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AIM: The main objective of the study was to develop and validate a model for the growth of Aspergillus brasiliensis on surfaces, specifically on agar culture medium. An additional aim was to determine conditions for complete growth inhibition of this micromycete using two different nonthermal plasma (NTP) sources. METHODS AND RESULTS: The developed model uses two key parameters, namely the growth rate and growth delay, which depend on the cultivation temperature and the amount of inoculum. These parameters well describe the growth of A. brasiliensis and the effect of NTP on it. For complete fungus inactivation, a single 10-minute exposure to a diffuse coplanar surface barrier discharge was sufficient, while a point-to-ring corona discharge required several repeated 10-minute exposures at 24-h intervals. CONCLUSIONS: The article presents a model for simulating the surface growth of A. brasiliensis and evaluates the effectiveness of two NTP sources in deactivating fungi on agar media.

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Glucoamylases are exo-enzymes that cleave the ends of the starch chain, releasing glucose units. In the current work, we described a novel 1,4-α-glucoamylase from an A. brasiliensis strain isolated from an environmental sample. The purified glucoamylase, GlaAb, has a molecular mass of 69 kDa and showed a starch binding domain. GlaAb showed a similar sequence to other fungal glucoamylases, and the molecular 3D model analysis of GlaAb suggests an overall structure as described in the literature, except by elongation in the loop connecting the 4th and 5th α-helices. The enzyme showed activity over a wide range of pH and temperature, with maximum activity at pH 4.5 and 60 °C. GlaAb was stable at 50 °C for 7 h, maintaining 67% residual activity, and it was not inhibited by glucose up to 0.1 M. The glucoamylase was 65% more active in the presence of Mn2+ and showed a Km of 2.21 mg mL-1, Vmax of 155 U mg-1, Kcat 179 s-1, and Kcat/Km 81.06 mg mL-1 s-1 using potato starch as substrate. The results obtained are promising and provide the basis for the development of applications of GlaAb in the industrial process.

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The incidence of Candida glabrata infections has rapidly grown and this species is among those responsible for causing invasive candidiasis with a high mortality rate. The diterpene ent-hardwickiic acid is a major constituent in Copaifera pubiflora oleoresin and the ethnopharmacological uses of this oleoresin by people from Brazilian Amazonian region point to a potential use of this major constituent as an antimicrobial. Therefore, the goal of this study was to evaluate the antifungal activity of ent-hardwickiic acid against Candida species and to produce derivatives of this diterpene by using microbial models for simulating the mammalian metabolism. The microbial transformations of ent-hardwickiic acid were carried out by Aspergillus brasiliensis and Cunninghamella elegans and hydroxylated metabolites were isolated and their chemical structures were determined. The antifungal activity of ent-hardwickiic acid and its metabolites was assessed by using the microdilution broth method in 96-well microplates and compared with that of fluconazole. All the diterpenes showed fungistatic effects (ranging from 19·7 to 75·2 µmol l-1 ) against C. glabrata at lower concentrations than fluconazole (163·2 µmol l-1 ) and were more potent fungicides (ranging from 39·5 to 150·4 µmol l-1 ) than fluconazole, which showed fungicidal effect at the concentration of 326·5 µmol l-1 .

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In food industries UV-C irradiation is used to achieve decontamination of some packaging devices, such as plastic caps or laminated foils, and of those smooth surfaces that can be directly irradiated. Since its effectiveness can be checked by microbial validation tests, some ascospore-forming molds (Aspergillus hiratsukae, Talaromyces bacillisporus, Aspergillus montevidensis, and Chaetomium globosum) were compared with one of the target microorganisms actually used in industrial bio-validations (Aspergillus brasiliensis ATCC 16404) to find the species most resistant to UV-C. Tests were carried out with an UV-C lamp (irradiance = 127 µW/cm2; emission peak = 253.7 nm) by inoculating HDPE caps with one or more layers of spores. Inactivation kinetics of each strain were studied and both the corresponding 1D-values and the number of Logarithmic Count Reductions (LCR) achieved were calculated. Our results showed the important role played by the type of inoculum (one or more layers) and by the differences in cell structure (thickness, presence of protective solutes, pigmentation, etc.) of the strains tested. With a single-layer inoculum, Chaetomium globosum showed the highest resistance to UV-C irradiation (1D-value = 100 s). With a multi-layer inoculum, Aspergillus brasiliensis ATCC 16404 was the most resistant fungus (1D-value = 188 s), even if it reached a number of logarithmic reductions that was higher than those of some ascospore-forming mycetes (Aspergillus montevidensis, Talaromyces bacillisporus) tested.

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The preservative efficacy test is an important method for assessing the antimicrobial effect of cosmetic products. In this study, the optimum conditions for the efficient microbial enumeration of Aspergillus brasiliensis were investigated. Cosmetic products, inoculated with A. brasiliensis spore suspensions, were cultivated at 22.5°C, 32.5°C, or 40°C and the detection rate and the number of colonies were determined using the pour culture method. There was no difference in the viable counts of visible colonies among different temperature conditions. However, the viable counts after 3 days of culture were significantly greater for the cultures maintained at 32.5°C or 40°C compared with those maintained at 22.5°C. This effect was attenuated in products containing fatty acids, which could inhibit fungal growth. Overall, these results demonstrate that cultivating A. brasiliensis at 32.5°C reduces the time required for enumeration in the preservative efficacy test. Thus, the results of this study are expected to help improve and expedite microbiological quality control in the cosmetic industry.

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Parameters such as type and concentration of the active compound, exposure time, application temperature, and organic load presence influence the antimicrobial action of sanitizers, although there is little data in the literature. Thus, this study aimed to evaluate the antifungal efficacy of different chemical sanitizers under different conditions according to the European Committee for Standardization (CEN). Aspergillus brasiliensis (ATCC 16404) was exposed to four compounds (benzalkonium chloride, iodine, peracetic acid, and sodium hypochlorite) at two different concentrations (minimum and maximum described on the product label), different exposure times (5, 10, and 15 min), temperatures (10, 20, 30, and 40 °C), and the presence or absence of an organic load. All parameters, including the type of sanitizer, influenced the antifungal efficacy of the tested compounds. Peracetic acid and benzalkonium chloride were the best antifungal sanitizers. The efficacy of peracetic acid increased as temperatures rose, although the opposite effect was observed for benzalkonium chloride. Sodium hypochlorite was ineffective under all tested conditions. In general, 5 min of sanitizer exposure is not enough and >10 min are necessary for effective fungal inactivation. The presence of organic load reduced sanitizer efficacy in most of the tested situations, and when comparing the efficacy of each compound in the presence and absence of an organic load, a difference of up to 1.5 log CFU was observed. The lowest concentration recommended on the sanitizer label is ineffective for 99.9% fungal inactivation, even at the highest exposure time (15 min) or under the best conditions of temperature and organic load absence. Knowledge of the influence exerted by these parameters contributes to successful hygiene since the person responsible for the sanitization process in the food facility can select and apply a certain compound in the most favorable conditions for maximum antifungal efficacy.

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Onychomycosis is a common nail infection caused by dermatophytes, non-dermatophytic molds (NDMs) and yeast. Aspergillus spp. are emerging etiological agents of non-dermatophyte mold onychomycosis (NDMO). Though this is usually of cosmetic concern, it may also cause pain and discomfort to the patient. The toenail is more commonly involved as compared to fingernail. The nails are discoloured and disfigured. Onychomycosis may expose the patient to cellulitis of lower extremities. The clinical presentation of dermatophytic and NDM onychomycosis is more or less similar, which creates problem in the diagnosis. Fingernail infection may cause social and psychological problem to the patient if fingernail is involved. Incidence of onychomycosis has been seen more in immunosuppressed individuals, where it is of more serious medical concern. In the present study we are reporting a case of proximal subungual onychomycosis (PSO) due to Aspergillus brasiliensis.

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AIMS: Since mycosynthesis of metal nanoparticles (NPs) is advertised as a promising and ecofriendly approach. Thus, this study aims to investigate the capability of Aspergillus brasiliensis ATCC 16404 for mycosynthesis of silver NPs (AgNPs). METHODS AND RESULTS: One-factor-at-a-time-technique was used to study the effect of different physicochemical parameters: the reaction time, pH, temperature, different stirring rates, illumination, and finally, the different concentrations of silver nitrate and fungal biomass on the mycosynthesis of AgNPs. The visual observation showed the characteristic brown colour formation due to the bioreduction of Ag+ ions to Ag0 by the mycelial cell-free filtrate (MCFF). The UV/visible spectrophotometric technique displayed a characteristic sharp peak at ʎ440 confirming the mycosynthesis of AgNPs. The zeta potential value -16·7 mV assured the long-term stability of AgNPs and the dynamic light scattering analysis revealed good dispersion and average particle size 77 nm. The energy dispersive X-ray spectroscopy displayed a maximum elemental distribution of silver elements. The X-ray diffraction spectroscopy demonstrated the crystallinity of the mycosynthesized AgNPs. The field emission scanning electron microscope and high-resolution transmission electron microscope revealed monodispersed spherical shaped AgNPs with average particle size of 6-21 nm. The FTIR analysis showed the major peaks of proteins providing the possible role of MCFF in the synthesis and stabilization of the AgNPs. The mycosynthesized AgNPs expressed good biocidal activity against different pathogenic micro-organisms causing some water-related diseases and health problems to local residents. CONCLUSIONS: This study proved that A. brasiliensis ATCC 16404 MCFF has good potential for mycosynthesis of AgNPs, which exhibited good antimicrobial effect on different pathogenic micro-organisms; thus, it can be applied for water disinfection. SIGNIFICANCE AND IMPACT OF THE STUDY: This research provides a helpful insight into the development of a new mycosynthesized antimicrobial agent.

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An emulsifier protein (EP) was produced and easily separated from oil-contaminated water as an economical substrate when Aspergillus brasiliensis, pretreated in a solid state culture with a controlled electric field, was used in an airlift bioreactor. The hydrocarbon-EP comprised 19.5% of the total protein, its purification enhanced the specific emulsifying activity (EA) seven times. The influence of operational conditions (pH and salt concentration) on the EA were assessed to characterise the emulsion stability. The EA was increased by 19% in alkaline environments (pH 7-11), but it was not affected by the presence of salt (0-35 g L-1). On the other hand, preheating the EP samples (60 °C) enhanced the EA by 2.5 times. Based on analysis of its EA, this EP can be applied as a bioremediation enhancer in contaminated soils.

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The effects of electric current on membranes associated with metabolism modifications in Aspergillus brasiliensis (niger) ATCC 9642 were studied. A 450-mL electrochemical cell with titanium ruthenium-oxide coated electrodes and packed with 15g of perlite, as inert support, was inoculated with A. brasiliensis spores and incubated in a solid inert-substrate culture (12 d; 30°C). Then, 4.5days after starting the culture, a current of 0.42mAcm-2 was applied for 24h. The application of low-intensity electric current increased the molecular oxygen consumption rate in the mitochondrial respiratory chain, resulting in high concentrations of reactive oxygen species, promoting high lipoperoxidation levels, according to measured malondialdehyde, and consequent alterations in membrane permeability explained the high n-hexadecane (HXD) degradation rates observed here (4.7-fold higher than cultures without current). Finally, cell differentiation and spore production were strongly stimulated. The study contributes to the understanding of the effect of current on the cell membrane and its association with HXD metabolism.

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The combination of biological and electrochemical techniques enhances the bioremediation efficiency of treating oil-contaminated water. In this study a non-growing fungal whole cell biocatalyst (BC; Aspergillus brasiliensis attached to perlite) pretreated with an electric field (EF), was used to degrade a hydrocarbon blend (hexadecane-phenanthrene-pyrene; 100:1:1w/w) in an airlift bioreactor (ALB). During hydrocarbon degradation, all mass transfer resistances (internal and external) and sorption capacity were experimentally quantified. Internal mass transfer resistances were evaluated through BC effectiveness factor analysis as a function of the Thiele modulus (using first order reaction kinetics, assuming a spherical BC, five particle diameters). External (interfacial) mass transfer resistances were evaluated by kLa determination. EF pretreatment during BC production promoted surface changes in BC and production of an emulsifier protein in the ALB. The BC surface modifications enhanced the affinity for hydrocarbons, improving hydrocarbon uptake by direct contact. The resulting emulsion was associated with decreased internal and external mass transfer resistances. EF pretreatment effects can be summarized as: a combined uptake mechanism (direct contact dominant followed by emulsified form dominant) diminishing mass transfer limitations, resulting in a non-specific hydrocarbon degradation in blend. The pretreated BC is a good applicant for oil-contaminated water remediation.

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We investigated the enzymatic complex produced by selected fungi strains isolated from the environment using the agro-industrial residues rice husk, soybean hull, and spent malt as substrates. Microbial growth was carried out in solid-state cultivation (SSC) and in submerged cultivations (SC) and the enzymatic activities of xylanase, cellulase, ß-xylosidase, and ß-glucosidase were determined. All substrates were effective in inducing enzymatic activities, with one strain of Aspergillus brasiliensis BLf1 showing maximum activities for all enzymes, except for cellulases. Using this fungus, the enzymatic activities of xylanase, cellulase, and ß-glucosidase were generally higher in SSC compared to SC, producing maxima activities of 120.5, 25.3 and 47.4 U g-1 of dry substrate, respectively. ß-xylosidase activity of 28.1 U g-1 of dry substrate was highest in SC. Experimental design was carried out to optimize xylanase activity by A. brasiliensis BLf1 in SSC using rice husk as substrate, producing maximum xylanase activity 183.5 U g-1 dry substrate, and xylooligosaccharides were produced and characterized. These results suggest A. brasiliensis BLf1 can be used to produce important lytic enzymes to be applied in the preparation of xylooligosaccharides.

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Abstract Filamentous fungi are widely diverse and ubiquitous organisms. Such biodiversity is barely known, making room for a great potential still to be discovered, especially in tropical environments - which are favorable to growth and species variety. Filamentous fungi are extensively applied to the production of industrial enzymes, such as the amylases. This class of enzymes acts in the hydrolysis of starch to glucose or maltooligosaccharides. In this work twenty-five filamentous fungi were isolated from samples of decomposing material collected in the Brazilian Atlantic Forest. The two best amylase producers were identified as Aspergillus brasiliensis and Rhizopus oryzae. Both are mesophilic, they grow well in organic nitrogen-rich media produce great amounts of glucoamylases. The enzymes of A. brasiliensis and R. oryzae are different, possibly because of their phylogenetical distance. The best amylase production of A. brasiliensis occurred during 120 hours with initial pH of 7.5; it had a better activity in the pH range of 3.5-5.0 and at 60-75°C. Both fungal glucoamylase had wide pH stability (3-8) and were activated by Mn2+. R. oryzae best production occurred in 96 hours and at pH 6.5. Its amylases had a greater activity in the pH range of 4.0-5.5 and temperature at 50-65ºC. The most significant difference between the enzymes produced by both fungi is the resistance to thermal denaturation: A. brasiliensis glucoamylase had a T50 of 60 minutes at 70ºC. The R. oryzae glucoamylase only had a residual activity when incubated at 50°C with a 12 min T50.

Resumo Fungos filamentosos são organismos amplamente diversificados e ubíquos. Esta biodiversidade ainda é pouco caracterizada, desta forma, há um grande potencial a ser descoberto, sobretudo em biomas tropicais, que favorecem o crescimento e diversificação de espécies. Fungos filamentosos são extensivamente utilizados para a produção industrial de enzimas, como as amilases. Esta classe de enzimas atua na hidrólise do amido em glicose ou maltooligossacarídeos. Neste trabalho 25 cepas de fungos filamentosos foram isoladas a partir de amostras de material em decomposição coletados na Mata Atlântica Brasileira. As duas cepas que produziram mais amilases foram identificadas como Aspergillus brasiliensis e Rhizopus oryzae. Ambos os fungos são mesofílicos, crescem bem em meio de cultivo rico em nitrogênio orgânico, e produziram grande quantidade de glucoamilase. As enzimas de A. brasiliensis e R. oryzae possuem características distintas, possivelmente devido à distância filogenética das espécies. A produção de amilase mais expressiva de A. brasiliensis ocorreu em 120 horas de cultivo e pH inicial de 7,5; possui maior atividade em temperaturas entre 60-75ºC e pH entre 3,5-5,0. Ambas glucoamilases fúngicas obtiveram ampla estabilidade de pH (3-8) e foram ativadas por Mn2+. A melhor produção de R. oryzae ocorreu em 96 horas de cultivo e pH 6,5. Suas amilases são mais ativas na faixa de pH de 4,0-5,5 e temperatura entre 50-60ºC. A diferença mais significativa dentre as enzimas produzidas pelos fungos selecionados é a resistência à desnaturação térmica, tendo a glucoamilase de A. brasiliensis um T50 de 60 minutos a 70ºC, já a glucoamilase de R. oryzae somente obteve atividade residual quando incubada a 50°C, com um T50 de apenas 12 minutos.

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The genus Aspergillus is ubiquitous in nature and includes various species extensively exploited industrially due to their ability to produce and secrete a variety of enzymes and metabolites. Most processes are performed in submerged fermentation (SmF); however, solid-state fermentation (SSF) offers several advantages, including lower catabolite repression and substrate inhibition and higher productivity and stability of the enzymes produced. This study aimed to explain the improved metabolic behavior of A. brasiliensis ATCC9642 in SSF at high glucose concentrations through a proteomic approach. Online respirometric analysis provided reproducible samples for secretomic studies when the maximum CO2 production rate occurred, ensuring consistent physiological states. Extracellular extracts from SSF cultures were treated by SDS-PAGE, digested with trypsin, and analyzed by LC-MS/MS. Of 531 sequences identified, 207 proteins were analyzed. Twenty-five were identified as the most abundant unregulated proteins; 87 were found to be up-regulated and 95 were down-regulated with increasing glucose concentration. Of the regulated proteins, 120 were enzymes, most involved in the metabolism of carbohydrates (51), amino acids (23), and nucleotides (9). This study shows the high protein secretory activity of A. brasiliensis under SSF conditions. High glucose concentration favors catabolic activities, while some stress-related proteins and those involved in proteolysis are down-regulated.

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Microbial steroid biotransformations have found a wide-reaching application for the production of more precious and functionalized compounds due to their high regio-and stereo selectivity. In this study, the possibility of using filamentous fungi Aspergillus brasiliensiscells in the biotransformation of progesterone, a C-21 steroid hormone was studied for the first time. The fungal strain was inoculated into the transformation medium supplemented with progesterone as a substrate. Biotransformation of this steroid for 7 days afforded 3 different hydroxylated metabolites: 11α-hydroxy progesterone; 14α-hydroxyprogesteroneand21-hydroxyprogesterone. The metabolites were separated by thin layer chromatography. Structure determinations of the metabolites were performed by comparing NMR, MS and IR spectra of the starting compound with those of metabolites. These results may be of industrial importance because the metabolites can be used as precursor of some steroid drugs.

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AIM: The aim of this study was to investigate the effect of plasma-enhanced chemical vapour deposition (PECVD) treatment on selected bacteria and spores and to contribute to the understanding of the synergistic effect of UV-directed plasma. METHODS AND RESULTS: The experiments were conducted on pure cultures of Aspergillus brasiliensis and Escherichia coli and on naturally contaminated pistachios that were exposed to pure oxygen-, pure argon- and to a mixture of oxygen-argon-generated plasma for different treatment times and at different micro-organism concentrations. Optical emission spectroscopy (OES) measurements were performed to observe the active species in the plasma. After exposure, the effectiveness of decontamination was assessed through microbiological techniques by calculating the growth reduction on a logarithmic scale. A treatment time of 30 min resulted in a 3·5 log reduction of A. brasiliensis using pure oxygen or argon, while treatment times of 5 min, 1 min and 15 s resulted in a 5·4 log reduction using a mixture of argon and oxygen (10 : 1 v/v). Treatment times of 1 min and 30 s resulted in a 4 log reduction of E. coli with oxygen and argon, respectively, which led to a complete elimination of the micro-organisms. Two-log reductions of fungi were achieved for pistachios after a treatment time of 1 min. CONCLUSIONS: These results suggest that this newly designed plasma reactor offers good potential applications for the reduction in micro-organisms on heat-sensitive materials, such as foods. The plasma that was generated with Ar/O2 was more effective than that which was generated with pure oxygen and pure argon. SIGNIFICANCE AND IMPACT OF THE STUDY: An improvement in the knowledge about PECVD mechanisms was acquired from the chemical and biological points of view, and the suitability of the method for treating dry food surfaces was demonstrated.

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Statistics based optimization, Plackett-Burman design (PBD) and response surface methodology (RSM) were employed to screen and optimize the media components for the production of naringinase from Aspergillus brasiliensis MTCC 1344, using solid state fermentation. Cassava waste (CW) was used as both the solid support and carbon source for the growth of A. brasiliensis. Based on the positive influence of the Pareto chart obtained from PBD on naringinase activity, three media components--maltose, peptone and calcium chloride were screened. Box-Behnken design (BBD) was employed using these three factors at three levels, for further optimization, and the second order polynomial equation was derived, based on the experimental data. Derringer's desired function methodology showed that the concentrations of maltose (7.74 g/L), peptone (4.19 g/L) and calcium chloride (7.63 mM) were the optimal levels for maximal naringinase activity (889.91 U/mg) which were validated through experiments.

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