New brunswick innova 44

Biomass production was conducted using shake-flask cultivation in 25 mL stoppered conical flasks. The cultivation medium included: 1.5 g L⁻¹ (NH₄)₂SO₄, 0.2 g L⁻¹ MgSO₄·7H₂O, 2.5 g L⁻¹ NaCl, 1.5 g L⁻¹ KH₂PO₄, and 1.5 mL L⁻¹ trace element solution. The concentration of yeast extract and glucose-rich hydrolyzate and the medium pH were varied as per the model design (Table 2). All flasks were incubated at 37 °C in an orbital shaker (New Brunswick Innova 44, Eppendorf) at 200 rpm for the relevant times (Table 2). After incubation, a 1 mL aliquot was aspirated, and cell density was recorded as the optical density at 600 nm (OD₆₀₀) using a spectrophotometer (Varian Cary 60 UV/Vis spectrophotometer; Agilent Technologies) against a blank of the 1 mL respective uninoculated cultivation medium.

R. solanacearum isolates were propagated in liquid YPG broth and incubated for four days at 28 °C on a rotary shaker (New Brunswick Innova 44, Eppendorf, USA) at 150 rpm. Subsequently, the cultures were centrifuged at 10 °C for 10 min at 10,000 rpm using a centrifuge (Rotina 380 R, Hettich, Tuttlingen, Germany). The resulting bacterial pellets were optically calibrated to 10⁸ CFU/mL and suspended in distilled water. Measurements were taken using a spectrophotometer (BioSpec-mini, Shimadzu, Kyoto, Japan) at an optical density (OD) of 600.

An aliquot of 0.1 g sea buckthorn oil (1 g oil-in-water emulsion) in 6.15 mL water was combined with 4 mL of simulated gastric fluid (SGF), 1 mL of porcine pepsin solution in SGF (2000 U/mL in final digestion mixture) and 31 μL of CaCl₂ (0.03 M). HCl (1 M) was added to reduce the pH to 3.0 and water was added to a final volume of 12.5 mL. The mixture was homogenized and incubated at 37 °C for 2 h (95 rpm) in a shaking incubator (New Brunswick Innova 44, Eppendorf AG, Hamburg, Germany).

We tested the release of meloxicam from a variety of polymer-based matrices molded in the shape of arms for the solid arm dosage form or v-shaped arm dosage form. In both cases, drug-loaded polymer matrices were cast as arms as described in the previous section. For the in vitro drug release studies, only the drug-loaded arms were used, not the entire gastric resident dosage form.
To perform the drug release study, drug-loaded arms were placed in 20 mL SGF containing 3% Kollidon RH 40 in an incubator shaker at 150 RPM and 37 °C (New Brunswick Innova 44, Eppendorf, USA). At various times, a part of the release medium was collected and stored at −20 °C until further analysis. At the end of the study, the samples were thawed and analyzed using HPLC.

Bacterial growth time of the preinoculum added to all flasks was 40 h using a methanol concentration of 5.0 g/L. The inoculum was centrifuged at 10 956×g for 20 min, the supernatant was removed and the cells were resuspended in a mineral medium. These aliquots were centrifuged, then the supernatant was removed (residual methanol was sent for analysis) and the pellets were resuspended in 2 mL of distilled water for absorbance measurement at 600 nm in a spectrophotometer (UV-1800; Shimadzu, Kyoto, Japan). Growth assays were started by mixing 10 mL of the activated inoculum of the bacterial suspension with 190 mL of mineral medium (φ=5%) containing methanol concentrations of 1, 4, 7, 12 and 18 g/L in 500-mL conical flasks. The bacterial cultures were incubated at 30 °C and 4×g on a rotary shaker, for 84 h (New Brunswick™ Innova 44®; Eppendorf, Enfield, CT, USA).

E. coli BL21 (DE3) harboring pRhaBAD-GmSuSy_Trc-YjiC or pRhaBAD-GmSuSy_YjiC plasmid was grown overnight in a 100-mL Erlenmeyer flask containing 30 mL of LB supplemented with 50 mg/L gentamicin in an incubator shaker (New Brunswick Innova 44, Eppendorf, Vienna, Austria) at 37 °C and 120 rpm agitation. The next day, 500 mL of TB containing 50 mg/L gentamicin was inoculated with 10% (v/v) of overnight preculture and incubated in a baffled 2 L shaken flask at 37 °C and 120 rpm agitation until an OD₆₀₀ reached around 0.6–0.8 value. Protein expression was then induced by the addition of 10 mM of L-rhamnose and 1 mM of IPTG (pRhaBAD-GmSuSy_Trc-YjiC) or 25 mM of L-rhamnose (pRhaBAD-GmSuSy_YjiC). Expression and sonication was performed as described above in the enzyme expression screening section. The cell lysate was then recovered by centrifugation (14000 g, 30 min, 4 °C) and stored at 4 °C.

R. erythropolis JCM3201^T (DSM No. 43066, German Collection of Micro-Organisms and Cell Cultures GmbH, Braunschweig, Germany) was maintained using Luria–Bertani (LB) agar plates (10 g L⁻¹ of peptone, 5 g L⁻¹ of yeast extract, 10 g L⁻¹ of sodium chloride, and 14 g L⁻¹ of agar). For inoculum, single colonies were cultured in 500-milliliter baffled shake flasks holding 100 mL of LB medium at 120 rpm (New Brunswick InnovaTM 44, Eppendorf, Hamburg, Germany) for 48 h. Afterward, the inoculum was transferred to prospective optimization media.