Enhancement of ethanol production efficiency in repeated-batch fermentation from sweet sorghum stem juice: effect of initial sugar, nitrogen and aeration

BACKGROUND: Ethanol concentration (PE), ethanol productivity (QP) and sugar consumption (SC) are important values in industrial ethanol production. In this study, initial sugar and nitrogen (urea) concentrations in sweet sorghum stem juice (SSJ) were optimized for high PE (≥10%, v/v), QP, (≥2.5 g/L·h) and SC (≥90%) by Saccharomyces cerevisiae SSJKKU01. Then, repeated-batch fermentations under normal gravity (NG) and high gravity (HG) conditions were studied. RESULTS: The initial sugar at 208 g/L and urea at 2.75 g/L were the optimum values to meet the criteria. At the initial yeast cell concentration of ~1 × 108 cells/mL, the PE, QP and SC were 97.06 g/L, 3.24 g/L·h and 95.43%, respectively. Repeated-batch fermentations showed that the ethanol production efficiency of eight successive cycles with and without aeration were not significantly different when the initial sugar of cycles 2 to 8 was under NG conditions (~140 g/L). Positive effects of aeration were observed when the initial sugar from cycle 2 was under HG conditions (180­200 g/L). The PE and QP under no aeration were consecutively lower from cycle 1 to cycle 6. Additionally, aeration affected ergosterol formation in yeast cell membrane at high ethanol concentrations, whereas trehalose content under all conditions was not different. CONCLUSION: Initial sugar, sufficient nitrogen and appropriated aeration are necessary for promoting yeast growth and ethanol fermentation. The SSJ was successfully used as an ethanol production medium for a high level of ethanol production. Aeration was not essential for repeated-batch fermentation under NG conditions, but it was beneficial under HG conditions.

Influencia del campo magnético sobre el crecimiento de microorganismos patógenos ambientales aislados en el Archivo Nacional de la República de Cuba / Influence of magnetic field on the growth of pathogen microorganisms isolated from the indoor environment at the Archivo Nacional de la República de Cuba

Introducción. En el Archivo Nacional de la República de Cuba, existe contaminación electromagnética y la influencia del campo magnético oscilante de frecuencia extremadamente baja podría cuantificarse con microorganismos patógenos aislados de su ambiente interior. Objetivo. Cuantificar la influencia de este tipo de campo magnético sobre el crecimiento de microorganismos patógenos aislados del ambiente en el Archivo Nacional de la República de Cuba. Materiales y métodos. Se emplearon cinco microorganismos: Streptococcus sp. (1), Listeria sp. (2) y Candida guillermondii (3), aislados en el Archivo, así como Escherichia coli ATCC 25922 (4) y Saccharomyces cerevisiae (5), como referencia. Se les aplicó un campo magnético oscilante de frecuencia extremadamente baja de 60 Hz/220 V de 3 mT durante dos horas, en tres tubos de cultivo con agua destilada y con caldo nutriente. Después se inocularon 0,1 ml en placas de Petri con los medios de cultivo agar CromoCen SC (1 y 2), agar de dextrosa y papa (3), agar CromoCen CC 4227 (4) y agar con extracto de malta (5). Las colonias se contaron (log UFC/ml) mediante el procesamiento digital de las imágenes de las placas de Petri empleando el programa MatLab ® . Resultados. Se observó una estimulación significativa (p=0,05) de la cantidad de colonias tratadas con respecto a los controles, siendo mayor en el caldo nutriente que en el agua destilada y más en las bacterias (caldo nutriente-colonias tratadas: 9,43 a 10,62 UFC/ml) que en las levaduras (caldo nutriente-colonias tratadas: 8,31 a 8,79 UFC/ml). La estimulación se produjo en orden decreciente así: Listeria sp., E. coli ATCC 25922, Streptococcus sp., C. guillermondii y S. cerevisiae . Conclusión. Se concluyó que el campo magnético aplicado tuvo un efecto estimulante sobre los microorganismos estudiados, lo cual potencia el riesgo para la salud del personal y los visitantes del Archivo Nacional de la República de Cuba.

Introduction: Electromagnetic pollution has been detected at the Archivo Nacional de la República de Cuba and the influence of extremely low frequency magnetic fields could be quantified with pathogenic microorganisms isolated from the indoor environment. Objective: To quantify the influence of an extremely low frequency magnetic field on the growth of pathogenic microorganisms isolated from the environment at the Archivo Nacional. Materials and methods: We used five microorganisms isolated at the Archivo Nacional: Streptococcus sp. (1), Listeria sp. (2) and Candida guillermondii (3), and Escherichia coli ATCC 25922 (4) and Saccharomyces cerevisiae (5) as references. We applied this magnetic field of extremely low frequency, 60 Hz/220 V (3 mT), for two hours to these microorganisms on three culture tubes with distilled water and nutrient broth. Then we inoculated 0.1 mL in the following solid culture mediums on Petri dishes: CromoCen SC Agar (1 and 2), Potato Dextrose Agar (3), CromoCen DC 4227 (4) and Malt Extract Agar (5). The colonies were counted (log CFU/mL) by digital processing of the images of Petri dishes using the MatLab ® tool. Results: We observed a statistically significant stimulation (p=0.05) in the quantity of treated colonies as compared to controls, which was higher in nutrient broth than in distilled water, and in bacteria (nutrient broth and treated colonies: 9.43 to 10.62 CFU/mL) as compared with yeasts (nutrient broth-treated colonies: 8.31 to 8.79 CFU/mL). In decreasing order, stimulation was as follows: Listeria sp., E. coli ATCC 25922, Streptococcus sp., C. guillermondii and S. cerevisiae . Conclusion: We concluded that the magnetic field applied had a stimulating effect on the microorganisms under study, which increases the risk to the health of staff and visitors at the Archivo Nacional .

Cultivo de cepas de levaduras de suelo trumao en ®vinaza¼ / Cultivation of yeast strains of the trumao soil in ®vinasse¼

30 cepas de levaduras aisladas desde un suelo trumao (Hapludans) usado como pradera en rotación, se cultivaron individualmente (100 µl = a 102 ufc delevaduras/mL) en matraces con 50 mL de ®vinaza¼, estos fueron incubados en un agitador orbital a 150 rpm, 23 °C por 5 días, luego de la incubación el contenido de cada matraz se centrifugo a 3.500 rpm por 20 min., a los pellet obtenidos se les determino: el peso seco (PS); fosforo total (FT) por digestión ácida y posterior lectura a 400 nm; proteínas totales (PT) por colorimetría Biuret a 595 nm y lípidos totales (LT) mediante el método colorimétrico de la sulfo-fosfo vainillina a 520 nm. Las 30 cepas de levaduras crecieron en la vinaza. El mayor PS lo registro la cepa 25 (331 g de levadura L-1 de ®vinaza¼). FT lo registro la cepa 28 (4,8 mg g-1 de levadura seca). PT lo registro la cepa 24 (25,90 mg g-1 de levadura seca) y LT lo registro la cepa 18 (287,4 mg g-1 de levadura seca).

Thirty strains of yeast isolated from a volcanic ash soil (Hapludans), used as pasture rotation, were individually cultured (100 µl = 102 cfu of yeast cells mL-1) in flasks with 50 mL of ®vinasse¼, these were incubated at 23 °C for 5 day, after incubation the contents of each flask was centrifuged at 3500 rpm for 20 min, the pellet obtained was determined: dry weight (DW); total phosphorus (FT) by acid digestion and later reading at 400 nm; total protein (TP) by Biuret at 595 nm and total lipid (TL) by the colorimetric method of the sulfo-phospho-vanillin at 520 nm. The Thirty strains of yeast grown on vinasse. The best DW, was determined for strain 25 (331 g yeast L-1 ®vinasse¼). FT was determined for strain 28 (4,8 mg g-1 dry yeast). TP was determined for strain 24 (25, 90 mg g-1 dry yeast) and TL was determined for strain18 (287, 4 mg g-1 dry yeast).

Occurrence of Brettanomyces/Dekkera in Brazilian red wines and its correlation with ethylphenols

The yeast Brettanomyces/Dekkeracan cause important spoilage in wines, with the production of ethylphenols and other off-flavor compounds. This study aimed at determining the presence of this yeast and the ethylphenols produced by them in Brazilian red wines, establishing their relationship with other chemical characteristics. Isolates of Brettanomyces/Dekkerawere quantified by plating 126 samples of dry red wine in selective culture medium, while ethylphenols were analyzed by solid phase extraction and GC/FID. Free and total SO2, alcohol, total dry extract, residual sugar, total and volatile acidity, and pH were also determined. Brettanomyces/Dekkerawas present in 27% of samples. Ethylphenols were detected in most samples, with amounts higher than the threshold limit of 426 mg/L found in 46.03% of samples. The majority of wine samples showed inadequate levels of SO2and residual sugars, facts that might facilitate microbial spoilage. The passage in barrels and the grape varieties (Cabernet Sauvignon and Merlot), did not show any influence on the levels of contamination or ethylphenols contents. The prevalence of Brettanomyces/Dekkeraand the concentrations of ethylphenols were high considering the sensory impact they can cause. The growth of Brettanomyces/Dekkerawas dependent on the levels of SO2and alcohol of wines. Knowledge of the contamination, the presence of ethylphenols, and their relationship with the chemical characteristics of wines can entice effective measures to prevent Brettanomyces/Dekkeraand contribute to improve the general quality of Brazilian red wines.

Antifungal activity of lectins against yeast of vaginal secretion

Lectins are carbohydrate-binding proteins of non-imune origin. This group of proteins is distributed widely in nature and they have been found in viruses, microorganisms, plants and animals. Lectins of plants have been isolated and characterized according to their chemical, physical-chemical, structural and biological properties. Among their biological activities, we can stress its fungicidal action. It has been previously described the effect of the lectins Dviol, DRL, ConBr and LSL obtained from the seeds of leguminous plants on the growth of yeasts isolated from vaginal secretions. In the present work the experiments were carried out in microtiter plates and the results interpreted by both methods: visual observations and a microplate reader at 530nm. The lectin concentrations varied from 0.5 to 256µg/mL, and the inoculum was established between 65-70% of trammitance. All yeast samples isolated from vaginal secretion were evaluated taxonomically, where were observed macroscopic and microscopic characteristics to each species. The LSL lectin did not demonstrate any antifungal activity to any isolate studied. The other lectins DRL, ConBr and DvioL, showed antifungal potential against yeast isolated from vaginal secretion. These findings offering offer a promising field of investigation to develop new therapeutic strategies against vaginal yeast infections, collaborating to improve women's health.

Chlorhexidine: beta-cyclodextrin inhibits yeast growth by extraction of ergosterol

Chlorhexidine (Cx) augmented with beta-cyclodextrin (β-cd) inclusion compounds, termed Cx:β-cd complexes, have been developed for use as antiseptic agents. The aim of this study was to examine the interactions of Cx:β-cd complexes, prepared at different molecular ratios, with sterol and yeast membranes. The Minimal Inhibitory Concentration (MIC) against the yeast Candida albicans (C.a.) was determined for each complex; the MICs were found to range from 0.5 to 2 µg/mL. To confirm the MIC data, quantitative analysis of viable cells was performed using trypan blue staining. Mechanistic characterization of the interactions that the Cx:β-cd complexes have with the yeast membrane and assessment of membrane morphology following exposure to Cx:β-cd complexes were performed using Sterol Quantification Method analysis (SQM) and scanning electron microscopy (SEM). SQM revealed that sterol extraction increased with increasing β-cd concentrations (1.71 × 10³; 1.4 × 10³; 3.45 × 10³, and 3.74 × 10³ CFU for 1:1, 1:2, 1:3, and 1:4, respectively), likely as a consequence of membrane ergosterol solubilization. SEM images demonstrated that cell membrane damage is a visible and significant mechanism that contributes to the antimicrobial effects of Cx:β-cd complexes. Cell disorganization increased significantly as the proportion of β-cyclodextrin present in the complex increased. Morphology of cells exposed to complexes with 1:3 and 1:4 molar ratios of Cx:β-cd were observed to have large aggregates mixed with yeast remains, representing more membrane disruption than that observed in cells treated with Cx alone. In conclusion, nanoaggregates of Cx:β-cd complexes block yeast growth via ergosterol extraction, permeabilizing the membrane by creating cluster-like structures within the cell membrane, possibly due to high amounts of hydrogen bonding.

Presentación inusual de onicomicosis por candida albicans / Unusual presentation of onychomycosis caused by Candida albicans

Antecedentes: la onicomicosis es la infección de las uñas producida por hongos dermatofitos y no dermatofitos, como las levaduras y mohos. A los dermatofitos corresponde la inmensa mayoría de estas infecciones, pero informes recientes señalan el incremento de las infecciones producidas por los otros agentes referidos; dentro de éstos merecen especial atención el incremento reportado en diversos estudios de las especies del género Candida. La onicomicosis por Candida spp usualmente ocurre en inmunosupresos, siendo más frecuente en mujeres y en uñas de las manos. Caso clínico: masculino de 15 años con lesión en la uña del primer dedo de la mano derecha de un año de evolución, constituida por distrofia, cambios de coloración, opacidad y onicolisis, afecta el borde distal y medial; se consideró el diagnóstico clínico de onicomicosis, se tomó muestras para estudio microbiológico e indicó tratamiento con terbinafina oral mientras se esperaba el resultado del cultivo, sin obtener mejoría satisfactoria en 6 semanas. Se aisló Candida albicans, por lo que se indicó fluconazol vía oral 150mg/semana por 16 semanas, con lo que se logró la remisión completa. Conclusión: el paciente descrito es un masculino sin antecedentes personales patológicos u ocupacionales que favorescan la onicomicosis por Cándida, lo que nos demuestra la necesidad del aislamiento del agente etiológico de la onicomicosis para elegir la mejor opción terapéutica, y así obtener los mejores resultados. Dado que en nuestro medio los estudios de sensibilidad para antimicóticos son muy limitados, se necesita mejorar la disponibilidad de los mismos, lo cual nos ayudará a determinar la sensibilidad o resistencia a los agentes antimicóticos con que contamos en la actualidad...

Productividad y selectividad del medio de cultivo a partir de guayaba agria (psidium araca) en el crecimiento de levaduras nativas del género candida sp / Productivity and selectivity of culture medium from the sour guava (psidium araca) in the growth of native yeast candida sp

El presente trabajo se llevó a cabo para evaluar la eficiencia del medio de cultivo a partir de guayaba agria (Psidium araca) frente a medios comerciales en el crecimiento de tres cepas nativas: Candida guillermondii, Candida famita y Candida sp. Se evaluó el crecimiento microbiano a diferentes concentraciones de fruta, 5, 10, 25 y 50% p/v, tomando como control los medios comerciales: Malta, Sabouraud y agar papa dextrosa (PDA). La productividad y selectividad del medio de guayaba agria fue determinada mediante el método Ecométrico en un tiempo de 48 horas. Los análisis estadísticos aplicados para evaluar y comparar el crecimiento de las cepas en los medios comerciales y en el medio de guayaba agria a diferentes concentraciones demostraron lo siguiente: Candida guillermondii presentó crecimiento mayor o igual a 25 y 50% p/v comparado con los medios comerciales; Candida famata y Candida sp presentaron mejores crecimientos al 5% p/v, con respecto a los diferentes medios comerciales. Los resultados demostraron que el medio de cultivo es altamente productivo y no selectivo, lo que representa una alternativa en la conservación, el mantenimiento y el desarrollo de las levaduras estudiadas.

This work was carried out to evaluate the efficiency of the culture medium from sour guava (Psidium araca) against commercial media in the growth of three native strains: Candida guillermondii, Candida famata and Candida sp. Microbial growth was evaluated at different concentrations of fruit, 5, 10, 25, 50% w /v, using as control the commercial media: Malta, Sabouraud and PDA (Potato Dextrose Agar). The productivity and selectivity of the sour guava medium was determined by the Ecometric method in a time of 48 hours. The applied statistical analysis to evaluate and compare growth of strains in commercial culture medium and in the medium from sour guava at different concentrations showed: Candida guillermondii grew greater than or equal to 25 and 50% w / v compared with commercial medium, Candida famata and Candida sp showed better growth at 5% w / v, with respect to commercial medium. The results showed that the medium is highly productive and non-selective representing an alternative to the conservation, maintenance and development of the yeasts.

Diversidad de levaduras asociadas a chichas tradicionales de Colombia / Yeast diversity associated to Colombian traditional chichas

En Colombia el conocimiento de la comunidad levaduriforme ha sido limitado, ya que los estudios se han enfocado principalmente en especies de interés clínico. Las fermentaciones espontáneas a partir de diversos sustratos representan hábitats de gran importancia para el estudio de la dinámica de las poblaciones de levaduras nativas, por esta razón, en el presente estudio se aislaron e identificaron las levaduras asociadas a las chichas de maíz, piña y arracacha, que son bebidas fermentadas de manera artesanal en Colombia. Se realizó el aislamiento de las levaduras más representativas de la chicha durante sus tres fases de fermentación: inicial, tumultuosa y final. Inicialmente, se hizo una caracterización parcial de los aislados, que incluyó pruebas fisiológicas, y medición de su capacidad para producir filamentos y esporas. Sin embargo, debido a que estas técnicas no fueron suficientes para identificar los aislados hasta el nivel taxonómico de género o de especie, se complementó el estudio de cada aislado empleando técnicas moleculares basadas en el análisis de restricción del gen rRNA 5.8S y los espaciadores transcritos internos (ITS1 e ITS2). Cuando el empleo de esta técnica no permitió obtener resultados definitivos y para confirmar las asignaciones realizadas usando PCR-RFLPs, se secuenció el dominio D1/D2 del gen 26S rRNA de los aislados más representativos. Mediante estas técnicas se lograron identificar las especies más representativas de los tres tipos de chicha: Candida tropicalis, Pichia kluyveri, Pichia guilliermondii, Hanseniapora guilliermondii, Pichia fermentans, Saccharomyces cerevisiae, Candida maltosa, Rhodotorula glutinis, Torulaspora delbrueckii, Hanseniaspora uvarum, Kazachstania exigua, Kluyveromyces marxianus, Yarrowia lypolitica, Candida parapsilosis, Debaromyces hansenii, Cryptococcus arboriformis, Saccharomyces martiniae, Dekkera anomala, Aureobasidium pullulans y Candida pseudointermedia. La caracterización preliminar de los aislados...

In Colombia, knowledge about yeast communities has been limited because most reports have focused on yeast species with clinical relevance. The spontaneous fermentation of different substrates creates important habitats for analyzing wild yeast populations; for this reason, in this study we isolated and identified yeasts associated with the “chichas” of corn, pineapple, and “arracacha,” which are traditional fermented Colombian beverages. The most representative yeasts were isolated from “chicha” during its three phases of fermentation: initial, tumultuous and final. Initially, we made a partial characterization of isolated yeasts, including macroscopic and microscopic descriptions, physiological tests, and measurement of capacity for producing spores and filaments. However, because these techniques were not sufficient for identification of isolated yeasts to the level of genus and species, the study was complemented by using molecular techniques based on restriction analysis of the ITS1-5.8S rRNA gene-ITS2. When this technique did not permit us to obtain positive results and confirm the PCR-RFLP results, we used the sequence of the D1/D2 domain of the 26S rRNA gene instead for most representative isolates. With these techniques, we identified the most representative yeast species of the three classes of “chicha”: Candida tropicalis, Pichia kluyveri, Pichia guilliermondii, Hanseniapora guilliermondii, Pichia fermentans, Saccharomyces cerevisiae, Candida maltosa, Rhodotorula glutinis, Torulaspora delbrueckii, Hanseniaspora uvarum, Kazachstania exigua, Kluyveromyces marxianus, Yarrowia lypolitica, Candida parapsilosis, Debaromyces hansenii, Cryptococcus arboriformis, Saccharomyces martiniae, Dekkera anomala, Aureobasidium pullulans and Candida pseudointermedia. The preliminary characterization of isolated yeasts, based on ethanol-tolerance and salt-tolerance tests, permitted recognition of wild yeasts for possible biotechnological uses in industry.

Optimization of feeding strategy for the ergosterol production by yeasts saccharomyces cerevisiae / Optimización de la estrategia de alimentación para la producción de ergosterol por levaduras saccharomyces cerevisiae

Objective of this study was to optimize ergosterol production by yeast strain Saccharomyces cerevisiae with the use of computer controlled feeding of cultivation medium. Baker´s yeasts strain of Saccharomyces cerevisiae originally modified and selected as mutant D7 was further applied in an industrial scale and also in this investigation. Composition of cultivation medium was optimized with the use of a modified Rosenbrock´s method with regard to following components: glucose, yeast extract, ammonium sulphate, potassium dihydrogen phosphate, magnesium sulphate and calcium chloride. Cultivation of yeast culture was performed in 7 L laboratory bioreactor with a working volume of 5 L equipped with a control unit and linked to a computer, with dissolved oxygen tension measurement, oxygen and carbon dioxide analyzers. BIOGENES prototype software was created from the commercial control system Genesis for Windows 3.0 (GFW), from Iconics and CLIPS 6.04 for the PC-Windows platform. From various factors affecting sterol biosynthesis a specific growth rate was chosen. Feed rate was controlled according to mathematical model. In this case it dealt with a design of optimal profile of specific growth rate with consequent calculation of carbon dioxide profile. Sterol concentration in the dry biomass increased from 1.0 % up to 3 %.

El objetivo de este estudio fue optimizar la producción de ergosterol por una cepa de levadura Saccharomyces cerevisiae, controlando la alimentación de medio de cultivo por computadora. La cepa de levadura panadera Saccharomyces cerevisiae originalmente modificada y seleccionada como mutante D7 fue posteriormente utilizada a escala industrial y también para esta investigación. La composición del medio de cultivo fue optimizada usando el método modificado de Rosenbrock respecto a los siguientes componentes: glucosa, extracto de levadura, sulfato de amonio, fosfato dihidrógeno de potasio, sulfato de magnesio y cloruro de calcio. El cultivo de las células de levadura se llevó a cabo en un biorreactor de laboratorio de 7L con un volumen de trabajo de 5L, equipado con una unidad de control conectada a una computadora, con medición de la tensión de oxígeno disuelto y analizadores de oxígeno y dióxido de carbono. Un software prototipo BIOGENES fue creado a partir del sistema de control comercial Genesis para Windows 3.0 (GFW), de Iconics y CLIPS 6.04 para la plataforma de PC-Windows. A partir de varios factores que afectan la biosíntesis de esterol se escogió una tasa específica de crecimiento. La tasa de alimentación se controló mediante un modelo matemático. En este caso, se trató con un diseño de perfil óptimo de tasa de crecimiento específico con un consecuente cálculo del perfil de dióxido de carbono. La concentración de esterol en la biomasa seca se incrementó desde 1,0% hasta 3%.

Integrated control of penicillium digitatum by the predacious yeast Saccharomycopsis crataegensis and sodium bicarbonate on oranges

Our investigation of integrated biological control (IBC) started with an assay testing activity of the predacious yeast Saccharomycopsis crataegensis UFMG-DC19.2 against Penicillium digitatum LCP 4354, a very aggressive fungus that causes postharvest decay in oranges. Under unfavourable environmental conditions, the yeast showed a high potential for control (39.9 percent disease severity reduction) of this fungus. This result was decisive for the next step, in which S. crataegensis was tested in association with sodium bicarbonate salt, a generally regarded as safe (GRAS) substance. The yeast was able to survive at different concentrations of the salt (1 percent, 2 percent and 5 percent), and continued to grow for a week at the wound site, remaining viable at high population for 14 days on the fruit surface. The yeast alone reduced the severity of decay by 41.7 percent and sodium bicarbonate alone reduced severity of decay by 19.8 percent, whereas the application of both led to a delay in the development of symptoms from 2 to 10 days. Ingredients of the formulations were not aggressive to fruits since no lesions were produced in control experiments.

Construction of killer industrial yeast Saccharomyces cerevisiae HAU-1 and its fermentation performance

Saccharomyces cerevisiae HAU-1, a time tested industrial yeast possesses most of the desirable fermentation characteristics like fast growth and fermentation rate, osmotolerance, high ethanol tolerance, ability to ferment molasses, and to ferment at elevated temperatures etc. However, this yeast was found to be sensitive against the killer strains of Saccharomyces cerevisiae. In the present study, killer trait was introduced into Saccharomyces cerevisiae HAU-1 by protoplast fusion with Saccharomyces cerevisiae MTCC 475, a killer strain. The resultant fusants were characterized for desirable fermentation characteristics. All the technologically important characteristics of distillery yeast Saccharomyces cerevisiae HAU-1 were retained in the fusants, and in addition the killer trait was also introduced into them. Further, the killer activity was found to be stably maintained during hostile conditions of ethanol fermentations in dextrose or molasses, and even during biomass recycling.

Use of Pseudomonas stutzeri and Candida utilis in the improvement of the conditions of Artemia culture and protection against pathogens

To evaluate the effect of two bacterial strains isolated from Artemia cysts and yeast (Candida utilis) on the survival, growth and total biomass production of its larvae, challenge tests were performed with Candida utilis, Pseudomonas stutzeri and Pasteurella haemolityca. In addition, a pathogenic strain of Vibrio alginolyticus was tested for comparative purposes. Pseudomonas stutzeri and Candida utilis have no impact on survival, but enhance growth and total biomass production of the larvae. However, we noted that Pasteurella haemolityca affect negatively Artemia larvae. The adhesion and antagonism assay demonstrates that Candida utilis and Pseudomonas stutzeri are fairly adherent and play an important role in the enhancement of the protection of Artemia culture against pathogens. On the basis of these results, it's suggested that it's possible to use Candida utilis and Pseudomonas stutzeri, potential candidates, as probiotic for the culture of Artemia larvae.

Increased presevation of sliced mozzarella cheese by antimibrobial sachet incorporated with allyl isothiocyanate

There is an increasing tendency to add natural antimicrobials of plant origin into food. The objective of this work was to develop a microbial sachet incorporated with allyl isothiocyanate (AIT), a volatile compound of plant origin, and to test its efficiency against growth of yeasts and molds, Staphylococcus sp. and psychrotrophic bacteria on sliced mozzarella cheese. Another objective was to quantify the concentration of AIT in the headspace of cheese packaging. A reduction of 3.6 log cycles was observed in yeasts and molds counts in the mozzarella packed with the antimicrobial sachet over 15-day storage time. The sachet also showed an antibacterial effect on Staphylococcus sp., reducing 2.4 log cycles after 12-day storage. Psychrotrophic bacteria species were the most resistant to the antimicrobial action. The highest concentration of AIT (0.08µg.mL-1) inside the active packaging system was observed at the 6-day of storage at 12 ºC ± 2 ºC. At the end of the storage time, AIT concentration decreased to only 10 percent of the initial concentration. Active packaging containing antimicrobial sachet has a potential use for sliced mozzarella, with molds and yeasts being the most sensitive to the antimicrobial effects.

Chlorine dioxide against bacteria and yeasts from the alcoholic fermentation / Dióxido de cloro contra bactérias e leveduras da fermentação alcoólica

The ethanol production in Brazil is carried out by fed-batch or continuous process with cell recycle, in such way that bacterial contaminants are also recycled and may be troublesome due to the substrate competition. Addition of sulphuric acid when inoculum cells are washed can control the bacterial growth or alternatively biocides are used. This work aimed to verify the effect of chlorine dioxide, a well-known biocide for bacterial decontamination of water and equipments, against contaminant bacteria (Bacillus subtilis, Lactobacillus plantarum, Lactobacillus fermentum and Leuconostoc mesenteroides) from alcoholic fermentation, through the method of minimum inhibitory concentration (MIC), as well as its effect on the industrial yeast inoculum. Lower MIC was found for B. subtilis (10 ppm) and Leuconostoc mesenteroides (50 ppm) than for Lactobacillus fermentum (75 ppm) and Lactobacillus plantarum (125 ppm). Additionally, these concentrations of chlorine dioxide had similar effects on bacteria as 3 ppm of Kamoran® (recommended dosage for fermentation tanks), exception for B. subtilis, which could not be controlled at this Kamoran® dosage. The growth of industrial yeasts was affected when the concentration of chlorine dioxide was higher than 50 ppm, but the effect was slightly dependent on the type of yeast strain. Smooth yeast colonies (dispersed cells) seemed to be more sensitive than wrinkled yeast colonies (clustered cells/pseudohyphal growth), both isolated from an alcohol-producing unit during the 2006/2007 sugar cane harvest. The main advantage in the usage of chlorine dioxide that it can replace antibiotics, avoiding the selection of resistant populations of microorganisms.

A produção de etanol no Brasil é atualmente realizada pelo processo de fermentação em batelada alimentada ou contínuo, com reciclo de células de leveduras, de forma que contaminantes bacterianos são também reciclados e podem causar problemas devido à competição pelo mesmo substrato. O controle bacteriano é feito pela adição de ácido sulfúrico na lavagem das células do fermento ou utilizando-se biocidas. O objetivo do trabalho foi verificar o efeito do dióxido de cloro, um biocida muito utilizado para a descontaminação da água e equipamentos, contra bactérias contaminantes da fermentação alcoólica (Bacillus subtilis, Lactobacillus plantarum, Lactobacillus fermentum e Leuconostoc mesenteroides), através do método da concentração inibitória mínima (CIM), assim como seu efeito sobre o fermento industrial. Valores menores de CIM foram encontrados para Bacillus subtilis (10 ppm) e Leuconostoc mesenteroides (50 ppm) do que para Lactobacillus fermentum (75 ppm) e Lactobacillus plantarum (125 ppm). Estas concentrações tiveram o mesmo efeito inibidor que 3 ppm de Kamoran®, com exceção de B. subtilis, no qual não se observou inibição de crescimento à esta concentração. As leveduras industriais apresentaram inibição no crescimento em concentrações superiores a 50 ppm, porém esta pareceu ser dependente do tipo de linhagem de levedura. Colônias cremosas (células dispersas) foram ligeiramente mais sensíveis que as colônias rugosas (células agrupadas/pseudohifas), ambas isoladas de uma unidade produtora de álcool durante a safra de cana-de-açúcar 2006/2007. A principal vantagem na utilização deste produto está na eliminação do uso de antibióticos, evitando a geração de populações resistentes de microrganismos.

Sensibilidad in vitro a los distintos antifúngicos tópicos disponibles en otomicosis externa / In vitro sensitivity to the different typical antifungoids available in external otomycosis

La otitis externa es motivo de consulta frecuente. Las otomicosis son una fracción pequeña dentro de este grupo que, en ocasiones, suele ser de diagnóstico y manejo más complejo. Muchas veces se presenta en forma recurrente. Los objetivos son conocer el perfil de los agentes causales, lograr un método para estudio de sensibilidad de los hongos a los antimicóticos tópicos, y obtener las bases para guías clínicas. Presentamos un estudio prospectivo en 23 pacientes con un cuadro compatible con otomicosis, obteniendo muestras del conducto auditivo externo, observándolas al fresco y cultivándolas. Estudiamos susceptibilidad a los distintos antimicóticos mediante difusión en discos de Agar, midiendo el halo de inhibición a las distintas cepas. Del total de muestras, 16 (70 por ciento) fueron positivas. La especie más frecuente fue Aspergillus niger (81 por ciento). El antifúngico que logró mejores resultados en susceptibilidad fue timerosal. Otros, como ácido bórico y nistatina, no presentaron halo de inhibición para ninguna de las cepas. La observación al fresco es un método útil, barato y rápido, con buena correlación clínica con el cultivo. La técnica utilizada no permite hablar de sensibilidad propiamente tal. Sin embargo, permite una valoración más objetiva de la respuesta de los hongos antimicóticos. Probablemente sea recomendable usar agentes con mejor halo de inhibición, tales como timerosal y terbinafina.

Production of a monoclonal antibody against a yeast secreted antigen of Penicillium marneffei.

Monoclonal antibody against P. mameffei yeast secreted antigen was produced in order to develop a serological test for penicilliosis marneffei. The yeast form of P. marneffei was cultured in brain heart infusion broth at 37 degrees C for 7 days. A secreted antigen was prepared, partially purified from culture supernatant and subsequently immunized in a BALB/c mouse. Mouse monoclonal antibody was produced from immune spleen cells by a standard hybridoma technique. Specificity of the obtained monoclonal antibody was assessed with yeast secreted antigens for P. mameffei, C. alblicans, C. neoformans, and H. capsulatum by an indirect ELISA. Three of 46 hybrid clones (1 F1, 2G5, and 3G4) reacted positively with P mameffei secreted antigen. 1 F1 and 3G4 were cloned by two rounds of limiting dilution. Partially purified monoclonal antibody and rabbit polyclonal antibody against P. marneffei yeast secreted antigen were used to develop a double antibody sandwich ELISA to detect P. marneffei antigen in plasma or serum samples of 7 patients with penicilliosis marneffei and 5 healthy controls. The sandwich ELISA developed using monoclonal antibody as a capture antibody and rabbit polyclonal antibody as a detector was able to detect P. marneffei antigen in all the plasma and serum samples of penicilliosis marneffei patients, while negative in all the healthy controls. Thus, the monoclonal antibody produced in the present study appeared to be highly specific for P. marneffei and the double antibody sandwich ELISA developed using monoclonal and polyclonal antibodies against the yeast secreted antigen of P. marneffei showed a strong potential for the diagnosis of penicilliosis marneffei.

Distinct immunologic properties of Penicillium marneffei yeasts obtained from different in vitro growth conditions.

A dimorphic fungus Penicillium marneffei is a causative agent of penicilliosis, a life-threatening disseminated disease in immunocompromised hosts predominantly found in southeast Asia and southern China. P. marneffei is the only known Penicillium that possesses a dimorphic characteristic. Since it is difficult to produce large amount of P. marneffei yeasts in vivo for experimentation purpose, yeast cells were produced in different in vitro conditions as alternatives. We interested in investigating the immunologic properties of yeast cells from different culture preparations. It was found that yeast cells obtained from brain heart infusion broth and Sabouraud dextrose broth did not resemble those resided in clinical specimens. A solution of 1% peptone, on the other hand, could induce a direct conidial transition into fission yeasts. Ability of yeast cells in each preparation to activate macrophages was determined by analyzing surface expression of CD40 and CD86 co-stimulatory molecules after two days of co-cultivation. Every P. marneffei yeast cell preparation demonstrated such ability. However, the ones from Sabouraud dextrose broth seemed to induce less phagocytosis. Additionally, although distinct antigenic profiles and lack of conformity in antigenic expression were observed among yeast cells from different culture conditions, most major immunogenic bands were present when Western analysis was performed using polyclonal antisera from penicilliosis patients. The results of the study raise attention on immunological and biochemical characteristics of P. marneffei yeasts if such preparations are to be used in future laboratory investigations.

Levaduras asociadas con el hábitat natural de Histoplasma capsulatum en Guerrero, México / Yeasts associated to natural habitat of Histoplasma capsulatum in Guerrero, Mexico

Objetivo: En el presente trabajo, primero que sobre el tema se ha realizado en México, se registran las especies de levaduras encontradas en diferentes sustratos colectados en espacios cerrados y abiertos (que representan los nichos ecológicos en que se desarrolla Histoplasma capsulatum var. capsulatum). Material y métodos: Los sustratos a partir de los cuales se hizo el aislamiento de levaduras se obtuvieron en distintas localidades de los municipios de Quechultenango y Olinalá, en el estado de Guerrero. Resultados y discusión: De guano de murciélago muestreado en grutas y cuevas se aislaron Candida Catenulata, C. ciferrii, C famata, C. guillermondii y Rhodotorula spp.; del suelo de una mina únicamente se aisló C. ciferril. Las especies C. albicans, C. ciferrii y C. tropicalis se aislaron de suelo con excretas de gallináceas, y C. famata, Cryptococcus albidus var. albidus, Trichosporon beigelii t Trichosporon spp. de suelo con excretas de gallo. Del intestino de murciélagos insectívoros únicamente se aisló C. famata, y, de murciélagos polinívoros C. lipolytica, Cr. abidus var. albidus y Trichosporon spp. De cada una de estas especies se mencionan sus características distintivas, así como los diferentes ambientes y sustratos de los cuales han sido aisladas