Cimarec digital stirring hotplates

Uncontaminated leaves of M. tamaulipana (i.e., without external agents and physical contaminants) were exposed to sunlight to remove their moisture [33 (link)]. The dry leaf samples were ground into a fine powder by an electric mill (Cuisinart DBM-8, Stamford, CT, USA). The powder of M. tamaulipana (150 g) was mixed with 96% ethanol (500 mL) to prepare the extract that was kept under constant stirring on Thermo Scientific™ Cimarec™ Digital Stirring Hotplates for three days at room temperature (27 °C), in amber bottles covered with aluminum [34 ]. Subsequently, the solution was filtered using Whatman No. 4 paper. The solvent was removed from the extract by passing it through a vacuum system on a rotary evaporator at 60 °C for two hours (Yamato Scientific America Inc., Model-RE301, Santa Clara, CA, USA), and the rest of the solvent was dried in an oven chamber (Shel-lab Model 1535, Sheldon Manufacturing Inc., Cornelius, OR, USA) for 72 h. Finally, the extract was recovered in solid form to prepare 2000 μg/mL of a stock solution for the antibacterial activity tests and the phytochemical composition analysis [15 (link),19 (link),34 ,35 (link)].

Proteins were extracted sequentially based on solubility differences according to Osborne classification using a modification method reported by Vazquez-Ovando et al. [23 (link)]. A 100 g demucilage DCF was suspended in 500 mL of distilled water before being extracted on a magnetic stirrer (Cimarec™ Digital Stirring Hotplates, Thermo Scientific, Waltham, MA USA) for 2 h at 4 °C. The suspension was centrifuged (Allegra 64R Centrifuge, Beckman Coulter Inc., Indianapolis, IN, USA) for 30 min at 950.3× g. The supernatant was decanted (designated as albumin fraction), and the residue was further extracted with 10 mL of 100 g/kg NaCl for 2 h and centrifuged to yield the globulin fraction. The resulting residue was then further extracted with 10 mL of a 700 g/L aqueous isopropanol solution under constant stirring. The supernatant was collected and designated as prolamin. The residue was resuspended in 10 mL of 0.1 M NaOH solution, centrifuged, and the separated supernatant was collected as glutelin. The residue after extraction was oven-dried at 90 °C for 6 h. All the protein fractions were lyophilized (Scanvac CoolSafe Touch 110-4, Labogene, Allerod, Denmark) and stored at 4 °C for further analysis.

To improve the interaction between the resin and the nanoparticles, silanization of ZrO was conducted using silane coupling agent (3-trimethoxysilyl propyl methacrylate, 97% (TMSPM), Sigma-Aldrich, Co., St Louis, MO, USA). The process of coating involved dissolving 0.3 g of TMSPM in 100 mL of acetone, followed by adding 30 g ZrO to the TMSPM/acetone mix and stirring for 60 min with a magnetic stirrer (Cimarec Digital Stirring Hotplates, SP131320-33Q; Thermo Fisher Scientific, Waltham, MA, USA), as previously reported [26 ].
When the sample had dried, it was heated at 120 °C for 2 h and then naturally cooled to obtain the surface-treated ZrO sample, which was further cooled to obtain silanized ZrO [27 (link)].