Hd3200

ZrO₂-acetylacetonate was synthesized by a sol-gel route according to previous work as a gel coarse powder [12 (link),17 (link)]. Briefly, the hybrid gel was obtained by adding acac and 1-propanol to Zr(IV) propoxide in a ratio of 0.5 mol acac per mol of Zr, followed by the addition of a distilled water–propanol solution and letting the gel dry in a ventilated oven at 40 °C. Twenty milligrams of the powder were dispersed in 40 mL of ethanol and subjected to ultrasounds by an ultrasonic homogenizer (Bandelin, HD3200) equipped with an MS73 probe. Ultrasounds were applied in pulse mode (30 s pulse and 30 s pause) for 2 h to reduce the particle size, guaranteeing that the high temperature does not disrupt the matrix [31 (link)]. The material was then diluted to 200 mL and left to settle overnight. The supernatant of the sedimentation was centrifuged at 15,000 rpm and 15 °C for 10 min, and the NPs were recovered by pulling the pellets in a unique sample. The NPs (ZrO₂-acac NPs) were weighed and stored in ethanol at a concentration of 1 mg mL⁻¹ until further use.

PVA granules were dissolved in distilled water to form 10% (w/v) PVA solutions by stirring at 90 °C for 3 h. Meanwhile, psyllium husk powders were dispersed in 10 mL distilled water using an ultrasonic homogenizer (Bandelin/Sonoplus HD3200) to obtain a 1% polymer solution. Afterward, PVA and psyllium husk polymer solutions were mixed to prepare blend solutions (4/1, v/v). The modified MCC (0.012 g) was added to the blend polymer solution. Then, D-limonene was introduced to be 2%, 4%, and 6% of the total polymer solutions. The nanofibers were electrospun at a high voltage of 25 kV and a flow rate of 1.25 mL/h, with a spinning drum at 250 rpm (Figure 3). The drum was coated with aluminum foil, and the distance between the tip and the collector was 88 mm. These samples were coded in Table 1.

For each plasmid transformed, a recombinant colony was transferred from the agar plate to 20 ml of LB medium (1% tryptone, 0.5% yeast extract, 1% NaCl) completed with 100 μg/ml of ampicillin and incubated overnight at 37°C, 180 rpm. The culture was diluted at an OD₆₀₀ value of 0.08 with 200 ml of LB, supplemented with the same antibiotic, and further incubated at 37°C. To induce the recombinant protein expression, 1 mM IPTG (isopropyl-β-d-thiogalacto-pyranoside) was added when the culture reached an OD₆₀₀ value of 0.5. After 3 h of induction, cells were collected by centrifugation at 3,300 × g at 4°C for 15 min. The obtained pellet was resuspended in a lysis buffer (100 mM Tris HCl, 10 mM EDTA, 2 M urea, and 2% Triton X-100 pH 8.0), at a final concentration of 20 OD/ml, and subjected to a sonication process (Bandelin, HD3200, MS 72 probe, running at 40% amplitude) for 30 min (30″ ON and 30″ OFF) in an ice bath. The inclusion bodies were collected by a centrifugation step at 3,300 × g for 15 min and then washed three times with the lysis buffer to remove contaminants. Next, the pellets of the inclusion bodies were dissolved in a denaturing buffer (100 mM Tris HCl, 10 mM EDTA, 8 M urea, and 10 mM DTT, pH 8) and incubated for 1 h at 37°C under stirring. The supernatants were collected after centrifugation at 3,300 × g at 4°C for 15 min.