Chicken viscera meal as substrate for the simultaneous production of antioxidant compounds and proteases by Aspergillus oryzae.

The use of chicken waste can contribute to the development of new processes and obtaining molecules with high added value. An experimental design was applied to evaluate the effect of moisture, temperature, and inoculum size on the production of antioxidant peptides and proteases by A. oryzae IOC3999 through solid-state fermentation (SSF) of chicken viscera meal. As a result, the process conditions strongly influenced protease production and antioxidant activity of the fermented products. A global analysis of the results indicated that the most adequate conditions for SSF were (assay 9): 40% initial moisture, 30 °C as the incubation temperature, 5.05 × 106 spores/g as the inoculum size, and 48-h fermentation as the fermentation time. Under this condition, the antioxidant activities for the ABTS- and DPPH-radicals inhibition and ferric reducing antioxidant power (FRAP) methods were 376.16, 153.29, and 300.47 (µmol TE/g), respectively, and the protease production reached 428.22 U/g. Ultrafiltration of the crude extract obtained under optimized fermentation conditions was performed, and the fraction containing peptides with molecular mass lower than 3 kDa showed the highest antioxidant activity. The proteases were biochemically characterized and showed maximal activity at pH values ranging from 5.0 to 6.0 and a temperature of 50 °C. The thermodynamic parameters indicated that the process of thermal protease inactivation is not spontaneous (ΔG*d > 88.78 kJ/mol), increasing with temperature (ΔH*d 27.01-26.88 kJ/mol), and with reduced disorder in the system (ΔS*d < - 197.74 kJ/mol) probably caused by agglomeration of partially denatured enzymes.

Development of a biocomposite based on alginate/gelatin crosslinked with genipin for ß-galactosidase immobilization: Performance and characteristics.

In this work, we studied the development of a biocomposite formulated with alginate and gelatin, crosslinked with genipin for application as support for ß-galactosidase immobilization. Also, the biocomposites with the immobilized enzyme were characterized by thermal analyses and SAXS (size, density, and interconnectivity of alginate rods) for a detailed analysis of the microstructure, as well as the thermal and operational stabilities of the enzyme. The structural modifications of the biocomposite determined by SAXS demonstrate that the addition of both genipin and enzyme produced a significant reduction in size and density of the Ca(II)-alginate rods. Immobilized ß-galactosidase could be stored for 175 days under refrigeration maintaining 80% of its initial activity. Moreover, 90% of its relative activity was kept after 11 reuses in a batch process of lactose hydrolysis. Thus, the biocomposite proved to be effective as support for enzyme immobilization.

L-asparaginase from Aspergillus oryzae spp.: effects of production process and biochemical parameters.

L-asparaginases prevent the formation of acrylamide, a substance commonly found in foods subjected to heat and that also contains reducing sugars and L-asparagine. This work aimed to select a strain of Aspergillus spp. able to produce L-asparaginase and to optimize the fermentation parameters, the partial purification and biochemical characterization were also performed. The Aspergillus oryzae IOC 3999 was selected due to its greater enzymatic activity: 1443.57 U/mL of L-asparaginase after 48 h of fermentation. The optimized conditions allowed for an increase of 223% on the L-asparaginase production: 2.9% lactose, 2.9% L-asparagine and 0.7% hydrolyzed casein, 0.152% KH2PO4, 0.052% KCl and MgSO4, 0.001% of CuNO3.3H2O, ZnSO4.7H2O and FeSO4.7H2O adjusted to pH 7.0; added a concentration of 5.05x106 spores/mL at 30 °C for 100 rpm. A purification factor of 2.11 was found and the molecular mass was estimated at 20.8 kDa. The enzyme showed optimum activity at 60 °C and pH 5 and stability at 50 °C for 1 h. The enzyme presented desirable biochemical characteristics, mainly the acid pH stability, indicating that the enzyme would work well in food matrices due to the closeness of pH, meaning that it could be a potential option for use in the food industry.

Analysis of phenanthrene degradation by Ascomycota fungi isolated from contaminated soil from Reynosa, Mexico.

Polycyclic aromatic hydrocarbons (PAHs) are organic compounds generated mainly by anthropogenic sources. They are considered toxic to mammals, since they have carcinogenic, mutagenic and genotoxic properties, among others. Although mycoremediation is an efficient, economical and eco-friendly technique for degrading PAHs, the fungal degradation potential of the phylum Ascomycota has not been widely studied. In this work, we evaluated different fungal strains from the polluted soil of 'La Escondida' lagoon in Reynosa, Mexico to know their potential to degrade phenanthrene (PHE). Forty-three soil isolates with the capacity to grow in the presence of PHE (0·1% w/v) were obtained. The fungi Aspergillus oryzae MF13 and Aspergillus flavipes QCS12 had the best potential to degrade PHE. Both fungi germinated and grew at PHE concentrations of up to 5000 mg l-1 and degraded 235 mg l-1 of PHE in 28 days, with and without an additional carbon source. These characteristics indicate that A. oryzae MF13 and A. flavipes QCS12 could be promising organisms for the remediation of sites contaminated with PAHs and detoxification of recalcitrant xenobiotics.

Soybean (Glycine max) hull valorization through the extraction of polyphenols by green alternative methods.

Soybean is one of the greatest crops in the world, with about 348.7 million tons being produced in 2018. Soybean hull is a by-product produced during the processing of soybean to obtain flour and oil. Though not being actually exploited, it is a source of polyphenols with antioxidant activity. Here, the extraction of polyphenols from soybean hull was performed by means of an alkaline hydrolysis treatment, which was optimized by the response surface methodology. At the optimal region, a total phenolic content of 0.72 g gallic acid equivalents per 100 g of soybean hull was obtained with an antioxidant activity of 2.17 mmoles of Trolox equivalents. Polyphenols responsible for the antioxidant activities were identified by LC-MS, including phenolic acids, anthocyanins, stilbenes, and the two main isoflavones of soybean, daidzein and genistein, in their non-glycosylated form. Other alternative extraction methods based on Aspergillus oryzae fermentation and α-amylase hydrolysis are also proposed.

Biosynthesis of oxygenated brasilane terpene glycosides involves a promiscuous N-acetylglucosamine transferase.

Investigation of the metabolome of the ascomycete Annulohypoxylon truncatum led to the identification of novel oxygenated brasilane glycosides and the revision of the stereochemistry of the brasilane A octahydro-1H-indene core scaffold to trans. The bra biosynthetic gene cluster containing five genes (braA-braE) was identified and verified by heterologous expression experiments in Aspergillus oryzae demonstrating that BraC is a multifunctional P450 monooxygenase. In vitro studies of BraB revealed it to be a very rare fungal UDP-GlcNAc dependent N-acetylglucosamine transferase. UDP-glucose is also accepted as a donor, and a broad acceptor substrate tolerance for various primary and secondary alcohols was observed.

Heterologous expression and functional characterization of the ligand-binding domain of oxysterol-binding protein from Aspergillus oryzae.

Oxysterol-binding proteins (OSBPs) comprise a family of sterol-binding proteins. In this study, we focused on AoOSBP1, one of the five OSBP proteins identified from the industrial fungus Aspergillus oryzae. The temporal expression pattern analysis showed that the expression of AoOSBP1, in both gene and protein levels, was stably expressed throughout the developmental stages, while was upregulated during the accelerated growth stage. The immunofluorescence observation revealed that AoOSBP1 protein was mainly distributed in the conidiophore, indicating its underlying role in spore formation. The ligand-binding domain of AoOSBP1, namely OSBP-related domain (ORD), was heterologously expressed in Escherichia coli and purified. The binding assay carried out using microscale thermophoresis showed that the recombinant AoORD protein exhibited binding affinity for ergosterol, and exhibited much higher affinity to oxysterols (25-hydroxycholesterol and 7-ketocholesterol) and phytosterols (ß-sitosterol and stigmasterol). By contrast, MBP tag as the negative control showed no binding affinity for sterols. The present work demonstrates that AoORD domain in AoOSBP1 is capable of binding sterols, plays an underlying role in sterols transportation, and may participate in spore formation.

Gene transcription profiling of Aspergillus oryzae 3.042 treated with ergosterol biosynthesis inhibitors.

Ergosterol, a unique component of fungal cells, is not only important for fungal growth and stress responses but also holds great economic value. Limited studies have been performed on ergosterol biosynthesis in Aspergillus oryzae, a safe filamentous fungus that has been used for the manufacture of oriental fermented foods. This study revealed that the ergosterol biosynthesis pathway is conserved between Saccharomyces cerevisiae and A. oryzae 3.042 by treatment with ergosterol biosynthesis inhibitors and bioinformatics analysis. However, the ergosterol biosynthesis pathway in A. oryzae 3.042 is more complicated than that in S. cerevisiae as there are multiple paralogs encoding the same biosynthetic enzymes. Using RNA-seq, this study identified 138 and 104 differentially expressed genes (DEG) in response to the ergosterol biosynthesis inhibitors tebuconazole and terbinafine, respectively. The results showed that the most common DEGs were transport- and metabolism-related genes. There were only 17 DEGs regulated by both tebuconazole and terbinafine treatments and there were 256 DEGs between tebuconazole and terbinafine treatments. These results provide new information on A. oryzae ergosterol biosynthesis and regulation mechanisms, which may lay the foundation for genetic modification of the ergosterol biosynthesis pathway in A. oryzae.

One-pot strategy for on-site enzyme production, biomass hydrolysis, and ethanol production using the whole solid-state fermentation medium of mixed filamentous fungi.

The efficient use of renewable lignocellulosic feedstocks to obtain biofuels and other bioproducts is a key requirement for a sustainable biobased economy. This requires novel and effective strategies to reduce the cost contribution of the cellulolytic enzymatic cocktails needed to convert the carbohydrates into simple sugars, in order to make large-scale commercial processes economically competitive. Here, we propose the use of the whole solid-state fermentation (SSF) medium of mixed filamentous fungi as an integrated one-pot strategy for on-site enzyme production, biomass hydrolysis, and ethanol production. Ten different individual and mixed cultivations of commonly used industrial filamentous fungi (Aspergillus niger, Aspergillus oryzae, Trichoderma harzianum, and Trichoderma reesei) were performed under SSF and the whole media (without the extraction step) were used in the hydrolysis of pretreated sugarcane bagasse. The cocultivation of T. reesei with A. oryzae increased the amount of glucose released by around 50%, compared with individual cultivations. The release of glucose and reducing sugars achieved using the whole SSF medium was around 3-fold higher than obtained with the enzyme extract. The addition of soybean protein (0.5% w/w) during the hydrolysis reaction further significantly improved the saccharification performance by blocking the lignin and avoiding unproductive adsorption of enzymes. The results of the alcoholic fermentation validated the overall integrated process, with a volumetric ethanol productivity of 4.77 g/L.h, representing 83.5% of the theoretical yield. These findings demonstrate the feasibility of the proposed one-pot integrated strategy using the whole SSF medium of mixed filamentous fungi for on-site enzymes production, biomass hydrolysis, and ethanol production. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:671-680, 2018.

Comparison between the univariate and multivariate analysis on the partial characterization of the endoglucanase produced in the solid state fermentation by Aspergillus oryzae ATCC 10124.

Endoglucanase production by Aspergillus oryzae ATCC 10124 cultivated in rice husks or peanut shells was optimized by experimental design as a function of humidity, time, and temperature. The optimum temperature for the endoglucanase activity was estimated by a univariate analysis (one factor at the time) as 50°C (rice husks) and 60°C (peanut shells), however, by a multivariate analysis (synergism of factors), it was determined a different temperature (56°C) for endoglucanase from peanut shells. For the optimum pH, values determined by univariate and multivariate analysis were 5 and 5.2 (rice husk) and 5 and 7.6 (peanut shells). In addition, the best half-lives were observed at 50°C as 22.8 hr (rice husks) and 7.3 hr (peanut shells), also, 80% of residual activities was obtained between 30 and 50°C for both substrates, and the pH stability was improved at 5-7 (rice hulls) and 6-9 (peanut shells). Both endoglucanases obtained presented different characteristics as a result of the versatility of fungi in different substrates.

Production of alpha-amylase from Aspergillus oryzae for several industrial applications in a single step.

A one-step method as a strategy of alpha-amylase concentration and purification was developed in this work. This methodology requires the use of a very low concentration of biodegradable polyelectrolyte (Eudragit(®) E-PO) and represents a low cost, fast, easy to scale up and non-polluting technology. Besides, this methodology allows recycling the polymer after precipitation. The formation of reversible soluble/insoluble complexes between alpha-amylase and the polymer Eudragit(®) E-PO was studied, and their precipitation in selected conditions was applied with bioseparation purposes. Turbidimetric assays allowed to determine the pH range where the complexes are insoluble (4.50-7.00); pH 5.50 yielded the highest turbidity of the system. The presence of NaCl (0.05M) in the medium totally dissociates the protein-polymer complexes. When the adequate concentration of polymer was added under these conditions to a liquid culture of Aspergillus oryzae, purification factors of alpha-amylase up to 7.43 and recoveries of 88% were obtained in a simple step without previous clarification. These results demonstrate that this methodology is suitable for the concentration and production of alpha-amylase from this source and could be applied at the beginning of downstream processing.

Enhancement of fructosyltransferase and fructooligosaccharides production by A. oryzae DIA-MF in Solid-State Fermentation using aguamiel as culture medium.

The aim of this work was to improve the production of fructosyltransferase (FTase) by Solid-State Fermentation (SSF) using aguamiel (agave sap) as culture medium and Aspergillus oryzae DIA-MF as producer strain. SSF was carried out evaluating the following parameters: inoculum rate, incubation temperature, initial pH and packing density to determine the most significant factors through Box-Hunter and Hunter design. The significant factors were then further optimized using a Box-Behnken design and response surface methodology. The maximum FTase activity (1347U/L) was obtained at 32°C, using packing density of 0.7g/cm(3). Inoculum rate and initial pH had no significant influence on the response. FOS synthesis applying the enzyme produced by A. oryzae DIA-MF was also studied using aguamiel as substrate.

Support networks and people with physical disabilities: social inclusion and access to health services / Redes de apoio e pessoas com deficiência física: inserção social e acesso aos serviços de saúde

This study seeks to identify the formation of social support networks of people with physical disabilities, and how these networks can help facilitate access to health services and promote social inclusion. It is a cross-sectional study, with data collected via a form applied to physically disabled persons over eighteen years of age registered with the Family Health Teams of the municipal district of João Pessoa in the state of Paraíba. It was observed that the support networks of these individuals predominantly consist of family members (parents, siblings, children, spouses) and people outside the family (friends and neighbors). However, 50% of the interviewees declared that they could not count on any support from outside the family. It was observed that the support network contributes to access to the services and participation in social groups. However, reduced social inclusion was detected, due to locomotion difficulties, this being the main barrier to social interaction. Among those individuals who began to interact in society, the part played by social support was fundamental.

Este estudo objetiva identificar a constituição das redes de apoio social das pessoas com deficiência física e como estas podem contribuir para facilitar o acesso aos serviços de saúde e a inclusão social das mesmas. Trata-se de um estudo transversal, com dados coletados através de um formulário, aplicado em pessoas com deficiência física maiores de dezoito anos, cadastradas nas Equipes de Saúde da Família do município de João Pessoa (PB). Constatou-se que as redes de apoio dessas pessoas estão constituídas principalmente pelos componentes da dimensão familiar (pais, irmãos, filhos, cônjuges) e extrafamiliar (amigos e vizinhos). No entanto, 50% dos entrevistados relataram não contar com qualquer apoio fora da família. Verificou-se que a rede de apoio contribui para o acesso aos serviços e para a participação em grupos sociais. Evidenciou-se, porém, uma reduzida inserção social, decorrente da dificuldade de locomoção, sendo esta a principal barreira para a interação social. Entre as pessoas que começaram a interagir na sociedade o apoio social foi fundamental.

Optimisation of soy flour fermentation parameters to produce ß-glucosidase for bioconversion into aglycones.

The solid state fermentation (SSF) parameters of defatted soybean flour (DSF) with Aspergillus oryzae IOC 3999/1998 or Monascus purpureus NRRL 1992 was evaluated using a rotational central composite experimental design to optimise the production of ß-glucosidase and convert glycosidic isoflavones in aglycones. Variables investigated were initial pH of DSF, volume of water added to 10 g of DSF and incubation temperature. ß-Glucosidase activity was measured using the synthetic substrate, p-nitrophenyl-ß-D-glucoside. The content of isoflavones was determinate by ultra performance liquid chromatography. The highest production of ß-glucosidase for both strains occurred when adding 10 mL of water to the DSF, incubating at 30 °C and using 6.0 as the initial DSF pH. A. oryzae IOC 3999/1998 expressed ß-glucosidase activity at 10.7 times higher than M. purpureus NRRL 1992. The DSF fermentation was more efficient in converting isoflavones with M. purpureus NRRL 1992.

Fed-batch synthesis of galacto-oligosaccharides with Aspergillus oryzae ß-galactosidase using optimal control strategy.

Fed-batch synthesis of galacto-oligosaccharides (GOS) from lactose with ß-galactosidase from Aspergillus oryzae was evaluated experimentally and reaction yield was maximized via optimal control technique. The optimal lactose and enzyme feed flow rate profiles were determined using a model for GOS synthesis previously reported by the authors. Experimentally it was found that fed-batch synthesis allowed an increase on the maximum total GOS concentration from 115 (batch synthesis) to 218 g L(-1) as consequence of the increase in total sugars concentration from 40 to 58% w/w. Such high concentration of total sugars was not attainable in batch operation because of the low solubility of lactose at the reaction temperature (40°C). Simulations predicted a GOS yield of 32.5 g g(-1) in fed-batch synthesis under optimal conditions, while experimentally the same yield as in batch synthesis was obtained (28 g g(-1) ). Besides, an enrichment of total oligosaccharides in GOS with a high polymerization degree (GOS-5 and GOS-6) was observed in the fed-batch synthesis. Experimental profiles for all sugars were similar to the ones predicted by simulation, which supports the use of this methodology for the optimization of GOS synthesis.

Kinetic modeling of fructooligosaccharide production using Aspergillus oryzae N74.

In this study, the kinetic for the bioconversion of sucrose to fructooligosaccharides (FOS) by free cells of Aspergillus oryzae N74 was modeled. In addition, the effect of immobilized glucose isomerase (IGI) on FOS production yield was evaluated and considered in the kinetic model. The selected kinetic models were based on a proposed reaction mechanism described by elementary rate equations and modified Michaelis-Menten kinetic equations. The use of IGI allowed to increase the FOS production yield (FOS(Yield)) and to decrease the glucose/fructose (G/F) ratio. At shake flask scale, the FOS(Yield) was increased in 4.7 % (final yield 58.3 %), while the G/F ratio was reduced 6.2-fold. At bench scale, the FOS(Yield) was increased in 2.2 % (final yield 57.3 %), while the G/F ratio was reduced 4.5-fold. The elementary rate equation model was the one that best adjusted experimental data for FOS production using either the fungus biomass or the mixture fungus biomass-IGI, with an overall average percentage error of 7.2. Despite that FOS production yield was not highly improved by the presence of IGI in the reaction mixture, it favored the reduction of residual glucose in the mixture, avoiding the loss of material owe to glucose transformation to fructose that can be used in situ for FOS production by the fructosyltransferase.

Synthesis of galacto-oligosaccharides by ß-galactosidase from Aspergillus oryzae using partially dissolved and supersaturated solution of lactose.

The effect of enzyme to substrate ratio, initial lactose concentration and temperature has been studied for the kinetically controlled reaction of lactose transgalactosylation with Aspergillus oryzae ß-galactosidase, to produce prebiotic galacto-oligosaccharides (GOS). Enzyme to substrate ratio had no significant effect on maximum yield and specific productivity. Galacto-oligosaccharide syntheses at very high lactose concentrations (40, 50 and 60%, w/w, lactose monohydrate) were evaluated at different temperatures (40, 47.5 and 55°C). Within these ranges, lactose could be found as a supersaturated solution or a heterogeneous system with precipitated lactose, resulting in significant effect on GOS synthesis. An increase in initial lactose concentration produced a slight increase in maximum yield as long as lactose remained dissolved. Increase in temperature produced a slight decrease in maximum yield and an increase in specific productivity when supersaturation of lactose occurred during reaction. Highest yield of 29 g GOS/100 g lactose added was obtained at a lactose monohydrate initial concentration of 50% (w/w) and 47.5°C. Highest specific productivity of 0.38 g GOSh(-1) mg enzyme(-1) was obtained at lactose monohydrate initial concentration of 40% (w/w) and 55°C, where a maximum yield of 27 g GOS/100 g lactose added was reached. This reflects the complex interplay between temperature and initial lactose concentration on the reaction of synthesis. When lactose precipitation occurred, values of yields and specific productivities lower than 22 g GOS/100 g lactose added and 0.03 gGOSh(-1) mg enzyme(-1) were obtained, respectively.

Bioconversion and saccharification of some lignocellulosic wastes by aspergillus oryzae ITCC-4857.01 for fermentable sugar production

The recent interest in bioconversion of agricultural and industrial wastes to chemical feedstock has led to extensive studies on cellulolytic enzymes produced by microorganisms. In the present study three lignocellulosic substrates viz. sugarcane bagasse, sawdust and water hyacinth were pre-treated with alkali and enzyme and their effect on bioconversion has been investigated. The ability of selected substrates for induction of cellulase enzyme by A. oryzae ITCC 4857.01 and for the potentiality of the induced enzyme to saccharify the substrates were also assessed. The maximum degree of conversion of substrate (0.415 percent) and improved specific substrate consumption (0.99 g substrate/g dry biomass) was exhibited in sugarcane bagasse after alkali treatment at 96 hrs. Both alkali-treatment and enzyme-treatment, water hyacinth was the best for cellulase induction and showed maximum endoglucanase activity of 11.42 U/ml. Reducing sugar yield ranged from 1.12 mg/ml for enzyme treated sawdust at 48 hrs to 7.53 mg/ml for alkali treated sugarcane bagasse at 96 hrs. Alkali-treated sugarcane bagasse gave the highest saccharification rate of 9.03 percent after 96 hrs. The most resistant substrate was sawdust which produced 5.92 percent saccharification by alkaline treatment. The saccharification of lignocellulosic substrates by enzyme produced by A. oryzae ITCC 4857.01 indicates the enzymes specificity towards the substrates. The use of such enzyme in lingo-cellulose hydrolysis will lead to efficient conversion of cellulose materials to other important products.

Bioremediation of dyes in textile effluents by Aspergillus oryzae.

In this study Aspergillus oryzae was utilized to remove azo dyes from aqueous solution. Physically induced in its paramorphogenic form to produce standardized mycelial pellets, the non-autoclaved and autoclaved hyphae biomass was applied to biosorb the reactive dyes Procion Red HE7B (PR-HE7B) and Procion Violet H3R (PV-H3R) at different pH values (2.50, 4.50, and 6.50). The best pH for biosorption was 2.50, though the autoclaved demonstrated a higher biosorption capacity than the non-autoclaved pellets. The toxicity level was determined using the Trimmed Spearman-Karber method with Daphnia similis in all bioassays. The calculated toxicity of PV-H3R (LC100 62.50 microg mL(-1)) was higher than to PR-HE7B (LC100 300.00 microg mL(-1)), and its results brought out that the decrease of toxicity levels to zero might be accomplished by adding small quantities of pelletized A. oryzae to the solutions.

Biosensor based on laccase and an ionic liquid for determination of rosmarinic acid in plant extracts.

Novel biosensors based on laccase from Aspergillus oryzae and the ionic liquids (ILs) 1-n-butyl-3-methylimidazolium hexafluorophosphate (BMIPF(6)) and 1-n-butyl-3-methylimidazolium tetrafluoroborate (BMIBF(4)) were constructed for determination of rosmarinic acid by square-wave voltammetry. The laccase catalyzes the oxidation of rosmarinic acid to the corresponding o-quinone, which is electrochemically reduced back to rosmarinic acid at +0.2V vs. Ag/AgCl. The biosensor based on BMIPF(6) showed a better performance than that based on BMIBF(4). The best performance was obtained with 50:20:15:15% (w/w/w/w) of the graphite powder:laccase:Nujol:BMIPF(6) composition in 0.1mol L(-1) acetate buffer solution (pH 5.0). The rosmarinic acid concentration was linear in the range of 9.99 x 10(-7) to 6.54 x 10(-5)mol L(-1) (r=0.9996) with a detection limit of 1.88 x 10(-7)mol L(-1). The recovery study for rosmarinic acid in plant extract samples gave values from 96.1 to 105.0% and the concentrations determined were in agreement with those obtained using capillary electrophoresis at the 95% confidence level. The BMIPF(6)-biosensor demonstrated long-term stability (300 days; 920 determinations) and reproducibility, with a relative standard deviation of 0.56%.