Action of mycocins produced by Wickerhamomyces anomalus on Malassezia pachydermatis isolated from the ear canal of dogs.

This study aims to examine the effectiveness of mycocins produced by Wickerhamomyces anomalus in inhibiting Malassezia pachydermatis, a yeast commonly found in the ear canal of dogs. M. pachydermatis has a zoophilic origin and can be found in mammals, and frequently in dogs, where it mainly colonizes the ear canal region and the skin, leading to lesions that are difficult to treat. The antimicrobial mechanism was evaluated using dilutions of supernatant with enzymatic activity, which may include ß-glucanases, glycoproteins known to act on microorganism cell walls. However, it is important to note that this supernatant may contain other compounds as well. ß-glucanases in the mycocins supernatant were found at a concentration of 0.8 U/mg. The susceptibility of M. pachydermatis isolates was tested using the microdilution method. The isolates suffered 100% inhibition when tested with the culture supernatant containing mycocins. In the proteinases production test, 44% of the isolates tested were strong proteinases producers. Subsequently all these isolates suffered inhibition of their activity when tested in research medium containing mycocins supernatant at a subinhibitory concentration of ß-glucanases. This shows that mycocins can inhibit the production of proteinases, a virulence factor of M. pachydermatis. The viability test showed the antifungal action of mycocins in inhibiting the viability of M. pachydermatis cells after a period of 8  hours of contact. These results support the antimicrobial potential of mycocins and their promise as a therapeutic option.

Killer Yeasts for the Biological Control of Postharvest Fungal Crop Diseases.

Every year and all over the world the fungal decay of fresh fruit and vegetables frequently generates substantial economic losses. Synthetic fungicides, traditionally used to efficiently combat the putrefactive agents, emerged, however, as the cause of environmental and human health issues. Given the need to seek for alternatives, several biological approaches were followed, among which those with killer yeasts stand out. Here, after the elaboration of the complex of problems, we explain the hitherto known yeast killer mechanisms and present the implementation of yeasts displaying such phenotype in biocontrol strategies for pre- or postharvest treatments to be aimed at combating postharvest fungal decay in numerous agricultural products.

Saccharomyces cerevisiae: A novel and efficient biological control agent for Colletotrichum acutatum during pre-harvest.

In this study, we evaluated the efficiency of six isolates of Saccharomyces cerevisiae in controlling Colletotrichum acutatum, the causal agent of postbloom fruit drop that occur in pre-harvest citrus. We analyzed the mechanisms of action involved in biological control such as: production of antifungal compounds, nutrient competition, detection of killer activity, and production of hydrolytic enzymes of the isolates of S. cerevisiae on C. acutatum and their efficiency in controlling postbloom fruit drop on detached citrus flowers. Our results showed that all six S. cerevisiae isolates produced antifungal compounds, competed for nutrients, inhibited pathogen germination, and produced killer activity and hydrolytic enzymes when in contact with the fungus wall. The isolates were able to control the disease when detached flowers were artificially inoculated, both preventively and curatively. In this work we identified a novel potential biological control agent for C. acutatum during pre-harvest. This is the first report of yeast efficiency for the biocontrol of postbloom fruit drop, which represents an important contribution to the field of biocontrol of diseases affecting citrus populations worldwide.

Production of functional killer protein in batch cultures upon a shift from aerobic to anaerobic conditions

The aim of this work was to study the production of functional protein in yeast culture. The cells of Saccharomyces cerevisiae Embrapa 1B (K+R+) killed a strain of Saccharomyces cerevisiae Embrapa 26B (K-R-)in grape must and YEPD media. The lethal effect of toxin-containing supernatant and the effect of aeration upon functional killer production and the correlation between the products of anaerobic metabolism and the functional toxin formation were evaluated. The results showed that at low sugar concentration, the toxin of the killer strain of Sacch. cerevisiae was only produced under anaerobic conditions . The system of killer protein production showed to be regulated by Pasteur and Crabtree effects. As soon as the ethanol was formed, the functional killer toxin was produced. The synthesis of the active killer toxin seemed to be somewhat associated with the switch to fermentation process and with concomitant alcohol dehydrogenase (ADH) activity.

Optimization of killer assays for yeast selection protocols / Optimización de la actividad killer para protocolos de selección de levaduras

A new optimized semiquantitative yeast killer assay is reported for the first time. The killer activity of 36 yeast isolates belonging to three species, namely, Metschnikowia pulcherrima, Wickerhamomyces anomala and Torulaspora delbrueckii, was tested with a view to potentially using these yeasts as biocontrol agents against the wine spoilage species Pichia guilliermondii and Pichia membranifaciens. The effectiveness of the classical streak-based (qualitative method) and the new semiquantitative techniques was compared. The percentage of yeasts showing killer activity was found to be higher by the semiquantitative technique (60%) than by the qualitative method (45%). In all cases, the addition of 1% NaCl into the medium allowed a better observation of the killer phenomenon. Important differences were observed in the killer capacity of different isolates belonging to a same killer species. The broadest spectrum of action was detected in isolates of W. anomala NPCC 1023 and 1025, and M. pulcherrima NPCC 1009 and 1013. We also brought experimental evidence supporting the importance of the adequate selection of the sensitive isolate to be used in killer evaluation. The new semiquantitative method proposed in this work enables to visualize the relationship between the number of yeasts tested and the growth of the inhibition halo (specific productivity). Hence, this experimental approach could become an interesting tool to be taken into account for killer yeast selection protocols.

En este trabajo se presenta un nuevo ensayo semicuantitativo que optimiza la detección de actividad killer en levaduras. Se evaluó la actividad killer de 36 cepas pertenecientes a las especies Metschnikowia pulcherrima, Wickerhamomyces anomala y Torulaspora delbrueckii, en vista del potencial uso de estas levaduras como agentes de biocontrol frente a las especies contaminantes de vinos Pichia guilliermondii y Pichia membranifaciens. Se comparó la efectividad de la técnica clásica basada en estrías (método cualitativo) con la del nuevo método semicuantitativo. El porcentaje de levaduras que mostraron actividad killer fue más alto cuando se utilizó el método semicuantitativo (60%) que con el método cualitativo (45%). En todos los casos, el agregado de 1% de NaCl en el medio permitió una mejor observación del fenómeno killer. Se observaron importantes diferencias en la capacidad killer de diferentes cepas dentro de la misma especie. Se detectaron dos cepas de W. anomala (NPCC 1023 y 1025) y dos cepas de M. pulcherrima (NPCC 1009 y 1013) con un amplio espectro de acción, ya que fueron capaces de inhibir el desarrollo de las tres levaduras sensibles evaluadas. La evidencia experimental demuestra la importancia de una adecuada selección de la cepa sensible al evaluar la actividad killer. El nuevo método semicuantitativo propuesto en este trabajo permite visualizar la relación entre el número de levaduras sembradas y el halo de inhibición del crecimiento (productividad específica). En conclusión, este método resulta una herramienta interesante para ser tenida en cuenta en los protocolos de selección de levaduras killer.

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.

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.

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.

Killer yeasts inhibit the growth of the phytopathogen Moniliophthora perniciosa, the causal agent of Witches' Broom disease / Leveduras micocinogênicas inibem o crescimento do fitopatógeno Moniliophthora perniciosa, o agente causador da doença Vassoura-de-bruxa

Fruit and soil yeasts isolated from the Amazon, Atlantic Rainforests and an organic farm were screened for killer activity against yeasts. Killer yeasts were then tested against the phytopathogen Moniliophthora perniciosa (syn. Crinipellis perniciosa) and a Dipodascus capitatus strain and a Candida sp strain inhibited its growth.

Leveduras de frutas e de solo isoladas da Floresta Amazônica, Mata Atlântica e de uma fazenda orgânica foram selecionadas em uma triagem para atividade micocinogênica. As estirpes micocinogênicas foram posteriormente testadas frente a Moniliophthora perniciosa (syn. Crinipellis perniciosa). Uma estirpe de Dipodascus capitatus e outra de Candida sp.inibiram o crescimento deste fitopatógeno.

Killer yeasts inhibit the growth of the phytopathogen Moniliophthora perniciosa, the causal agent of Witches' Broom disease.

Fruit and soil yeasts isolated from the Amazon, Atlantic Rainforests and an organic farm were screened for killer activity against yeasts. Killer yeasts were then tested against the phytopathogen Moniliophthora perniciosa (syn. Crinipellis perniciosa) and a Dipodascus capitatus strain and a Candida sp strain inhibited its growth.

Killer yeasts inhibit the growth of the phytopathogen Moniliophthora perniciosa, the causal agent of Witches' Broom disease

Fruit and soil yeasts isolated from the Amazon, Atlantic Rainforests and an organic farm were screened for killer activity against yeasts. Killer yeasts were then tested against the phytopathogen Moniliophthora perniciosa (syn. Crinipellis perniciosa) and a Dipodascus capitatus strain and a Candida sp strain inhibited its growth.

Leveduras de frutas e de solo isoladas da Floresta Amazônica, Mata Atlântica e de uma fazenda orgânica foram selecionadas em uma triagem para atividade micocinogênica. As estirpes micocinogênicas foram posteriormente testadas frente a Moniliophthora perniciosa (syn. Crinipellis perniciosa). Uma estirpe de Dipodascus capitatus e outra de Candida sp.inibiram o crescimento deste fitopatógeno.

Comportamiento de una cepa salvaje de Saccharomyces cerevisiae killer y su isogénica sensible respecto de diferentes fuentes de nitrógeno en cultivos mixtos / Behaviour of a wild Saccharomyces cerevisiae killer yeast and its isogenic sensitive one with respect to different nitrogen sources in mixed cultures

La composición química del vino constituye el fundamento de la posterior respuesta sensorial del producto y está determinada por varios factores, como las relaciones levadura-levadura. Se denomina fenómeno killer a la secreción por parte de ciertas cepas de levadura de una proteína tóxica que mata a células denominadas sensibles. El conocimiento del comportamiento en condición aeróbica de cultivos mixtos killer-sensible es útil para relacionarlo con la primera fase de la fermentación enológica, ya que en ella puede definirse la prevalencia o no de la cepa killer. Además, el empleo de mutantes con el plásmido curado permite comparaciones más precisas. El objetivo fue analizar el mecanismo de competencia por sustrato en levaduras killer de Saccharomyces cerevisiae y su mutante sensible con el plásmido curado, empleando distintas fuentes de nitrógeno. Si las muestras se incuban a temperatura de inactivación de la toxina, se evita la infraestimación de células sensibles. Los resultados del co-cultivo de las cepas en proporciones iguales muestran el rol desempeñado por la fuente de nitrógeno en la actividad killer. Cuando el inóculo es 10%K-90%S, el modelo de exclusión competitiva planteado para levaduras killer deja paso a otras variables de competencia.

Wine chemical composition is the outcome of complex chemosensory interactions that are difficult to predict because of the influences of many variables, like as yeast-yeast interactions. Killer phenomenon implicates the secretion of a toxic protein by some yeasts, that kill other yeasts called sensitive. The knowledge of the behaviour of killer-sensitive mixed cultures in aerobic conditions is useful to be related with the first stages of oenological fermentation. In these stages it can be defined the killer prevalence in the medium. Also, the use of cured plasmid mutants allows better comparisons. The objective was to analyse the mechanism of substrate competition in Saccharomyces cerevisiae killer strains and its sensitive cured plasmid mutant, using different nitrogen sources. When samples were incubated at the toxin inactivation temperature, the infraestimation of sensitive cells is avoided. Results obtained in co-cultures (50%K-50%S) show the role of the nitrogen source in killer activity. Results obtained with 10%K-90%S inoculum, show that there are another competence variables than the competitive exclusion model for killer yeasts.

Characterization of the Saccharomyces cerevisae Y500-4L killer toxin

The strain Saccharomyces cerevisiae Y500-4L, selected from the must of alcohol producing plants, liberates a toxin which is lethal to the commercial yeast produced by Fleischmann Royal Nabisco and other strains of yeast. This toxin was characterized, and the maximum production was obtained after 24 hours of incubation at 25ºC in YEPD medium. The maximum activity was achieved between pH 4.1 and 4.5 and between 22 and 25ºC and maximum stability in the pH range 3.8 to 4.5 at -10ºC. The killer toxin was inactivated by heating at 40ºC for 1 hour at pH 4.1. After concentration by ultrafiltration of culture supernatants and purification by gel filtration chromatography, the molecular weight of the purified toxin was estimated by SDS-PAGE to be about 18-20 kDa.

A linhagem de Saccharomyces cerevisiae Y500-4L, selecionada de mosto de fermentação de usina de álcool, produz toxina "killer", letal à levedura comercial Fleischmann Royal Nabisco e outras linhagens de leveduras. Esta proteína foi caracterizada, verificando-se que a produção máxima foi obtida após 24 horas de incubação a 25ºC em meio YEPD. A toxina "killer" apresentou maior atividade na faixa de pH 4,1-4,5 e temperatura de 22-25ºC; e maior estabilidade na faixa de pH 3,8-4,5 a -10ºC, sendo totalmente inativada após 1 hora de incubação a 40ºC em pH 4,1. Após concentração a partir do sobrenadante do meio de cultura através de ultrafiltração e purificação por cromatografia de filtração em gel, estimou-se, através de SDS-PAGE, que o peso molecular desta toxina é cerca de 18 a 20 kDa.