Which are the best murine models to study Eosinophilic Chronic Rhinosinusitis? A contemporary review.

OBJECTIVE: Mechanisms that lead to Eosinophilic Chronic Rhinosinusitis (ECRS) are not fully established in the literature. It is desirable to assess ECRS in a model that embraces most of the related events. This article reviewed the murine models for ECRS and compared them regarding eosinophilic polypoid formation. METHODS: The authors reviewed the articles that included the terms "chronic rhinosinusitis" OR "chronic sinusitis" AND "animal model". We analyzed articles in English that evaluated both the number of polyps and the number of eosinophils in the sinus mucosa of mouse models. RESULTS: We identified a total of 15 articles describing different models of ECRS that used BALB/c or C57BL/6 mice, and different triggers/stimulants such as Staphylococcus aureus Enterotoxin B (SEB) + Ovalbumin (OVA); House Dust Mite (HDM) ±â€¯Ovalbumin (OVA); and Aspergillus oryzae Protease (AP) + Ovalbumin (OVA). OVA associated with SEB was the commonest protocol to induce ECRS in both BALB/c and C57BL/6 mice, and it produced a robust response of eosinophilic nasal polyps in both. AP + OVA protocol also led to a good ECRS response. The other models were not considered adequate to produce eosinophilic polyps in mice. CONCLUSION: In conclusion, OVA associated with SEB seems to produce the most robust eosinophilic sinonasal inflammation.

Which are the best murine models to study Eosinophilic Chronic Rhinosinusitis? A contemporary review

Abstract Objective: Mechanisms that lead to Eosinophilic Chronic Rhinosinusitis (ECRS) are not fully established in the literature. It is desirable to assess ECRS in a model that embraces most of the related events. This article reviewed the murine models for ECRS and compared them regarding eosinophilic polypoid formation. Methods: The authors reviewed the articles that included the terms "chronic rhinosinusitis" OR "chronic sinusitis" AND "animal model". We analyzed articles in English that evaluated both the number of polyps and the number of eosinophils in the sinus mucosa of mouse models. Results: We identified a total of 15 articles describing different models of ECRS that used BALB/c or C57BL/6 mice, and different triggers/stimulants such as Staphylococcus aureus Enterotoxin B (SEB) + Ovalbumin (OVA); House Dust Mite (HDM) ± Ovalbumin (OVA); and Aspergillus oryzae Protease (AP) + Ovalbumin (OVA). OVA associated with SEB was the commonest protocol to induce ECRS in both BALB/c and C57BL/6 mice, and it produced a robust response of eosinophilic nasal polyps in both. AP + OVA protocol also led to a good ECRS response. The other models were not considered adequate to produce eosinophilic polyps in mice. Conclusion: In conclusion, OVA associated with SEB seems to produce the most robust eosinophilic sinonasal inflammation.

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.

Successive Fermentation of Aguamiel and Molasses by Aspergillus oryzae and Saccharomyces cerevisiae to Obtain High Purity Fructooligosaccharides.

Fructooligosaccharides (FOS) are usually synthesized with pure enzymes using highly concentrated sucrose solutions. In this work, low-cost aguamiel and molasses were explored as sucrose alternatives to produce FOS, via whole-cell fermentation, with an Aspergillus oryzae DIA-MF strain. FOS production process was optimized through a central composite experimental design, with two independent variables: initial sucrose concentration in a medium composed of aguamiel and molasses (AgMe), and inoculum concentration. The optimized process-165 g/L initial sucrose in AgMe (adjusted with concentrated molasses) and 1 × 107 spores/mL inoculum concentration-resulted in an FOS production of 119 ± 12 g/L and a yield of 0.64 ± 0.05 g FOS/g GFi. Among the FOSs produced were kestose, nystose, 1-fructofuranosyl-nystose, and potentially a novel trisaccharide produced by this strain. To reduce the content of mono- and disaccharides in the mixture, run a successive fermentation was run with two Saccharomyces cerevisiae strains. Fermentations run with S. cerevisiae S227 improved FOS purity in the mixture from 39 ± 3% to 61.0 ± 0.6% (w/w) after 16 h of fermentation. This study showed that agro-industrial wastes such as molasses with aguamiel are excellent alternatives as substrate sources for the production of prebiotic FOS, resulting in a lower-cost process.

In silico investigation of riboswitches in fungi: structural and dynamical insights into TPP riboswitches in Aspergillus oryzae.

Riboswitches are RNA sensors affecting post-transcriptional processes through their ability to bind to small molecules. Thiamine pyrophosphate (TPP) riboswitch plays a crucial role in regulating genes involved in synthesizing or transporting thiamine and phosphorylated derivatives in bacteria, archaea, plants, and fungi. Although TPP riboswitch is reasonably well known in bacteria, there is a gap in the knowledge of the fungal TPP riboswitches structure and dynamics, involving mainly sequence variation and TPP interaction with the aptamers. On the other hand, the increase of fungal infections and antifungal resistance raises the need for new antifungal therapies. In this work, we used computational approaches to build three-dimensional models for the three TPP riboswitches identified in Aspergillus oryzae, in which we studied their structure, dynamics, and binding free energy change (ΔGbind) with TPP. Interaction patterns between the TPP and the surrounding nucleotides were conserved among the three models, evidencing high structural conservation. Furthermore, we show that the TPP riboswitch from the A. oryzae NMT1 gene behaves similarly to the E. coli thiA gene concerning the ΔGbind. In contrast, mutations in the fungal TPP riboswitches from THI4 and the nucleoside transporter genes led to structural differences, affecting the binding-site volume, hydrogen bond occupancy, and ΔGbind. Besides, the number of water molecules surrounding TPP influenced the ΔGbind considerably. Notably, our ΔGbind estimation agreed with previous experimental data, reinforcing the relationship between sequence conservation and TPP interaction.

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.

Purification and characterization of an FeII- and α-ketoglutarate-dependent xanthine hydroxylase from Aspergillus oryzae.

XanA is an FeII- and α-ketoglutarate-dependent enzyme responsible for the conversion of xanthine to uric acid. It is unique to fungi and it was first described in Aspergillus nidulans. In this work, we present the preliminary characterization of the XanA enzyme from Aspergillus oryzae, a relevant fungus in food production in Japan. The XanA protein (GenBank BAE56701.1) was expressed as a recombinant protein in Escherichia coli BL21 (DE3) Arctic cells. Initial purification assays showed low protein solubility; therefore, the buffer composition was optimized using a fluorescence-based thermal shift assay. The protein was stabilized in solution in the presence of either 600 µM xanthine, 1 M NaCl, 600 µM α-ketoglutarate or 20% glycerol, which increases the melting temperature (Tm) by 2, 4, 5 and 6 °C respectively. The XanA protein was purified by following a three-step purification protocol. The nickel affinity purified protein was subjected to ion-exchange chromatography once the N-terminal 6XHis-tag had been successfully removed, followed by size-exclusion purification. Dynamic light scattering experiments showed that the purified protein was monodisperse and behaved as a monomer in solution. Preliminary activity assays in the presence of xanthine, α-ketoglutarate, and iron suggest that the enzyme is an iron- and α-ketoglutarate-dependent xanthine dioxygenase. Furthermore, the enzyme's optimum activity conditions were determined to be 25 °C, pH of 7.2, HEPES buffer, and 1% of glycerol. In conclusion, we established the conditions to purify the XanA enzyme from A. oryzae in its active form from E. coli bacteria and determined the optimal activity conditions.

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.

Detoxification of Ochratoxin A by a novel Aspergillus oryzae strain and optimization of its biodegradation.

The mycotoxin Ochratoxin A (OTA) causes serious health risks and is found in food products throughout the world. The most promising method to detoxify this compound is biodegradation. In this study, Aspergillus oryzae strain M30011 was isolated and characterized based on its considerable capacity to degrade OTA. The degradation product (compound I) of A. oryzae-treated OTA was isolated, and its toxicity response was also evaluated. Furthermore, the relationships between three key cultivation condition factors affecting the OTA degradation rate were examined using the response surface methodology (RSM). Compound I was identified as ochratoxin α (C11H9O5Cl), and the toxicity response experiments indicated that A. oryzae detoxified OTA to a great extent. A maximum degradation rate of 94% was observed after 72h. This study demonstrates the potential for using A. oryzae to detoxify OTA and suggests that it could be applied in the food industry to improve food safety and quality.

Immobilization of Aspergillus oryzae ß-galactosidase in cation functionalized agarose matrix and its application in the synthesis of lactulose.

Aspergillus oryzae ß-galactosidase was immobilized in in-house quaternary ammonium agarose (QAA) and used for the first time in the synthesis of lactulose. A biocatalyst was obtained with a specific activity of 24,690 IUH∙g-1; protein immobilization yield of 97% and enzyme immobilization yield of 76% were obtained at 30 °C in 10 mM phosphate buffer pH 7 for standard size agarose at 100 mgprotein∙gsupport-1 which the maximum protein load of QAA. Highest yield and specific productivity of lactulose were 0.24 g∙g-1 and 9.78 g∙g-1 h-1 respectively, obtained at pH 6, 100 IUH∙g lactose-1 enzyme/lactose ratio and 12 lactose/fructose molar ratio. In repeated-batch operation with the immobilized enzyme, the cumulative mass of lactulose per unit mass of contacted protein and cumulative specific productivity were higher than obtained with the soluble enzyme since the first batch. After enzyme activity exhaustion, the enzyme was desorbed and QAA support was reused without alteration in its maximum enzyme load capacity and without detriment in yield, productivity and selectivity in the batch synthesis of lactulose with the resulting biocatalyst. This significantly decreases the economic impact of the support, presenting itself as a distinctive advantage of immobilization by ionic interaction.

Optimization of media composition and growth conditions for production of milk-clotting protease (MCP) from Aspergillus oryzae DRDFS13 under solid-state fermentation.

This study reports the optimization of milk-clotting protease production from Aspergillus oryzae DRDFS13 under solid-state fermentation (SSF) in both one-variable-at-a-time and response surface methodology (RSM). The production and optimization of milk-clotting protease obtained from Aspergillus oryzae DRDFS13 under solid-state fermentation (SSF) using different agro-industrial wastes as solid substrates were studied. The agro-industrial wastes used included wheat bran, rice bran, pea bran, and grass pea bran. The chemical composition of the best solid substrate was tested using standard methods. Others cultivation parameters were studied, and the results showed that the optimum fermentation medium composed of wheat bran, casein (1% w/w), and glucose (0.5% w/w) and the conditions for maximum milk-clotting protease production were at the moisture content of 55.0%, inoculum of 0.5*106 spores/mL, incubation temperature of 30 °C, pH of 6.0, and fermentation time of 5 days. The highest milk-clotting activity was obtained from the crude enzyme extracted using 0.1 M NaCl and partial purification of the crude enzyme using chilled acetone, and 80% (NH4)2SO4 increased the ratio of MCA/PA from 0.56 to 1.30 and 0.65, respectively. Moreover, the highest MCA (137.58 U/mL) was obtained at a casein concentration of 0.5%, pH 4.0, and 25 °C, using RSM. Thus, results from the present study showed that the optimization of milk-clotting protease production from A. oryzae DRDFS 13 under SSF by both one-variable-at-a-time and RSM significantly increased the milk-clotting activity. This is the first report from a fungus in the Ethiopian setting and a modest contribution to highlight the potential of harnessing microbial protease enzymes for industrial applications.

Improved protease activity of Pixian broad bean paste with cocultivation of Aspergillus oryzae QM-6 and Aspergillus niger QH-3

BACKGROUND: The preparation of broad bean koji is a key process in the production of Pixian broad bean paste (PBP). Protease is essential for the degradation of proteins during PBP fermentation. To obtain broad bean koji with high protease activity using the cocultivated strains of Aspergillus oryzae QM-6 (A. oryzae QM-6) and Aspergillus niger QH-3 (A. niger QH-3), the optimization of acid and neutral protease activities was carried out using Box­Behnken design with response surface methodology (RSM). RESULTS: The optimum conditions were found to be as follows: inoculation proportion (X1), 3:1 (A. oryzae QM-6: A. niger QH-3, w/w); culture temperature (X2), 33°C; inoculum size (X3), 0.5% (w/w); incubation time (X4), 5 d. The acid and neutral protease activities were 605.2 ± 12.4 U/g and 1582.9 ± 23.7 U/g, respectively, which were in good agreement with the predicted values. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles revealed that the broad bean koji extracellular proteins in the case of cocultivation were richer compared to those in the case of A. oryzae QM-6 or A. niger QH-3 strain only. In addition, the free amino acids (FAAs) in the fermentation product were 55% higher in the cocultivation process than in that involving only A. oryzae QM-6, further confirming the diversity of proteases in the fermentation products. CONCLUSIONS: The optimal conditions of koji-making in PBP were obtained using RSM. The cocultivation of A. oryzae and A. niger increases the overall enzyme activities in the culture medium and the FAAs content, which would thus have potential application in the PBP industry.

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.

Desarrollo y capacidad de remoción de los pesticidas atrazina, clorpirifós y endosulfán por parte de cepas de Aspergillus sección Flavi no toxigénicas / Development and removal ability of non-toxigenic Aspergillus section Flavi in presence of atrazine, chlorpyrifos and endosulfan

This study evaluated the in vitro effect of three concentrations of atrazine, chlorpyrifos and endosulfan on the growth parameters of four non-toxigenic Aspergillus section Flavi strains. The ability of the strains to remove these pesticides in a synthetic medium was also determined. Growth parameters were measured on soil extract solid medium supplied with 5,10 and 20mg/l of each pesticide, and conditioned to -0.70, -2.78, -7.06 and -10.0 water potential (MPa). Removal assays were performed in Czapek Doc medium (CZD) supplied with 20mg/l of each pesticide under optimal environmental conditions (-2.78 of MPa and 25 °C). The residual levels of each pesticide were detected by the reversed-phase HPLC/fluorescence detection system. The lag phases of the strains significantly decreased in the presence of the pesticides with respect to the control media. This result indicates a fast adaptation to the conditions assayed. Similarly, the mycelial growth rates in the different treatments increased depending on pesticide concentrations. Aspergillus oryzae AM 1 and AM 2 strains showed high percentages of atrazine degradation (above 90%), followed by endosulfan (56 and 76%) and chlorpyrifos (50 and 73%) after 30 days of incubation. A significant (p <0.001) correlation (r = 0.974) between removal percentages and growth rate was found. This study shows that non-toxigenic Aspergillus section Flavi strains from agricultural soils are able to effectively grow in the presence of high concentrations of atrazine, chlorpyrifos and endosulfan under a wide range of MPa conditions. Moreover, these strains have the ability to remove high levels of these pesticides in vitro in a short time.

En este estudio se evaluó los efectos in vitro de 3 concentraciones de atrazina, clorpirifós y endosulfán sobre los parámetros de crecimiento de 4 cepas no toxigénicas de Aspergillus sección Flavi. También se evaluó la capacidad de las cepas de remover los pesticidas. Los parámetros de crecimiento se ensayaron en medio agar extracto de suelo suplementado con 5, 10 y 20mg/l de cada pesticida y acondicionado a -0.70, -2.78, -7.06 y -10.0 de potencial de agua (MPa). Los ensayos de remoción se realizaron en medio Czapek Dox con 20mg/l de cada pesticida bajo condiciones óptimas de crecimiento (-2.78 de MPa y 25 °C). Los niveles residuales de atrazina, clorpirifós y endosulfán se detectaron en un sistema HPLC con detección por fluorescencia. La fase de latencia de las cepas disminuyó significantemente en presencia de los pesticidas, indicando una rápida adaptación a dichas condiciones. La velocidad de crecimiento se incrementó considerablemente dependiendo de la concentración de pesticida. Las cepas Aspergillus oryzae AM1 y AM2 mostraron porcentajes elevados de degradación de atrazina (aproximadamente el 90%), seguidos por endosulfán (56 y 76%) y clorpirifós (50 y 73%). Se observó una correlación (r = 0.974) significante (p <0.001) entre el porcentaje de pesticida removido y la velocidad de crecimiento. Este estudio muestra que cepas no-toxigénicas de Aspergillus sección Flavi aisladas de suelos agrícolas desarrollan eficientemente en presencia de altas concentraciones de atrazina, clorpirifós y endosulfán en un amplio rango de MPa. Además, presentan capacidad de remover in vitro altos niveles de pesticidas en corto tiempo.

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.

Development and removal ability of non-toxigenic Aspergillus section Flavi in presence of atrazine, chlorpyrifos and endosulfan.

This study evaluated the in vitro effect of three concentrations of atrazine, chlorpyrifos and endosulfan on the growth parameters of four non-toxigenic Aspergillus section Flavi strains. The ability of the strains to remove these pesticides in a synthetic medium was also determined. Growth parameters were measured on soil extract solid medium supplied with 5, 10 and 20mg/l of each pesticide, and conditioned to -0.70, -2.78, -7.06 and -10.0 water potential (MPa). Removal assays were performed in Czapek Doc medium (CZD) supplied with 20mg/l of each pesticide under optimal environmental conditions (-2.78 of MPa and 25°C). The residual levels of each pesticide were detected by the reversed-phase HPLC/fluorescence detection system. The lag phases of the strains significantly decreased in the presence of the pesticides with respect to the control media. This result indicates a fast adaptation to the conditions assayed. Similarly, the mycelial growth rates in the different treatments increased depending on pesticide concentrations. Aspergillus oryzae AM 1 and AM 2 strains showed high percentages of atrazine degradation (above 90%), followed by endosulfan (56 and 76%) and chlorpyrifos (50 and 73%) after 30 days of incubation. A significant (p<0.001) correlation (r=0.974) between removal percentages and growth rate was found. This study shows that non-toxigenic Aspergillus section Flavi strains from agricultural soils are able to effectively grow in the presence of high concentrations of atrazine, chlorpyrifos and endosulfan under a wide range of MPa conditions. Moreover, these strains have the ability to remove high levels of these pesticides in vitro in a short time.

Fed-batch operation for the synthesis of lactulose with ß-galactosidase of Aspergillus oryzae.

Fed-batch synthesis of lactulose from lactose and fructose with Aspergillus oryzae ß-galactosidase was evaluated, obtaining a concentration of 40.4g·L-1, which is 20% higher than obtained in batch, while the concentration of transgalactosylated oligosaccharides (TOS) was reduced by 98%. Therefore, selectivity of lactulose synthesis can be significantly higher by operating in fed-batch mode. The enzyme-limiting substrate mass ratio (E/S) is a critical variable in fed-batch operation. Higher values favor lactose hydrolysis over transgalactosylation, being 400IU/g the limit for proper lactulose synthesis in fed-batch operation. Selectivity of lactulose synthesis increased with E/S being quite high at 800IUH·g-1 or higher. However, this increase was obtained at the expense of lactulose yield. Lactulose synthesis in fed-batch operation was a better option than conventional batch synthesis, since higher product concentration and selectivity of lactulose over TOS synthesis were obtained.

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.

Solid-state fermentation of Acanthogobius hasta processing by-products for the production of antioxidant protein hydrolysates with Aspergillus oryzae

Functional properties and antioxidative activity of a protein hydrolysate prepared from Acanthogobius hasta processing by-product protein during solid-state fermentation with Aspergillus oryzae were investigated. Overall, protease activity increased with the degree of hydrolysis (DH) decreased during solid-state fermentation. All the protein hydrolysate had excellent solubility, possessed interfacial properties, and varying degrees of antioxidant activity which were governed by their concentrations and DH, molecular weight distribution and amino acid composition. After 5 days fermentation, the DH of the protein hydrolysate was 31.23%. The protein hydrolysate had the highest total hydrophobic amino acid content, the highest DPPH scavenging activity, reducing power, and the chelating activity. The radical-scavenging activity of the hydrolysates at 6 mg/mL was 78.6%. The reducing power of protein hydrolysate at the range of 0-6 mg/mL was lower than that of BHA at the range of 0-60 µg/mL, while the chelating activity of APs was similar to that of BHA at the range of 0-60 µg/mL. Moreover, the protein hydrolysate showed good emulsifying and foaming properties over a wide pH range from 2 to 12. Therefore, solid state fermentation provided a suitable and low-cost method for converting Acanthogobius hasta processing by-product protein into antioxidant protein hydrolysates.