Production and immobilization of pectinases from Penicillium crustosum in magnetic core-shell nanostructures for juice clarification.

Global market of food enzymes is held by pectinases, mostly sourced from filamentous fungi via submerged fermentation. Given the one-time use nature of enzymes to clarify juices and wines, there is a crucial need to explore alternatives for enzyme immobilization, enabling their reuse in food applications. In this research, an isolated fungal strain (Penicillium crustosum OR889307) was evaluated as a new potential pectinase producer in submerged fermentation. Additionally, the enzyme was immobilized in magnetic core-shell nanostructures for juice clarification. Findings revealed that Penicillium crustosum exhibited enzymatic activities higher than other Penicillium species, and pectinase production was enhanced with lemon peel as a cosubstrate in submerged fermentation. The enzyme production (548.93 U/mL) was optimized by response surface methodology, determining the optimal conditions at 35 °C and pH 6.0. Subsequently, the enzyme was covalently immobilized on synthesized magnetic core-shell nanoparticles. The immobilized enzyme exhibited superior stability at higher temperatures (50 °C) and acidic conditions (pH 4.5). Finally, the immobilized pectinases decreased 30 % the orange juice turbidity and maintained 84 % of the enzymatic activity after five consecutive cycles. In conclusion, Penicillium crustosum is a proven pectinase producer and these enzymes immobilized on functionalized nanoparticles improve the stability and reusability of pectinase for juice clarification.

Microbial communities present in Sargassum spp. leachates from the Mexican Caribbean which are involved in their degradation in the environment, a tool to tackle the problem.

The Sargassum phenomenon is currently affecting the Caribbean in several ways; one of them is the increase of greenhouse gases due to the decomposition process of this macroalgae; these processes also produce large amounts of pollutant leachates, in which several microbial communities are involved. To understand these processes, we conducted a 150-day study on the Sargassum spp environmental degradation under outdoor conditions, during which leachates were collected at 0, 30, 90, and 150 days. Subsequently, a metagenomic study of the microorganisms found in the leachates was carried out, in which changes in the microbial community were observed over time. The results showed that anaerobic bacterial genera such as Thermofilum and Methanopyrus were predominant at the beginning of this study (0 and 30 days), degrading sugars of sulfur polymers such as fucoidan, but throughout the experiment, the microbial communities were changed also, with the genera Fischerella and Dolichospermum being the most predominant at days 90 and 150, respectively. A principal component analysis (PCA) indicated, with 94% variance, that genera were positively correlated at 30 and 90 days, but not with initial populations, indicating changes in community structure due to sargassum degradation were present. Finally, at 150 days, the leachate volume decreased by almost 50% and there was a higher abundance of the genera Desulfobacter and Dolichospemum. This is the first work carried out to understand the degradation of Sargassum spp, which will serve, together with other works, to understand and provide a solution to this serious environmental problem in the Caribbean.

Metagenomic analysis and antimicrobial activity of two fermented milk kefir samples.

In recent years, the fermented milk product kefir has been intensively studied because of its health benefits. Here, we evaluated the microbial consortia of two kefir samples, from Escarcega, Campeche, and Campeche (México). We considered a functional comparison between both samples, including fungal and bacterial inhibition; second, we applied shotgun metagenomics to assess the structure and functional diversity of the communities of microorganisms. These two samples exhibited antagonisms against bacterial and fungal pathogens. Bioactive polyketides and nonribosomal peptides were identified by LC-HRMS analysis. We also observed a high bacterial diversity and an abundance of Actinobacteria in both kefir samples, and a greater abundance of Saccharomyces species in kefir of Escarcega than in the Campeche kefir. When the prophage compositions were evaluated, the Campeche sample showed a higher diversity of prophage sequences. In Escarcega, we observed a prevalence of prophage families that infect Enterobacteria and Lactobacillus. The sequences associated with secondary metabolites, such as plipastatin, fengycin, and bacillaene, and also bacteriocins like helveticin and zoocin, were also found in different proportions, with greater diversity in the Escarcega sample. The analyses described in this work open the opportunity to understand the microbial diversity in kefir samples from two distant localities.

Decolorization of Textile Effluent by Trametes hirsuta Bm-2 and lac-T as Possible Main Laccase-Contributing Gene.

The decolorization of dye and textile effluent by Trametes hirsuta was studied in both induced and non-induced media. A removal of 70-100% of the color was achieved through adsorption and the action of laccases. Laccase activity was increased significantly with the addition of grapefruit peel (4000 U/mL) and effluent with grapefruit peel (16,000 U/mL) in comparison with the basal medium (50 U/mL). Analysis of the expression of laccase isoenzymes lac-B and lac-T revealed clear differences in the expression of these genes. The low levels of expression of lac-B in all media suggest a basal or constitutive gene expression, whereas lac-T was over-expressed in the media with effluent, and showed an up/down regulation depending on culture conditions and time. The results obtained suggest that the lac-T gene of T. hirsuta is involved in the decolorization of dyes.

Molecular characterization of laccase genes from the basidiomycete Trametes hirsuta Bm-2 and analysis of the 5' untranslated region (5'UTR).

The aim of this study was to identify and characterize laccase genes produced by Trametes hirsuta Bm-2 in a liquid medium, both with and without induction. The amplification of 5'and 3'regions of laccase sequences was obtained by the RACE-PCR method, and these were assembled to obtain a cDNA of total length. Two new laccase genes were isolated from basal medium (lac-B) and lignocellulosic grapefruit substrate (lac-T), both encoding open reading frames of 2566 bp. Both laccase-predicted proteins consisted of 521 amino acids, four copper-binding regions, a signal peptide, and five potential glycosilation sites (Asn-Xaa-Ser/Tre). Moreover, the deduced amino acid sequences share about 76-85% identity with other laccases of WRF. Sequence comparison showed 47 synonymous point mutations between lac-B and lac-T. In addition, 5' untranslated regions (UTR) of laccase genes lac-B and lac-T showed differences in length and number of regulatory elements that may affect transcriptional or translational expression of these genes.

Inhibición del crecimiento y modificación genética de Phytophthora capsici usando quitosano de bajo grado de polimerización / Growth inhibition and genetic modification of Phytophthora capsici using chitosan with low degree of polymerization

Phytophtora capsici es un patógeno que incide sobre cultivos de la familia de las solanáceas causando pérdidas económicas en cultivos de pimientos, tomates, berenjenas y cur-cubitáceas. En este trabajo evaluamos el efecto del quitosano de bajo grado de polimerización (QBP) sobre el crecimiento de P. capsici y sobre la regulación génica de este fitopatógeno a nivel transcripcional. A una concentración de 0,4mg/l de QBP se obtuvo un 88% de inhibición en el crecimiento; concentraciones superiores a 1,6 mg/l inhibieron el crecimiento en un 100%. Mediante ensayos de cambio en la movilidad electroforética de ácidos nucleicos se comprobó que el quitosano interactúa con el ADN y el ARN del hongo frente a concentraciones entre 2 y 4 mg/l de ADN y entre 0,5 y 3 mg/l de ARN. Además, se efectuó un análisis de despliegue diferencial de los productos de amplificación por RT-PCR de los ARN mensajeros de P. capsici obtenidos en presencia o ausencia de QBP; este mostró cambios en el perfil de expresión inducidos por el tratamiento con quitosano. El análisis bioinformático de las secuencias de los transcritos expresados diferencialmente sugiere que el QBP afectó la regulación génica de elementos involucrados en la síntesis de quitina y de proteínas de unión a hidratos de carbono.

Phytophthora blight of peppers, caused by oomycete Phytophthora capsici, currently causes economic losses in crops such as peppers, tomatoes, eggplant and cucurbits. In this work, we evaluated the effect of chitosan with low degree of polymerization (LDP) on growth and gene expression of P. capsici cultures. LDP chitosan inhibited 88% of P. capsici mycelial growth at concentrations up to 0,4 mg/l, whereas at concentrations higher than 1,6 mg/l it completely inhibit growth. Gel mobility shift assays demonstrated that chitosan interacts with DNA and RNA of the fungus at concentrations ranging from 2 to 4 mg/l for DNA and 0,5 to 3 mg/l for RNA. The differential display analysis of RT-PCR-amplification products of P. capsici messenger RNA revealed changes in gene expression profiles after the chitosan treatment. Bioinformatic analysis of sequences from selected differentially-expressed bands showed the gene regulation of elements involved in chitin synthesis and carbohydrate-binding proteins.

[Growth inhibition and genetic modification of Phytophthora capsici using chitosan with low degree of polymerization]. / Inhibición del crecimiento y modificación genética de Phytophthora capsici usando quitosano de bajo grado de polimerización.

Phytophthora blight of peppers, caused by oomycete Phytophthora capsici, currently causes economic losses in crops such as peppers, tomatoes, eggplant and cucurbits. In this work, we evaluated the effect of chitosan with low degree of polymerization (LDP) on growth and gene expression of P. capsici cultures. LDP chitosan inhibited 88% of P. capsici mycelial growth at concentrations up to 0,4 mg/l, whereas at concentrations higher than 1,6 mg/l it completely inhibit growth. Gel mobility shift assays demonstrated that chitosan interacts with DNA and RNA of the fungus at concentrations ranging from 2 to 4mg/l for DNA and 0,5 to 3mg/l for RNA. The differential display analysis of RT-PCR-amplification products of P. capsici messenger RNA revealed changes in gene expression profiles after the chitosan treatment. Bioinformatic analysis of sequences from selected differentially-expressed bands showed the gene regulation of elements involved in chitin synthesis and carbohydrate-binding proteins.

Laccase-mediator system produced by Trametes hirsuta Bm-2 on lignocellulosic substrate improves dye decolorization.

Lignin is a source for obtaining natural phenols with high commercial value that can act as redox mediators enhancing effects in dye decolorization. In this study Trametes hirsuta Bm-2 was grown on wheat bran to produce laccases and phenol extracts (PE). Ultrafiltered phenols obtained at different times were evaluated in their potential as redox mediators of laccase activity and indigo carmin decolorization. Laccase activity (L) on ABTS increased up to 12.4 times with L/PE72 compared with laccase alone and L/PE48 showed the highest level of dye decolorization (97%) compared with laccase (12%). The chromatographic analysis by HPLC showed variation in the profile and concentration of phenols at different times of culture. Stability of the laccase mediator system (LMs) in dye decolorization was maintained over 3 months. Our results suggest the use of natural mediators as a strategy for improving efficiency in dye biodegradation by laccase-producing fungi.

Laccase Gene Expression and Vinasse Biodegradation by Trametes hirsuta Strain Bm-2.

Vinasse is the dark-colored wastewater that is generated by bioethanol distilleries from feedstock molasses. The vinasse that is generated from molasses contains high amounts of pollutants, including phenolic compounds and melanoindin. The goal of this work was to study the expression of laccase genes in the Trametes hirsuta strain Bm-2, isolated in Yucatan, Mexico, in the presence of phenolic compounds, as well as its effectiveness in removing colorants from vinasse. In the presence of all phenolic compounds tested (guaiacol, ferulic acid, and vanillic acid), increased levels of laccase-encoding mRNA were observed. Transcript levels in the presence of guaiacol were 40 times higher than those in the control. The lcc1 and lcc2 genes of T. hirsuta were differentially expressed; guaiacol and vanillin induced the expression of both genes, whereas ferulic acid only induced the expression of lcc2. The discoloration of vinasse was concomitant with the increase in laccase activity. The highest value of enzyme activity (2543.7 U/mL) was obtained in 10% (v/v) vinasse, which corresponded to a 69.2% increase in discoloration. This study demonstrates the potential of the Bm-2 strain of T. hirsuta for the biodegradation of vinasse.