Capacidad antifúngica de biopelículas de quitosán conteniendo bacterias ácido lácticas / Antifungal ability of chitosan biofilms containing lactic acid bacteria

INTRODUCCIÓN: Existen reportes del uso de biopelículas como soporte para la incorporación de microorganismos benéficos, sin embargo, son pocos los reportes donde se evalúe la capacidad antimicrobiana de las biopelículas conteniendo bacterias ácido lácticas (BAL). OBJETIVO: Optimizar los componentes de una biopelícula comestible basada en quitosán para conservar la viabilidad y la capacidad antifúngica de la BAL Lactobacillus plantarum CDBB-B-1091 durante 28 días. MÉTODOS: Bajo un diseño de 8 tratamientos tipo Plackett-Burman se evaluaron dos niveles de 7 factores (glucosa, lactosa, glicerol, almidón, humedad relativa del ambiente, pH, concentración de BAL). De los factores (componentes) que mostraron efecto, se optimizó la concentración mediante la metodología de superficie de respuesta basada en un arreglo de Box-Benhken. RESULTADOS: Se encontró que la concentración de células (A), concentración de almidón (B) y concentración de glucosa (C) son los componentes de la biopelícula más determinantes para mantener la viabilidad y la capacidad antifúngica contra el hongo fitopatógeno Colletotrichum gloeosporioides. Mediante análisis de superficie de respuesta se obtuvieron los valores óptimos para mantener la viabilidad de las bacterias por 28 días, siendo los valores de 7,009164 log UFC/g película para el factor A, 1,997712% para B y 0,10750016 M para el factor C. De acuerdo al análisis de la varianza la concentración de células el factor más influyente. Sin embargo, para la capacidad antifúngica solamente fue posible obtener inhibición del 100% con películas recién elaboradas, siendo para este día los valores óptimos de 8,9004 log (UFC/g) para el factor A, 2,0% para B y 0,0850143 M para C. CONCLUSIÓN: La capacidad antifúngica de las biopelículas conteniendo BAL fue decreciendo a medida que transcurrió el almacenamiento de las biopelículas. Aún con lo anterior, se proponen los modelos de regresión para predecir los valores de viabilidad y la capacidad antifúngica de biopelículas conteniendo la bacteria Lactobacillus plantarum CDBB-B-1091

INTRODUCTION: There are reports of the use of biofilms as a support for the incorporation of beneficial microorganisms, however, there are scarce the reports where the antimicrobial capacity of biofilms containing lactic acid bacteria (LAB) is evaluated. OBJECTIVE: Optimize the components of an edible biofilm based on chitosan to preserve the viability and antifungal capacity of the LAB Lactobacillus plantarum CDBB-B-1091 for 28 days. METHODS: Through a design Plackett-Burman of 8 treatments, two levels of 7 factors (componente) were evaluated (glucose, lactose, glycerol, starch, relative humidity, pH, BAL concentration). Of the factors that showed effect, the concentration was optimized using the response surface methodology based on a Box-Benhken arrangement. RESULTS: It was found that cell concentration (A), starch concentration (B) and glucose concentration (C) are the most determining biofilm components to maintain viability and antifungal ability against the phytopathogenic fungus Colletotrichum gloeosporioides. Optimal values were obtained by response surface analysis to maintain the viability of the bacteria for 28 days, the values being 7.009164 log CFU/g film for factor A, 1.997712% for B and 0.10750016 M for factor C. According to ANOVA the concentration of cells being the most influential factor. However, for the antifungal capacity it was only possible to obtain 100% inhibition with freshly made films, for this day the optimal values of 8.9004 log (CFU/g) for factor A, 2.0% for B and 0.0850143 M for C. CONCLUSION: The antifungal capacity of the biofilms containing BAL was decreasing as the storage of the biofilms passed. Even with the above, regression models are proposed to predict the viability values and the antifungal capacity of biofilms containing the bacterium Lactobacillus plantarum CDBB-B-1091

The metabolism and genetic regulation of lipids in the oleaginous yeast Yarrowia lipolytica.

The biotechnological potential of Yarrowia lipolytica, as a single cell oil-producing microorganism, is presented in this review. Although initially this yeast species was considered as a lipid-degrading, recently, it was reclassified as a lipid-producing microorganism, since it has been reported to be capable of accumulating diverse desirable fatty acids after metabolic pathway engineering. In the first part of the present document, a general revision of the oil metabolic pathways and the capacity of oil production in Y. lipolytica is presented. The single cell oil produced by these metabolic engineering strategies has been designed by optimization, introduction, or suppression of new pathways to increase yield on lipid production. Later on, the genetic regulation systems and the lipid composition generated by this yeast for industrial purposes are discussed. These lipids could be safely used in the chemical food and biofuel industries, due to their high proportion of oleic acid. This document emphasizes in the overviewing at Y. lipolytica as an ideal oil cell factory, and as an excellent model to produce single cell oil.

Isolation and identification of marine strains of Stenotrophomona maltophilia with high chitinolytic activity.

Chitin is the second most abundant organic compound in nature and represents a rich carbon and nitrogen source that is primarily transformed by bacterial communities. Bacteria capable of gradually hydrolyzing chitin into N-acetylglucosamine monomers can have applications in the transformation of residues from shrimp and other crustaceans. The objective of the present study was to isolate, characterize and identify microorganisms with high chitinolytic activity. These microorganisms were isolated and characterized based on macro- and microscopic morphological traits. Strains were selected on colloidal chitin agar medium primarily based on a hydrolysis halo larger than 2 mm and a growing phase no longer than 6 days. Secondary selection consisted of semi-quantitative evaluation of chitinolytic activity with a drop dilution assay. From the above, ten strains were selected. Then, strain-specific activity was evaluated. The B4 strain showed the highest specific activity, which was 6,677.07 U/mg protein. Molecular identification indicated that the isolated strains belong to the species Stenotrophomonas maltophilia.

Characterization of Aloe vera-chitosan composite films and their use for reducing the disease caused by fungi in papaya Maradol.

Composite films with Aloe vera (A), chitosan (Ch) and essential oils (EOs) were formulated. Six of the twelve combinations tested formed films: A70Ch30, A70Ch30-15, A60Ch40, A60Ch40-15, A50Ch50, and A50Ch50-15. The A60Ch40-15 film showed the best physicochemical characteristics as well as the greatest in vitro antifungal activity. Although the A90Ch10 and A80Ch20-15 mixtures did not form films, their solutions showed high antifungal activity in vitro. Based on multivariate analysis of the data, A60Ch40-15, A90Ch10 and A80Ch20-15 films were selected as coating treatments for papaya during storage at 30 ± 2 °C and 80% RH. Uncoated fruits (control 1) and treated with synthetic fungicide (control 2) were used as control. Coated fruits showed lower respiration rate, greater firmness and fewer changes in external coloration compared to control. Furthermore, these coatings reduced the incidence and severity of fungal disease by 40-50% compared to control 2. Aloe vera-chitosan films (A90Ch10 and A60Ch40-15), enriched with the EOs of cinnamon (10 mL L-1) and thyme (10 mL L-1), improved quality of the fruit (higher firmness, lower CO2 content, less internal color change) with 50% less disease incidence during storage at room temperature.

The UmGcn5 gene encoding histone acetyltransferase from Ustilago maydis is involved in dimorphism and virulence.

We isolated a gene encoding a histone acetyltransferase from Ustilago maydis (DC.) Cda., which is orthologous to the Saccharomyces cerevisiae GCN5 gene. The gene was isolated from genomic clones identified by their specific hybridization to a gene fragment obtained by the polymerase chain reaction (PCR). This gene (Umgcn5; um05168) contains an open reading frame (ORF) of 1421bp that encodes a putative protein of 473 amino acids with a Mr. of 52.6kDa. The protein exhibits a high degree of homology with histone acetyltransferases from different organisms. Null a2b2 ΔUmgcn5 mutants were constructed by substitution of the region encoding the catalytic site with a hygromycin B resistance cassette. Null a1b1 ΔUmgcn5 mutants were isolated from genetic crosses of a2b2 ΔUmgcn5 and a1b1 wild-type strains in maize. Mutants displayed a slight reduction in growth rate under different conditions, and were more sensitive than the wild type to stress conditions, but more important, they grew as long mycelial cells, and formed fuzz-like colonies under all conditions where wild-type strains grew in the yeast-like morphology and formed smooth colonies. This phenotype was not reverted by cAMP addition. Mutants were not virulent to maize plants, and were unable to form teliospores. These phenotypic alterations of the mutants were reverted by their transformation with the wild-type gene.

Functional analysis of the MATB mating-type idiomorph of the dimorphic fungus Yarrowia lipolytica.

The whole MATA cassette from Yarrowia lipolytica, a dimorphic fungus, was replaced by the URA3 gene through a double homologous recombination. This MAT-less strain lost its mate capacity with A or B Y. lipolytica strains. Introduction of polymerase chain reaction-synthesized idiomorph MATB in a null strain of A locus by double homologous recombination gave rise to a "transsexual" B strain. Mating capacity of this engineered mutant was assayed using Y. lipolytica strains of either A or B mating type. Mating took place only with an A strain, demonstrating the MATB idiomorph functionality in a MATA phenotype. Our data suggest that specific downstream genes are responsible for the final A or B phenotypes present in all Y. lipolytica cells, independent of their MAT idiomorph phenotype.

Isolation and molecular analysis of Umhda2 a gene encoding a histone deacetylase from Ustilago maydis.

By use of the polymerase chain reaction and synthetic oligonucleotides designed from conserved regions, we amplified a fragment of a gene from Ustilago maydis encoding a putative histone deacetylase. With this probe we isolated the full gene from a minigenomic library. The gene (designated as Umhda2) contains an open reading frame (ORF) of 1701bp encoding a protein of 566 amino acids. Multiple comparison analysis with other histone deacetylases suggests that the Umhda2 gene product belongs to the Rpd3-related family of proteins. The highest degree of homology with histone deacetylases from other organisms corresponded to Hdalp of Schizosaccharomyces pombe and Rpd3p of Saccharomyces cerevisiae with 64.2 and 62.2% of sequence similarity, respectively. It displayed a substantially lower similarity with another histone deacetylase from U. maydis (Hdalp, 52.4%). Semi-quantitative RTPCR results indicate that the gene is transcriptionally up-regulated during the in vitro yeast-to-mycelium dimorphic transition.

Loss of virulence in Ustilago maydis by Umchs6 gene disruption.

A gene encoding a sixth chitin synthase (Umchs6, sequence GenBank accession No. ) from the plant pathogenic hemibasidiomycete Ustilago maydis (DC.) Cda. was isolated and characterized. The predicted protein is 1103 amino acids in length with a calculated molecular mass of 123.5 kDa. a2b2 null mutants were obtained by substitution of a central fragment of the Umchs6 gene with the hygromycin resistance cassette, and a1b1 null mutants were obtained by genetic recombination in plants of an a2b2deltach6 and a wild-type a1b1 strain. The mutation had no effect on the dimorphic transition in vitro or on mating, and growth rate of the mutants was only slightly reduced. On the other hand, they displayed important alterations in cell morphology, particularly at the mycelial stage, and in the staining pattern with calcofluor white. Levels of chitin synthase activity in vitro and chitin content were reduced. The most noticeable characteristic of the mutants was their almost complete loss of virulence to maize (Zea mays L.). This was a recessive character. Microscopic observations during the infectious process suggest that chitin synthase 6 activity is very important for growth of the fungus into the plant. Transformation of a2b2deltach6 mutants with an autonomous replicating plasmid carrying the full Umchs6 gene restored their normal morphological phenotype and virulence. These results are evidence that the mutation in the Umchs6 gene was solely responsible for the phenotypic alterations observed.