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White-rot fungi are efficient degraders of the lignin moiety due to the production of extracellular enzymes that includes the laccases which are able to depolymerize lignin oxidative processes to low-molecular weight phenolic and aromatic compounds. These end-products have important industrial applications, as building blocks for chemical syntheses of high-value. Thus, the objective of this work was to optimize the culture conditions for laccase production by Pleurotus ostreatus Florida grown on Kraft lignin, and to evaluate lignin biodegradation products. The fungal strain produced optimum laccase titers after evaluation in a 33-factorial statistical design by varying: lignin concentration, yeast extract and time of cultivation. The extracellular fluid obtained under the optimized culture condition was treated by liquid-liquid partition with ethyl acetate in order to determine the lignin degradation products by LC-MS/MS. Concentration of lignin was the main variable that increased laccase production assayed against substrate 2,6-dimethoxyphenol. Optimal laccase production predicted by the model was 5.73 U/mL under the condition: 0.87% (w/v) lignin, 4.5% (w/v) yeast extract and 10 days cultivation. The experimental laccase titer obtained following optimal conditions was 5.68 U/mL. The lignin metabolites identified were 4-methoxycinnamaldehyde and cuminaldehyde. In addition, abscisic acid was produced; considered a stress-related hormone product of the fungus. These findings show a suitable source of fungal laccase for obtaining new metabolites from the biodegradation of Kraft lignin by fermentation. more...

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. more...

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. more...

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. more...

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. more...

In this work, a modification of a glassy carbon electrode (GCE) is presented for the first time using the polysaccharide carboxymethyl-botryosphaeran (CMB) and nanostructured carbon black (CB). The combination of the carbohydrate biopolymer and the carbonaceous nanomaterial (CMB-CB/GCE) showed improvements in the voltammetric responses of dopamine (DOP) and paracetamol (PAR) when compared to unmodified GCE. The morphology of the proposed sensor was characterized by scanning electron microscopy. The electrochemical characterization was performed by cyclic voltammetry and electrochemical impedance spectroscopy using the redox pair [Fe(CN)6]3−/4–. Using phosphate buffer solution (pH 7.0) and differential pulse voltammetry, DOP and PAR were determined simultaneously in the concentration range 0.099–2.9, and 0.70–19 μmol L–1, with limits of detection of 0.013, and 0.11 μmol L–1, respectively. The developed method with the fabricated electrochemical sensor is simple, fast, and sensitive. The proposed sensor was successfully applied to the individual and simultaneous determination of DOP and PAR in various samples, showing the practical feasibility of the proposed sensor as a novel platform for the sensitive determination of these phenolic compounds. more...

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. more...

A photoelectrochemical biosensing strategy for the highly sensitive detection of the flavonoid rutin was developed by synergizing the photoelectrocatalytic properties of hematite (α-Fe2O3) decorated with palladium nanoparticles (PdNPs) and the biocatalysis towards laccase-based reactions. The integration of α-Fe2O3.PdNPs with a polyphenol oxidase as a biorecognition element yields a novel biosensing platform. Under visible light irradiation, the photoactive biocomposite can generate a stable photocurrent, which was found to be directly dependent upon the concentration of rutin. Under the optimal experimental conditions, the cathodic photocurrent, measured at 0.33 V vs. Ag/AgCl, from the square-wave voltammograms presented a linear dependence on the rutin concentration within the range of 0.008–30.0 × 10−8 mol L−1 (sensitivity: 1.7 μA·(× 10−8 M−1)·cm−2), with an experimental detection limit (S/N = 3) of 8.4 × 10−11 mol L−1. The proposed biosensor device presented good selectivity towards rutin in the presence of various organic compounds and inorganic ions, demonstrating the potential application of this biosensing platform in complex matrices. This bioanalytical device also exhibited excellent operational and analytical properties, such as intra-day (standard deviation, SD = 0.21%) and inter-day (SD = 1.30%) repeatability, and long storage stability (SD = 2.80% over 30 days). Graphical abstract more...

A hydrogel containing exocellular (1 → 6)-β-D-glucan (lasiodiplodan, LAS) was developed and its wound healing potential was evaluated. β-Glucans have attracted much interest by the cosmetic industry sector because of their bioactive and functional properties and in promoting skin health. In the present work an β-glucan was studied as a healing biomaterial that has not hitherto been reported in the scientific literature. LAS produced by the ascomycete Lasiodiplodia theobromae MMPI was used in the formulation of a healing hydrogel. Physicochemical and microbiological quality parameters, antioxidant potential and stability of the formulation was evaluated. FTIR, thermal analysis and SEM techniques were also employed in the characterization. Wistar rats were used as a biological model to investigate the wound healing potential. Histological analyses of cutaneous tissue from the dorsal region were conducted after 4, 7, 10 and 14 days of treatment, and evaluated re-epithelialization, cell proliferation and collagen production. Physicochemical stability, microbiological quality and antioxidant potential, especially in relation to its ability to scavenge hydroxyl radicals were found. The hydrogel stimulated cell re-epithelialization and proliferation during all days of the treatment, and stimulated an increase of collagen fibers. Lasiodiplodan showed immunomodulatory activity in wound healing and this biomacromolecule could be an alternative compound in wound care. more...

Studies demonstrate that obesity can increase tumor development. Botryosphaeran, a fungal (1→3)(1→6)-β-D-glucan, presents antimutagenic, antiproliferative and pro-apoptotic activities. This study evaluated the effects of botryosphaeran on tumor development and metabolic and hematological parameters in tumor-bearing obese and non-obese rats. Obesity was induced by a high-fat and high-sugar diet, while control rats received standard diet and water without sugar for 10 weeks. On 8th-week, Walker-256 tumor cells were inoculated in the rats, and treatment with botryosphaeran (12 mg/Kg b.w.) started. Groups:control tumor-CT, control tumor botryosphaeran-CTB, obese tumor-OT and obese tumor botryosphaeran-OTB. On 10th-week, tumor development, cachexia, metabolic and hematological parameters were analyzed. Tumor development and cachexia were significantly higher in the OT group compared to the CT group, and botryosphaeran attenuated these parameters. OT rats presented accumulation of adipose tissue, reduced muscle mass, glucose intolerance, insulin resistance, hyperglycemia, anemia, leukocytosis, and thrombocytopenia. Botryosphaeran corrected insulin resistance and hyperglycemia, modulated cholesterol levels, and increased leukocyte and lymphocytes in obese rats, which can be attributable to an inflammatory response against the Walker-256 tumor, contributing to a lower tumor development. Our data demonstrated that botryosphaeran was effective in attenuating tumor growth and in improving the metabolic and hematological profiles of the tumor-bearing rats, demonstrating its potential role in the cancer's management. more...

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. more...

Surface-enhanced Raman spectroscopy (SERS) became a useful analytical technique with the development of appropriate metallic substrates. The need for SERS substrates that immobilize metallic nanoparticles prompted this work to search for an appropriate material. This work presents the preparation, characterization and application of a SERS substrate for crystal violet (CV) detection, as the probe molecule. The inner layer of the substrate is a thin film of the fungal β-D-glucan, botryosphaeran, covered by a thin layer of silver nanoparticles (AgNPs). The nanoparticles were produced by laser ablation, a fast and clean method for their preparation, and the layers were assembled by casting. Scanning electron and atomic force microscopies, UV–VIS and Raman spectroscopy and X-ray diffraction allowed the characterization of the surface of the substrate. Analysis by Raman spectroscopy showed promising results for SERS amplification on the substrate. Detection of CV reached enhancement factors up to 106 orders of magnitude, compared to normal Raman spectra. Linearity was observed for analyses on the SERS substrate at concentration ranges of 0.005 to 1 µmol L-1. The assembly reached the detection of 12 pmol cm−2 of CV, which corresponds to 96 fg of the probe molecule contained in the area of the substrate effectively interacting with the laser. The substrate was more efficient than silver colloids to perform SERS. more...

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. more...

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. more...

Okara is a by-product of soybean processing produced in large quantities, but under-used by the food industry. Strategies to increase its nutritional quality could contribute to the usage of this biomass. Fresh and heat-treated okara were subjected to solid-state fermentation (SSF) using S. cerevisiae and physicochemical parameters of quality, total phenolics, β-glucosidase activity, antioxidant activity and the biotransformation of isoflavone were evaluated. Fermentation contributed to the improvement of nutritional quality, promoting an increase in protein content, and increases in total phenolics, and antioxidant activity. SSF also promoted the bioconversion of isoflavone β-D-glucosides to aglycone forms through the action of β-glucosidases. Furthermore, SSF led to the reduction of raw fiber content with a potential improvement in digestibility. SSF was demonstrated to be a valuable tool in improving quality and functional attributes, and in adding value to okara, thus contributing to its better usage in the development of food products and supplements. more...

The synthesis of transformed products from 2,6-dimethoxyphenol (2,6-DMP) can be selectively obtained through controlled oxidative coupling in laccase-catalyzed reactions, and the products formed are dependent upon the enzyme source, as well as, upon the reactions conditions applied. The laccase from Botryosphaeria rhodina MAMB-05 was investigated for its ability to catalyze the oxidative transformation of 2,6-DMP. Studies conducted in aqueous medium at pH 3.0 and 6.5, for reaction times of 48, 96 and 144 h, led to the formation of three dimers (I, II, III) via carbon-carbon and carbon-oxygen coupling reactions depending upon the pH and reaction time. Dimer I was identified as 3,3′,5,5′-tetramethoxydiphenylquinone by 13C NMR, while dimer II was identified by 1H NMR as 3,3′,5,5′-tetramethoxybiphenyl-4,4′-diol (TMBP). Dimer III was synthesized at pH 3.0 and was influenced by the reaction time (96h), and is predicted to be formed via carbon-oxygen coupling. The chemical structure of III (4-(2,6-dimethoxy-phenoxy)-2,6-dimethoxyphenol) was identified by ESI-Q-TOF HRMS. TMBP (II) was the only product obtained at pH 6.5 independent of the reaction times. TMBP synthesis at pH 6.5 was optimized by design-matrix using a three-factor Box-Behnken incomplete factorial-design that defined the parameters: 2,6-DMP concentration (2.6–3.4 mmol) laccase activity (1.35–3.38 U) and reaction time (120 h) to give a yield of 11.93 ± 0.49%, which agreed with the mathematical model value of 12%. TMBP was evaluated as an antioxidant to stabilize biodiesel, and showed an efficacy similar to the commercial antioxidant standard, butyl hydroxytoluene, indicating that TMBP could serve as an alternative antioxidant to stabilize biodiesel. more...

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. more...

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. more...

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. more...

Laccase from Botryosphaeria rhodina MAMB-05 was covalently immobilized on carboxymethyl-botryosphaeran by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide (EDC/NHS) in aqueous solution. This approach was employed to fabricate a novel laccase-based biosensor to electrochemically quantify quercetin (QCT), using a simple carbon black paste electrode as a transducer. The proposed biosensor was characterized by electrochemical impedance spectroscopy and Nyquist plots were used to evaluate the immobilization of the enzyme. For determining QCT, variables were optimized, that included experimental conditions for laccase immobilization, pH of the supporting electrolyte, and instrumental parameters of the electroanalytical technique. From square-wave-voltammograms, a linear dependence between the cathodic current peak and QCT concentration was observed within the range 4.98–50.0 × 10−8 mol L−1, with a theoretical detection limit of 2.6 × 10−8 mol L−1. The proposed method was successfully applied to determine QCT in beverages, pharmaceuticals, and biological samples. The proposed biosensor device presented good selectivity in the presence of uric acid, various inorganic ions, as well as other phenolic compounds, demonstrating the potential application of this biosensing platform in chemically complex solutions. Operational and analytical stability of the laccase-biosensor were evaluated, and good intra-day (SD = 1.23%) and inter-day (SD = 2.32%) repeatability, and long storage stability (SD = 3.47%) are presented. more...

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. more...

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. more...

&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. more...

β-Glucans are biomacromolecules of industrial interest for its biological and technological properties, including ability to modify the rheology of food systems and different biological functionalities. In this study, lasiodiplodan an exocellular polysaccharide of the (1→6)-β-D-glucan type produced by the fungus Lasiodiplodia theobromae MMPI was obtained in a bench-scale bioreactor operated in discontinuous mode. Lasiodiplodan produced was derivatized by carboxymethylation and characterized by Fourier Transform Infrared spectroscopy (FT-IR), Thermogravimetry and Differential Thermal Analysis (TG-DTA), X-Ray Diffraction (XRD), scan electron microscopy (SEM) and their antioxidant and antimicrobial potential also was assessed. Two strong absorption bands in the regions of 1422 to 1598 cm-1, resulting from symmetric and asymmetric stretching vibrations of the -COO- group respectively, were observed in the FT-IR spectrum of the derivatized sample and indicated the carboxymethylation of the macromolecule. TG-DTA curves indicated that native (LAS-N) and carboxymethylated (LAS-C) lasiodiplodan did not suffer significant changes in relation to the heating rates. Native and carboxymethylated lasiodiplodan demonstrated high thermal stability considering the usual standards of the pharmaceutical industry. The final temperature of thermal decomposition increased when the heating rate was increased and less number of steps for thermal decomposition was observed when air was substituted for nitrogen. XRD analysis showed the LAS-N and LAS-C have no crystalline structure, but carboxymethylation led to the arising of regions with certain molecular orientation in biopolymer structure. SEM analysis showed that the carboxymethylation promoted changes in biopolymer macrostructure, including breaking of the polymeric structure and arising of bubbles on the surface area. Carboxymethylation contributed to improving the polysaccharide's antioxidant capacity and LAS-C demonstrated antimicrobial activity against Candida tropicalis. more...

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 more...

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. more...

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). more...

Sulfated polysaccharides are known to display activity against enveloped viruses, such as herpes and dengue. The aim of this work was to assess the antiviral activity of botryosphaeran, a fungal exocellular (1 → 3)(1 → 6)-β- d -glucan devoid of sulfate groups, and its chemically sulfonated derivatives, against herpes simplex virus (HSV), dengue virus (DENV) and poliovirus (PV). The natural parent polysaccharide inhibited acyclovir-sensitive HSV (HSV-KOS) infection in Vero cells (IC 50 of 39.3 μg mL −1 ), while the IC 50 against acyclovir-resistant HSV (HSV-AR) was 47.5 μg mL −1 . Botryosphaeran was derivatized by sulfonylation with chlorosulfonic acid to prepare two sulfonated derivatives, S1 and S2, with degrees of substitution (DS) of 0.4 and 1.1, respectively. Antiviral evaluation of S1 and S2 gave the IC 50 of 3.0 and 2.4 μg mL −1 against HSV-KOS, and 7.3 and 2.7 μg mL −1 against HSV-AR, respectively. This study demonstrated for the first time that native botryosphaeran inhibited HSV infection, albeit moderately, while its sulfonated derivatives developed high activity against viral infection. DENV inhibition was weak for botryosphaeran, but remarkably stronger for S1 and S2. All compounds were inactive against PV, as it lacked a viral envelope. The presence of sulfate groups and the DS were confirmed to be important features for antiviral activity. more...

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. more...

Laccase was immobilized on a glassy carbon electrode layered with multi-walled carbon nanotubes using a film of botryosphaeran, a fungal (1 → 3)(1 → 6)-β-D-glucan. This novel biosensing platform was characterized by electrochemical impedance spectroscopy and scanning electron microscopy, and applied for the determination of dopamine. Experimental variables such as enzyme concentration, pH value and operational parameters of the electroanalytical technique were optimized. Using square-wave voltammetry, there was a linear dependence of peak current and dopamine concentration within the range of 2.99–38.5 μmol L −1 with a limit of detection of 0.127 μmol L −1 . The biosensor was successfully applied in the determination of dopamine in pharmaceutical injection and synthetic biological samples, and presented good selectivity even in the presence of uric acid and ascorbic acid, as well as other phenolic compounds. The different aspects regarding the operational stability of the laccase biosensor were evaluated, demonstrating good intra-day and inter-day repeatability, and long-storage stability. Furthermore, this biosensor was evaluated in the indirect determination of spironolactone by using the analytical signal of dopamine, presenting a limit of detection of 0.94 μmol L −1 . The results obtained in the analysis of spironolactone in commercial pharmaceutical samples were satisfactory. more...

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. more...

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. more...

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. more...

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 more...

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). more...

Gourmet vinegars are versatile culinary ingredients that have aroused the interest of consumers not only for their unusual taste, but also for their health benefits. In this work, blueberry and honey wine was first obtained using a bench-scale bioreactor. This wine had high concentrations of phenolic compounds (706.18 mg GAE/mL) and appreciable free radical scavenging ability against DPPH (161.42 µmol trolox equivalent/100 mL) and ABTS•+ (356.41 µmol trolox equivalent/100 mL), as well as ferric-ion reducing antioxidant capacity (1419.86 µmol FeSO4/100 mL). In sequence, vinegar was produced from the wine in wooden barrels using successive acetification cycles. The average acetic acid concentration found in the vinegars was 4.4 g/100 mL, and the average acetic acid yield and volumetric productivity were 52.69% and 0.38 g/(L.h), respectively. Appreciable polyphenolic compounds (681.87 to 847.98 µg GAE/mL) and anthocyanin (12.8 to 15.39 mg/L) contents were found and high antioxidant activity. In addition, the vinegars demonstrated antimicrobial ability against Bacillus subtilis and Salmonella enterica Typhimurium. more...

Aims Obesity is associated with comorbidities such as diabetes and hepatic steatosis. β-Glucans have been described as effective in treating conditions including dyslipidaemia and diabetes. Thus, the objective of this study was to evaluate the effects of botryosphaeran [(1 → 3)(1 → 6)-β-D-glucan] on obesity and its comorbidities, and understand its mechanism of action. Main methods Obesity was induced in adult male Wistar rats by ingestion of a high-fat diet and water with sucrose (300 g/L) for 8 weeks. Control rats received standard diet. After six weeks, treatment commenced with botryosphaeran (12 mg/kg.b.w., via gavage, 15 days), respective controls received water. Rats were divided into 3 groups: control (C), obese (O), and obese + botryosphaeran (OB). In the 8th week, obesity was characterized. Feed-intake, glucose and lipid profiles, glucose tolerance, and concentrations of glycogen and lipids in liver were analyzed. Protein expression was determined by Western blotting. Key findings Obese rats showed significant increases in weight gain and adipose tissue, presented glucose intolerance, dyslipidaemia, and hepatic steatosis. Botryosphaeran significantly reduced feed intake, weight gain, periepididymal and mesenteric fat, and improved glucose tolerance. Botryosphaeran also reduced triglyceride and VLDL, and increased HDL levels. Furthermore, botryosphaeran increased glycogen and reduced total lipids, triglycerides and cholesterol in liver, and increased AMP-activated protein kinase(AMPK) activity and Forkhead transcription factor 3a(FOXO3a) protein expression in adipose tissue. Significance This study demonstrated that botryosphaeran was effective in reducing obesity, hepatic steatosis, dyslipidaemia insulin resistance and glucose intolerance in diet-induced obese rats, and these effects were, at least in part, associated with reduced feed intake, and AMPK and FOXO3a activities. more...

A rapid and sensitive method has been devised in order to detect and estimate the synthesis of the enzyme alcohol oxidase (AOX) by fungi, by way of the use of two chemical complexes, namely, acetylacetone (3,5-diacetyl-1,4-dihydrolutidine) and acetylacetanilide (3,5-di- N-phenylacetyl-1,4-dihydrolutidine). This method involves the use of the AOX enzyme that could specifically oxidize methanol, giving rise to equimolar equivalents each of formaldehyde (HCHO) and hydrogen peroxide (H2O2) as the end products. Further, the formaldehyde, thus produced was allowed to interact with the neutral solutions of acetylacetone and the ammonium salt, gradually developing a yellow color, owing to the synthesis and release of 3,5-diacetyl-1,4-dihydrolutidine (yellow product; λ = 420 nm; λex/em = 390/470 nm) and the product, so generated was quantified spectrophotometrically by measureing its absorbance at 412 nm. In another set up, the amount of formaldehyde produced as a sequel to the oxidation of methanol by the AOX enzyme was determined by allowing it to react with the acetylacetanilide reagent, after which the volume of the fluorescent product - 3,5-di- N-phenylacetyl-1,4-dihydrolutidine (colorless product; λex/em = 390/470 nm) that was generated was estimated by measuring its emission at 460 nm (excitation wavelength at 360 nm) in a spectrophotometer. Of the various substrates tested, a commercial source of the AOX enzyme appreciably oxidizes methanol, thereby generating formaldehyde, and further reacts with acetylacetone, to give rise to a bright yellow complex, displaying a maximum activity of 1402 U/mL. Determination of the AOX activity by the use of acetylacetone and acetylacetanilide could serve as a viable alternative to the conventional alcohol oxidase-peroxidase-2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid (AOX-POD-ABTS) based method. In view of this, this method appears to be invaluable for application at the various food, pharmaceutical, fuel, biosensor, biorefinery, biopolymer , biomaterial, platform chemical, and biodiesel industries. more...

β-Glucans are biomacromolecules that present biological properties of medical and pharmacological interest. The chemical modification of the primary structure of these carbohydrate biopolymers is a way to enhance or achieve new biological properties. Acetylated derivatives of (1→6)-β-D-glucan (lasiodiplodan) with different degrees of substitution (0.48, 0.66, 1.03 and 1.26) were obtained and characterized by infra-red and NMR (nuclear magnetic resonance) spectroscopy, thermal analysis, X-ray diffraction and antioxidant capacity. Acetylation was confirmed by FT-IR, and 13C NMR spectroscopy. Thermal analysis indicated that unmodified lasiodiplodan and the O-acetylated -glucan derivative of degree of substitution 0.48 presented three stages of mass-loss, whereas acetylated derivatives of DS (degree of substitution) of 0.66, 1.03 and 1.26 presented four stages of mass-loss. X-ray diffractograms demonstrated that both native and acetylated lasiodiplodan presented crystalline regions in an amorphous polymeric matrix. Scanning electron microscopy revealed that O-acetylation promoted morphologic changes in the biopolymer according to the DS. Acetylation also contributed to improve antioxidant capacity. more...

Fungal β- d -glucans of the (1 → 3)-type are known to exhibit direct antitumor effects, and can also indirectly decrease tumor proliferation through immunomodulatory responses. The underlying molecular mechanisms involved in decreasing tumor formation, however, are not well understood. In this study, we examined the antiproliferative role and mechanism of action of three different fungal exocellular β-glucans in MCF-7 breast cancer cells. The β-glucans were obtained from Botryosphaeria rhodina MAMB-05 [two botryosphaerans; (1 → 3)(1 → 6)-β- d -glucan; one produced on glucose, the other on fructose] and Lasiodiplodia theobromae MMPI [lasiodiplodan; (1 → 6)-β- d -glucan, produced on glucose]. Using the cell proliferation-MTT assay, we showed that the β-glucans exhibited a time- and concentration-dependent antiproliferative activity (IC 50 , 100 μg/ml). Markers of cell cycle, apoptosis, necrosis and oxidative stress were analyzed using flow cytometry, RT-PCR and Western blotting. Exposure to β-glucans increased apoptosis, necrosis, oxidative stress, mRNA expression of p53, p27 and Bax; the activity of AMP-activated protein-kinase, Forkhead transcription factor FOXO3a, Bax and caspase-3; and decreased the activity of p70S6K in MCF-7 cells. In the presence of hydrogen peroxide, the fungal β-glucans increased oxidative stress, which was associated with reduced cell viability. We showed that these β-glucans exhibited an antiproliferative effect that was associated with apoptosis, necrosis and oxidative stress. This study demonstrated for the first time that the apoptosis induced by β-glucans was mediated by AMP-activated protein-kinase and Forkhead transcription factor, FOXO3a. Our findings provide novel mechanistic insights into their antiproliferative roles, and compelling evidence that these β-glucans possess a broad range of biomodulatory properties that may prove useful in cancer treatment. more...

In this study on the valorization of hemicelluloses (a co-product generated during cellulosic bioethanol production), prehydrolyzates obtained from poplar woodchips pretreated in an industrial experimental steam-explosion pilot-plant facility were evaluated for the production of bioxylitol using the yeast, Candida guilliermondii FTI 20037, employing both batch and fed-batch fermentation modes in shake flasks on defined nutrient medium. The prehydrolyzates consisted of monosaccharides (pentose and hexose sugars) as well as xylo-oligosaccharides and undegraded hemicellulose. Xylose (31.6 ± 0.57 g/L) was the major sugar in the prehydrolyzates that also contained acetic acid and degradation products of lignin and sugars (phenolic and furanic compounds). Xylose in the prehydrolyzates could be further increased (106.4 ± 0.02 g/L) through an acid hydrolysis step (0.6 % (w/v) H2SO4). Compounds of a toxic nature in both the acid hydrolyzates and prehydrolyzates were removed by treatment with Amberlite IRA-400 resin (chloride form). Batch fermentation of pure xylose and poplar prehydrolyzate resulted in bioxylitol production of 9.9 ± 0.01 and 4.9 ± 0.17 g/L, respectively, indicating that the poplar prehydrolyzates exhibited an inhibitory effect on fermentation. After detoxification of the poplar prehydrolyzates, bioxylitol production increased to 8.9 ± 0.01 g/L. Fed-batch fermentation of the prehydrolyzate increased the bioxylitol production to 12.39 ± 0.33 g/L, while acid hydrolysis followed by detoxification resulted in a maximum bioxylitol production of 22.0 ± 0.01 g/L, a 348 % increase. The results demonstrated that acid hydrolysis and detoxification followed by fed-batch fermentation was an efficient way to produce bioxylitol from poplar prehydrolyzates. more...

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. more...

There is growing interest in the anticancer and immunomodulatory potential of fungal β-d-glucans. In the present study, the modulation of gene expression via RT-qPCR and cell cycle kinetics via flow cytometry were assessed in human normal and tumor (Jurkat) lymphocytes after treatment with botryosphaeran (a fungal (1→3)(1→6)-β-d-glucan) from Botryosphaeria rhodina MAMB-05. Cell cultures were treated with botryosphaeran either alone, or in combination with doxorubicin (DXR), in a post-treatment protocol. The expression of genes involved in immunomodulatory processes, apoptosis and cell cycle control, as well as β-d-glucans cell receptors were assessed. Flow cytometry analysis identified tetraploid Jurkat cells in G1 phase when treated with botryosphaeran combined with DXR. This antiproliferative effect in G1 may be associated with down-regulation of the expression of genes involved in the G1 checkpoint. The repression of the CCR5 gene following botryosphaeran treatment, either alone or in combination with DXR, in tumor lymphocytes indicates a possible affinity of this particular (1→3)(1→6)-β-d-glucan for the receptor CCR5. Therefore, botryosphaeran action appears to be involved in the repression of genes related to the G1 phase of the cell cycle and possibly in the interaction of the botryosphaeran, either alone, or in combination with DXR, with the CCR5 receptor. more...

Development of high-valued products through an integrated biorefinery approach is essential for the revival of the Canadian forest industry. Utilization of tree bark is one such example with considerable potential. The bark of trees contains a reservoir of phytochemicals that could be harnessed as value-added co-products. The objective of this study was to identify, characterize, and seek commercial potential of phytochemicals present in poplar bark. Industrially harvested poplar bark (Populus tremuloides) was procured from Quebec. Water-soaked poplar bark was subjected to three steam-explosion treatments (treatments 1 to 3) using a steam-explosion reactor. During the experiment, extracts such as air purge, pre-steam purge, cooked purge, and pressed liquid were collected and subjected to compositional analysis. The results showed that the leftover bark residue even after the treatments was still fit for regular combustion for generating heat. Among the several identified phytochemicals (>100), furfural, 5-HMF, benzoic acid, salicylic acid, and catechol can be obtained as bulk chemicals, and sakuranin present at 15.9 % (extracted with acetone) as a high-valued and fine chemical. Considering the increasing demand for plant-based ingredients, there exists enormous potential to utilize these phytochemicals in agro-pharmaceutical industries. Further end-use studies, however, are required to ascertain the true economic potential of poplar bark phytochemicals. more...

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. more...

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. more...

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. more...