Voriconazole-loaded self-nanoemulsifying drug delivery system (SNEDDS) to improve transcorneal permeability.

The aim of this study was to develop self- nanoemulsifying drug delivery system (SNEDDS) to improve the transcorneal permeability of voriconazole. A 'mixture design around a reference mixture' approach was applied. This latter included four components, namely, isopropyl myristate, PEG 400, Tween® 80 and Span® 80 as oil, co-solvent, surfactant and co-surfactant, respectively. Droplet size was selected as response. The effect of mixture components on droplet size was analyzed by means of response trace method. Optimal formulation was subjected to stability studies and characterized for droplet size, polydispersity index (PDI), pH, osmolarity, viscosity and percentage of transmittance. Ex-vivo transcorneal permeation of the optimal and the marketed formulations was carried out on excised bovine cornea using Franz cell diffusion apparatus. Optimal voriconazole loaded-SNEDDS showed moderate emulsification efficiency and was characterized by a droplet size of 21.447 ± 0.081 nm, a PDI of 0.156 ± 0.004, a pH of 7.205 ± 0.006, an osmolarity of 310 mosmol/Kg and a viscosity of 8.818 ± 0.076 cP. Moreover, it presented an excellent stability and exhibited a significant improvement (p < 0.05) in apparent permeability coefficient (1.982 ± 0.187 × 10-6 cm/s) when compared to commercialized formulation (1.165 ± 0.106 × 10-6 cm/s). These results suggest that SNEDDS is a promising carrier for voriconazole ocular delivery.

Combinatorial effect of mutagenesis and medium component optimization on Bacillus amyloliquefaciens antifungal activity and efficacy in eradicating Botrytis cinerea.

This work is directed towards Bacillus amyloliquefaciens strain BLB371 metabolite production for biocontrol of fungal phytopathogens. In order to maximise antifungal metabolite production by this strain, two approaches were combined: random mutagenesis and medium component optimization. After three rounds of mutagenesis, a hyper active mutant, named M3-7, was obtained. It produces 7 fold more antifungal metabolites (1800AU/mL) than the wild strain in MC medium. A hybrid design was applied to optimise a new medium to enhance antifungal metabolite production by M3-7. The new optimized medium (35g/L of peptone, 32.5g/L of sucrose, 10.5g/L of yeast extract, 2.4g/L of KH2PO4, 1.3g/L of MgSO4 and 23mg/L of MnSO4) achieved 1.62 fold enhancement in antifungal compound production (3000AU/mL) by this mutant, compared to that achieved in MC medium. Therefore, combinatory effect of these two approaches (mutagenesis and medium component optimization) allowed 12 fold improvement in antifungal activity (from 250UA/mL to 3000UA/mL). This improvement was confirmed against several phytopathogenic fungi with an increase of MIC and MFC over than 50%. More interestingly, a total eradication of gray mold was obtained on tomato fruits infected by Botrytis cinerea and treated by M3-7, compared to those treated by BLB371. From the practical point of view, combining random mutagenesis and medium optimization could be considered as an excellent tool for obtaining promising biological products useful against phytopathogenic fungi.

A new raw-starch-digesting α-amylase: production under solid-state fermentation on crude millet and biochemical characterization.

A new Bacillus strain degrading starch, named Bacillus sp. UEB-S, was isolated from a southern Tunisian area. Amylase production using solid-state fermentation on millet, an inexpensive and available agro-resource, was investigated. Response surface methodology was applied to establish the relationship between enzyme production and four variables: inoculum size, moisture-to-millet ratio, temperature, and fermentation duration. The maximum enzyme activity recovered was 680 U/g of dry substrate when using 1.38 × 10(9) CFU/g as inoculation level, 5.6:1 (ml/g) as moisture ratio (86%), for 4 days of cultivation at 37 degrees C, which was in perfect agreement with the predicted model value. Amylase was purified by Q-Sepharose anion-exchange and Sephacryl S-200 gel filtration chromatography with a 14-fold increase in specific activity. Its molecular mass was estimated at 130 kDa. The enzyme showed maximal activity at pH 5 and 70 degrees C, and efficiently hydrolyzed starch to yield glucose and maltose as end products. The enzyme proved its efficiency for digesting raw cereal below gelatinization temperature and, hence, its potentiality to be used in industrial processes.

Decolorization of Solophenyl Red 3BL Polyazo Dye by Laccase-Mediator System: Optimization through Response Surface Methodology.

The decolorization of direct Solophenyl red 3BL (SR), a polyazo dye extensively used in textile industry was studied. The Fomes fomentarius laccase alone did not decolorize SR. The natural redox mediator, acetosyringone (AS), was necessary for decolorization to occur. Box-Behnken design was used to evaluate the effects of three parameters, namely, enzyme concentration (0.5-2.5 U mL(-1)), redox mediator concentration (3-30 µM), and incubation time (1-24 h), on the SR decolorization yield. The fitted mathematical model allowed us to plot response surfaces as well as isoresponse curves and to determine optimal decolorization conditions. The results clearly indicated that the AS concentration was the main factor influencing the SR decolorization yield. The selected optimal conditions were enzyme concentration 0.8 U mL(-1), mediator concentration 33 µM, and time 14 h 30 min. These conditions allowed 79.66% of SR decolorization versus 80.70% for the predicted value. These results showed a promising future of applying laccase-AS system for industrial wastewater bioremediation.

Improvement of Bacillus thuringiensis bioinsecticide production by sporeless and sporulating strains using response surface methodology.

Statistical experimental designs, involving a Plackett-Burman design followed by a rotatable central composite design were used to optimize the culture medium constituents for Bacillus thuringiensis bioinsecticide production. This was carried out by using firstly an asporogenic strain and extrapolated to some sporeless and sporulating strains. Initial screening of production parameters was performed and the variables with statistically significant effects on delta-endotoxin production were identified: glucose, glycerol, yeast extract and MnSO(4). These variables were selected for further optimization by response surface methodology. The obtained results revealed that the optimum culture medium for delta-endotoxin production consists of 22.5 g/l of glucose, 4.8g/l of glycerol, 5.8 g/l of yeast extract and 0.008 g/l of MnSO(4). Under these conditions, delta-endotoxin production was 2,130 and 2,260 mg/l into 250 and 1,000 ml flask respectively, which represent more than 38% improvement in toxin production over the basal medium (1,636 mg/l). Such medium composition was shown to be suitable for overproducing delta-endotoxins by sporeless and sporulating strains.

Application of Asymetrical and Hoke Designs for Optimization of Laccase Production by the White-Rot Fungus Fomes fomentarius in Solid-State Fermentation.

Statistical approaches were employed for the optimization of different cultural parameters for the production of laccase by the white rot fungus Fomes fomentarius MUCL 35117 in wheat bran-based solid medium. first, screening of production parameters was performed using an asymmetrical design 2(5)3(3)//16, and the variables with statistically significant effects on laccase production were identified. Second, inoculum size, CaCl(2) concentration, CuSO(4) concentration, and incubation time were selected for further optimization studies using a Hoke design. The application of the response surface methodology allows us to determine a set of optimal conditions (CaCl(2), 5.5 mg/gs, CuSO(4), 2.5 mg/gs, inoculum size, 3 fungal discs (6 mm Ø), and 13 days of static cultivation). Experiments carried out under these conditions led to a laccase production yield of 150 U/g dry substrate.

Optimization of lipase-catalyzed synthesis of acetylated tyrosol by response surface methodology.

The ability of a noncommercial immobilized lipase from Staphylococcus xylosus (SXLi) to catalyze the transesterification of tyrosol and ethyl acetate was investigated. Response surface methodology was used to evaluate the effects of the temperature (40-60 degrees C), the enzyme amount (50-500 UI), and the ethyl acetate/hexane volume ratio (0.2-1) on the tyrosol acetylation conversion yield. Two independent replicates were carried out under the optimal conditions predicted by the model (reaction temperature 54 degrees C, enzyme amount 500 UI, and volume ratio ethyl acetate/hexane 0.2). The maximum conversion yield reached 95.36 +/- 3.6%, which agreed with the expected value (96.8 +/- 3.7%). The ester obtained was characterized by spectroscopic methods. Chemical acetylation of tyrosol was performed, and the products were separated using HPLC. Among the eluted products from HPLC, mono- and diacetylated derivatives were identified by positive mass spectrometry. Tyrosol and its monoacetylated derivative exert similar antiradicalar activity on 2,2-diphenyl-1-picrylhydrazyle.