Determinación de la contaminación con Zygosaccharomyces rouxii durante la elaboración de jugos de uva concentrados

El jugo de uva concentrado (JUC) es un commodity y por su carácter natural se utiliza para elaborar jugos mezclas, golosinas, dulces, mermeladas, jaleas, galletitas, pan, como edulcorante de bebidas gaseosas, y también en la industria farmacéutica. La producción de JUC constituye una parte importante de la industria vitivinícola argentina, siendo nuestro país el mayor exportador mundial de JUC durante el año 2014. El comercio internacional del JUC forma parte de mercados con una demanda que crece en forma sostenida. La industrialización de la uva para la obtención de jugos concentrados presenta varias etapas de procesamiento que incluyen tratamientos térmicos que afectan la microbiota presente en la materia prima. A pesar de esto, los productos no están exentos de presentar problemas microbiológicos que deterioran la calidad del mismo. El JUC es un alimento de humedad intermedia (aw 0,7-0,8), con elevada concentración de azúcares y bajo pH. La alteración de estos sustratos es causada por levaduras osmófilas, dentro de este grupo el género que se aísla con mayor frecuencia es Zygosaccharomyces sp. El objetivo del presente trabajo fue la identificación de puntos críticos de contaminación con levaduras osmófilas en plantas elaboradoras, identificando las especies presentes en los jugos de uva y las superficies asociadas a su concentrado. El conocimiento de los puntos críticos de contaminación permitiría la aplicación de medidas preventivas para aumentar la estabilidad microbiana de los JUC. Para ello se eligieron tres plantas concentradoras de jugo de uva y se muestrearon los jugos de uva pre-concentrados y concentrados y las superficies asociadas a su elaboración. Se realizó el recuento de levaduras osmófilas en el medio MY50G y la posterior identificación molecular de las levaduras presentes en todas las muestras mediante secuenciación del fragmento amplificado ITS1-5.8S-ITS2. Los resultados mostraron que Z. rouxii fue la especie encontrada en todas las muestras de jugo de uva pre-concentrado y concentrado y en la mayoría de los casos representó el 100% de las levaduras aisladas. También se evidenció que los períodos de almacenamiento del jugo de uva pre-concentrado y concentrado fueron claves para que la población de Z. rouxii aumentara. Por lo cual constituyen puntos críticos en la elaboración y deberán ser cuidadosamente controlados para evitar el deterioro del producto. Por otro lado, se concluyó que en las superficies limpias, antes que entren en contacto con los jugos de uva pre-concentrados o concentrados, no hubo incidencia de Z. rouxii. Este hecho sugiere que las prácticas sanitarias utilizadas en las tres plantas serían capaces de eliminar las poblaciones de Z. rouxii de las superficies, siempre y cuando los restos de mosto sean completamente removidos de todas las áreas en contacto con el producto. Siete especies de levaduras fueron identificadas en las superficies: Wickerhamomyces anomalus, Torulaspora delbrueckii, Citeromyces matritensis, Lachancea thermotolerans, Metschnikowia pulcherrima, Candida orthopsilosis y Candida apícola. El hallazgo de estas especies osmotolerantes, sugiere que las mismas presentan características que les permitieron persistir en las superficies higienizadas, los recuentos obtenidos fueron en muchos casos muy elevados. Estas especies han sido descritas como asociadas a los ambientes de las plantas elaboradoras de productos azucarados pero no han sido clasificadas como alterantes del producto per se.

Is there a common water-activity limit for the three domains of life?

Archaea and Bacteria constitute a majority of life systems on Earth but have long been considered inferior to Eukarya in terms of solute tolerance. Whereas the most halophilic prokaryotes are known for an ability to multiply at saturated NaCl (water activity (a_w) 0.755) some xerophilic fungi can germinate, usually at high-sugar concentrations, at values as low as 0.650–0.605 a_w. Here, we present evidence that halophilic prokayotes can grow down to water activities of <0.755 for Halanaerobium lacusrosei (0.748), Halobacterium strain 004.1 (0.728), Halobacterium sp. NRC-1 and Halococcus morrhuae (0.717), Haloquadratum walsbyi (0.709), Halococcus salifodinae (0.693), Halobacterium noricense (0.687), Natrinema pallidum (0.681) and haloarchaeal strains GN-2 and GN-5 (0.635 a_w). Furthermore, extrapolation of growth curves (prone to giving conservative estimates) indicated theoretical minima down to 0.611 a_w for extreme, obligately halophilic Archaea and Bacteria. These were compared with minima for the most solute-tolerant Bacteria in high-sugar (or other non-saline) media (Mycobacterium spp., Tetragenococcus halophilus, Saccharibacter floricola, Staphylococcus aureus and so on) and eukaryotic microbes in saline (Wallemia spp., Basipetospora halophila, Dunaliella spp. and so on) and high-sugar substrates (for example, Xeromyces bisporus, Zygosaccharomyces rouxii, Aspergillus and Eurotium spp.). We also manipulated the balance of chaotropic and kosmotropic stressors for the extreme, xerophilic fungi Aspergillus penicilloides and X. bisporus and, via this approach, their established water-activity limits for mycelial growth (~0.65) were reduced to 0.640. Furthermore, extrapolations indicated theoretical limits of 0.632 and 0.636 a_w for A. penicilloides and X. bisporus, respectively. Collectively, these findings suggest that there is a common water-activity limit that is determined by physicochemical constraints for the three domains of life.

Cuéntame: análisis de imagen aplicado al crecimiento de colonias de levadura

Algunas levaduras son capaces de producir deterioro en alimentos desarrollándose en su superficie como colonias. La medida del crecimiento de éstas evaluando el aumento de células viables es una técnica laboriosa y tediosa, mientras que la medida del aumento de su radio proporciona un resultado inmediato. En este trabajo, como alternativa a la medición manual del radio de la colonia, se plantea el empleo de técnicas de análisis de imagen que permiten automatizar el proceso de medición. A partir de las imágenes escaladas digitales, adquiridas en escala de gris de las colonias en crecimiento se ha desarrollado un algoritmo de análisis de imagen con el software MATLAB®. Esta herramienta se ha utilizado para procesar diariamente las imágenes de colonias de cuatro especies de levaduras deteriorantes: Zygosaccharomyces rouxii, Debaryomyces hansenii, Saccharomyces cerevisiae y Rhodotorula glutinis. El error de predicción del tamaño de la colonia al aplicar el algoritmo es comparable con el cometido en la medición manual, no superando en ambos casos el 3-4% y obteniéndose un ajuste medio (R2) entre ambas mediciones de 0.99, ajuste consistente e independiente de la especie de levadura estudiada. La observación de que el crecimiento bifásico del radio está correlacionado con las fases de aumento de células viables hace de este algoritmo una excelente herramienta.

Utilización del análisis hiperespectral como método para la detección de levaduras en alimentos deteriorados

La microbiología predictiva desarrolla modelos matemáticos que describen el comportamiento microbiano en alimentos. Su eficiencia es limita da en alimentos con algún grado de estructura, que son la mayoría, porque estos modelos están basados en datos experimentales obtenidos en cultivos líquidos, mucho más homogéneos. En este trabajo se evalúa la técnica de análisis hiperespectral como una nueva tecnología para la detección precoz de levaduras en alimentos sólidos mediante análisis de componentes principales. Las imágenes hiperespectrales (400-1000 nm) de placas de Petri inoculadas con Zygosaccharomyces rouxii Bch a diferentes tiempos, muestran que el primer componente discrimina entre el agar y las colonias, mientras que el segundo revela una mayor variabilidad en las colonias. Esta herramienta no invasiva caracteriza el estado de desarrollo, número y tamaño de las colonias, por lo que permitiría obtener datos para el aborar modelos más fiables para predecir el riesgo de deterioro y la vida útil de los alimentos.

A simple mathematical model that describes the growth of the area and the number of total and viable cells in yeast colonies

We propose a model, based on the Gompertz equation, to describe the growth of yeasts colonies on agar medium. This model presents several advantages: (i) one equation describes the colony growth, which previously needed two separate ones (linear increase of radius and of the squared radius); (ii) a similar equation can be applied to total and viable cells, colony area or colony radius, because the number of total cells in mature colonies is proportional to their area; and (iii) its parameters estimate the cell yield, the cell concentration that triggers growth limitation and the effect of this limitation on the specific growth rate. To elaborate the model, area, total and viable cells of 600 colonies of Saccharomyces cerevisiae, Debaryomyces fabryi, Zygosaccharomyces rouxii and Rhodotorula glutinis have been measured. With low inocula, viable cells showed an initial short exponential phase when colonies were not visible. This phase was shortened with higher inocula. In visible or mature colonies, cell growth displayed Gompertz-type kinetics. It was concluded that the cells growth in colonies is similar to liquid cultures only during the first hours, the rest of the time they grow, with near-zero specific growth rates, at least for 3 weeks.

Pesquisa de antifúngicos de origem vegetal

Dissertação mest., Engenharia Biológica, Universidade do Algarve, 2009

Yeast diversity in the Mediterranean strawberry tree (Arbutus unedo L.) fruits' fermentations

In the Mediterranean region the fruits of the strawberry tree (Arbutus unedo L.) may be fermented and distilled to produce a traditional beverage very much appreciated in Southern Europe. The aim of the present work was to study the diversity of the yeast population and the killer activity of the isolates identified as Saccharomyces cerevisiae, obtained during solid state industrial fermentations of the arbutus berries. The identification of the isolates was performed by the 5.8S rRNA-ITS region restriction analysis and by sequencing the D1/D2 region of the large subunit of the rRNA gene. At the start of the fermentations, various non-Saccharomyces species were detected including Aureobasidium pullulans, Dothichiza pithyophila, Dioszegia zsoltii, Hanseniaspora uvarum and yeasts belonging to the genera Metschnikowia, Cryptococcus and Rhodotorula. However, as the biological processes progressed the number of different species decreased with S. cerevisiae and Pichia membranaefaciens becoming dominant at advanced stages of the must fermentation that is characterized by high concentrations of ethanol. Forty three isolates identified as S. cerevisiae were tested for killer activity against two sensitive reference strains and Zygosaccharomyces bailii. Their killer sensitivity in relation to five killer referenced toxins (K2, K5, K8, K9 and K10) was also studied. Out of the isolates analyzed, 95.3% were sensitive and 4.7% were tolerant against the killer toxins tested. Only three isolates revealed killer activity against one sensitive strain and two of them against the spoiler yeast Z. bailii. The microbiota obtained revealed an interesting potential to be used as starter cultures to overcome unpredictable uncontrolled fermentations of the arbutus fruits as well as in other applications of biotechnological interest. (C) 2012 Elsevier Ltd. All rights reserved.

Screening of filamentous fungi for production of enzymes of biotechnological interest

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Yeast biodiversity from oleic ecosystems: Study of their biotechnological properties

The aim of this study was to know the yeast biodiversity from fresh olive (Olea europaea L.) fruits, olive paste (crush olives) and olive pomace (solid waste) from Arbequina and Cornicabra varieties. Yeasts were isolated from fruits randomly harvested at various olive groves in the region of Castilla La Mancha (Spain). Olive paste and pomace, a byproduct of the processing of this raw material, were also collected in sterile flasks from different oil mills. Molecular identification methodology used included comparison of polymerase chain reaction (PCR) amplicons of their 5.8S rRNA gene and internal transcribed spacers ITS1 and ITS2 followed by restriction pattern analysis (RFLP). For some species, sequence analysis of the 5.8S rDNA gene was necessary. The results were compared to sequences held in public databases (BLAST). These techniques allowed to identify fourteen different species of yeasts, belonging to seven different genera (Zygosaccharomyces, Pichia, Lachancea, Kluyveromyces, Saccharomyces, Candida, Torulaspora) from the 108 yeast isolates. Species diversity was thus considerable: Pichia caribbica, Zygosaccharomyces fermentati (Lachancea fermentati) and Pichia holstii (Nakazawaea holstii) were the most commonly isolated species, followed by Pichia mississippiensis, Lachancea sp., Kluyveromyces thermotolerans and Saccharomyces rosinii. The biotechnological properties of these isolates, was also studied. For this purpose, the activity of various enzymes (beta-glucosidase, beta-glucanase, carboxymethylcellulase, polygalacturonase, peroxidase and lipase) was evaluated. It was important that none of species showed lipase activity, a few had cellulase and polygalacturonase activities and the majority of them presented beta-glucanase, beta-glucosidase and peroxidase activities. (C) 2010 Elsevier Ltd. All rights reserved.

Isolamento de leveduras em indústria de refrigerante e avaliação da susceptibilidade à ação antimicrobiana dos agentes sanificantes de uso industrial

Pós-graduação em Microbiologia - IBILCE

Assessment of the stereoselective fungal biotransformation of albendazole and its analysis by HPLC in polar organic mode

A high-performance liquid chromatographic method using polar organic mode was developed to analyze albendazole (ABZ), albendazole sulfone (ABZSO(2)) and the chiral and active metabolite albendazole sulfoxide (ABZSOX, ricobendazole) that was further applied in stereoselective fungal biotransformation studies. The chromatographic separation was performed on a Chiralpak AS column using acetonitrile:ethanol (97:3, v/v) plus 0.2% triethylamine and 0.2% acetic acid as the mobile phase at a flow rate of 0.5 mL min(-1). The present study employed hollow fiber liquid-phase microextraction as sample preparation. The method showed to be linear over the concentration range of 25-5000 ng mL(-1) for each ABZSOX enantiomer, 200-10,000 ng mL(-1) for ABZ and 50-1000 ng mL(-1) for ABZSO(2) metabolite with correlation coefficient (r)> 0.9934. The mean recoveries for ABZ, rac-ABZSOX and ABZSO(2) were, respectively, 9%, 33% and 20% with relative standard deviation below 10%. Within-day and between-day precision and accuracy assays for these analytes were studied at three concentration levels and were lower than 15%. This study opens the door regarding the possibility of using fungi in obtaining of the active metabolite ricobendazole. Nigrospora sphaerica (Sacc.) E. W. Mason (5567), Pestalotiopsis foedans (VR8), Papulaspora immersa Hotson (SS13) and Mucor rouxii were able to stereoselectively metabolize ABZ into its chiral metabolite. Among them, the fungus Mucor rouxii was the most efficient in the production of (+)-ABZSOX. (C) 2011 Elsevier B.V. All rights reserved.

Stepwise functional evolution in the Ffz sugar transporter family

Abstract of the poster presented at the Microbiotec’15 Congress, 10-12 December 2015, University of Évora, Portugal.

Studio di termoresistenza di lieviti fermentativi

In anni recenti il potenziale degradativo dei lieviti nelle bevande ha assunto un’importanza crescente a causa degli importanti danni economici ad essi associati e i produttori sono sempre più attenti a queste problematiche, anche in relazione alla tendenza di ridurre i trattamenti termici, in una strategia di risparmio energetico e di riduzione del danno termico organolettico. L’obiettivo dell’elaborato è stato quello di testare la termoresistenza di diversi ceppi di lieviti che possono costituire un rischio per bevande di fantasia: sette appartenenti alla specie Saccharomyces cerevisiae, tre Zygosaccharomyces, un ceppo di Saccharomycodes ludwigii, uno di Schizosaccharomyces pombe e uno di Kluyveromyces marxianus. Le prove di termoresistenza sono state condotte in vitro utilizzando terreno di coltura impiegando temperature di 55°C, 60°C e 65°C e i dati sono stati modellati con l’equazione di Weibull per ottenere le curve di morte termica dei diversi ceppi. Questo modello permette di tenere conto dell’approccio vitalistico dell’interazione tra temperatura e cellula e descrive l’inattivazione microbica come risultato del fallimento delle cellule nel contrastare le condizioni ostili dovute all’alta temperatura. I risultati hanno dimostrato che i ceppi erano caratterizzati da termoresistenza molto variabile. In generale, tra le specie non Saccharomyces, i più resistenti erano K. marxianus e Sch. Pombe mentre tra i Saccharomyces il più resistente era il ceppo SPA, isolato in uno stabilimento di bevande e responsabile di un incidente industriale che ha portato all’alterazione di oltre 500000 bottiglie di bibite gassate. Questo screening preliminare per la valutazione della termoresistenza ad un potenziale trattamento industriale ha permesso di individuare i lieviti più resistenti che saranno poi oggetto di un successivo studio in cui verranno tenuti presenti altri fattori tecnologici e di prodotto per poi potersi muovere in un ambiente industriale.

Avaliação microbiológica de vinho engarrafado

Mestrado em Engenharia Alimentar - Instituto Superior de Agronomia - UL