Study of operational variables in the submerged growth ofPleurotus ostreatus mushroom mycelium

Pleurotus ostreatus mushroom mycelium was cultivated in submerged culture in shake-flask experiments with acid extract from peat and yeast extract as nutrient sources. Different concentrations of water-diluted peat extract were tested in an attempt to overcome the effect of growth inhibitors apparently present in nondiluted peat extracts. The best results were obtained with a ratio of one part of peat extract diluted with one part of water. Several operating variables were studied to optimize the growth of mycelial biomass ofP. ostreatus. The best results produced approximately 5 g/L dry biomass with a yield of 60% and an efficiency of 33%. These results were obtained in 8 d at 5% (v/v) inoculum ratio, 28°C, pH of 5.0, and 150 rpm.     

Study of biodegradation products from azo dyes in fungal degradation by capillary electrophoresis/electrospray mass spectrometry

Biodegradation products from four model sulfonated azo dyes Orange II, Acid Orange 8, Food Yellow 3, and 4-[(4-hydroxyphenyl)azo]-benzenesulfonic acid, sodium salt (4HABA), during fungal degradation were determined by capillary electrophoresis coupled with ion trap mass spectrometry (CE-MS) with electrospray ionization and a coaxial sheath flow interface. The development and optimization of this analytical method including the sheath liquid composition and flow rate, nebulizing gas flow rate, carrier electrolyte, and MS voltage are described herein. Detection of unknown biodegradation products was carried out under negative ion mode with base peak electrophorogram (BPE) or extractive ion electrophorogram (EIE) monitoring. A volatile ammonium acetate buffer (10 mM) without organic modifier and a shealth liquid made from 2-propanol and water (80:20, v/v) were suited for the separation and ESI interface. The sulfonated ion was the base peak for model azo dyes and their metabolites containing sulfonic group. Results showed that the tested azo dyes were degraded quickly in the culture of white rot fungus, Pleurotus ostreatus in 3 days with the major biodegradation products being 4-hydroxy-benzenesulfonic acid, 3-methyl-4-hydroxy-benzenesulfonic acid, benzenesulfonic acid, 1,2-naphthoquinone-6-sulfonic acid and 3-methyl-benzenesulfonic acid.     

Fusaroside, a unique glycolipid from Fusarium sp., an endophytic fungus isolated from Melia azedarach

Fusaroside (1), a unique trehalose-containing glycolipid composed of the 4-hydroxyl group of a trehalose unit attached to the carboxylic carbon of a long-chain fatty acid, was isolated from the organic extract of fermentation broths of an endophytic fungus, Fusarium sp. LN-11 isolated from the leaves of Melia azedarach. Six known compounds, phalluside (2), (9R*,10R*,7E)-6, 9,10-trihydroxyoctadec-7-enoic acid (3), porrigenic acid (4), (9Z)-2,3-dihydroxypropyl octadeca-9-enoate (5), cerevisterol (6) and ergokonin B (7), were also isolated from this fungus. The glycolipid contains a rare branched long-chain fatty acid (C(20:4)) with a conjugated diene moiety and a conjugated ketone moiety. The structure of the new compound 1 was elucidated by spectroscopic methods (1D and 2D NMR experiments, MS) and chemical degradations. The metabolites 1-5 were shown to have moderate to weak active against the brine shrimp larvae. To our knowledge, this is the first report of isolation of the first representative of a new family of glycolipids from natural sources.     

Secondary Metabolites from the Endophytic Fungus Xylaria cubensis

Seven new metabolites, including three sesquiterpenoids, 10‐hydroxythujopsene ( 1 ), akotriol ( 2 ), and xylaritriol ( 3 ), one diterpenoid, cubentriol ( 4 ), one aliphatic derivative, akoenic acid ( 5 ), one alkaloid, akodionine ( 6 ), and one isocoumarin, akolitserin ( 7 ), together with seven known compounds, 8 – 14 , were isolated from the AcOEt‐soluble fraction of the fermentation broth of the endophytic fungus Xylaria cubensis, derived from the leaves of Litsea akoensis Hayata (Lauraceae). Their structures were elucidated by spectroscopic analyses, including 1D‐ and 2D‐NMR experiments, and by HR‐ESI‐MS mass spectrometry. Among the isolates, (?)‐(R)‐7‐hydroxymellein showed IL‐6 inhibitory activity with an IC₅₀ value of 9.41 μM .     

Arsenic and cadmium content in edible mushrooms from São Paulo,Brazil determined by INAA and GF AAS

Arsenic and cadmium contents in eight edible mushroom species (Agaricus bisporus, Agaricus sp, Pleurotus ostreatus, Pleorotus florida, Pleorotus eryngui, Pleurotus osteatus, Pleurotus salmoneostramineus, Lentinula edodes) consumed by Brazilian population were determined by instrumental neutron activation analysis (INAA) and graphite furnace atomic absorption spectrometry (GF AAS), respectively. Arsenic concentrations varied from 0.009 mg/kg in P. eryngui to 0.210 mg/kg dry weight in L. edodoes and Cd from 0.011 g/kg in P. eryngui to 0.229 mg/kg dw in P. salmoneostramineus. The consumption of mushrooms in São Paulo-Brazil may be considered safe from a toxicological point of view as As and Cd presented levels of ingestion are below the maximum levels recommended by the World Health Organization.     

Carbohydrate compositions of preparations of fungal origin

The fractional composition of the polysaccharides formed by some local strains of basidial fungi (Panus tigrinus, Pleurotus ostreatus, Fomes formentarius, andPhanarechaeta chrysosporium) in a submerged medium on various plant wastes (spent cottonseed pulp and cottonplant stems) has been investigated. Water-soluble polysaccharides, pectin substances, and hemicelluloses have been isolated from the products synthesized by the fungi, and their qualitative and quantitative monosaccharide compositions have been determined.Materials presented at the Second International Symposium of the Chemistry of Natural Compounds (SCNC, Eskiehir, Turkey, October 22–24, 1996).Translated from Khimiya Prirodnykh Soedinenii, No. 3, pp. 352–357, May–June, 1977.     

Investigation of the response of wood-rotting fungi to copper stress by size-exclusion chromatography and capillary zone electrophoresis with ICP MS detection

A method based on the coupling of size-exclusion chromatography (SEC) with inductively coupled plasma mass spectrometry (ICP MS) was developed for screening the changes in the bioligand composition of wood-rotting fungi as a function of their exposure to copper stress. Strains of four different species of wood-rotting fungi: Phanerochaete chrysosporium, Schizophyllum commune, Daedalea quercina and Pleurotus ostreatus were examined. Only one, namely Ph. chrysosporium, showed any significant difference in terms of the fingerprint of Cu-binding ligands between control and exposed cells which suggest trapping of Cu(II) by cell walls as the only resistance mechanisms. In the case of Ph. chrysosporium the bioinduction of a new Cu-binding ligand was demonstrated. The presence of a new compound in the SE chromatographic fraction of interest was confirmed by CZE-ICP MS. Attempts to identify the new compound by electrospray MS/MS failed because of insufficient sensitivity.     

<Emphasis Type="Italic">Arcopilus aureus</Emphasis>, a Resveratrol-Producing Endophyte from <Emphasis Type="Italic">Vitis vinifera</Emphasis>

Due to great interest on producing bioactive compounds for functional foods and biopharmaceuticals, it is important to explore the microbial degradation of potential sources of target biomolecules. Gallotannins are polyphenols present in nature, an example of them is tannic acid which is susceptible to enzymatic hydrolysis. This hydrolysis is performed by tannase or tannin acyl hydrolase, releasing in this way, biomolecules with high-added value. In the present study, chemical profiles obtained after fungal degradation of tannic acid under two bioprocesses (submerged fermentation (SmF) and solid state fermentation (SSF)) were determined. In both fermentation systems (SmF and SSF), Aspergillus niger GH1 strain and tannic acid as a sole carbon source and inducer were used (the presence of tannic acid promotes production of enzyme tannase). In case of SSF, polyurethane foam (PUF) was used like as support of fermentation; culture medium only was used in case of submerged fermentation. Fermentation processes were monitored during 72 h; samples were taken kinetically every 8 h; and all extracts obtained were partially purified to obtain polyphenolic fraction and then were analyzed by liquid chromatography-mass spectrometry (LC-MS). Molecules like gallic acid and n-galloyl glucose were identified as intermediates in degradation of tannic acid; during SSF was identified ellagic acid production. The results obtained in this study will contribute to biotechnological production of ellagic acid.     

Production of lovastatin by wild strains of Aspergillus terreus

A wild fungal strain of Aspergillus terreus, labeled as PM3, was isolated by using the Candida albicans bioassay and confirmed by 18S r DNA analyses. Lovastatin was produced by submerged and solid state fermentations. Of the 30 isolated fungal strains, 11 showed lovastatin production with Aspergillus terreus PM3 being the best with a yield of 240 mg/L at the 10th day of submerged fermentation. Carboxymethylcellulose had a stimulatory effect on lovastatin production. It restricted uncontrolled filamentous growth, induced pellet formation and, thereby, improved lovastatin yield. In solid state fermentation (SSF), of the agro wastes from five crops (bran of wheat and rice, husks of red gram and soybean, and green gram straw), wheat bran showed maximum lovastatin production (12.5 mg/g of dry substrate) at pH 7.1 and a temperature of 30 +/- 2 degrees C. Development of a lovastatin production process based on wheat bran as a substrate in SSF is economically attractive as it is a cheap and readily available raw material in agriculture-based countries.     

Solid state fermentation of plant residues for improved animal feed byPleurotus sp.

In laboratory-scale experiments, studies were made on the solid state fermentation of plant residues—rice straw and the upper soft portion of the stems of sarkanda (Saccharum munja)—by selected cultures of white-rot fungi,Pleurotus sajor-caju andPleurotus ostreatus. These cultures were selected after preliminary screening of their lignin-degrading capacities on lignin-agar medium. Their lignin degrading and (cellulose + hemicellulose) sparing, along with protein improving capacities, were studied for their potential application in animal feed production. A 100 g quantity of presoaked and sterilized residues was inoculated with wheat spawn of the two cultures and incubated at 25‡C. It was observed that, after 25 d, the crude protein contents (N × 6.25) of rice straw increased from 3 to 17.0% in the case of P.sajor-caju and to 19.2% in case of P. ostreatus. The percent removal values of cellulose, hemicellulose, and lignin were found to be as follows: 45.8, 16.8, and 47.1%, respectively, in the case ofP. sajor-caju and 56.5, 40.4, and 50%, respectively, in the case of P.ostreatus. After solid state fermentation of sarkanda for 25 d, its protein content increased from 3 to 12.8% in the case ofP. sajor-caju and to 14.5% in the case ofP. ostreatus. The percent removal of cellulose, hemicellulose, and lignin was found to be as follows: 31.2, 7.1, and 19%, respectively, in the case ofP. sajor-caju and 34.4, 7.1, and 14.3%, respectively, in the case ofP. ostreatus. The results obtained after solid state fermentation of the two residues by the mixed culture of these two basidiomycetes was also presented.     

A New Glycine Derivative and a New Indole Alkaloid from the Fermentation Broth of the Plant Endophytic Fungus Pestalotiopsis podocarpi Isolated from the Chinese Podocarpaceae Plant Podocarpus macrophyllus

A new glycine derivative, podocarpiamide ( 1 ), a new indole alkaloid, 1‐methoxy‐1H‐indol‐3‐ethanol ( 2 ), together with two known compounds, 1‐methoxy‐1H‐indole‐3‐acetic acid ( 3 ) and methyl 1‐methoxy‐1H‐indole‐3‐acetate ( 4 ), were isolated from the fermentation broth of the plant endophytic fungus Pestalotiopsis podocarpi. Their structures were elucidated by extensive spectroscopic analysis including 1D‐ and 2D‐NMR (HSQC, HMBC, and COSY) and MS experiments. Compound 1 has an interesting unusual carbamic acid structure.     

Screening and Production of Ligninolytic Enzyme by a Marine-Derived Fungal Pestalotiopsis sp. J63

Marine-derived fungi are prone to produce structurally unique secondary metabolites, a considerable number of which display the promising biological properties and/or industrial applications. Among those, ligninolytic enzymes have attracted great interest in recent years. In this work, about 20 strains were isolated from sea mud samples collected in the East China Sea and then screened for their capacity to produce lignin-degrading enzymes. The results showed that a strain, named J63, had a great potential to secrete a considerable amount of laccase. Using molecular method, it was identified as an endophytic fungus, Pestalotiopsis sp. which was rarely reported as ligninolytic enzyme producer in the literature. The production of laccase by Pestalotiopsis sp. J63 was investigated under submerged fermentation (SF) and solid state fermentation (SSF) with various lignocellulosic by-products as substrates. The SSF of rice straw powder accumulated the highest level of laccase activity (10,700 IU/g substrate), whereas the SF of untreated sugarcane bagasse provided the maximum amount of laccase activity (2,000 IU/ml). The value was far higher than those reported by other reports. In addition, it produced 0.11 U/ml cellulase when alkaline-pretreated sugarcane bagasse was used as growth substrate under SF. Meanwhile, the growth of fungi and laccase production under different salinity conditions were also studied. It appeared to be a moderately halo-tolerant organism.     

Determination of the site of glucuronidation in an N-(3,5-dichlorophenyl)succinimide metabolite by electrospray ionization tandem mass spectrometry following derivatization to picolinyl esters

Derivatization using 3-pyridylcarbinol coupled with liquid chromatography electrospray ionization tandem mass spectrometry (LC/MS/MS) was used to characterize a novel Phase II metabolite of the nephrotoxic agricultural fungicide, N-(3,5-dichlorophenyl)succinimide (NDPS). A glucuronide conjugate of N-(3,5-dichlorophenyl)-2-hydroxysuccinamic acid (2-NDHSA) was identified in the urine from a rat dosed with [14C]NDPS. However, 2-NDHSA contains an aliphatic hydroxyl group and a carboxylic acid group, both of which are potential sites for glucuronidation. Mass spectrometry alone was unable to distinguish between these possibilities. Since the position of glucuronidation may be important in the mechanism of NDPS-induced nephrotoxicity, chemical derivatization in conjunction with mass spectrometry was used to characterize the glucuronide. The 2-NDHSA glucuronide conjugate was isolated from rat urine, derivatized with 3-pyridylcarbinol, and the derivatized metabolite was then analyzed by LC/MS/MS. Two known NDPS metabolites, 2-NDHSA and N-(3,5-dichlorophenyl)succinamic acid (NDPSA), were also isolated from rat urine and derivatized similarly. 3-Pyridinylcarbinol reacted rapidly with the carboxylic acid groups and formation of the picolinyl esters increased the ionization potential under positive ion conditions. The urinary glucuronide of 2-NDHSA was identified as an alcohol-linked glucuronide by examination of the molecular ions and the collision-induced dissociation (CID) product ion spectra of the derivatized products. When used in combination with mass spectrometry, derivatization of carboxylic acids with 3-pyridylcarbinol provided useful mass fragmentations and is a rapid way to obtain structural information about the position of glucuronidation of NDPS metabolites.     

Determination of the degradation products of selected sulfonated phenylazonaphthol dyes treated by white rot fungus Pleurotus ostreatus by capillary electrophoresis coupled with electrospray ionization ion trap mass spectrometry

The removal of water-soluble sulphonated phenylazonaphthol dye effluents generated by textile industries is an important issue in wastewater treatment. Microbial treatment of environmental pollutants including dyes, with white rot fungi has received wide attention as a potential alternative for conventional methods in wastewater treatment. Three sulphonated phenylazonaphthol dyes with similar molecular structures Acid Orange 7, Acid Orange 8 and Mordant Violet 5 were selected and degraded by the white rot fungus Pleurotus ostreatus. Chemical instrumental analysis methods such as high-performance liquid chromatography (HPLC) and capillary electrophoresis combined with electrospray ionization mass spectrometry (CE-ESI-MS) were used to identify the degraded dyes. Mordant Violet 5 had two degradation pathways when degraded by P. ostreatus. The first degradation pathway for Mordant Violet 5 was for trans structure and the cis-Mordant Violet 5 followed the second pathway. Acid Orange 8 and Acid Orange 7 had the same degradation mechanism as the first degradation mechanism for Mordant Violet 5, that is cleavage of azo bond at the naphthalene ring side where benzenesulfonic acid and 1,2-naphthoquinone are formed.     

Oxygenated hexylitaconates from a marine sponge-derived fungus Penicillium sp

In the course of our search for bioactive metabolites from marine organisms, new hexylitaconic acid derivatives (1-4), along with (3S)-hexylitaconic acid (5), were isolated from a sponge-derived fungus Penicillium sp. Based on the NMR and MS data, the structures of compounds 1-4 were defined as α,β-dicarboxylic acid derivatives, such as hexylitaconic acid and tensyuic acids which were previously reported as metabolite of Aspergillus niger, Penicillium striatisporum, or Apiospora montagnei. The isolated compounds were evaluated for cytotoxicity against a panel of five human solid tumor cell lines, and for anti-inflammatory activity gauged by their inhibitory effects on the production of major pro-inflammatory mediators (nitric oxide (NO), interleukin (IL)-6, tumor necrosis factor (TNF)-α, and IL-1β) in murine macrophage cells. Compounds 1 and 4 showed weak inhibition of IL-1β production at the concentration of 200 μM.     

Fatty acid derivatives from the halotolerant fungus Cladosporium cladosporioides

Halotolerant fungus Cladosporium cladosporioides OUCMDZ‐187 was isolated from the mangrove plant Rhizophora stylosa collected in Shankou, Guangxi Province of China. Three new fatty acid esters cladosporesters A–C ( 1 – 3 ) and 5 new fatty acids cladosporacids A–E ( 4 – 8 ) were isolated from the ethyl acetate extract of the fermentation broth of OUCMDZ‐187 in a hypersaline (10% salt) medium. Their structures were elucidated by UV, IR, MS, specific rotation, and 1D and 2D NMR data.     

Emerging Approaches in Fermentative Production of Statins

Microbial metabolites have many important applications in pharmaceutical and health-care industry. The products of microbial origin are usually produced by submerged fermentation. The solid-state fermentation represents an alternative mode of fermentation, which is increasingly being employed as an alternative to submerged fermentation for metabolite production. The prospect of producing high-value product using low-value raw material offers a substantial premium to switch to these technologies. The cost of statins being one major factor, solid-state fermentation with agro-industrial residues as carbon, nitrogen and support matrix, promises to substantially lower the cost of production. Hence, newer approaches are required to exploit the agro-industrial residues for statin production. The development of these technologies offers an opportunity to exploit low-cost substrates without substantial investment in newer production methodologies. The emerging evidence of beneficial effect of statins in applications other than lipid lowering such as in Alzheimer disease, HIV, age-related dementia, and cancer chemotherapy makes it very important to develop methods for economic production of statins.     

Determination of energy metabolites in cancer cells by porous graphitic carbon liquid chromatography electrospray ionization mass spectrometry for the assessment of energy metabolism

A high performance liquid chromatography (HPLC) tandem mass spectrometric (MS/MS) method has been developed for the simultaneous determination of fifteen glucose, or acetate derived metabolites isolated from tumor cells. Glycolytic and tricarboxylic acid (TCA) cycle metabolites as well as acidic amino acids were separated on a HPLC porous graphitic carbon (PGC) column and simultaneously determined by means of triple quadrupole MS/MS using multiple reaction monitoring (MRM). Target compounds were eluted within 10 min with 8% v/v formic acid as an electronic modifier added to a 4:1 v/v methanol water mobile phase. The calibration is linear in the 1–100 μM concentration range for each analyte. The limit of detection ranges between 0.39 and 2.78 μM for the analytes concerned. To test the PGC–HPLC–MS/MS method in metabolomic studies, ZR-75.1 human mammary adenocarcinoma cells were labeled with U-¹³C glucose or 1-¹³C acetate. Applying the MRM mode, the incorporation of ¹³C into metabolites, isolated from the tumor cells, and derived from glucose or acetate, could be properly identified.     

1H and 13C NMR assignments for two new xanthones from the endophytic fungus Xylaria sp. FRR 5657

The complete 1H and 13C NMR assignments are reported for the new natural products, 7-hydroxy-3-(hydroxymethyl)-1-methoxy-9H-xanthen-9-one (1) and 2,5-dihydroxy-8-methoxy-6-methyl-9-oxo-9H-xanthene -1-carboxylic acid (2). Both of these secondary metabolites were isolated from the fermentation culture of the endophytic fungus Xylaria sp. FRR 5657. 1D and 2D NMR experiments that included 1H, gCOSY, gHSQC and gHMBC were used for the determination of the structure and assignment of these xanthones.     

Cytotoxic fatty acid from Pleurocybella porrigens

The new conjugated ketonic fatty acid, porrigenic acid (1), was isolated as a cytotoxic constituent of Pleurocybella porrigens. The structure of 1 was elucidated using spectroscopic methods including 1D and 2D NMR and MS. The absolute stereochemistry of 1 was determined by application of the exciton chirality method. Compound 1 exhibited cytotoxic activity against myeloma THP-1 cells, but did not show any significant toxicity against B16F1 melanoma. This is the first report of the isolation and structural elucidation of the new cytotoxic constituent porrigenic acid (1) from the edible mushroom P. porrigens.