Search

Little information is available in the literature concerning the kinetics of the production of exopolysaccharides (EPS). The kinetics of growth, substrate consumption and lasiodiplodan (LAS) production by the filamentous fungus Lasiodiplodia theobromae MMPI were studied in a new bioreactor design (Low-Shear Aerated and Agitated Bioreactor; LSAAB) using media based on glucose and glycerol. The kinetic parameters of cultivation were compared with fermentations conducted in a stirred tank bioreactor (STR) and in shake flasks. Cultivation in LSAAB using glucose resulted in 3.17 ± 0.16 g.L−1 of LAS, a value lower than that obtained in shake-flasks (6.49 ± 0.03 g.L−1), but somewhat higher than obtained in STR (0.70 ± 0.12 g.L−1). When glycerol was used as substrate, the production of LAS was less effective than glucose. From the fungal biomass produced, as well as the related growth kinetic parameters (YX/S and QX), fungal growth was more efficient in LSAAB. Lasiodiplodan production by L. theobromae MMPI from glycerol was accelerated by an increase in broth pH during cultivation, and the behavior was opposite (decreased pH) when using glucose. LSAAB proved to be promising for the production of LAS by the fungus studied. Although glucose was shown to be the better substrate for the production of LAS, glycerol, as a fermentation substrate, has potential of a less-expensive medium for cultivating the fungus.

Soybean molasses was studied for the first time as a raw material for the fermentative production of (1 → 6)-β-d-glucan (lasiodiplodan) by Lasiodiplodia theobromae MMPI fungus. The work included characterization of soybean molasses, assessment of cultivation medium and nutritional supplementation, saccharification, fermentation of unhydrolyzed and hydrolyzed molasses, and characterization of lasiodiplodan. Crude soybean molasses showed high concentrations of monosaccharides (glucose: 13.26 g/L, galactose: 9.53 g/L and fructose: 1.99 g/L) and oligosaccharides (sucrose: 74.51 g/L, raffinose: 96.58 g/L, and stachyose: 114.02 g/L). Clarification of molasses promoted reduction of 60% of the total phenolic contents, and showed high-nutritional quality not requiring medium supplementation for fungal growth and lasiodiplodan production. Lasiodiplodia theobromae was able to assimilate the sugars from molasses (YC: 98.25%), including the oligosaccharides, thus no need for prior acid-hydrolysis for saccharification. Maximum production (1.06 g/L), yield (0.13 g/g) and volumetric productivity of lasiodiplodan (0.01 g/L h) were verified in fermentations using unhydrolyzed molasses. Both lasiodiplodan samples demonstrated high thermal stability, and presented three stages of mass-loss; the first at 150 oC, followed by two events between 250 and 525 °C. FT-IR spectra showed characteristic signs of lasiodiplodan including bands at 1060 and 1600 cm−1 corresponding to symmetrical stretching of C–O–C (characteristic group of sugars) and glucose ring vibration, respectively.

The ascomycete Neodeightonia phoenicum CMIB-151 was isolated from bracts of a palm tree (Syagrus romanzoffiana) in Brazil, and identified by molecular techniques. This is the first report of the isolation of this fungus in South America, and for the first time this fungus has been described as producing an exopolysaccharide (EPS). Different carbon (glucose, fructose, sucrose, maltose, lactose, starch) and nitrogen (yeast extract, peptone, urea, ammonium sulfate) sources were evaluated for the production of EPS. Sucrose and peptone resulted in high production and yield of EPS. High production of EPS occurred at pH 6.0 and 8.0, and acidic conditions (initial pH 5.0) promoted higher mycelial growth. The EPS produced showed a high degree of purity, emulsifying activity and a high water- and oil-holding capacity; important technological properties for industrial applications. FT-IR and 13C-NMR (CP/MAS) analyzes showed typical spectra of carbohydrates containing six-carbon sugar in its structure, and the presence of β-glycosidic configuration with 3-O-substitution. The isolated EPS exhibited remarkable thermal stability.

Purpose Flubendiamide is a highly toxic and persistent insecticide that causes loss of insect muscle functions leading to paralysis and death. The objective was to screen for filamentous fungi in soils where insecticides had been applied, to isolate entomopathogenic fungi from insect larva (Anticarsia gemmatalis) that infest soybean crops, and to use these in biodegradation of insecticides. Method Filamentous fungi were isolated from soils, and growth inhibition was evaluated on solid medium containing commercial insecticides, Belt® (flubendiamide) and Actara® (thiamethoxam). A total of 133 fungi were isolated from soil and 80 entomopathogenic fungi from insect larva. Based on growth inhibition tests, ten soil fungi, 2 entomopathogenic fungi, and Botryosphaeria rhodina MAMB-05 (reference standard) were selected for growth on commercial insecticides in solid media. Fungi were grown in submerged fermentation on media containing commercial insecticides and assayed for laccase activity. Result Isolates JUSOLCL039 (soil), JUANT070 (insect), and MAMB-05 performed best, and were respectively inhibited by 48.41%, 75.97%, and 79.23% when cultivated on 35 g/L Actara®, and 0.0, 5.42%, and 43.39% on 39.04 g/L Belt®. JUSOLCL039 and JUANT070 were molecularly identified as Trichoderma koningiopsis and Neurospora sp., respectively. The three fungal isolates produced laccase constitutively, albeit at low activities. Fungal growth on pure flubendiamide and thiamethoxam resulted in only thiamethoxam inducing high laccase titers (10.16 U/mL) by JUANT070. Neurospora sp. and B. rhodina degraded flubendiamide by 27.4% and 9.5% in vivo, while a crude laccase from B. rhodina degraded flubendiamide by 20.2% in vitro. Conclusion This is the first report of fungi capable of degrading flubendiamide, which have applications in bioremediation.

Wood-rot fungi from the Boreal Forest of Canada were studied for their ability to demethylate Kraft lignin (KL). Demethylation by the action of enzymes (O-demethylases) removed the O-methyl/methoxyl groups of lignin liberating methanol, and produced a demethylated KL enriched in vicinal-hydroxyl groups with potential to serve as lignin-based phenol-formaldehyde polymers. Screening experiments identified the liberation of methanol (measured by selected-ion flow-tube mass spectrometry), lignin-demethylating enzymes, alcohol oxidase, and other ligninolytic enzymes. Highest amounts of headspace methanol (parts-per-billion) were detected in the genus Aspergillus, Ctenomyces, Cunninghamella, Penicillium, and Sporobolomyces. Methanol generated from lignin demethylation induced alcohol oxidase activity, but which was higher in Aspergillus, Ctenomyces, Entoloma, and non-sporulating fungi. Among the fungi tested, three brown-rot, i.e., Fomitopsis pinicola and Galerina autumnalis and a mitosporic Aspergillus sp.3 BRI 270, were cultured solely on KL, and lignin model compounds (LMCs) to determine lignin demethylation. Various carbohydrate supplements added to nutrient media containing KL significantly influenced demethylating activity. Aspergillus sp.3 BRI 270 showed the highest degree of lignin demethylation (30.1%) which no evidence presented for this comment occurred when cultivated on KL media supplemented with birchwood xylan as analyzed by 1H NMR following O-acetylation of modified KLs. All fungi demonstrated considerable demethylating activity by utilizing softwood KL, but OrganoSolv lignins (poplar, willow, wheat straw, and mixed agricultural wastes), because of their harsh chemical treatments of extraction, affected the microbial and enzymatic demethylation. Extracellular demethylating enzymes from Aspergillus sp.3 BRI 270 generated high vicinal-diol content (measured by the pyrocatechol titanium(III)-nitrilotriacetate method) in LMCs: 4-hydroxy-3-methoxy cinnamaldehyde (326.00 μmol/mL), syringaldehyde (102.67 μmol/mL), and Kraft lignin (397.46 μmol/mL) as analyzed by 1H NMR analysis.

A cellular immobilization technique was developed as a new proposal for the maintenance and preservation of microbial cultures. Cells of enterobacteria, Escherichia coli (ATCC 25922) and Enterobacter aerogenes (ATCC 13048) were entrapped in calcium alginate beads, which were subjected to different storage conditions. Non-dehydrated beads crude devoid of bacterial cells were kept under refrigeration at 5 °C, or frozen at −60 °C, and freeze-dried beads were stored at −18 °C. The viability and biochemical stability of the entrapped cells during storage time was assessed by the Petrifilm™ procedure and VITEK 2 system. Morphological aspects of the beads were evaluated by optical and scanning electron microscopy. Cell entrapment within alginate beads was a promising technique for maintaining E. coli and E. aerogenes stock cultures. E. aerogenes cells entrapped within calcium alginate beads and maintained under refrigeration or frozen at −18 °C were viable for 240 days, and maintained their biochemical characteristics. Encapsulated E. aerogenes cells maintained at −60 °C remained viable for up to 150 days. The viability of the E. coli cells was maintained throughout the storage period under all storage conditions studied, but this was accompanied by a loss of synthesis capacity of the enzymes, β-galactosidase and β-glucuronidase.

Obesity is an important risk factor in tumor development. Botryosphaeran, a (1 → 3)(1 → 6)-β-D-glucan, produced by the fungus Botryosphaeria rhodina (MAMB-05), is a high molecular mass, water-soluble exopolysaccharide. It consists of a main chain of (1 → 3)-linked β- d -glucose units, with a degree of branching of ~22% at carbon-6 with glucose and gentiobiose residues linked through β-(1 → 6)-bonds, and presents a triple helix conformation. Botryosphaeran presents anticlastogenic, antiproliferative, pro-apoptotic and anti-obesogenic activities. This study evaluated the effects of botryosphaeran on tumor development in obesity and analyzed its mechanism of action. Obesity was induced in male Wistar rats by a high-fat/high-sugar diet. After 9 weeks, rats were divided into two groups: Obese Tumor (OT) and Obese Tumor Botryosphaeran (OTB), and inoculated with 1 × 107 Walker-256 tumor cells, and treatment with botryosphaeran (30 mg/kg b.w./day via gavage for 15 days) commenced. On the 11th week, biological parameters, tumor development, metabolic profile, erythrogram and protein expression were evaluated. Botryosphaeran significantly reduced tumor growth, body-weight loss and cachexia. Furthermore, botryosphaeran decreased mesenteric fat and insulin resistance, corrected macrocytic anemia, and increased Forkhead transcription factor-3a (FOXO3a) activity. Our study demonstrated the potential role of botryosphaeran in the management of cancer in tumor-bearing obese rats by increasing insulin sensitivity and FOXO3a activity.

In this work a crude laccase preparation from Botryosphaeria rhodina MAMB-05 was employed to construct an electrochemical biosensor using nanostructured carbon black. The biosensor was evaluated by differential pulse voltammetry under optimized conditions and was utilized in the determination of epinephrine (EPN). The biosensor’s storage stability was evaluated and was found to be stable over seven consecutive days with a reduction of only 7.0% in the EPN response. Under the optimum conditions developed, the biosensor provided a linear current response for the concentration of EPN in the range from 4.98 to 285 μM. The biosensor was successfully applied for determining EPN in pharmaceutical-grade EPN injection samples, and in synthetic cerebrospinal fluid, demonstrating its promising analytical performance in drug analysis.

The antimutagenic effect of botryosphaeran, an exocellular (1 → 3)(1 → 6)-β- d -glucan, from the ascomyceteous and plant-borne endophytic fungus, Botryosphaeria rhodina MAMB-05, was evaluated in young (6–8 weeks) and elderly (18 months) Swiss albino mice of both genders. The hypolipidemic, hypoglycemic and antiatherogenic potential was also evaluated in 18-month old male LDL receptor knockout (LDLr −/− ) mice. Administration of botryosphaeran by gavage (doses: 7.5, 15, 30 mg/kg b.w./day) in a 30-day pretreatment protocol (young mice), or 15-day protocol (older mice), did not cause genotoxicity as assessed by the micronucleus test in peripheral blood (PB) and bone marrow cells (BMCs). Furthermore, there was no cytotoxic effect of this β- d -glucan in the treatments. A lower frequency of micronuclei was observed in BMCs from young and old mice that received botryosphaeran, indicating its antimutagenic effect. Botryosphaeran (30 mg/kg b.w./day) promoted 102.22% (young) and 103.45% (elderly) reductions in cyclophosphamide-induced damage in male mice. Botryosphaeran also exerted chemoprotective effects in LDLr −/− and wild-type (C57BL/6) mice. Botryosphaeran treatment for 15 days at a dose of 30 mg/kg b.w./day improved the lipidic profile (reductions of 53.8–84.3%), and decreased aortic lipid deposition (32.8%) in the LDLr −/− atherosclerotic mice. The results indicate botryosphaeran has relevant biologic effects, making it a promising candidate for the development of new therapeutic agents.

The coconut kernel-associated fungus, Lasiodiplodia theobromae VBE1, was grown on coconut cake with added coconut oil as lipase inducer under solid-state fermentation conditions. The extracellular-produced lipases were purified and resulted in two enzymes: lipase A (68,000 Da)—purified 25.41-fold, recovery of 47.1%—and lipase B (32,000 Da)—purified 18.47-fold, recovery of 8.2%. Both lipases showed optimal activity at pH 8.0 and 35 °C, were activated by Ca2+, exhibited highest specificity towards coconut oil and p-nitrophenyl palmitate, and were stable in iso-octane and hexane. Ethanol supported higher lipase activity than methanol, and n-butanol inactivated both lipases. Crude lipase immobilized by entrapment within 4% (w/v) calcium alginate beads was more stable than the crude-free lipase preparation within the range pH 2.5–10.0 and 20–80 °C. The immobilized lipase preparation was used to catalyze the transesterification/methanolysis of coconut oil to biodiesel (fatty acyl methyl esters (FAMEs)) and was quantified by gas chromatography. The principal FAMEs were laurate (46.1%), myristate (22.3%), palmitate (9.9%), and oleate (7.2%), with minor amounts of caprylate, caprate, and stearate also present. The FAME profile was comparatively similar to NaOH-mediated transesterified biodiesel from coconut oil, but distinctly different to petroleum-derived diesel. This study concluded that Lasiodiplodia theobromae VBE1 lipases have potential for biodiesel production from coconut oil.

Linseed and okra mucilages, the fungal exopolysaccharide botryosphaeran, and commercial fructo-oligosaccharides (FOS) were used to microencapsulate Lactobacillus casei LC-01 and L. casei BGP 93 in sodium alginate microspheres by the extrusion technique in calcium chloride. The addition of carbohydrate biopolymers from linseed, okra and the fungal exocellular (1 → 3)(1 → 6)-β-D-glucan, named botryosphaeran provided higher encapsulation efficiency (EE) (>93% and >86%) for L. casei LC 01 and L. casei BGP 93, respectively. The use of linseed, okra and botryosphaeran improved the stability of probiotics encapsulated in the microspheres during the storage period over 15 d at 5 °C when compared to microspheres formulated with sodium alginate alone as the main encapsulating agent (p ≤ 0.05). In in vitro gastrointestinal simulation tests, the use of FOS combined with linseed mucilage was shown to be more effective in protecting L. casei cells LC-01 and L. casei BGP 93.

Aims Cancer is a multifactorial disease characterized by an uncontrolled growth of cells that can lead to cachexia-anorexia syndrome. Botryosphaeran, a fungal (1 → 3)(1 → 6)-β-D-glucan produced by Botryosphaeria rhodina MAMB-05, has presented antimutagenic, antiproliferative, pro-apoptotic, hypoglycemic and hypocholesterolemic effects. This study evaluated the effects of botryosphaeran (30 mg/kg b.w./day) on tumor development and cachexia syndrome in Walker-256 tumor-bearing rats, and also the metabolic and hematological profiles of these animals. Materials and methods Male Wistar rats were divided into 3 groups: control (C), control tumor (CT) and control tumor botryosphaeran (CTB). On the first day, 1 × 107 Walker-256 tumor cells were inoculated subcutaneously into the right flank of the CT and CTB rats, and concomitantly treatment with botryosphaeran (30 mg/kg b.w./day) started. After the 15th day of treatment, biological parameters, tumor development, cachexia, glucose and lipid profiles, hemogram and protein expression were analyzed. Key findings Botryosphaeran significantly reduced tumor development (p = 0.0024) and cancer cachexia, modulated the levels of glucose, triglycerides and HDL-cholesterol, and corrected macrocytic anemia. Botryosphaeran also increased significantly the bax expression in the tumor tissue (p = 0.038) demonstrating that this (1 → 3)(1 → 6)-β-D-glucan is increasing the apoptosis of tumor cells. p53, p27, bcl-2, caspase-3 and Forkhead transcription factor 3a (FOXO3a) protein expression were similar among the groups. Significance This study demonstrated that botryosphaeran was effective in decreasing tumor development and cachexia by direct and indirect mechanisms increasing apoptosis and improving the metabolic and hematological profiles.

In this work, two types of biomass preparations (VMSM and M3) from the filamentous fungus Botryosphaeria rhodina MAMB-05, which were previously used in a process of production of β-glucan, were assessed as biosorbents of lead. The operating conditions, optimized through response surface methodology and experimental design, were shown to be pH 5.29 and a biosorbent dose of 0.23 g/L for the VMSM biomass type, and pH 5.06 and a dose of biosorbent of 0.60 g/L for the M3 biomass type, at a constant temperature of 27 °C. Fourier transform-infrared spectroscopy analyzed the presence of functional groups on the biomass surface. In addition to give an extra value to the by-product biomass, the VMSM-type from B. rhodina MAMB-05 showed an excellent lead biosorption capacity (qm) with a value of 403.4 mg/g for the Langmuir model, comparing favorably with literature results, while the M3 subtype biomass showed a value of 96.05 mg/g.

&beta, Glucans are biomacromolecules well known, among other biological activities, for their immunomodulatory potential. Similarly, extracts of Baccharis dracunculifolia also possess biological properties and are used in folk medicine for the treatment of inflammation, ulcers, and hepatic diseases. Microparticles containing (1&rarr, 6)-&beta, d-glucan (lasiodiplodan) and B. dracunculifolia extract were produced and characterized. A 23 factorial design was employed to define the conditions of production of microparticles by atomization. Lasiodiplodan associated with maltodextrin and gum arabic was studied as a matrix material. Microparticles of 0.4 &mu, m mean size and high phenolics content (3157.9 &mu, g GAE/g) were obtained under the optimized conditions. The microparticle size ranged from 0.23 to 1.21 &micro, m, and the mathematical model that best represented the release kinetics of the extract was the Korsmeyer-Peppas model. Diffusional exponent (n) values of 0.64 at pH 7.7 and 1.15 at pH 2.61 were found, indicating particles with a non-Fickian or anomalous transport system, and Super Case II transport, respectively. Thermal analysis indicated that the microparticles demonstrated high thermal stability. The X-ray diffraction analyses revealed an amorphous structure, and HPLC-DAD analysis showed microparticles rich in phenolic compounds: caffeic acid, p-coumaric acid, and catechin. The microparticles obtained comprise a new biomaterial with biological potential for applications in different fields.

Cultures of 23 indigenous yeast strains (22 Saccharomyces cerevisiae and a non-Saccharomyces, Torulaspora delbrueckii), isolated from fermentation tanks at wineries in Castilla-La Mancha (Spain), and were performed under winemaking conditions using a synthetic must. Polysaccharide analysis and turbidity assays were conducted so as to observe the capacity of the released mannoproteins against protein haze formation in white wine, and 3 strains (2 Saccharomyces cerevisiae and T. delbrueckii) were chosen for further experiments. The action of a commercial b-glucanolytic enzyme preparation (Lallzyme BETA®), and a β-(1→3)-glucanase preparation from Trichoderma harzianum Rifai were evaluated to release polysaccharides from the different yeast strains’ cell walls. Protection against protein haze formation was strain dependent, and only two strains (Sc2 and Sc4) presented >50% stabilization in comparison to controls. Addition of β-glucanases did not increase the concentrations of polysaccharides in the fermentation musts; however, a significant increase of polymeric mannose (mannoproteins) was detected using an enzymatic assay following total acid hydrolysis of the soluble polysaccharides. Enzymatic treatment presented positive effects and decreased protein haze formation in white wine. DOI http://dx.doi.org/10.17807/orbital.v8i6.869

Carbohydrate biopolymers of fungal-origin are an important natural resource in the search for new bioagents with therapeutic and nutraceutical potential. In this study the mutagenic, genotoxic, antigenotoxic and antioxidant properties of the fungal exopolysaccharide botryosphaeran, a (1→3)(1→6)-β-D-glucan, from Botryosphaeria rhodina MAMB-05, was evaluated. The mutagenicity was assessed at five concentrations in Salmonella typhimurium by the Ames test. Normal and tumor (Jurkat cells) human T lymphocyte cultures were used to evaluate the genotoxicity and antigenotoxicity (Comet assay) of botryosphaeran alone and in combination with the mutagen methyl methanesulfonate (MMS). The ability of botryosphaeran to reduce the production of reactive oxygen and nitrogen species (RONS) generated by hydrogen peroxide was assessed using the CM-H2DCFDA probe in lymphocyte cultures under different treatment times. None of the evaluated botryosphaeran concentrations were mutagenic in bacteria, nor induced genotoxicity in normal and tumor lymphocytes. Botryosphaeran protected lymphocyte DNA against damage caused by MMS under simultaneous treatment and post-treatment conditions. However, botryosphaeran was not able to reduce the RONS generated by H2O2. Besides the absence of genotoxicity, botryosphaeran exerted a protective effect on human lymphocytes against genotoxic damage caused by MMS. These results are important in the validation of botryosphaeran as a therapeutic agent targeting health promotion.

Baccharis dracunculifolia has been used in popular medicine because of its bioactive properties, which include anti-inflammatory, antiulcerative and hepatoprotective potentials. A 2³ central composite rotational design was used to define the best conditions for extracting the bioactive compounds from the leaves of Baccharis dracunculifolia. The parameters studied included the solvent concentration, time and temperature of extraction and the influence of these on the total phenolics content and antioxidant activity. The best conditions were 95 % methanol (v/v), 20 min extraction time, and 70 °C temperature. Under the optimized conditions, high contents of total phenolic compounds (72.5 mg GAE g-1) and high DPPH (865.6 µmol TE g-1) and ABTS (331.11 μmol TE g-1) free radical scavenging capacity and a high ferric reducing antioxidant power (1558.00 μmol FeSO4.7H2O g-1), were obtained. The extract also showed promising anti-microbial activity, demonstrating fungistatic action against C. tropicalis and C. albicans (5 mg mL-1 and 7 mg mL-1, respectively) and bacteriostatic effect against Escherichia coli (60 e 70 mg mL-1), Salmonella enterica typhimurium (40; 60 and 70 mg mL-1) and Staphylococcus aureus (4 mg mL-1). HPLC-DAD analysis showed an extract rich in the phenolic compounds catechin (0.659 mg g-1), caffeic acid (1.135 mg g-1), p-coumaric acid (0.772 mg g-1), ferulic acid (6.24 mg g-1), pinocembrina (0.155 mg g-1) and rutin (0.037 mg g-1).

In this work we propose the use of statistical mixture design in the construction of a biosensor device based on graphite oxide, platinum nanoparticles and biomaterials obtained from Botryosphaeria rhodina MAMB-05. The biosensor was characterized by electrochemical impedance spectroscopy. Under optimized experimental parameters by factorial design, the biosensor was applied to the voltammetric determination of chlorogenic acid (CGA) measured as 5-O-caffeoylquinic acid (5-CQA). The biosensor response was linear (R2 = 0.998) for 5-CQA in the concentration range 0.56–7.3 µmol L−1, with limit of detection and quantification of 0.18 and 0.59 µmol L−1, respectively. The new biosensing device was applied to quality control analysis based upon the determination of CGA content in specialty and traditional coffee beverages. The results indicated that specialty coffee had a significantly higher content of CGA. Principal component analysis of the voltammetric fingerprint of brewed coffees revealed that the laccase-based biosensor can be used for their discrimination.

The present study reports on the remediation of an effluent from the wood-laminate industry using Pleurotus ostreatus EB 016 in combination with photo-Fenton oxidation. Fermentation of the effluent with P. ostreatus EB-016 was carried out in agitated flasks to evaluate the influence of pH, and concentrations of carbon and nitrogen sources by a multivariate approach. Subsequently, bioassays was conducted in an air-lift bioreactor using the pre-optimized conditions. In addition, photo-Fenton oxidative treatment was employed to degrade recalcitrant compounds, and the ecotoxicity of the effluents were evaluated using Escherichia coli as a biological model. The crude effluent presented high contents of total phenolics (1,220 mg/L), solids (18.45 g/L) and color intensity (8,333 CU), besides high values of chemical (COD 2,477 mg O2./L) and biochemical (BOD5, 8,450 mg O2/L) oxygen demand. Another feature was the high inhibition on Escherichia coli (71%). Reduction of 64% COD was obtained under optimized conditions (pH5.7, 7.5 g/L sucrose, 4.0 g/L ammonium nitrate) in agitated flasks after 10 days treatment. In the air-lift reactor, 50.6% COD and 29.9% total phenols were removed after 10 days. Combination of biotreatment with photo-Fenton oxidation resulted in removal of 99.2% COD and 92.2% phenolics and absence of inhibition on Escherichia coli.

A fungal isolate (MAMB-05) from a stem canker on a eucalypt tree, molecularly classified as Botryosphaeria rhodina, produced an exopolysaccharide (EPS) of the β-D-glucan type when cultivated on glucose by submerged fermentation. Isolation and elucidation of the chemical structure demonstrated that the β-glucan was of the (1→3)(1→6)-linked type and was named botryosphaeran. When cultivated on different carbohydrates, B. rhodina MAMB-05 produced a family of structurally related botryosphaerans sharing the same backbone chain of (1→3)-linked D-glucosidic residues, but varied in the degree of branching on C-6 with glucose and gentiobiose. A branch point occurred on every fifth glucose residue along the backbone chain for the botryosphaeran produced on glucose (EPSGLC) and sucrose (EPSSUC) and one in every three glucose residues when produced on fructose (EPSFRU). Details of the chemical structures were resolved by methylation, Smith degradation, FT-IR, and 13C NMR. Membrane-modifying agents (soybean oil, Tween 80) enhanced botryosphaeran production without altering its chemical structure. Sulfonylation of botryosphaeran increased water solubility and conferred new functions, anticoagulation and antiviral activity. Through extensive screening, botryosphaeran was found to be non-mutagenic and non-genotoxic and is regarded as having Generally Recommended As Safe (GRAS) status. The biopolymer exhibited strong antimutagenic/anticlastogenic activity being chemoprotective against several cancer chemotherapy drugs. Botryosphaeran demonstrated hypoglycemia in diabetes-induced rats, and hypocholesterolemia in mice and rats conditioned on a high-lipid ration. Antiproliferative activity was demonstrated in breast cancer MCF-7 cells, was associated with apoptosis, necrosis, and oxidative stress, and mediated by AMP-activated protein kinase and forkhead transcription factor, FOXO3a. This chapter addresses the studies carried out with botryosphaeran.

Glucans find a wide range of commercial applications in different industrial sectors, food, pharmaceuticals, and fine chemicals. Commercially available β-glucans are obtained chiefly through complex extraction procedures of the cell walls of microorganisms and cereals that are time-consuming processes. Some microorganisms also produce β-glucans exocellularly, which have been exploited commercially for this purpose. In this context, exopolysaccharides such as lasiodiplodan (a linear (1 → 6) β-glucan) standout because of the greater ease of their production by submerged fermentation and recovery from the cell-free fermentation broth by precipitation methods. Lasiodiplodan from the fungus Lasiodiplodia theobromae MMPI was first described in 2008, and studies have since addressed its production, chemical derivatization, and chemical and biological characterization. Lasiodiplodan presents biological functions that include antioxidant, hypoglycemic, antiproliferative, and transaminase activities, as well as protective activity against doxorubicin-induced DNA damage. The different biological properties of lasiodiplodan make it a biomolecule attractive for commercial exploitation. Medicines for treatments of different conditions of human diseases, containing lasiodiplodan as one of the active or co-adjuvant principles, could be developed by the pharmaceutical industry. Antiaging creams and ointments, as well as self-healing hydrogels, are also good possibilities for commercial applications. Another attractive possibility of use is as a prebiotic agent in food products. Chemical modifications in the lasiodiplodan structure by O-acetylation, carboxymethylation, phosphorylation, or sulfonylation have been shown to be a potentiating mechanism of some of its properties. This chapter addresses the studies that have been carried out with lasiodiplodan and future production perspectives and applications.

Eight vegetable oils and glycerol were examined as sole carbon source to screen nine isolates of Botryosphaeria spp. Among these, B. rhodina MAMB-05 produced highest laccase titres on all carbon sources evaluated followed by B. rhodina MMLR isolated (from orange, Citrus sp.). The influence of inorganic nitrogen (NH4Cl, NaNO3, (NH4)2SO4, and NH4NO3), organic nitrogen (yeast extract (YE), urea, peptone, and corn steep liquor) sources, inorganic phosphate (KH2PO4, Na2HPO4, NaH2PO4, K2HPO4), as well as increasing concentrations of copper (from zero to 100 µg mL-1), as nutritional requirements for enhancing laccase production by B. rhodina MAMB-05 were also evaluated. Soybean oil was the only carbon source in a central composite factorialdesign (23) to optimize laccase production with the selected variables NaNO3, KH2PO4 and copper (CuSO4.5H2O), using the response surface methodology. The optimal conditions obtained were NaNO3 (100 g L-1), KH2PO4 (125 g L-1) and 50 µg mL-1 of CuSO4.5H2O. The specific laccase activity obtained (2.95 U mg-1) in the validation experiments using the optimal condition for laccase production by B. rhodina MAMB-05 was in agreement with the predicted value (3.07 U mg-1), and demonstrated the fitness of the proposed model. DOI: http://dx.doi.org/10.17807/orbital.v10i7.1206

The ascomycetous fungus, Botryosphaeria rhodina MAMB-05, secretes a (1→3)(1→6)-β-D-glucan, and the scaled-up production of this β-glucan results in large amounts of mycelial biomass being produced that represent a potentially cost-effective biosorbent for rare-earth elements. High sorption capacities for both La(III) and Sm(III) were demonstrated for viable and autoclaved lyophilized fungal mycelium. Fourier-transformed infra-red absorption spectra and the point of zero charge (PZC) were similar for the viable and inactive fungal mycelia. The rare-earth lanthanide elements (La and Sm) binding increased at initial pH values greater than 5.0, which was also observed for the PZC determination. The maximum La(III) uptake capacity was observed at lower amounts of La(III) ions in solution, decreasing from 100.0 to 25.3% when the initial lanthanide concentration increased from 15 to 100 mg/L. Lanthanide biosorption by B. rhodina MAMB-05 mycelia followed the Langmuir model, and the affinity of biosorbent functional groups was similar for La(III) and Sm(III).

d-Glucans possess immunomodulatory activities and potential for the development of new therapeutic agents. Biological activities can be enhanced in these biopolymers through chemical derivatization, e.g., carboxymethylation. This work presents the carboxymethylation, characterization and the evaluation of antioxidant activities of the exocellular (1→6)-β-d-glucan produced by Lasiodiplodia theobromae MMPI. Thermal analysis indicated that the native and carboxymethylated polysaccharides presented four stages of mass-loss. The first stage occurred at 125°C (loss of water) with two consecutive events of mass loss (200–400°C) attributed to polymer degradation and the fourth stage between 425 and 620°C (final decomposition). Scanning electron microscopy analysis indicated that the gross morphological features of lasiodiplodan were ruptured following carboxymethylation. X-ray diffractometry analysis demonstrated that the native and carboxymethylated polysaccharides presented a non-crystalline structure. Carboxymethylation contributed to improving the polysaccharide's water solubility and antioxidant capacity.

A total of 95 yeast strains were isolated from the microbiota of different grapes collected at vineyards in southern Brazil. The yeasts were screened for β-(1 → 3)-glucanases using a newly developed zymogram method that relies upon the appearance of clearance zones around growing colonies cultured on agar–botryosphaeran medium and also by submerged fermentation on nutrient medium containing botryosphaeran, a (1 → 3),(1 → 6)-β-d-glucan. Among 14 β-(1 → 3)-glucanase-positive yeasts identified, four strains produced the highest β-glucanolytic activities and were evaluated for enzyme production on cellobiose, botryosphaeran, and mycelial biomass from Botryosphaeria rhodina (MAMB-05). Yeast strain 1WA1 produced the highest β-(1 → 3)-glucanase and β-glucosidase activities and was identified by molecular characterization as Aureobasidium pullulans. The physicochemical properties of the crude β-glucanolytic enzyme preparation were characterized, and the preparation was used to hydrolyze several β-d-glucans (laminarin, botryosphaeran, lasiodiplodan, pustulan, and curdlan). The production and physicochemical properties of the β-glucanolytic preparation enable its potential applications in wine enology and production of prebiotics through hydrolysis of β-d-glucans.

β-D-Glucans are known to present antitumor, anticancer, and anti-inflammatory activities that are influenced by their own antioxidant capacity. The antioxidant activity of botryosphaeran, an exopolysaccharide of the (1 → 3;1 → 6)-β-D-glucan type produced by the Botryosphaeria rhodina MAMB-05was evaluated and compared to some other β-D-glucans (lasiodiplodan an exocellular (1 → 6)-β-D-glucan from Lasiodiplodia theobromae , laminarin and curdlan), and oligosaccharides, disaccharides, and monosaccharides in a study of scavenging activities of free radicals in-vitro . Botryosphaeran displayed high total antioxidant activity (80%) as well as good scavenging activity against hydroxyl radical (90.6%), superoxide anion (37%), hydrogen peroxide (38%), and nitric oxide radical (90%). No reducing power, metal-chelating capacity or inhibition of lipid peroxidation was observed for these β-D-glucans. The results demonstrated that botryosphaeran exhibited effective antioxidant activity as supported by many different assays, suggesting that this β-D-glucan may serve as a source of a new bioactive compound with effective antioxidant activity.

Fast-growing hybrid poplar species are attractive as feedstocks for the production of bio-based chemicals including biofuels. Pretreatment makes cellulose accessible to enzymatic hydrolysis, producing hemicellulose and lignin as major co-products. Poplar woodchips pretreated hydrothermally in an experimental industrial pilot-plant generated two prehydrolyzate streams; purge-condensate collected from cooking the woodchips and press-filtrate collected after the fiber explosion step. Both prehydrolyzates contain hemicellulose sugars (pentoses, hexoses, oligosaccharides), acetic acid, and degradation products from hemicellulose and lignin (furanic and phenolic compounds, respectively). Purge prehydrolyzate was highly diluted, of low sugar content and high biochemical/chemical oxygen demand, and constituted a process waste that was addressed for value-adding through production of enzymes by fermentation. Wood-decay fungi were screened for laccases on purge prehydrolyzate, with Botryosphaeria rhodina producing highest titers. B. rhodina detoxified the prehydrolyzate but at expense of sugar consumption. Adding xylose and glycerol to purge did not inhibit fungal growth or impede laccase production. Phenolics in the purge had inducing effect on enhancing laccase titers. Laccase production was optimized by Box-Behnken-(33)-factorial design varying the concentration of xylose in the purge, together with adding copper as laccase inducer and glycerol to increase the level of fermentable substrate, resulting in optimal enzyme titers (36.37 ± 3.52U/mL; validated). The crude laccase preparation was employed to detoxify the more concentrated press prehydrolyzate, reducing the overall content of phenolics by ∼30 %. Chromatographic (high-performance liquid chromatography-ultraviolet, gas chromatography/mass spectrometry) analysis identified various phenolic compounds present in the press prehydrolyzate following laccase treatment, and in the presence of the mediator, ABTS, the phenolics content decreased further.

Plant oil-extracted seed-cakes are good fermentation substrates for producing lipases that find application in transesterification of seed oils into biodiesel. This work describes the production of lipases by five lipolytic, oil-seed associated fungi (Aspergillus niger, Chalaropsis thielavioides, Colletotrichum gloeosporioides, Lasiodiplodia theobromae, and Phoma glomerata) by Solid-State Fermentation (SSF) on eight plant oil-seed cakes. The highest lipase activity was from the Coelomycete Lasiodiplodia theobromae VBE-1 grown on coconut kernel-cake, and was selected to optimize lipase production. The effects of supplementing coconut kernel-cake with mineral salts and coconut oil on lipase production by L. theobromae VBE-1 resulted in enhanced lipase activity. The effects of time of growth, moisture content, initial pH, temperature, as well as nutritional factors (carbon, nitrogen, vegetable oils, surfactants) when added to coconut kernel-cake, on lipase production were examined by a one-factor-at-a-time approach, and identified key variables for optimization by Response Surface Methodology (RSM). A 26 factorial central-composite experimental design with eight starting points and six replicates at the central point was used for lipase optimization. After validating the predicted levels of the factors, lipase production rose to 698 U/g Dry Substrate (DS) over un-optimized conditions (450 U/g DS).

This study provides a comparison of fermentation conditions for the production lipases by Fusarium sp. (Gibberella fujikuroi complex) isolate FCLA-MA-41 in submerged (SmF) and solid-state fermentation (SSF) using agro-industrial residues. To assess SmF, a univariate study of seven carbon sources (crambe, corn, linseed, olive, palm (dende), soybean oils and chicken fat) and four nitrogen sources (ammonium sulfate, sodium nitrate, urea, yeast extract) was performed. The relationship among the concentrations of carbon and nitrogen sources, Triton X-100 and yeast extract was studied using a Central Composite Design (CCD). To assess SSF, different residues (sugarcane bagasse) and oil-seed meals (castorbean, corn, crambe, soybean) with additional supplements added (minimum salts, seed oils) were examined. Based on the CCD matrix, a medium containing crambe oil (17.5 mL/L), Triton X-100 (5 g/L), ammonium sulfate (5 g/L) and yeast extract (1 g/L) was proposed, resulting in a lipase titer of 3.0 ± 0.25 U/mL. The cost/production relationship was 7.73% less expensive than SmF with corn oil. SSF produced a maximum lipase titer of 5.0 ± 0.25 U/gds on crambe meal moistened with phosphate buffer. The best cost/enzyme activity estimate was for SSF with crambe meal as substrate in only distilled water (87.27% less expensive than SmF).

Botryosphaeran (BOT) is an exocellular β- d -glucan (carbohydrate biopolymer) of the (1 → 3;1 → 6)-linked type produced by Botryosphaeria rhodina MAMB-05. The cytotoxic, mutagenic, genotoxic, and protective effects of this substance were evaluated in Chinese hamster lung fibroblasts (V79) and rat hepatocarcinoma cells (HTC) by the micronucleus test (MN) and the comet assay. BOT was not genotoxic in either cell line; it decreased the clastogenic effects of doxorubicin, H2O2, and benzo[a]pyrene. These results indicate that BOT may have potential as a therapeutic agent.

The exopolysaccharide botryosphaeran (EPS(GLC); a (1--> 3)(1-->6)-β-D-glucan from Botryosphaeria rhodina MAMB- 05) was sulfonated to produce a water-soluble fraction (EPS(GLC)-S) using pyridine and chlorosulfonic acid in formamid. This procedure was then repeated twice to produce another fraction (EPSGLC-RS) with a higher degree of substitution (DS, 1.64). The purity of each botryosphaeran sample (unsulfonated and sulfonated) was assessed by gel filtration chromatography (Sepharose CL-4B), where each polysaccharide was eluted as a single symmetrical peak. The structures of the sulfonated and re-sulfonated botryosphaerans were investigated using ultraviolet-visible (UV-Vis), Fourier-transform infrared (FT-IR), and (13)C nuclear magnetic resonance ((13)C NMR) spectroscopies. EPS(GLC) and EPS(GLC)-RS were also assayed for anticoagulation activity, and EPS(GLC)-RS was identified as an anticoagulant.

Botryosphaeran, a water-soluble exopolysaccharide of the β-(1 → 3;1 → 6)-D-glucan type that has been isolated from the culture medium of Botryosphaeria rhodina MAMB-05 grown in submerged fermentation using glucose as the sole carbon source, was previously demonstrated to be non-genotoxic in peripheral blood and bone marrow, and exhibited strong anticlastogenic activity. In the present study, the effects of botryosphaeran were investigated in streptozotocin-induced diabetic rats as well as in high-fat diet-fed hyperlipidemic Wistar rats. The plasma glucose level was reduced by 52% in the diabetic group of rats after administration of 12 mg botryosphaeran/kg body weight of the rats (b.w.)/day by gavage over 15 days. A reduction in the median ration intake was accompanied by an increase in the median body weight gain, as well as the efficiency of food conversion. These results demonstrate that botryosphaeran has protective effects by reducing the symptoms of cachexia in Diabetes mellitus. Botryosphaeran administered by gavage at a concentration of 12 mg botryosphaeran/kg b.w./day over 15 days also reduced the plasma levels of total cholesterol and low density lipoprotein-cholesterol by 18% and 27%, respectively, in hyperlipidemic rats. Based on these findings, we conclude that botryosphaeran possesses hypoglycemic and hypocholesterolemic properties in conditions of diabetes mellitus and hyperlipidemia, respectively, and may be used as an oral anti-diabetic agent.

Made available in DSpace on 2013-08-28T14:07:22Z (GMT). No. of bitstreams: 1 WOS000295211000023.pdf: 422179 bytes, checksum: 9e96ed2bf3237d547956e48f80dcd461 (MD5) Made available in DSpace on 2013-09-30T18:09:29Z (GMT). No. of bitstreams: 2 WOS000295211000023.pdf: 422179 bytes, checksum: 9e96ed2bf3237d547956e48f80dcd461 (MD5) WOS000295211000023.pdf.txt: 37101 bytes, checksum: 468b5bec7bde38bd684ddc8137cdd0a4 (MD5) Previous issue date: 2011-09-01 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T13:23:09Z No. of bitstreams: 2 WOS000295211000023.pdf: 422179 bytes, checksum: 9e96ed2bf3237d547956e48f80dcd461 (MD5) WOS000295211000023.pdf.txt: 37101 bytes, checksum: 468b5bec7bde38bd684ddc8137cdd0a4 (MD5) Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T13:23:09Z No. of bitstreams: 2 WOS000295211000023.pdf: 422179 bytes, checksum: 9e96ed2bf3237d547956e48f80dcd461 (MD5) WOS000295211000023.pdf.txt: 37101 bytes, checksum: 468b5bec7bde38bd684ddc8137cdd0a4 (MD5) Made available in DSpace on 2014-05-20T13:23:09Z (GMT). No. of bitstreams: 2 WOS000295211000023.pdf: 422179 bytes, checksum: 9e96ed2bf3237d547956e48f80dcd461 (MD5) WOS000295211000023.pdf.txt: 37101 bytes, checksum: 468b5bec7bde38bd684ddc8137cdd0a4 (MD5) Previous issue date: 2011-09-01 Made available in DSpace on 2014-05-20T13:23:09Z (GMT). No. of bitstreams: 2 WOS000295211000023.pdf: 422179 bytes, checksum: 9e96ed2bf3237d547956e48f80dcd461 (MD5) WOS000295211000023.pdf.txt: 37101 bytes, checksum: 468b5bec7bde38bd684ddc8137cdd0a4 (MD5) Previous issue date: 2011-09-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) PROPPG-UEL Differential scanning calorimetry (DSC), thermogravimetry (TG) and Fourier-transform infra-red spectroscopy (FT-IR) analyses were performed to investigate changes in the physico-chemical properties of botryosphaerans, a family of exopolysaccharides (EPS) produced by the fungus Botryosphaeria rhodina MAMB-05 grown on glucose (EPS(GLC)), sucrose (EPS(SUC)) and fructose (EPS(FRU)). A slight endothermic transition and small mass loss attributable to the removal of water of hydration were observed in the DSC and TG analyses, respectively, for the three EPS samples. The FT-IR spectra confirmed no structural changes occurred during thermal treatment. Viscometry was utilized to obtain information on the rheological behaviour of the EPS in aqueous solutions. The Power Law and Cross Equations determined the natural pseudoplastic characteristics of the EPS. Comparatively, results obtained for EPS produced when B. rhodina MAMB-05 was grown on each of the three carbohydrate sources demonstrated similar apparent viscosity values for EPS(GLC) and EPS(SUC), while EPS(FRU) displayed the lowest apparent viscosity of the three botryosphaerans, suggesting a higher degree of ramification and lower Mw. EPS(GLC) and EPSSUC possessed similar degrees of ramification. The slight differences found in their viscosities can be explained by the differences in the type of branching among the three botryosphaerans, thus varying the strength of intermolecular interactions and consequently, consistency and viscosity. The physico-chemical studies of botryosphaerans represent the originality of this work, and the knowledge of these properties is an important criterion for potential applications. Univ Estadual Paulista, Fac Sci & Technol, Dept Phys Chem & Biol, BR-19060900 Presidente Prudente, Brazil Lakehead Univ, Biorefining Res Initiat, Thunder Bay, on P7B 5E1, Canada Univiversidade Estadual de Londrina (UEL), Dept Biochem & Biotechnol, CCE, BR-86051990 Londrina, Brazil Univ Fed Parana, Dept Biochem & Mol Biol, BR-81531990 Curitiba, Parana, Brazil Univ Estadual Paulista, Fac Sci & Technol, Dept Phys Chem & Biol, BR-19060900 Presidente Prudente, Brazil

Commercial oil-yielding seeds (castor, coconut, neem, peanut, pongamia, rubber and sesame) were collected from different places in the state of Tamil Nadu (India) from which 1279 endophytic fungi were isolated. The oil-bearing seeds exhibited rich fungal diversity. High Shannon-Index H' was observed with pongamia seeds (2.847) while a low Index occurred for coconut kernel-associated mycoflora (1.018). Maximum Colonization Frequency (%) was observed for Lasiodiplodia theobromae (176). Dominance Index (expressed in terms of the Simpson's Index D) was high (0.581) for coconut kernel-associated fungi, and low for pongamia seed-borne fungi. Species Richness (Chao) of the fungal isolates was high (47.09) in the case of neem seeds, and low (16.6) for peanut seeds. All 1279 fungal isolates were screened for lipolytic activity employing a zymogram method using Tween-20 in agar. Forty isolates showed strong lipolytic activity, and were morphologically identified as belonging to 19 taxa (Alternaria, Aspergillus, Chalaropsis, Cladosporium, Colletotrichum, Curvularia, Drechslera, Fusarium, Lasiodiplodia, Mucor, Penicillium, Pestalotiopsis, Phoma, Phomopsis, Phyllosticta, Rhizopus, Sclerotinia, Stachybotrys and Trichoderma). These isolates also exhibited amylolytic, proteolytic and cellulolytic activities. Five fungal isolates (Aspergillus niger, Chalaropsis thielavioides, Colletotrichum gloeosporioides, Lasiodiplodia theobromae and Phoma glomerata) exhibited highest lipase activities, and the best producer was Lasiodiplodia theobromae (108 U/mL), which was characterized by genomic sequence analysis of the ITS region of 18S rDNA.

Nine isolates of Botryosphaeria spp. were screened for lipases when cultivated on eight different plant seed oils and glycerol, and all produced lipases. Botryosphaeria ribis EC-01 produced highest lipase titres on soybean oil and glycerol, while eight isolates of Botryosphaeria rhodina produced significantly lower enzyme titres. B. ribis EC-01 produced lipase when grown on different fatty acids, surfactants, carbohydrates and triacylglycerols, with highest enzyme titres produced on Triton X-100-emulsified stearic (316.7 U/mL), palmitic (283.5 U/mL) and oleic (247.4 U/mg) acids, and soybean oil (105.6 U/mL), as well as castor oil (191.2 U/mg); an enhancement of 9-fold over soybean oil-grown cultures. Glycerol was also a good substrate for lipase production. The crude lipase extract was optimally active at pH 8.0 and 55 °C, stable between 30 and 55 °C and pH 1–10, and tolerant to 50% (v/v) glycerol, methanol and ethanol. The crude lipase showed affinity for substrates of short, average and long-chain fatty acids (different esters of p -nitrophenol and triacylglycerols). Zymograms developed with 4-methylumbelliferyl-butyrate showed two bands of lipolytic activity at 45 and 15 kDa. This is the first report on the production of lipases by B. ribis grown on these different carbon sources.

Botryosphaeran (EPS(FRU)), an exopolysaccharide of the beta-(1-->3,1-->6)-d-glucan type with 31% branching at C-6, is produced by the fungus Botryosphaeria rhodina MAMB-05 when grown on fructose as carbon source. Botryosphaeran was derivatized by sulfonation to induce anticoagulant activity. The effectiveness of the sulfonation reaction by chlorosulfonic acid in pyridine was monitored by the degree of substitution and FT-IR analysis of the sulfonated EPS(FRU) (once sulfonated, EPS(FRUSULF); and re-sulfonated, EPS(FRURESULF)). Activated partial thromboplastin time (APTT) and thrombin time (TT) tests of EPS(FRURESULF) indicated significant in vitro anticoagulant activity that was dose-dependent. EPS(FRU) did not inhibit any of the coagulation tests.

Botryosphaeran, a (1 → 3;1 → 6)-β- d -glucan produced by Botryosphaeria rhodina MAMB-05, was found to be present in a triple helix conformation from helix–coil transition studies using Congo Red. The triple helix conformation was disrupted at increasing alkali concentrations. Conformational changes were also observed using phenanthrene as a fluorescent probe, and the fluorescence intensity decreased 80% in the presence of dimethyl sulfoxide. The results confirmed the triple helix conformation of botryosphaeran, an important property manifesting biological response modifying activity.

Four exopolysaccharides (EPS) obtained from Botryosphaeria rhodina strains isolated from rotting tropical fruit (graviola, mango, pinha, and orange) grown on sucrose were purified on Sepharose CL-4B. Total acid hydrolysis of each EPS yielded only glucose. Data from methylation analysis and 13C NMR spectroscopy indicated that the EPS from the graviola isolate consisted of a main chain of glucopyranosyl (1→3) linkages substituted at O-6 as shown in the putative structure below: Download : Download full-size image The EPS of the other fungal isolates consisted of a linear chain of (1→6)-linked glucopyranosyl residues of the following structure: Download : Download full-size image FTIR spectra showed one band at 891 cm−1, and 13C NMR spectroscopy showed that all glucosidic linkages were of the β-configuration. Dye-inclusion studies with Congo Red indicated that each EPS existed in a triple-helix conformational state. β-(1→6)- d -Glucans produced as exocellular polysaccharides by fungi are uncommon.

Three d -glucans were isolated from the mycelium of the fungus Botryosphaeria rhodina MAMB-05 by sequential extraction with hot-water and hot aqueous KOH (2% w/v) followed by ethanol precipitation. Following their purification by gel permeation chromatography on Sepharose CL-4B, the structural characteristics of the d -glucans were determined by FT-IR and 13C NMR spectroscopy and, after methylation, by GC–MS. The hot-water extract produced a fraction designated Q1A that was a β-(1→6)- d -glucan with the following structure: Download : Download full-size image The alkaline extract, when subjected to repeated freeze–thawing, yielded two fractions: K1P (insoluble) that comprised a β-(1→3)- d -glucan with β- d -glucose branches at C-6 with the structure: Download : Download full-size image and K1SA (soluble) consisting of a backbone chain of α-(1→4)-linked d -glucopyranosyl residues substituted at O-6 with α- d -glucopyranosyl residues: Download : Download full-size image

Nine isolates of Botryosphaeria spp. were evaluated for their growth and the production of cell wall-lytic enzymes (laccase, pectinase and β-1,3-glucanase) when grown on basal medium in the absence and presence of the laccase inducer, veratryl alcohol (VA). The genetic relationship among the nine isolates collected from different host plants was determined by RAPD analyses. ITS sequence analysis showed eight closely related isolates classified as Botryosphaeria rhodina, and one isolate classified as Botryosphaeria ribis. RAPD analysis resolved the isolates into three main clusters based upon levels of laccase and β-1,3-glucanase activity. There appears to be no correlation between pectinase production and genetic diversity among the nine isolates. However, the strain characterized as B. ribis, positioned out of the main cluster, was found to be the highest producer of pectinases in the presence of VA.

Botryosphaeran is an exopolysaccharide (EPS) of the (1"3;1"6)-β-D-glucan type produced by the fungus Botryosphaeria rhodina MAMB-05. In attempts to enhance its extracellular production, the quantitative effects of different nitrogen sources, fermentation time, inoculum size, C/N ratio, and initial pH were evaluated on the yields of botryosphaeran. Ammonium nitrate was selected as the best nitrogen source in examining these effects using a 2³-factorial central-composite experimental design, and analysis by the response surface method. According to a factorial design, the most important variables influencing both botryosphaeran and mycelium biomass production was inoculum concentration and the time of growth. Under these conditions, optimum botryosphaeran production occurred at 88 h of growth, 0.88 g of mycelium L-1 of nutrient mediumat C/N ratio of 30, and resulted in a 5.2 g L-1 yield of EPS in the fermentation broth. For production of the mycelium biomass, the optimum condition occurred at the same time of growth and inoculum concentration; however the best C/N ratio was 75, and resulted in a yield of 33.8 g L-1 of biomass. The results also indicated that the maximal mycelial growth is not associated to the yields of botryosphaeran production by Botryosphaeria rhodina MAMB-05. DOI: http://dx.doi.org/10.17807/orbital.v7i1.637

Botryosphaeran, a (1 → 3;1 → 6)-β- d -glucan produced by Botryosphaeria rhodina , and laminarin were hydrolysed by two fungal β-glucanases predominantly of the 1,3-type produced by B. rhodina and Trichoderma harzianum Rifai grown on botryosphaeran as sole carbon source. Both β-glucanase preparations presented different modes of attack on botryosphaeran and laminarin. Laminarin was hydrolysed to the extent of ∼50% in 1 h and 100% within 24 h, and its hydrolysis products were mainly glucose and gentiobiose, and lesser amounts of laminaribiose and oligosaccharides of DP 3–4 during the early stages of hydrolysis, while botryosphaeran yielded mainly glucose and gentiobiose with some trisaccharide, but no laminaribiose or tetrasaccharide when hydrolysed by the T. harzianum enzyme. By contrast, B. rhodina β-1,3-glucanases produced predominantly glucose during all stages of botryosphaeran hydrolysis. Some physicochemical properties of the 1,3- and 1,6-β-glucanases, and β-glucosidases contained in the two fungal β-glucanase preparations are also described for the first time.

Botryosphaeria rhodina and Trichoderma harzianum Rifai were grown on botryosphaeran (an exopolysaccharide (EPS) of the β-1,3;1,6- d -glucan type produced by B. rhodina ) as sole carbon source with the objective of producing β-glucanases of the 1,3-type. Conditions for β-1,3-glucanase production by T. harzianum were examined by a statistical response surface method, and showed maximal enzyme production at 5 days growth in media containing 1.5 g/l of EPS. Good agreement was obtained between the experimental values of β-1,3-glucanase activity and the corresponding values predicted by the mathematical model. The crude β-1,3-glucanase preparations were active towards a number of different β-1,3-glucans and β-glucosides. The mycelium of B. rhodina also proved to be a good substrate for β-1,3-glucanase production by both fungal species.

The herbicide, Scepter, whose active principle is imazaquin, is commonly used in soybean farming to combat wide-leaf weeds. The basidiomycete, Pleurotus ostreatus , and the ascomycete, Botryosphaeria rhodina , were evaluated for their growth and laccase production when cultured on basal media containing Scepter. Both fungi could grow on the herbicide when cultivated in solid and submerged liquid culture in the presence of Scepter at concentrations of 0-6% (v/v) for P. ostreatus , and up to 0-50% (v/v) for B. rhodina , and in each case produced laccases when assayed against ABTS [2,2(1)-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)] and 2,6-dimethoxyphenol. P . ostreatus could tolerate up to 6% of Scepter before it became toxic to the fungus, while in the case of B. rhodina , 50% (v/v) Scepter was the highest amount that supported grow, and laccase activity was detectable up to 25% (v/v). An inverse relationship existed between the level of Scepter in the culture medium that supported fungal growth and laccase production. Analysis of the results showed that the fungi studied presented different behaviour towards Scepter in the culture environment.

The influence of glucose concentration and other carbohydrates (monosaccharides: fructose, galactose, mannose; polyols: mannitol and sorbitol; disaccharides: lactose, sucrose and commercial sucrose; and industrial sugarcane molasses) were compared as sole carbon sources for the production of Botryosphaeran, an exopolysaccharide (EPS) produced by Botryosphaeria sp. The optimum glucose concentration for EPS production was 50 g l(-1). With the exception of mannitol, the fungus produced EPS on all carbon sources studied, with highest yields occurring with sucrose followed by glucose. All EPS showed exclusively glucose after acid hydrolysis and monosaccharide analysis. FTIR spectroscopy demonstrated the presence of beta-anomers indicating that all the EPS produced by Botryosphaeria sp. on the different carbon sources were essentially of the beta-D-glucan type.

The exopolysaccharide, Botryosphaeran, produced by the ligninolytic, ascomyceteous fungus Botryosphaeria sp., was isolated from the extracellular fluid by precipitation with ethanol, and purified by gel permeation chromatography to yield a carbohydrate-rich fraction (96%) composed mainly of glucose (98%). Infra-red and 13 C NMR spectroscopy showed that all the glucosidic linkages were in the β-configuration. Data from methylation analysis and Smith degradation indicated that Botryosphaeran was a (1→3)-β- d -glucan with approx 22% side branching at C-6. The products obtained from partial acid hydrolysis demonstrated that the side branches consisted of single (1→6)-β-linked glucosyl, and (1→6)-β-linked gentiobiosyl residues.

Demethylation of industrial lignin has been for long coveted as a pathway to the production of an abundant natural substitute for fossil-oil derived phenol. In an attempt to possibly identify a novel Kraft lignin-demethylating enzyme, we surveyed a collection of fungi by using selected ion flow tube-mass spectrometry (SIFT-MS). This method readily identifies methanol resulting from lignin demethylation activity. Absidia cylindrospora, and unidentified Cylindrocladium sp. and Aspergillus sp. were shown to metabolize lignin via different pathways, based on the HPLC analysis of lignin fragments. Of these three, Cylindrocladium and Aspergillus were shown to retain most of the lignin intact after 3 weeks in culture, while removing about 40% of the available methoxy groups. Our results demonstrate that after optimization of culture and lignin recovery methods, biological modification of Kraft lignin may be a feasible pathway to obtaining demethylated lignin for further industrial use.

Four virulent strain isolates of the fungus, Bipolaris euphorbiae (previously identified as a Helminthosporium sp.), isolated from host plants in four states within Brazil were screened for the production of phytotoxins that promoted wilting and defoliation of the Brazilian weed, Euphorbia heterophylla, commonly found growing among soyabean crops. Only one isolate, B. euphorbiae Strain I (EUPH petropar from Mato Grosso state), produced phytotoxin in-vitro when grown in stationary culture for 7 d at 28 ° C on minimum salts medium supplemented with 1.5 % glucose as the sole carbon source. Phytotoxin was also produced when the fungal strain was grown on fructose, galactose, mannose, xylose and sucrose. The addition of nitrogen source (yeast extract, peptone or malt extract) to the culture medium did not influence phytotoxin production. The phytotoxin produced by Strain I was most active at pH 6.0, stable between pH 3-9, and was highly thermostable, remaining fully active when heated at 90 ° C for 1 h.

The concentration at which 50% inhibition (I50) of fungal growth occurred for various lignin-related compounds was evaluated on Botryosphaeria sp. MAMB-5 cultivated on agar medium. A trend for inhibition of growth on lignin-related aromatic acids and phenolic compounds was observed and was based upon chemical structural features: benzoic acids containing di-methoxyl substituents on the aromatic ring were less inhibitory than the parent unsubstituted compound; p-phenolic benzoates containing a single methoxyl group were less inhibitory than those containing a di-methoxyl group; methoxylated derivatives of p-phenolic cinnamic acids were less inhibitory than the unsubstituted parent compound; but un-methylated hydroxyls on di-phenols allowed growth compared to the mono- and di-O-methylated derivatives, which were inhibitory. Botryosphaeria sp. grew on basal media containing up to 10% (w/v) lignosulfonate and kraft lignin in stationary liquid culture. In submerged liquid cultivation, Botryosphaeria sp. grew on basal medium containing abietic acid, catechol, 4-chlorophenol, guaiacol, vanillyl alcohol, veratraldehyde and lignosulfonate, and in each case produced laccases. The production of laccases implicated a possible role for their involvement in the biodegradation of aromatic compounds by Botryosphaeria sp. 4-Chlorophenol was the most toxic of the compounds examined.

The chemical composition and structural characterization of exopolysaccharides from the fungus Lasiodiplodia theobromae MMBJ are described, and the immunomodulatory activity of a purified β-glucan was evaluated. L. theobromae MMBJ produced three different β-glucans. One, fraction PEPS, was a branched (1→3)(1→6)-β-glucan and was insoluble in cold water. The other two, fractions SEPS-005R and SEPS-10E, were characterized as linear (1→6)-β-glucans with molar mass of 1.8 × 10 6 Da and 7.0 × 10 3 Da, respectively. From a total of 2.2 g/L of EPS produced by L. theobromae through submerged fermentation, 1.5 g/L (67%) was of the branched (1→3)(1→6)-β-glucan, while 25% (w/w) were linear (1→6)-β-glucans. Tests conducted with macrophages showed that the high molar mass (1→6)-β-glucan fraction (SEPS-005R) induced a pro-inflammatory response pattern.