Overproduction of Glucose Oxidase by Aspergillus tubingensis CTM 507 Randomly Obtained Mutants and Study of Its Insecticidal Activity against Ephestia kuehniella.

In order to enhance the production of glucose oxidase (GOD), random mutagenesis of Aspergillus tubingensis CTM 507 was performed using the chemical and physical mutagens: nitric acid and UV irradiation, respectively. The majority of the isolated mutants showed good GOD production, but only some mutants presented a significant overproduction, as compared with the parent strain. The selected mutants (19 strains), showing an overproduction larger than 200%, are quite stable after three successive subcultures. Among these, six strains revealed an important improvement in submerged fermentation. The insecticidal activity of GOD produced by the wild and the selected mutant strains was evaluated against the third larval instars of E. kuehniella. Mutant strains U11, U12, U20, and U21, presenting the most important effect, displayed an LC50 value of 89.00, 88.51, 80.00, and 86.00 U/cm2, respectively, which was 1.5-fold more important than the wild strain (61 U/cm2). According to histopathology observations, the GOD enzyme showed approximately similar damage on the E. kuehniella midgut including rupture and disintegration of the epithelial layer and cellular vacuolization. The data supports, for the first time, the use of GOD as a pest control agent against E. kuehniella.

A New Endophytic Fusarium Oxysporum Gibberellic Acid: Optimization of Production Using Combined Strategies of Experimental Designs and Potency on Tomato Growth under Stress Condition.

This study reports the potential of the endophytic fungi identified as a Fusarium oxysporum to produce gibberellic acid (GA3). The GA3 production was confirmed by high performance liquid chromatography. To improve the production of this phytohormone under solid state fermentation (SSF), successive optimization strategies were used. Firstly, Plackett-Burman design was applied for screening medium components and culture condition. Under the optimized condition, GA3 yield (7.14 g/kg) was 2.62-fold higher than by the use of the initial condition (2.72 g/kg). The concentration of the most influential parameters and their interaction were optimized with a Box-Behnken experimental design. The optimized condition led to a 1.14-fold enhancement in GA3 production, reaching 8.16 g/kg. The GA3 crude extract obtained by SSF was then used to study its ameliorative role on adverse salinity effect on tomato plants (Solanum lycopersicum L.). The interactive effects of different GA3 concentrations were examined on morphological and physiological parameters of tomato plants. The application of GA3 (10-6 M) under salt stress condition (100 mM) was found to improve growth and physiological parameters including plant height, total chlorophyll, starch, and proline contents. The exogenous application of GA3 is a potent strategy to reverse abiotic stress that affect the agricultural productivity and limit plant growth and yield.

Optimization of enzymatic extraction of pectin from Opuntia ficus indica cladodes after mucilage removal.

In this study, pectin was isolated from Opuntia ficus indica (OFI) cladodes after removing mucilage using the xylanase and cellulase. The process variables were optimized by the Box Behnken design with three factors at three levels. The optimal extraction condition obtained was: liquid to solid (LS), cellulase to xylanase and enzymes to matter ratios of 22ml/g, 2:1U/U and 4U/g, respectively. The simulated extraction yield of 17.91% was validated by the experimental result (16.67±0.30). The enzyme-extracted pectin from OFI cladodes (EAEPC) was low methylated, with a high uronic acid content, a water and oil holding capacity of 5.42g/g and 1.23g/g, respectively, a good foam and emulsion stability and important DPPH radical scavenging activity. Both the OFI cladodes and enzymatic process present promising alternatives to traditional sources and extraction processes of pectin, respectively. EAEPC thus represents a promising additive in food industries.

Ultrasonic extraction of pectin from Opuntia ficus indica cladodes after mucilage removal: Optimization of experimental conditions and evaluation of chemical and functional properties.

Ultrasonic assisted extraction (UAE) of pectin from Opuntia ficus indica (OFI) cladodes after mucilage removal was attempted using the response surface methodology. The process variables were optimized by the isovariant central composite design in order to improve the pectin extraction yield. The optimum condition obtained was: sonication time 70min, temperature 70°C, pH 1.5 and the water-material ratio 30ml/g. This condition was validated and the performance of experimental extraction was 18.14%±1.41%, which was closely linked to the predicted value (19.06%). Thus, UAE present a promising alternative to conventional extraction process thanks to its high efficiency which was achieved in less time and at lower temperatures. The pectin extracted by UAE from OFI cladodes (UAEPC) has a low degree of esterification, high uronic acid content, important functional properties and good anti-radical activity. These results are in favor of the use of UAEPC as potential additive in food industry.

Extraction and characterization of three polysaccharides extracted from Opuntia ficus indica cladodes.

The chemical extraction and the characterization of polysaccharides from mucilage (MC), pectin (PC) and total pectic mucilage fraction (TFC) of Opuntia ficus indica cladodes as well as the evaluation of their antioxidant activities was investigated. The FTIR spectroscopic analysis revealed the presence of carboxyl and hydroxyl groups corresponding to polysaccharides. Uronic acid and the total sugar contents of PC were higher than those of TFC and MC whereas ash content of MC was considerably more important. In addition, the findings showed that all the samples had little protein content and low average molecular weight compared to the results mentioned in literature. Furthermore, MC reached not only the highest water (WHC) and oil holding (OHC) capacities (7.81g/g and 1.34g/g, respectively) but also the highest antioxidant properties (DPPH and ABTS scavenging activities, ß-carotene bleaching inhibition activity and reducing power). However, PC had the strongest emulsifying and foaming properties. As for TFC, it had low WHC, OHC and emulsifying properties whereas it had higher foaming properties than MC and greater antioxidant properties compared to PC. These outcomes can encourage the use of PC as a surfactant and MC and TFC as natural antioxidants in food and pharmaceutical industries.

Synergistic effect of Aspergillus tubingensis CTM 507 glucose oxidase in presence of ascorbic acid and alpha amylase on dough properties, baking quality and shelf life of bread.

The impact of Aspergillus tubingensis glucose oxidase (GOD) in combination with α-amylase and ascorbic acid on dough properties, qualities and shelf life of bread was investigated. Regression models of alveograph and texture parameters of dough and bread were adjusted. Indeed, the mixture of GOD (44 %) and ascorbic acid (56 %) on flour containing basal improver showed its potential as a corrective action to get better functional and rheological properties of dough and bread texture. Furthermore, wheat flour containing basal additives and enriched with GOD (63.8 %), ascorbic acid (32 %) and α- amylase (4.2 %) led to high technological bread making parameters, to decrease the crumb firmness and chewiness and to improve elasticity, adhesion, cohesion and specific volume of bread. In addition to that, the optimized formulation addition significantly reduced water activity and therefore decreased bread susceptibility to microbial spoilage. These findings demonstrated that GOD could partially substitute not only ascorbic acid but also α-amylase. The generated models allowed to predict the behavior of wheat flour containing additives in the range of values tested and to define the additives formula that led to desired rheological and baking qualities of dough. This fact provides new perspectives to compensate flour quality deficiencies at the moment of selecting raw materials and technological parameters reducing the production costs and facilitating gluten free products development. Graphical abstractᅟ.

Enzymatically hydrolysed, acetylated and dually modified corn starch: physico-chemical, rheological and nutritional properties and effects on cake quality.

Corn starch was treated by enzymatic hydrolysis with Aspergillus oryzae S2 α-amylase, acetylation with vinyl acetate, and dual modification. The dual modified starch displayed a higher substitution degree than the acetylated starch and lower reducing sugar content than the hydrolysed starch. The results revealed that the cooling viscosity and amylose content of those products decrease (P < 0.05). An increase in moisture, water, and oil absorption capacity was observed for the acetylated starch and, which was less pronounced for the enzymatically hydrolysed starch but more pronounced for the enzymatically hydrolysed acetylated product. The latter product underwent an increase in resistant starch content, which is induced by a rise in hydrolysis time to attain about 67 % after 1 h of reaction. The modified starch samples were added to cake formulations at 5 and 10 % concentrations on a wheat flour basis and compared to native starch. The results revealed that when applied at 5 % concentrations, the modified starches reduced the hardness, cohesion, adhesion and chewiness of baked cakes and enhanced their elasticity, volume, height, crust color, and appearance as compared to native starch. These effects were more pronounced for the cake incorporating the dually modified starch.

Optimization of submerged Aspergillus oryzae S2 α-amylase production.

Use of 4 agro-industrial by products and organic materials as nitrogen sources for production of Aspergillus oryzae S2 α-amylase in liquid culture was investigated. The 2 agro-industrial byproducts maltose and saccharose, and also lactose and starch were individually evaluated for use as carbon sources. A Box-Behnken experimental design was used to determine optimal conditions for production of α-amylase. A maximum amylase activity of 750 U/mL was obtained at a temperature of 24°C, a urea concentration of 1 g/L, and a C/N ratio of 2. Laboratory scale application of optimal conditions in a 7 L fermentor produced a final α-amylase activity of 770 U/mL after 3 days of batch cultivation. Addition of 10% starch to the culture medium each 12 h immediately after the stationary phase of cell growth led to a production yield of 1,220 U/mL at the end of fed-batch cultivation.

Optimization of Aspergillus oryzae S2 α-amylase, ascorbic acid, and glucose oxidase combination for improved French and composite Ukrainian wheat dough properties and bread quality using a mixture design approach.

A simplex-centroid experimental design was used for the optimization of both reducing and oxidizing improvers, namely Aspergillus oryzae S2 α-amylase (Amy), ascorbic acid (Asc), and glucose oxidase (GOD). This optimization was performed to enhance the dough and breadmaking qualities of soft French wheat flour and a composite counterpart that contained 30% Ukrainian wheat flour. Statistically significant correlations were calculated between the W index and textural parameters (e.g., dough chewiness and bread cohesiveness). The findings revealed that while the best mixture for French flour comprised 21.8% of Amy, 41.2% of Asc, and 37% of GOD, for the composite counterpart, it comprised 2.3% of Amy, 66% of Asc, and 31.7% of GOD. These optimized mixtures rearranged soft French wheat flour and its composite counterpart to a good quality and an improved flour texture, respectively. Additionally, they increased the loaf specific volumes of the breads made from soft French wheat flour and its counterpart by 25.8 and 45.43%, respectively, significantly decreased the breads' susceptibility to microbial contamination, and reclassified the breads as "good" in terms of sensory attributes.

Biocontrol of tomato plant diseases caused by Fusarium solani using a new isolated Aspergillus tubingensis CTM 507 glucose oxidase.

The present study focuses on the potential of glucose oxidase (GOD) as a promising biocontrol agent for fungal plant pathogens. In fact, a new GOD producing fungus was isolated and identified as an Aspergillus tubingensis. GOD (125 AU) has been found to inhibit Fusarium solani growth and spore production. Indeed, GOD caused the reduction of spores, the formation of chlamydospores, the induction of mycelial cords and the vacuolization of mycelium. In vivo assays, GOD acted as a curative treatment capable of protecting the tomato plants against F. solani diseases. In fact, the incidence was null in the curative treatment with GOD and it is around 45% for the preventive treatment. The optimization of media composition and culture conditions led to a 2.6-fold enhancement in enzyme activity, reaching 81.48U/mL. This study has demonstrated that GOD is a potent antifungal agent that could be used as a new biofungicide to protect plants from diseases.

Purification, biochemical characterization and antifungal activity of a novel Aspergillus tubingensis glucose oxidase steady on broad range of pH and temperatures.

This study was carried out to evaluate the in vitro and in vivo antifungal efficiency of Aspergillus tubingensis CTM 507 glucose oxidase (GOD) against plant pathogenic fungi. GOD displayed a wide inhibitory spectrum toward different fungi at a concentration of 20 AU. The GOD had a strong inhibitor effect on mycelia growth and spore germination of Pythium ultimum. Interestingly, the GOD exhibited a potent in vivo antifungal effect against P. ultimum responsible for potato plants disease. The antifungal GOD was purified 13-fold with 27 % yield and a specific activity of 3435 U/mg. The relative molecular mass of the GOD was 180 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The GOD activity was optimum at pH 4.5 and 60 °C. It was found to be stable over a large pH range (3-9). It also displayed a marked thermostability with a 50-min half-life at 65 °C. The 10 residues of the N-terminal sequence of the purified GOD (S-K-G-S-A-V-T-T-P-D) showed no homology to the other reported GOD, identifying a novel GOD. FTIR spectroscopic analysis revealed the presence of C-O and C=O groups corresponding to a D-glucono-lactone. The findings indicated that GOD is the first A. tubingensis-produced fungicide ever reported to exhibit such promising biological properties. It could become a natural alternative to synthetic fungicides to control certain important plant microbial diseases.

Aspergillus oryzae S2 alpha-amylase production under solid state fermentation: optimization of culture conditions.

Aspergillus oryzae S2 was assayed for alpha-amylase production under solid state fermentation (SSF). In addition to AmyA and AmyB already produced in monitored submerged culture, the strain was noted to produce new AmyB oligomeric forms, in particular a dominant tetrameric form named AmyC. The latter was purified to homogeneity through fractional acetone precipitation and size exclusion chromatography. SDS-PAGE and native PAGE analyses revealed that, purified AmyC was an approximately 172 kDa tetramer of four 42 kDa subunits. AmyC was also noted to display the same NH2-terminal amino acid sequence residues and approximately the same physico-chemical properties of AmyA and AmyB, to exhibit maximum activity at pH 5.6 and 60 °C, and to produce maltose and maltotriose as major starch hydrolysis end-products. Soyabean meal was the best substitute to yeast extract compared to fish powder waste and wheat gluten waste. AmyC production was optimized under SSF using statistical design methodology. Moisture content of 76.25%, C/N substrate ratio of 0.62, and inoculum size of 10(6.87) spores allowed maximum activity of 22118.34 U/g of dried substrate, which was 33 times higher than the one obtained before the application of the central composite design (CCD).

Purification and biochemical characterization of a novel thermostable lichenase from Aspergillus niger US368.

New ß-1,3;1,4-glucanase was purified from Aspergillus niger US368. The pure glucanase has a molecular mass of about 32 kDa. The N-terminal sequence of the purified enzyme (A-G-T-N-P-P-I-G-V) was determined. The optimum pH and temperature recorded for enzyme activity were 5 and 60 °C, respectively. It also displayed marked thermostability with a half-life of 30 min at 70 °C. At 37 °C, the enzyme showed 100% stability from pH 3 to 10. The Km and Vmax values exhibited by the enzyme on barley ß-glucan were 0.62 mg ml(-1) and 34.46 U ml(-1), respectively. The enzyme is a retaining-one and was only active toward glucan containing ß-1,3;1,4-linkages. The production of ß-glucanase with barley flour as the sole carbon source was optimized. This is the first report on the purification and characterization of a ß-1,3;1,4-glucanase from A. niger. This lichenase could be considered as a candidate for future application particularly in the animal feed industry.

Encapsulation in alginate and alginate coated-chitosan improved the survival of newly probiotic in oxgall and gastric juice.

This study was undertaken to develop an optimum composition model for the microencapsulation of a newly probiotic on sodium alginate using response surface methodology. The individual and interactive effects of three independent variables, namely sodium alginate concentration, biomass concentration, and hardening time, were investigated using Box-Behnken design experiments. A second ordered polynomial model was fitted and optimum conditions were estimated. The optimal conditions identified were 2% for sodium alginate, 10(10)UFC/ml for biomass, and 30 min for hardening time. The experimental value obtained for immobilized cells under these conditions was about 80.98%, which was in close agreement with the predicted value of 82.6%. Viability of microspheres (96%) was enhanced with chitosan as coating materials. The survival rates of free and microencapsulated Lactobacillus plantarum TN8 during exposure to artificial gastrointestinal conditions were compared. The results revealed that the encapsulated cells exhibited significantly higher resistances to artificial intestinal juice (AIJ) and artificial gastric juice (AGJ). Microencapsulation was also noted to effectively protect the strain from heating at 65 °C and refrigerating at 4 °C. Taken together, the findings indicated that microencapsulation conferred important protective effects to L. plantarum against the gastrointestinal conditions encountered during the transit of food.

Heterologous expression and optimization using experimental designs allowed highly efficient production of the PHY US417 phytase in Bacillus subtilis 168.

To attempt cost-effective production of US417 phytase in Bacillus subtilis, we developed an efficient system for its large-scale production in the generally recognized as safe microorganism B. subtilis 168. Hence, the phy US417 corresponding gene was cloned in the pMSP3535 vector, and for the first time for a plasmid carrying the pAMß1 replication origin, multimeric forms of the resulting plasmid were used to transform naturally competent B. subtilis 168 cells. Subsequently, a sequential optimization strategy based on Plackett-Burman and Box-Behnken experimental designs was applied to enhance phytase production by the recombinant Bacillus. The maximum phytase activity of 47 U ml-1 was reached in the presence of 12.5 g l-1 of yeast extract and 15 g l-1 of ammonium sulphate with shaking at 300 rpm. This is 73 fold higher than the activity produced by the native US417 strain before optimization. Characterization of the produced recombinant phytase has revealed that the enzyme exhibited improved thermostability compared to the wild type PHY US417 phytase strengthening its potential for application as feed supplement. Together, our findings strongly suggest that the strategy herein developed combining heterologous expression using a cloning vector carrying the pAMß1 replication origin and experimental designs optimization can be generalized for recombinant proteins production in Bacillus.

Secretion of cyclodextrin glucanotransferase in E. coli using Bacillus subtilis lipase signal peptide and optimization of culture medium.

The cyclodextrin glycosyltransferase (CGTase) of Paenibacillus pabuli US132 was fused to the secretive lipase signal peptide of B. subtilis. This leads to an efficient secretion of the recombinant enzyme into the culture medium of E. coli as an active and soluble form contrasting with the native construction leading to a periplasmic production. In order to enhance the yield of CGTase production, an experimental design methodology was applied for the optimization of the culture composition. Hence, the media components were submitted to preliminary screening using a Plakett-Burman design. The concentrations of the major operating ones were then optimized to enhance the secretion of CGTase using response surface methodology. The findings revealed that concentrations of 0.5% potato starch, 3% yeast extract, 3% tryptone, 1.5% casein hydrolysate, 0.5% NaCl, 0.2% KH2PO4, and 0.02% MgSO4 were the optimal conditions for CGTase production. The experimental value (9.43 U/mL) obtained for CGTase activity was very close to the predicted value (9.27 U/mL).

Application of a statistical design to the optimization of parameters and culture medium for alpha-amylase production by Aspergillus oryzae CBS 819.72 grown on gruel (wheat grinding by-product).

The production optimization of alpha-amylase (E.C.3.2.1.1) from Aspergillus oryzae CBS 819.72 fungus, using a by-product of wheat grinding (gruel) as sole carbon source, was performed with statistical methodology based on three experimental designs. The optimisation of temperature, agitation and inoculum size was attempted using a Box-Behnken design under the response surface methodology. The screening of nineteen nutrients for their influence on alpha-amylase production was achieved using a Plackett-Burman design. KH(2)PO(4), urea, glycerol, (NH(4))(2)SO(4), CoCl(2), casein hydrolysate, soybean meal hydrolysate, MgSO(4) were selected based on their positive influence on enzyme formation. The optimized nutrients concentration was obtained using a Taguchi experimental design and the analysis of the data predicts a theoretical increase in the alpha-amylase expression of 73.2% (from 40.1 to 151.1 U/ml). These conditions were validated experimentally and revealed an enhanced alpha-amylase yield of 72.7%.