Supercritical CO2 extraction of secondary metabolites from Agaricus blazei: experiments and modelling

The mycelium and young fruiting bodies of Agaricus blazei were submitted to supercritical CO2 extraction, in a modified commercial flow apparatus, at temperatures from 40 to 80 ºC, pressures up to 600 bar and CO2 flow-rates from 2.0 to 9.0 g.min-1. The best extraction conditions of secondary metabolites, whereby the degree of solubilization (g extract/100 g of fungi) is the highest, was obtained with pure CO2 at 400 bar, 70 ºC and a CO2 flow rate of 5.7g.min-1. The extract in that conditions were analysed by GC-Ms. In order to increase the extraction yield of secondary metabolites, which are mostly present in glycolipid fractions, a polar compound (ethanol) was used as co-solvent in the proportions of 5 and 10 % (mol/mol). The presence of ethanol increased the yield when compared with the extraction with pure CO2. Moreover, a simple model was applied to the supercritical CO2 extraction of secondary metabolites from Agaricus blazei.

Methyl syringate: An efficient phenolic mediator for bacterial and fungal laccases

The aim of the present work is to provide insight into the mechanism of laccase reactions using syringyl-type mediators. We studied the pH dependence and the kinetics of oxidation of syringyl-type phenolics using the low CotA and the high redox potential TvL laccases. Additionally, the efficiency of these compounds as redox mediators for the oxidation of non-phenolic lignin units was tested at different pH values and increasing mediator/non-phenolic ratios. Finally, the intermediates and products of reactions were identified by LC-MS and H-1 NMR. These approaches allow concluding on the (1) mechanism involved in the oxidation of phenolics by bacterial laccases, (2) importance of the chemical nature and properties of phenolic mediators, (3) apparent independence of the enzyme's properties on the yields of non-phenolics conversion, (4) competitive routes involved in the catalytic cycle of the laccase-mediator system with several new C-O coupling type structures being proposed.

Essential oils of Origanum vulgare L. subsp glandulosum (Desf.) letswaart from Tunisia: chemical composition and antioxidant activity

BACKGROUND: Characterisation of the essential oils from O. glandulosum collected in three locations of Tunisia, chemical composition and the evaluation of their antioxidant activities were carried out. RESULTS: The essential oils from Origanum vulgare L. subsp. glandulosum (Desf.) letswaart collected from three localities of north Tunisia - Krib, Bargou and Nefza - were obtained in yields of 2.5, 3.0 and 4.6% (v/w), respectively. The essential oils were analysed by GC and GC/MS and assayed for their total phenolics content, by the Folin-Ciocalteu method, and antioxidant effectiveness, using the 2,2-diphenyl-1-picrylhydrazil (DPPH) radical scavenging assay. The main components of these essential oils, from Nefza, Bargou and Krib, were p-cymene (36%, 40% and 46%), thymol (32%, 39% and 18%), gamma-terpinene (24%, 12% and 16%) and carvacrol (2%, 2% and 15%), respectively). The ability to scavenge the DPPH radicals, expressed by IC50, ranged from 59 to 80 mg L-1. The total phenolic content, expressed in gallic acid equivalent (GAE) g kg(-1) dry weight, varied from 9.37 to 17.70 g kg(-1) dw. CONCLUSIONS: A correlation was identified between the total phenolic content of the essential oils and DPPH radical scavenger capacity. The occurrence of a p-cymene chemotype of O. glandulosum in the northern region of Tunisia is demonstrated.

Are air-borne mycotoxins a public health concern in Portugal?

Introduction - Microscopic filamentous fungi, under suitable environmental conditions, can lead to the production of highly toxic chemical substances, commonly known as mycotoxins. The most widespread and studied mycotoxins are metabolites of some genera of moulds such as Aspergillus, Penicillium and Fusarium. Quite peculiar conditions may influence mycotoxin biosynthesis, such as climate, geographical location, cultivation practices, storage and type of substrate. Toxicity has been extensively investigated for the most important mycotoxins, such as aflatoxins, ochratoxin A and Fusarium toxins, and much information derived from toxicokinetics in animal models has also been obtained. The adverse effects are mainly related to genotoxicity, carcinogenicity, mutagenicity, teratogenicity and immunotoxicity. Aim of the study - To identify fungal species able to produce important mycotoxins in different Portuguese settings.

Fungal contamination of poultries litter: a public health problem

Exposure to certain fungi can cause human illness. Fungi cause adverse human health effects through three specific mechanisms: generation of a harmful immune response (e.g., allergy or hypersensitivity pneumonitis); direct infection by the fungal organism; by toxic-irritant effects from mold byproducts, such as mycotoxins. In Portugal there is an increasingly industry of large facilities that produce whole chickens for domestic consumption and only few investigations have reported on fungal contamination of the poultry litter. The material used for poultry litter is varied but normally can be constitute by: pine shavings; sawdust of eucalyptus; other types of wood; peanut; coffee; sugar cane; straw; hay; grass; paper processed. Litter is one of the most contributive factors to fungal contamination in poultries. Spreading litter is one of the tasks that normally involve higher exposure of the poultry workers to dust, fungi and their metabolites, such as VOC’s and mycotoxins. After being used and removed from poultries, litter is ploughed into agricultural soils, being this practice potentially dangerous for the soil environment, as well for both humans and animals. The goal of this study was to characterize litter’s fungal contamination and also to report the incidence of keratinophilic and toxigenic fungi.

Exposure to microbial volatile organic compounds in a waste-handling unit

The production of MVOC by fungi has been taken into account especially from the viewpoint of indoor pollution with microorganisms but the relevance of fungal metabolites in working environments has not been sufficiently studied. The purpose of this study was to assess exposure to MVOCs in a waste-handling unit. It was used Multirae equipment (RAE Systems) to measured MVOCs concentration with a 10.6 eV lamps. The measurements were done near workers nose and during the normal activities. All measurements were done continuously and had the duration of 5 minutes at least. It was consider the higher value obtained in each measurement. In addition, for knowing fungi contamination, five air samples of 50 litres were collected through impaction method at 140 L/minute, at one meter tall, on to malt extract agar with the antibiotic chloramphenicol (MEA). MVOCs results range between 4.7 ppm and 8.9 ppm in the 6 locations consider. These results are eight times higher than normally obtained in indoor settings. Considering fungi results, two species were identified in air, being the genera Penicillium found in all the samples in uncountable colonies and Rhizopus only in one sample (40 UFC/m3). These fungi are known as MVOCs producers, namely terpenoids, ketones, alcohols and others. Until now, there has been no evidence that MVOCs are toxicologically relevant, but further epidemiological research is necessary to elucidate their role on human’s health, particularly in occupational settings where microbiological contamination is common. Additionally, further research should concentrate on quantitative analyses of specific MVOCs.

Occupational exposure to Aflatoxin B1 in Portuguese swine farms

Aflatoxins were first isolated about 40 years ago afier outbreaks of disease and death in turkeys and cancer in rainbow trout fed with rations formulated from peanut and cottonseed meals. These toxins are secondary metabolites produced under certain conditions of temperature, p14 and humidity predominantiy by Aspergilius flavus and Aspergilius parasiticus fungi species. Among 18 different types of aflatoxins identified, major members are aflatoxin B1, B2, G1 and G2. Aflatoxin B1 (AFB1) is normaily predominant in cultures as well as in food products. AFB1 was shown to be genotoxic and a potent hepatocarcinogen. This mycotoxin is metabolized by the mixed function oxidase system to a number of hydroxylated metabolites including the 8,9-epoxide. The latter is considered to be the ultimate carcinogen that reacts with cellular deoxyribonucleic acid (DNA) and proteins to form covalent adducts.

Occupational exposure to toxigenic Aspergillus versicolor in Portuguese swines

Biological factors associated with airbome dust are the most important hazards in pig buildings and include allergenic and/or toxic compounds, as well as infectious agents such as fungi and their metabolites, like mycotoxins. Inhalation of such agents can be a potential occupationai treat. Exposure of workers from swine confinement buildings to respiratory hazards has been reported elsewhere in Europe, Asia and America. Analogous data has not been reported for Portugal and this omission has hindered the development of policies in the area of occupational health and farm safety. Aspergilius versicolor is known as being the major producer of the hepatotoxic and carcinogenic mycotoxin sterigmatocystin. The toxicity of this mycotoxin is manifested primarily in liver and kidney. This study aimed to determine occupational exposure treat due to fungal contamination caused by A. versicolor in seven Portuguese swine.

Fungal contamination: comparation between Portuguese poultries and swines

Animal confinement tends to increase the overall microbial load in the production environment caused by high amounts of feed and organic residuals (manure and wastewater) present in those environments. The number of animais and the handling and management required to work in these settings also contribute to enhance that microbial ioad. Animal housing typically exposes workers to substantial concentrations of bioaerosols, such as fungi and their metabolites. Therefore, agricultural workers, and especially pig and poultry farmers, are at increased risk of occupational respiratory diseases. Exposure to bioaerosols in poultries and swines may vary depending upon the stage of the animals' growth, density, manure management procedures, litter type and used floor coverage, among others. Gathering temporal information about the quantity and the composition of fungal load is necessary to better understand the relationship between these factors and adverse health symptoms of workers. This study aimed to characterize and compare fungal contamination between these two different settings.

Nanofiltration of Cork Wastewaters and Their Possible Use in Leather Industry as Tanning Agents

Cork processing wastewater is a very complex mixture of vegetal extracts and has, among other natural compounds, a very high content of phenolic/tannic colloidal matter that is responsible for severe environmental problems. In the present work, the concentration of this wastewater by nanofiltration was investigated with the aim of producing a cork tannin concentrate to be utilized in tanning. Permeation results showed that the permeate fluxes are controlled by both osmotic pressure and fouling/gel layer phenomena, leading to a rapid decrease of permeate fluxes with the concentration factor. The rejection coefficients to organic matter were higher than 95%, indicating that nanofiltration has a very good ability to concentrate the tannins and produce a permeate stream depleted from organic matter. The cork tannin concentrate obtained by nanofiltration and evaporation had total solids concentration of 34.8 g/l. The skins tanned by this concentrate were effectively converted to leather with a shrinking temperature of 7 degrees C.

Cork industry wastewater partition by ultra/nanofiltration: A biodegradation and valorisation study

Wastewater from cork processing industry present high levels of organic and phenolic compounds, such as tannins, with a low biodegradability and a significant toxicity. These compounds are not readily removed by conventional municipal wastewater treatment, which is largely based on primary sedimentation followed by biological treatment. The purpose of this work is to study the biodegradability of different cork wastewater fractions, obtained through membrane separation, in order to assess its potential for biological treatment and having in view its valorisation through tannins recovery, which could be applied in other industries. Various ultrafiltration and nanofiltration membranes where used, with molecular weight cut-offs (MWCO) ranging from 0.125 to 91 kDa. The wastewater and the different permeated fractions were analyzed in terms of Total Organic Carbon (TOC), Chemical Oxygen Demand (COD), Biochemical Oxygen Demand (BOD), Total Phenols (TP), Tannins, Color, pH and Conductivity. Results for the wastewater shown that it is characterized by a high organic content (670.5-1056.8 mg TOC/L, 2285-2604 mg COD/L, 1000-1225 mg BOD/L), a relatively low biodegradability (0.35-0.38 for BODs/COD and 0.44-0.47 for BOD20/COD) and a high content of phenols (360-410 mg tannic acid/L) and tannins (250-270 mg tannic acid/L). The results for the wastewater fractions shown a general decrease on the pollutant content of permeates, and an increase of its biodegradability, with the decrease of the membrane MWCO applied. Particularly, the permeated fraction from the membrane MWCO of 3.8 kDa, presented a favourable index of biodegradability (0.8) and a minimized phenols toxicity that enables it to undergo a biological treatment and so, to be treated in a municipal wastewater treatment plant. Also, within the perspective of valorisation, the rejected fraction obtained through this membrane MWCO may have a significant potential for tannins recovery. Permeated fractions from membranes with MWCO lower than 3.8 kDa, presented a particularly significant decline of organic matter and phenols, enabling this permeates to be reused in the cork processing and so, representing an interesting perspective of zero discharge for the cork industry, with evident environmental and economic advantages. (C) 2010 Elsevier Ltd. All rights reserved.

Accessing indoor fungal contamination using conventional and molecular methods in Portuguese poultries

Epidemiological studies showed increased prevalence of respiratory symptoms and adverse changes in pulmonary function parameters in poultry workers, corroborating the increased exposure to risk factors, such as fungal load and their metabolites. This study aimed to determine the occupational exposure threat due to fungal contamination caused by the toxigenic isolates belonging to the complex of the species of Aspergillus flavus and also isolates fromAspergillus fumigatus species complex. The study was carried out in seven Portuguese poultries, using cultural and molecularmethodologies. For conventional/cultural methods, air, surfaces, and litter samples were collected by impaction method using the Millipore Air Sampler. For the molecular analysis, air samples were collected by impinger method using the Coriolis μ air sampler. After DNA extraction, samples were analyzed by real-time PCR using specific primers and probes for toxigenic strains of the Aspergillus flavus complex and for detection of isolates from Aspergillus fumigatus complex. Through conventional methods, and among the Aspergillus genus, different prevalences were detected regarding the presence of Aspergillus flavus and Aspergillus fumigatus species complexes, namely: 74.5 versus 1.0% in the air samples, 24.0 versus 16.0% in the surfaces, 0 versus 32.6% in new litter, and 9.9 versus 15.9%in used litter. Through molecular biology, we were able to detect the presence of aflatoxigenic strains in pavilions in which Aspergillus flavus did not grow in culture. Aspergillus fumigatus was only found in one indoor air sample by conventional methods. Using molecular methodologies, however, Aspergillus fumigatus complex was detected in seven indoor samples from three different poultry units. The characterization of fungal contamination caused by Aspergillus flavus and Aspergillus fumigatus raises the concern of occupational threat not only due to the detected fungal load but also because of the toxigenic potential of these species.

Evaluation of coumarins effect in saccharomyces cerevisiae

Coumarin and derivates (coumarins) are phenolic compounds widely distributed in the plant kingdom, as for example in tonka beam and cassia cinnamon. These compounds are involved in various processes such as the defense against phytopathogens, the response to abiotic stress and the regulation of oxidative stress. Coumarins can be produced synthetically and are broadly used as additives in the food, perfumes and cosmetics and pharmaceutical industry due th their vast array of biological activities, including anticoagulant, analgesic, anti-inflammatory and anti-microbial.

Occupational exposure to toxigenic fungi from Aspergillus flavus complex

Bioaerosols are mainly composed of fungal particles, bacteria and plant spores, being fungi responsible for the release of VOCs and micotoxins into indoor environments. Aspergillus flavus is a common opportunistic pathogen causing human infections and is involved in the production of aflatoxin and other secondary metabolites associated with toxic and allergic reactions. Poultry workers are exposed to high concentrations of fungi and are therefore more prone to develop associated pathologies. To evaluate occupational exposure of the workers to Aspergillus flavus and aflatoxins, six animal production facilities were selected, including 10 buildings, from which indoor air samples and outdoor reference samples were obtained. Twenty-five duplicate samples were collected by two methodologies: impactation onto malt extract agar of 25L air samples using a Millipore Air Tester were used to evaluate quantitative (CFU/m3) and qualitative (species identification, whenever possible) sample composition; 300 L air samples collected with the Coriolis Air Sampler into phosphate–saline buffer were used to isolate DNA, following molecular identification of Aspergillus section flavi using nor-1 specific primers by real-time PCR.