Diversity of yeasts during fermentation of cocoa from two sites in the Brazilian Amazon

The purpose of this study was to identify the yeasts involved in spontaneous fermentation of cocoa from the Brazilian Amazon region. The fermentation process was carried out experimentally with cocoa seeds from two sites (Medicilândia and Tucumã), State of Pará, northern Brazil, during a six-day period. Totals of 44 yeasts were isolated from Medicilândia and 29 from Tucumã. Molecular identification was carried out by sequencing the D1/D2 region fragment of the rRNA 26S gene, expanded with universal primers for the NL1GC and LS2 eukaryotes. Pichia manshurica and Saccharomyces cerevisiae were identified in Medicilândia and five yeast species (Pichia fermentans, P. kudriavzevii, P. manshurica, S. cerevisiae and Zygosaccharomyces bailii) were identified in Tucumã. The results showed that P. manshurica and S. cerevisiae may have potential for use as starter cultures in future studies to improve the quality of cocoa seeds fermented in the Brazilian Amazon region. (AU)

A proposta deste estudo foi identificar as leveduras envolvidas na fermentação espontânea de cacau da Amazônia brasileira. A fermentação foi realizada em Medicilândia e Tucumã, Pará, Brasil, durante 6 dias. Em total foram obtidos 44 isolados de leveduras de Medicilândia e 29 de Tucumã. A identificação molecular foi realizada por sequenciamento do fragmento da região D1/D2 do gene rRNA 26S, amplificado com primers universais para eucariotos NL1GC e LS2. Em Medicilândia, foram identificadas Pichia manshurica e Saccharomyces cerevisiae. Em Tucumã foram identificadas cinco espécies (Pichia fermentans, P. kudriavzevii, P. manshurica, S. cerevisiae e Zygosaccharomyces bailii). Os resultados sugerem que P. manshurica e S. cerevisiae podem ter potencial para uso como culturas starter em estudos futuros, para melhorar a qualidade das sementes de cacau fermentadas na Amazônia brasileira.(AU)

Caracterización molecular y fisiológicas de levaduras de suelo trumao / Molecular and physiological characterization of yeasts of trumao soil

Las levaduras juegan un importante rol en la naturaleza siendo el mayor reservorio de ellas el suelo. Mediante el método de las diluciones seriadas y posterior siembra en agar Sabouraud se aislaron en cultivo puro 77 cepas de levaduras desde un mismo suelo trumao del sur de Chile, usado como pradera permanente (30 cepas), pradera en rotación (30 cepas) y como control bosque nativo (17 cepas), estas cepas se identificaron molecularmente por PCR-RFLP en conjunto con secuenciación del rDNA de ITS-5.8S, además se realizo una caracterización fisiológica (asimilación fuente de carbono, de nitrógeno y fermentación de azucares) a cada cepa. Mediante las técnicas moleculares las 77 cepas se reunieron en 10 grupos, de estos solamente tres grupos se pudieron identificar a nivel de especie y uno hasta género: Devariomyces hansenii. Pichia fermentan. Kazachstania exigua., Candida sp.

Yeasts plays an important role in nature, It is the largest reservoir of soil them. By the method of serial dilutions and subsequent planting in Sabouraud agar were isolated in pure culture 77 strains of yeast from the same volcanic ash soil of southern Chile, used as permanent pasture (30 strains), rotation pasture (30 strains) and native forest as a control (17 strains), these strains were identified molecularly by PCR-RFLP in conjunction with rDNA sequencing ITS-5.8S, physiological characterization addition was performed to each strain (carbon and nitrogen source assimilation and fermentation of sugars). Using molecular techniques met the 77 strains in 10 groups; only three groups could be identified to species level and one to gender: Devariomyces hansenii; Pichia fermented; Kazachstania exigua; Candida sp.

Lactobacillus plantarum: Effect of a protective biofilm on the surface of olives during storage

Abstract The aim of the present study was to investigate the effect of Lactobacillus plantarum adhesion to the surface of olives during storage through studying the interaction between the surfaces of the olives and L. plantarum. The results showed that the total number of adherent L. plantarum increased exponentially from 1.2 × 106 to 1.3 × 108 cfu/g. Images obtained using environmental scanning electron microscopy (ESEM) after 4 days of storage revealed that the olive surface was covered with a uniform and compact biofilm constituted of L. plantarum and yeast. Physicochemical analysis of surface of L. plantarum revealed that it was hydrophilic (Giwi > 0 mJ/m2). The surface of the olives also appeared to be hydrophilic (Giwi = 3.28 mJ/m2). The electron-donor characteristics of the surfaces of L. plantarum and olive were γ− = 53.1 mJ/m2 and γ− = 28.1 mJ/m2, respectively. The formation of a protective biofilm of L. plantarum increased the hydrophilicity (from 3.28 to 46.14 mJ/m2) and the electron-donor capacity (from 28.1 to 67.2 mJ/m2) of the olive surface by 1 day of storage. Analysis of the impact of the biofilm that formed on the surface of the olives during storage showed a reduction in the content of undesirable planktonic microorganisms, such as fungi, which could have occurred due to competition for nutrients and oxygen or modifications in the physicochemical properties of the olives. Thus, coating the surface of olives with a natural material, such as L. plantarum, may be a first step in developing strategies to prevent their microbial colonization.

Suplementação e cultura de levedura na alimentação de bezerros de corte em pastagem de aveia e azevém / Supplementation and yeast culture on feeding of beef calves grazing on oat and ryegrass

Avaliaram-se os efeitos da suplementação e o uso de cultura de levedura sobre o ganho médio diário (GMD), os parâmetros ruminais e a concentração de ureia plasmática em bezerros sob pastejo de aveia preta (Avena strigosa) e azevém (Lolium multiflorum), e estudaram-se a produção e a composição química da forragem. Utilizaram-se 32 bezerros, em pastejo contínuo e lotação variável. Os tratamentos foram: apenas mistura mineral (MM); mistura mineral mais levedura (MML); mistura mineral mais ração (MMR); e mistura mineral mais ração mais levedura (MMRL). A MM contendo casca de soja foi ofertada em 150g/animal/dia, e a levedura foi adicionada à MM na quantidade de 10g/animal/dia. A ração contendo 75% de NDT e 23% de PB foi fornecida para um consumo diário médio de 12g/kg de peso corporal. O GMD, para os respectivos tratamentos, foi 0,610; 0,599; 0,809 e 0,818kg/animal, na ordem citada. A concentração de amônia ruminal e a de ureia plasmática não foram influenciadas pelos tratamentos. A biomassa forrageira diminuiu ao longo do ciclo vegetativo, com rendimentos inicial e final de 1.364 e 879kg de MS/ha, e os teores médios de NDT e PB de 71,7 e 11,7%, respectivamente. O fornecimento do suplemento melhorou o desempenho dos animais, e a adição de levedura não influenciou as variáveis estudadas.

We evaluated the effects of supplementation and use of yeast culture on the average daily gain (ADG), ruminal parameters and plasma urea nitrogen in calves grazing oat (Avena strigosa) and ryegrass (Lolium multiflorum) and studied the production and chemical composition of forage. 32 calves were used in continuous grazing with variable stocking rate. The treatments administered were: only mineral (MM), administration of mineral with yeast (MML), mineral plus ration (MMR) and mineral plus ration with yeast (MMRL). MM added with soybean hulls was supplied at 150 g/animal/day and yeast added to the MM in the amount of 10 g/animal/day. The ration containing 75% of TDN and 23% of PB was supplied for an average daily intake of 12 g /kg of body weight. The ADG for the respective treatments were 0.610, 0.599, 0.809 and 0.818kg/animal, respectively. The concentration of ruminal ammonia and plasma urea was not affected by treatments. Forage biomasses declined throughout the growing season, with initial and final yields of 1.364 and 879kg DM/ha, and TDN and CP levels of 71.7 and 11.7%, respectively. The supply of the supplement improved animal performance, and the addition of yeast did not influence the variables studied.

Kinetic analysis of gene expression during mycelium to yeast transition and yeast to mycelium germination in Paracoccidioides brasiliensis / Análisis de la cinética de expresión de genes durante la transición de micelio a levadura y la germinación levadura a micelio en Paracoccidioides brasiliensis

Introduction: Paracoccidioidomycosis is an endemic systemic mycosis caused by Paracoccidioides brasiliensis, a thermally dimorphic fungus that in tissues and cultures at 37°C grows as a yeast while at lower temperatures (less than 24°C) it becomes a mold; however the genes that rule these processes and their expression are poorly understood. Objective: This research focused on the kinetic expression of certain genes in P. brasiliensis throughout the dimorphic process, one that involves the transition from the mycelium to yeast forms and the germination from the yeast to mycelium form. Materials and methods: A real-time quantitative polymerase chain reaction (RT-qPCR) was optimized to measure the expression of ten genes connected with diverse cellular functions including cell synthesis and wall structure, oxidative stress response, heat shock response, metabolism, proteins’ processing, solute transport across the cell membrane and signal transduction pathways at different time points during the mycelia to yeast transition, as well as in the yeast to mycelia germination processes. Results: Genes involved in cell synthesis and wall structure, metabolism and signal transduction were differentially expressed and highly up-regulated during the yeast to mycelia germination process; on the other hand, genes involved in heat shock response, cell synthesis and wall structure were highly up-regulated during the mycelia to yeast transition process. The remaining genes were differentially regulated during both processes. Conclusion: In this work the up-regulation of certain genes involved in the morphological changes occurring in P. brasiliensis yeast and mycelia forms were confirmed, indicating that these biological processes play an important role during the host-pathogen interactions, as well as in the fungus adaptation to environmental conditions.

Introducción. La paracoccidioidomicosis es una micosis sistémica causada por el hongo termodimorfo Paracoccidioides brasiliensis. En tejidos y cultivos a 37°C crece como levadura, mientras que a temperaturas menores de 24°C crece como un moho. Sin embargo, se conoce poco sobre los genes que regulan estos procesos. Objetivo. Se evaluó la cinética de expresión de algunos genes en P. brasiliensis mediante el proceso de dimorfismo incluida la transición del micelio a levadura y de la germinación de levadura a micelio. Materiales y métodos. Se optimizó una PCR cuantitativa en tiempo real (RT-qPCR) para medir la expresión de diez genes relacionados con diversas funciones celulares que incluyeron: síntesis de pared, respuesta al estrés oxidativo, respuesta al choque térmico, metabolismo, procesamiento de proteínas, trasporte de solutos a través de membranas y transducción de señales, todo ello a diferentes tiempos durante la transición de micelio a levadura, así como de la germinación de levadura a micelio. Resultados. Se encontró que los genes relacionados con síntesis de pared, metabolismo y transducción de señales, se expresaban de manera diferencial y con regulación positiva durante la germinaciónlevadura a micelio, mientras que algunos genes relacionados con respuesta a choque térmico y a síntesis de pared estaban sobreexpresados en la transición de micelio a levadura. Los genes restantes se regularon de manera diferencial en ambos procesos. Conclusiones. En este trabajo se confirma la regulación positiva de algunos genes relacionados con los cambios morfológicos de las fases levadura y micelio en P. brasiliensis, procesos biológicos que juegan un papel de importancia durante la interacción huésped-parásito y durante la adaptación del hongo al ambiente, respectivamente.

Determinación de zearalenona por cromatografía líquida de alta resolución: normalización de un nuevo método para raciones especiales para cerdos / Zearalenone determination in high performance liquid chromatography: a new standardization method for commercial swine rations

The aim of this work was to offer a new method of high performance liquid chronomatography (HPLC) to evaluate commercial swine rations (CSR) contaminated by zearalenone (ZEA). After ZEA extraction and purification from CSR, the samples were eluted with acetonitrile, methanol and water solvent system. The results indicated that the proposed method showed to be rapid and efficient for the detection and quantification of ZEA in CSR, since its recovery was 102.62 percent, it offered excellent precision with a coefficient of variation of 0.9992. Furthermore, it is also proposed a as a biocontrol assay for micotoxigenic fungi isolated and maintained in the laboratory. The test was performed with the killer yeast Trichosporum insectorum CBS 10422 against Fusarium sp and Aspergillus flavus, which demonstrated to be effective against the latter.

El propósito de este articulo es ofrecer un nuevo método de cromatografía líquida de alta resolución (CLAR) para evaluar las raciones especiales para cerdos (REC) contaminado con zearalenona (ZEA). Después de la extracción y purificación de ZEA, las muestras se eluyeron con acetonitrilo, metanol y agua del sistema disolvente. Los resultados indican que el método propuesto demostró ser rápido y eficaz para la detección y cuantificación de ZEA en REC, ya que sus indicadores se presentan capaces de recuperación de 102,62 por ciento, además de ofrece una excelente precision, con un çõefiCiênte de variación de 0,9992. Por otra parte, también se propone una prueba de control biológico de hongos micotoxige-nic aislados y mantenidos en el laboratorio. La prueba se realizó con la levadura killer Trichosporum insectorum CBS 10422 contra Fusarium sp y Aspergillus flavus, mostrando eficaces sólo contra Aspergillus.

Expression of recombinant human serum albumin in the methylotrophic yeast; Hansenula polymorpha

Human serum albumin [HAS] is the major protein component of human plasma. It plays a very important role in transporting of macro molecules and maintaining the normal osmolarity. It is used as a therapeutical protein in patients with hypoalbuminemia and acute bleeding and burning. Albumin consumption in the world is about 500 ton/year. The aim of this research is to study the production of rHSA in shake flask culture by Hansenula polymorpha. H. polymorpha was used for the production of recombinant human serum albumin [rHSA] in several of shake flask culturing; expression of rHSA was investigated relating several parameters affecting the expression of HSA. To optimize the secretory expression of rHSA under the control of FMD promoter in H polymorpha RB-11 incubation time, culture media temperature and protease inhibitors were analyzed. This study not only established production of rHSA in yeast but also analyzed the correlation between affecting parameters and the level of HSA expression. Comparison of the HSA levels in the culture supernatants showed that the highest HSA yield was 17.6mg/l. The research shows that among three different temperatures [25°C,30°C and 37°C] 37°C was the best temperature and amongst three different incubation times [24h,48h and 72h] 48h was the optimum time and YNB 1% glycerol with buffer was the best derepression medium in comparison with others. Using these optimized conditions, stable production of rHSA of around 17.6mg/l was achieved. Our results suggest that affecting experssion factors improved in this study are suitable for production of recombinant albumin

Levaduras inhibidoras de Penicillium / Inhibitory Penicillium yeasts

El objetivo de este trabajo fue determinar la acción inhibitoria in vitro e in vivo de algunas cepas de levaduras de la zona citrícola jujeña sobre el crecimiento de los mohos patógenos post-cosecha y seleccionarlas para elaborar un producto de biocontrol. Se aislaron de frutos cítricos cepas de los mohos patógenos post-cosecha Penicillium digitatum, P. italicum,P. ulaiense, Phyllosticta sp. y Galactomyces geotrichum, así como de levaduras saprófítas de los géneros Brettanomyces, Candida, Cryptococcus, Kloeckera, Pichia y Rhodotorula. También se obtuvieron algunas levaduras de otras fuentes. Se identificaron las levaduras por las características macro y micromorfológicas y las pruebas fisiológicas. La actividad in vitro e in vivo de las diferentes cepas fue diferente según se enfrentaran a P. digitatum o P. ulaiense. Candida cantarellii y una cepa de Pichia subpelliculosa produjeron una reducción significativa del área de las lesiones provocadas por estas especies de Penicillium, y podrían ser empleadas en la formulación de un producto para biocontrol.

The objective of this work was to establish the in vitro and in vivo inhibition of post-harvest pathogenic moulds by yeasts in order to make a biocontrol product. Post-harvest pathogenic moulds Penicillium digitatumP. italicum, P. ulaiense, Phyllosticta sp., Galactomyces geotrichum and yeasts belonging to genera Brettanomyces, Candida, Cryptococcus, Kloeckera,Pichia, Rhodotorula were isolated from citrus fruits. Some yeasts strains were also isolated from other sources. The yeasts were identified by their macro and micro-morphology and physiological tests. The in vitro and in vivo activities against P. digitatum or P. ulaiense were different. Candida cantarellii and one strain of Pichia subpelliculosa produced a significant reduction of the lesion area caused by the pathogenic moulds P. digitatum and P. ulaiense, and could be used in a biocontrol product formulation.

Formación y regeneración de protoplastos de phaffia rhodozyma / Formation and regeneration of phaffia rhodozyma protoplasts

En este trabajo se describe la preparación y regeneración de protoplastos a partir de cultivos frescos de la levadura carotenogénica phaffia rhodozyma, en forma eficiente y con un alto grado de rendimiento en la sobrevida de las células tratadas. Para ello se han realizado una serie de experimentos para formar protoplastos de p.rhodozyma, utilizando tres cepas silvestres y cinco cepas afectadas en carotenogénesis. Se probó las enzimas bioglucanasa, biocelulasa, bioxilanasa, glucoronidasa, zimoliasa 100T, lisozima y novozima 234, encontrándose que novozima 234, es la que tiene mayor eficiencia. En las cepas silvestres UCD 67-210 y UCD 67-385 y las cinco cepas de color, se logra un 100 porciento de protoplastos entre 60 a 90 minutos de tratamiento con novozima a 37ºC. Sin embargo, no fue posible formar protoplastos en ninguna de las condiciones estudiadas con las cepas silvestres UCD 67-383. Tales resultados pueden ser devidos a diferencias en la pared celular entre dichas cepas. Además, se ha observado que la incubación a 37ºC reduce notablemente la sobrevida de las células tratadas. Para la formación y regeneración de protoplastos se utilizó una serie de condiciones, encontrando que un sorbitol 0.2 M la destrucción de las células es menor que a concentraciones de sorbitol 0.8 y 1 M. Sin embargo, el medio más adecuado para la formación y regeneración de los protoplastos debe contener KC1 0.8 M como agente osmolar, manteniendo la isotonía del medio y logrando que la destrucción celular sea menor