido analizadas aún en Uruguay. Se advierte un deterioro ambiental creciente desde el año 2000, pautado por el aumento de la biomasa de cianobacterias y las concentraciones de toxinas (microcistinas). Los nutrientes en el agua indican eutrofización avanzada, asociada al incremento del área agrícola de la cuenca. Los resultados experimentales demostraron el papel clave de los nutrientes y el tiempo de residencia en el favorecimiento de estos organismos. Las floraciones tóxicas de cianobacterias amenazan seriamente los múltiples servicios ecosistémicos que brinda el río, siendo indispensable la instrumentación de planes de monitoreo de cianobacterias y medidas de manejo para controlar la eutrofización a largo plazo

Blooms de cianobactérias tóxicas comprometem o uso da água do Rio Negro, Uruguai

Toxic cyanobacterial blooms generate negative environmental, economic and health impacts. We carried out an analysis of historical limnological data (1989-2020, n = 423) and we performed a nutrient enrichment experiment to study the cyanobacteria of Río Negro, the largest internal river of Uruguay. We defined four hazard levels of exposure to toxic cyanobacteria based on quantitative indicators (biovolume and abundance of cyanobacterial cells and visual observation). The most common cyanobacteria (Microcystis sp. and Dolichospermum sp.) are potential producers of various toxins, including some that have not yet been analyzed in Uruguay. Accelerating environmental deterioration since 2000 has been caused by an increase of cyanobacteria biomass and concentrations of toxins (microcystins). Nutrient concentrations indicated advanced eutrophication, associated with the increase in the area devoted to agriculture of ​​the watershed. Experimental results demonstrated the key role of nutrients and residence time in favoring these organisms. Toxic cyanobacterial blooms seriously threaten the multiple ecosystem services provided by the river, making it essential to implement cyanobacteria monitoring, and management plans to control eutrophication in the long term.  Las floraciones de cianobacterias tóxicas generan impactos negativos a nivel ambiental, económico, y en la salud humana y animal. Se realizó un análisis de datos históricos (1989-2020, n = 423) y un experimento de enriquecimiento de nutrientes para estudiar las cianobacterias del Río Negro, principal río interno del país. En base a indicadores cuantitativos (biovolumen, abundancia de células de cianobacterias y observación visual), se definieron cuatro niveles de peligrosidad de exposición a cianobacterias tóxicas. Las cianobacterias más frecuentes (Microcystis sp. y Dolichospermum sp.) son productoras potenciales de diversas toxinas, incluyendo algunas que no han sido analizadas aún en Uruguay. Se advierte un deterioro ambiental creciente desde el año 2000, pautado por el aumento de la biomasa de cianobacterias y las concentraciones de toxinas (microcistinas). Los nutrientes en el agua indican eutrofización avanzada, asociada al incremento del área agrícola de la cuenca. Los resultados experimentales demostraron el papel clave de los nutrientes y el tiempo de residencia en el favorecimiento de estos organismos. Las floraciones tóxicas de cianobacterias amenazan seriamente los múltiples servicios ecosistémicos que brinda el río, siendo indispensable la instrumentación de planes de monitoreo de cianobacterias y medidas de manejo para controlar la eutrofización a largo plazo.Blooms de cianobactérias tóxicas geram impactos ambientais e econômicos negativos, alem de danos à saúde humana e animal. Análise de dados históricos (1989-2020, n = 423) e um experimento de enriquecimento de nutrientes foram realizados para estudar as cianobactérias do Río Negro, o principal rio interno do Uruguai. Com base em indicadores quantitativos (biovolume e abundância de células cianobacterianas e observação visual) foram definidas quatro categorias de perigosidade de exposição a toxinas. As cianobactérias mais comuns (Microcystis sp. e Dolichospermum sp.) são produtoras potenciais de várias toxinas, algumas das quais nunca foram analisadas en Uruguai. Elevadas biomassas de cianobactérias  e concentrações das toxinas (microcistinas) foram encontradas desde 2000 com tendência a aumentar casos em categorias de alta perigosidade e alertando para a deterioração ambiental, a última década. Concentrações altas de nutrientes na água indicam eutrofização avançada associado com o aumento da área da bacia dedicada à agricultura. Os resultados experimentais demonstraram o papel fundamental dos nutrientes e do tempo de residencia no favorecimento de cianobactérias. A proliferação de cianobactérias tóxicas ameaçam seriamente os múltiplos serviços ecossistêmicos fornecidos pelo rio, tornando essencial a implementação de monitoramento de cianobactérias, planos de manejo e monitoreamento para controlar a eutrofização em longo prazo.

Synergistic effects of nutrients and light favor Nostocales over non-heterocystous cyanobacteria

Blooms of Nostocales (Cyanobacteria) are thought to be invading subtropical and temperate water bodies. According to nutrient stoichiometry and physiological differences between cyanobacterial groups, the replacement of non-heterocystous species by Nostocales is favored when dissolved inorganic nitrogen decreases. However, some studies have shown different trends. We used laboratory bioassays to evaluate the concomitant effects of light and nutrient enrichment on phytoplankton assemblages dominated by non-heterocystous filamentous cyanobacteria. Three nutrient conditions (no addition, addition of phosphate, and addition of nitrate and phosphate) and two light intensities (40 and 80 µmol photon m−2 s−1) were assayed. Nostocales replaced or co-dominated with non-heterocystous species in all treatments by the end of the study. The shift in community composition towards Nostocales dominance led to an increase in species richness, which suggests that species with different eco-physiological traits may have differential impacts on diversity. The highest saxitoxin concentrations were measured in no addition treatments, which could link production to nutritional stress. Nostocales featured high phenotypic plasticity in terms of changes in average trichome volume and growth rates. These results help to have a better understanding of the conditions under which Nostocales predominance can occur.Facultad de Ciencias Naturales y Muse

Floración excepcional de cianobacterias tóxicas en la costa de Uruguay, verano 2019 = Exceptional bloom of toxic cyanobacteria on the Uruguayan coast, summer 2019

Las floraciones de cianobacterias tóxicas son un problema mundial. La eutrofización antrópica y la generación de embalses han promovido su desarrollo en la Cuenca del Plata, favoreciendo principalmente al complejo Microcystis aeruginosa (CMA). En este trabajo describimos las floraciones excepcionales de CMA ocurridas en el verano 2019 en la costa del Río de la Plata y Atlántica de Uruguay y discutimos sus mecanismos moduladores en base a datos biológicos, meteorológicos y oceanográficos de muestreos, imágenes satelitales y re-análisis. Las floraciones fueron extensas (Carmelo a Rocha; 500 km) y persistentes (ca.: 4 meses). En todos los casos fueron organismos del CMA con estructura genética similar y su presencia estuvo asociada a niveles cuantificables y elevados de microcistina. En base a las características oceanográficas y la similitud genética entre los organismos, se hipotetiza que las floraciones tienen un origen común, probablemente asociado a tributarios con alta carga de nutrientes y alto tiempo de residencia del agua en la Cuenca baja del Plata. Las precipitaciones extremas generaron caudales récord que transportaron las cianobacterias hasta el Río de la Plata, donde las condiciones de viento y temperatura particulares facilitaron su transporte y arribo a la costa de Rocha, llegando a ambientes de relevancia para la conservación (e.g. Laguna de Rocha)

Cianobacterias y cianotoxinas en ecosistemas límnicos de Uruguay = Cyanobacteria and cyanotoxins in freshwaters of Uruguay

Las floraciones de cianobacterias en cuerpos de agua dulce constituyen un problema mundial, asociado a la eutrofización (enriquecimiento de nutrientes) y cambios hidrológicos de los ecosistemas. En este estudio se generó una base de datos históricos (n= 3061 para 64 ecosistemas, de 1980 a 2014), elaborada por un grupo de trabajo interinstitucional (OSE, DINAMA, IM, Universidad de la República e IIBCE), y se analizó la distribución de cianobacterias planctónicas y de cianotoxinas (microcistina, saxitoxina y cilindrospermopsina) en Uruguay. Los valores de clorofila ay nutrientes totales indicaron procesos de eutrofización en diversos ecosistemas. En dos mapas georreferenciados se visualiza la distribución de cianobacterias en el país, según indicadores cuantitativos globales, y de la microcistina (cianotoxina más frecuente). En dichos mapas se destacan los embalses (Río Uruguay y Río Negro) y las playas del Río de la Plata como las zonas de mayor riesgo de exposición a cianobacterias según las categorías de la Organización Mundial de la Salud para aguas recreacionales. Además, se analizaron muestras de floraciones (espuma) y se cuantificó 20 mg L-1de microcistinas totales y la presencia de genes que sugieren diferentes variedades de microcistinas. Se generó información básica que podrá ser útil para programas de monitoreo nacionales e investigación