Production of fibrolytic enzymes by Aspergillus japonicus C03 using agro-industrial residues with potential application as additives in animal feed

Solid-state fermentation obtained from different and low-cost carbon sources was evaluated to endocellulases and endoxylanases production by Aspergillus japonicus C03. Regarding the enzymatic production the highest levels were observed at 30 degrees C, using soy bran added to crushed corncob or wheat bran added to sugarcane bagasse, humidified with salt solutions, and incubated for 3 days (xylanase) or 6 days (cellulase) with 70% relative humidity. Peptone improved the xylanase and cellulase activities in 12 and 29%, respectively. The optimum temperature corresponded to 60 degrees C and 50-55 degrees C for xylanase and cellulase, respectively, both having 4.0 as optimum pH. Xylanase was fully stable up to 40 degrees C, which is close to the rumen temperature. The enzymes were stable in pH 4.0-7.0. Cu(++) and Mn(++) increased xylanase and cellulase activities by 10 and 64%, respectively. A. japonicus C03 xylanase was greatly stable in goat rumen fluid for 4 h during in vivo and in vitro experiments.

Production of fibrolytic enzymes by Aspergillus japonicus C03 using agro-industrial residues with potential application as additives in animal feed

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Optimization of fibrolytic enzyme production by Aspergillus japonicus C03 with potential application in ruminant feed and their effects on tropical forages hydrolysis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Production of fructooligosaccharides by the mycelia of Aspergillus japonicus immobilized in calcium alginate

This work describes fructose oligosaccharide (FOS) production by the immobilized mycelia (IM) of a strain of Aspergillus japonicus, isolated from soil. The microorganism was inoculated into 50 mi of medium composed of sugar cane molasses (5.0% of total sugars); yeast powder; 2.0%; K2HPO4, 0.5%; NaNO3, 0.2%; MgSO4. 7H(2)O, 0.05%; KCl, 0.05%, final pH 5.0, and the flasks were agitated in an orbital shaker at 200 rpm for 60 h, at 30 degrees C. The beta-fructofuranosidase activity (Uf), transfructosylating activity (Ut), hydrolyzing activity (Uh), and FOS production were analyzed by high performance liquid chromatography. FOS production was performed in a batch process in a 2-l jar fermenter by IM in calcium alginate beads. The optimum pH and temperature were 5.0-5.6 and 55 degrees C, respectively No loss of activity was observed when the mycelium was maintaned at 60 degrees C for 60 min. Maximum production was obtained using 5.75% (cellular weight/volume) of mycelia (122.4 Ut g(-1)) and 65% sucrose solution (w:v) for 4 h of reaction when the final product reached 61.28% of fetal FOS containing GF(2) (30.56%), GF(3) (26.45%), GF(4) (4.27%), sucrose (9.6%) and glucose (29.10%). In the assay conditions, 23 batches were performed without loss of activity of the IM, showing that the microorganism and the process utilized have potential for industrial applications. (C) 1998 Elsevier B.V. Ltd. All rights reserved.

Sugarcane molasses and yeast powder used in the Fructooligosaccharides production by Aspergillus japonicus-FCL 119T and Aspergillus niger ATCC 20611

Different concentrations of sucrose (3-25% w/v) and peptone (2-5% w/v) were studied in the formulation of media during the cultivation of Aspergillus japonicus-FCL 119T and Aspergillus niger ATCC 20611. Moreover, cane molasses (3.5-17.5% w/v total sugar) and yeast powder (1.5-5% w/v) were used as alternative nutrients for both strains' cultivation. These media were formulated for analysis of cellular growth, P-Fructosyltransferase and Fructooligosaccharides (FOS) production. Transfructosylating activity (U-t) and FOS production were analyzed by HPLC. The highest enzyme production by both the strains was 3% (w/v) sucrose and 3% (w/v) peptone, or 3.5% (w/v) total sugars present in cane molasses and 1.5% (w/v) yeast powder. Cane molasses and yeast powder were as good as sucrose and peptone in the enzyme and FOS (around 60% w/w) production by studied strains.

Optimization of xylanase biosynthesis by Aspergillus japonicus isolated from a Caatinga area in the Brazilian state of Bahia

The objective of this research was to investigate the potential of xylanase production by Aspergillus japonicus and to determine the effects of cultivation conditions in the process, aiming toward optimization of enzyme production. The best temperature, as well as the best carbon source, for biomass production was determined through an automated turbidimetric method (Bioscreen-C). The enzyme activity of this fungus was separately evaluated in two solid substrates (wheat and soybean bran) and in Vogel medium, adding other carbon sources. Temperature effects, cultivation time, and spore concentrations were also tested. The best temperature for enzyme and biomass production was 25°C; however, the best carbon source for growth (determined by the Bioscreen C) did not turn out to be a good inducer of xylanase production. Maximum xylanase activity was achieved when the fungus was cultivated in wheat bran (without the addition of any other carbon source) using a spore concentration of 1 × 107 spores/mL (25°C, pH 5.0, 120 h). A. japonicus is a good xylanase producer under the conditions presented in these assays. © 2006 Academic Journals.

Efeito de bebidas enriquecidas com frutooligossacarídeos (FOS), produzidos pela linhagem Aspergillus japonicus-119, no trato intestinal de ratos Wistar

Pós-graduação em Ciências Biológicas (Microbiologia Aplicada) - IBRC

Effects of Aspergillus spp. exogenous fibrolytic enzymes on in vitro fermentation of tropical forages

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effects of Aspergillus spp. exogenous fibrolytic enzymes on in vitro fermentation of tropical forages

BACKGROUND: Cellulose and hemicellulose are quantitatively the most important structural carbohydrates present in ruminant diets. Rumen micro-organisms produce enzymes that catalyse their hydrolysis, but the complex network formed by structural carbohydrates and lignin reduces their digestibility and restricts efficient utilisation of feeds by ruminants. This study aimed to produce two enzymatic extracts, apply them in ruminant diets to determine the best levels for ruminal digestibility and evaluate their effects on in vitro digestibility. RESULTS: In experiment 1 a two-stage in vitro technique was used to examine the effects of different enzymatic levels of Aspergillus japonicus and Aspergillus terricola on tropical forages. Enzyme addition had minor effects on corn silage at the highest enzymatic level. In experiment 2 an in vitro gas production (GP) technique was applied to determine apparent in vitro organic matter digestibility and metabolisable energy. The addition of enzymes in GP showed interesting results. Good data were obtained using sugar cane and Tifton-85 hay supplemented with extracts of A. japonicus and A. terricola respectively. CONCLUSION: Overall, the study suggests that addition of crude extracts containing exogenous fibrolytic enzymes to ruminant diets enhances the effective utilisation of ruminant feedstuffs such as forages. Copyright (c) 2012 Society of Chemical Industry

Optimização das condições de cultura da Saccharomyces Cerevisiae e de outros fungos produtores de oligossacáridos

Este estudo envolve o controlo e a optimização das condições de culturas dos microrganismos: Saccharomyces cerevisiae CCMI 396, S. cerevisiae v. lab., Aspergillus oryzae CCMI 125, Aspergillus japonicus CCMI 443, Fusarium oxysporum CCMI 866, Aspergillus niger CCMI 296 com vista à produção de oligossacáridos. Determinaram-se os parâmetros característicos das culturas de duas diferentes estirpes de Saccharomyces com diferentes fontes de carbono e em diferentes condições ambientais. O perfil de crescimento da S. cerevisiae CCMI 396 foi semelhante nos diferentes meios de cultura estudados, sendo a velocidade específica de crescimento mais elevada no meio com glucose a pH 5 e a 30°C (0,36h-1). A S. cerevisiae v. lab. Teve velocidade específica de crescimento idêntica nas mesmas condições da outra estirpe, no entanto, o perfil de crescimento foi diferente nos outros meios de cultura. Estudou-se o efeito da adição de sumo de laranja ou de tomate ao meio de cultura com sacarose e avaliou-se a evolução glucídica no meio de cultura durante o ensaio por HPLC com detector RI. Determinou-se a frutosiltransferase no sobrenadante e na fracção intracelular e determinou-se a evolução dos oligossacáridos. Numa segunda parte deste trabalho efectuaram-se culturas dos quatro fungos filamentosos com vista a avaliar a capacidade de produção, nomeadamente, de fruto­oligassacáridos. Os resultados mostraram que a espécie Aspergillus japonicus CCMI 443 originou, nas mesmas condições de cultura, valores superiores, sendo a percentagem de produção FOStotais/GluCtotais de 61% para as enzimas intracelulares e 40% para as enzimas no sobrenadante. ABSTRACT; This study involves control and optimization of the cultures of microorganisms: Saccharomyces cerevisiae CCMI 396, S. cerevisiae v. lab., Aspergillus oryzae CCMI 125, Aspergillus japonicus CCMI 443, Fusarium oxysporum CCMI 866, Aspergillus níger CCMI 296 for oligosaccharides production. Were determined the parameters characteristic of the cultures of two different strains of Saccharomyces with different sources of carbon and in different environmental conditions. The growth profile of S. cerevisiae CCMI 396 was similar in different cultures media, but the highest specific growth was obtained in a medium with glucose, pH 5, at 30°C (0.36h-1). S. cerevisiae v. lab. had similar growth profile in a medium with glucose but with others culture media was different. We studied the effect of adding orange juice or tomato to the culture medium with sucrose and evaluated the evolution glucidic in the culture medium during the test by HPLC with RI detector. Fructosyltransferase was determined in the extracellular and the intracellular fractions and determined the evolution of oligosaccharides. ln the second part of this work were carried out cultures of four filamentous fungi in order to assess production capacity, in particular, fructoligosaccharides. The results showed that the specie Aspergillus japonicus CCMI 443 originated in the same culture conditions, higher values and the percentage of production FOStotal/Guctotal of 61% for intracellular enzymes and 40% for extracellular enzymes.

UNDERSTANDING LATE SEASON FRUIT ROT PATHOSYSTEMS AND INSECT INTERACTIONS IN MID-ATLANTIC VINEYARDS

Fungal fruit rots and insect pests are among the most important problems negatively affecting the yield and quality of mid-Atlantic wine. In pathogenicity trials of fungi recovered from diseased Chardonnay and Vidal blanc grapes, Alternaria alternata, Pestalotiopsis telopeae, and Aspergillus japonicus were found to be unreported fruit rot pathogens in the region. Additionally, P. telopeae and A. japonicus had comparable virulence to the region’s common fruit rot pathogens. Furthermore, a timed-exclusion field study was implemented to evaluate vineyard insect-fruit rot relationships. It was found that clusters exposed to early-season insect communities that included Paralobesia viteana had a significantly greater incidence of sour rot than clusters protected from insects all season. These results were contrary to the current assumption that fall insects are the primary drivers of sour rot in the region. This research provides diagnostic tools and information to develop management-strategies against fungal and insect pests for mid-Atlantic grape growers.

Production of beta-fructofuranosidases by Aspergillus niveus using agroindustrial residues as carbon sources: Characterization of an intracellular enzyme accumulated in the presence of glucose

The production of beta-fructofuranosidases by Aspergillus niveus, cultivated under submerged fermentation using agroindustrial residues, was investigated. The highest productivity of beta-fructofuranosidases was obtained in Khanna medium supplemented with sugar cane bagasse as carbon source. Glucose enhanced the production of the intracellular enzyme, whereas that of the extracellular one was decreased. The intracellular beta-fructofuranosidase was a trimeric protein of approximately 141 kDa (gel filtration) with 53.5% carbohydrate content, composed of 57 kDa monomers (SDS-PAGE). The optimum temperature and optimum pH were 60 degrees C and 4.5, respectively. The purified enzyme showed good thermal stability and exhibited a half-life of 53 min at 60 degrees C. beta-Fructofuranosidase activity was slightly activated by Cu(2+), Mn(2+), Mg(2+), and Na(+) at 1 mM concentration. The enzyme hydrolyzed sucrose, raffinose, and inulin, with K(d) values of 5.78 mM, 5.74 mM, and 1.74 mM, respectively. (C) 2008 Elsevier Ltd. All rights reserved.

Biochemical properties of an extracellular beta-D-fructofuranosidase II produced by Aspergillus phoenicis under Solid-Sate Fermentation using soy bran as substrate

The filamentous fungus A. phoenicis produced high levels of beta-D-fructofuranosidase (FFase) when grown for 72 hrs under Solid-State Fermentation (SSF), using soy bran moistened with tap water (1:0.5 w/v) as substrate/carbon source. Two isoforms (I and II) were obtained, and FFase II was purified 18-fold to apparent homogeneity with 14% recovery. The native molecular mass of the glycoprotein (12% of carbohydrate content) was 158.5 kDa with two subunits of 85 kDa estimated by SDS-PAGE. Optima of temperature and pH were 55 degrees C and 4.5. The enzyme was stable for more than 1 hr at 50 degrees C and was also stable in a pH range from 7.0 to 8.0. FFase II retained 80% of activity after storage at 4 degrees C by 200 hrs. Dichroism analysis showed the presence of random and beta-sheet structure. A. phoenicis FFase II was activated by Mn(2+), Mg(2+) and Co(2+), and inhibited by Cu(2+), Hg(2+) and EDTA. The enzyme hydrolyzed sucrose, inulin and raffinose. K(d) and V(max) values were 18 mM and 189 U/mg protein using sucrose as substrate.

Production of Fructooligosaccharides by Aspergillus phoenicis Biofilm on Polyethylene as Inert Support

Aspergillus phoenicis biofilms on polyethylene as inert support were used to produce fructooligosaccharides (FOS) in media containing 25% (m/V) of sucrose as a carbon source. The maximum production of total FOS (122 mg/mL), with 68% of 1-kestose and 32% of nystose, was obtained in Khanna medium maintained at 30 degrees C for 48 h under orbital agitation (100 rpm). At high concentrations of sucrose (30%, m/V), the recovery of FOS was higher than that observed at a low concentration (5%, m/V). High levels of FOS (242 mg/mL) were also recovered when using the biofilm in sodium acetate buffer with high sucrose concentration (50%, m/V) for 10 h. When the dried biofilm was reused in a fresh culture medium, there was a recovery of approx. 13.7% of total FOS after 72 h of cultivation at 30 C, and 10% corresponded to 1-kestose. The biofilm morphology, analyzed by scanning electron microscope, revealed a noncompact mycelium structure, with unfilled spaces and channels present among the hyphae. The results obtained in this study show that A. phoenicis biofilms may find application for FOS production in a single-step fermentation process, which is cost-effective in terms of reusability, downstream processing and efficiency.

Effects Of High Pressure Homogenization On The Activity, Stability, Kinetics And Three-dimensional Conformation Of A Glucose Oxidase Produced By Aspergillus Niger.

High pressure homogenization (HPH) is a non-thermal method, which has been employed to change the activity and stability of biotechnologically relevant enzymes. This work investigated how HPH affects the structural and functional characteristics of a glucose oxidase (GO) from Aspergillus niger. The enzyme was homogenized at 75 and 150 MPa and the effects were evaluated with respect to the enzyme activity, stability, kinetic parameters and molecular structure. The enzyme showed a pH-dependent response to the HPH treatment, with reduction or maintenance of activity at pH 4.5-6.0 and a remarkable activity increase (30-300%) at pH 6.5 in all tested temperatures (15, 50 and 75°C). The enzyme thermal tolerance was reduced due to HPH treatment and the storage for 24 h at high temperatures (50 and 75°C) also caused a reduction of activity. Interestingly, at lower temperatures (15°C) the activity levels were slightly higher than that observed for native enzyme or at least maintained. These effects of HPH treatment on function and stability of GO were further investigated by spectroscopic methods. Both fluorescence and circular dichroism revealed conformational changes in the molecular structure of the enzyme that might be associated with the distinct functional and stability behavior of GO.