Dialysis sack

fIn the current study, Se enriched bacterial strains identified as Enterobacter cloacae (ADS1), Klebsiella pneumoniae (ADS2), and Stenotrophomonas maltophilia (ADS18) were used as a source of bacterial organic Se. The stock culture of ADS1, ADS2, and ADS18 strains prepared at the Laboratory of Microbiology, Department of Animal Science, Faculty of Agriculture, Universiti Putra Malaysia (UPM) and the sonicated Se-enriched bacterial cells were produced according to the procedure described by Dalia et al. [43 (link)]. The extraction of selenoprotein from Se-enriched bacterial cells was carried out using dialysis technique The dialysis process was performed using dialysis sacks of flat width 25 mm, 12,000 Da, (Sigma-Aldrich) against deionised water, which was changed every 12 h for a total of 96 hours to separate inorganic Se from organic form [44 (link)]. The content in the dialysis tube was lyophilised and then used as a source of bacterial Se.

CNCs were isolated from KW following the method described elsewhere with few modifications [7 (link), 30 (link)–33 (link)]. The cellulose fibers (C₄₀ and C₈₀) extracted from KW were first hydrolyzed with 64% (w/w) sulfuric acid (1:20 g/mL) at 45°C for 60 min under magnetic stirrer (the resulting NCs designated as CNCs₄₀ and CNCs₈₀). The mixture was then diluted 10-fold with ice cubes to stop the reaction, and washed by successive centrifugations at 4°C (Beckman Coulter Allegra 64R Refrigerated Centrifuge, USA) for 10 min each at 10,000 rpm. The mixture was also dialyzed against distilled water using dialysis sacks (Avg. flat width 35 mm, MWCO 12,000 Da, Sigma-Aldrich, USA) until neutral pH was reached (5 days). Subsequently, the resulting suspension was homogenized using a disperser type UltraTurrax (Janke and Kunkel IKA-Labortechnik, Ultra-Turrax T50) for 5 min at 10,000 rpm twice and sonicated (Sonics and Materials Inc. Vibracell, VCX 750, Newtown CT, USA) in an ice bath for 5 min. The aqueous suspension was freeze-dried in a lyophilizer (Operon Co., Ltd.—Bio-Equip, Korea) and dried for 72 h to obtain CNCs powder. The yields of CNCs were estimated gravimetrically considering the initial weight of the extracted cellulose fibers (C₄₀ and C₈₀).

NPs were suspended in 5 ml of deionised water, placed in dialysis sacks with a cut-off at 12 kDa (Sigma-Aldrich) and dialysed against 50 ml of deionised water. At each time point the release medium was collected and replenished. All samples were evaporated under low pressure using rotary evaporator (vacuum 72 mbar, water bath 80 °C) until complete dryness. The samples were re-hydrated using 2 ml of methanol, and analysed using HP-TLC. Data was showed as an accumulation curve.