Cellulose acetate membrane

Caffeine, 5-CGA, and trigonelline from T0, TF, and R were identified using a Shimadzu liquid chromatography system (Shimadzu Corp., Kyoto, Japan) equipped with a C18 column following the protocol in Bressani et al. [18 (link)]. Ground whole fruits and beans (0.5 g) were placed in tubes containing 50 mL Milli-Q water and boiled for 3 min to extract the total compounds. Then, the resulting suspension was filtered through a 0.22 µm cellulose acetate membrane (Merck Millipore). Identification and quantitative analysis were performed by injecting caffeine, trigonelline, and 5-CGA (Sigma-Aldrich) standards and building calibration curves. All analyses were performed in duplicate.

The samples were aseptically collected after allowing the water to flow for 2 min. The water samples were filtered through a cellulose acetate membrane (0.45-μm pore size, Millipore, SP, Brazil) on a filtration ramp (Sartorius do Brasil Ltda). The membrane was placed on a plate containing R2A agar (Difco, Detroit, MI, USA) at 30°C for 7 to 14 days, to allow filamentous fungi and yeasts to grow. After the incubation period, the colonies were counted, and the results were expressed as CFU/mL. One of the colonies with the same morphology within the same board was subcultured for identification in Sabouraud Dextrose plus chloramphenicol.

The pectin samples were dissolved in 0.1 M NaCl solution to prepare a pectin concentration of 1 mg/mL. Before HPLC analysis, the supernatant was centrifuged at 10,000 × g for 30 s at 4 °C and subsequently filtered via a cellulose acetate membrane (0.45 μm of pore size, Millipore). Molecular parameters (Mw, Mn, and Mw/Mn) of pectin samples were determined by a HPLC system (Waters e2695, MA, USA) equipped with a multi-angle laser-light scattering (MALLS) and a differential refractive index detector (Waters, MA, USA). The flow rate was set to 0.8 mL/min. The mobile phase was 0.1 M NaCl solution. The data was processed by using ASTRA5.3.4.20 software [19] (link).

Water aliquots were collected from Conceição Lagoon and carefully filtered through a cellulose acetate membrane (Millipore 47 mm in diameter and 0.2 µm in pore size) in the laboratory. One liter of the filtered water or medium (dilution of the sample resulted in a reduction in the amount of grazers [14 (link)]) was transferred onto each of three sterile glass recipients resulting in 70% of filtered medium and 30% of inoculum (whole water—no previous filtration), equivalent to a volume of 1.43 L per replicate. Bacterioplankton cultures received the following labels according to the treatment: (1) light (in situ light intensity and photoperiod: 14 h light, 10 h dark, room temperature); (2) control (dark, room temperature); and (3) nutrients (C + N + P = glucose, NH₄Cl, and K₂HPO₄ additions at final concentrations of 100 µM C : 10 µM N : 1 µM P, dark, room temperature). All treatments were kept under the described conditions for 72 h. The reason why control and nutrients' treatments were kept in the dark was to prevent the accumulation of autotrophic biomass (presence of photoautotrophic bacteria and picoeukaryotes) throughout the experiment, which would interfere with the results by competition or release of extracellular DOC. On the other hand, light treatment was important for the effects of light/dark cycles on bacterial activity when compared to complete darkness.

In vitro release study of formulated hydrogel was carried out using Franz Diffusion cell with effective diffusional surface area of 0.77 cm² and a receptor cell volume of 5 mL. The receptor compartment was filled with dissolution medium consisting of phosphate buffer pH 5.5 and 1.5% polysorbate 80, and temperature was maintained at 32 °C (in-simulation to skin pH and temperature) [14 (link)]. The cellulose acetate membrane (Sigma Aldrich, Germany) with a pore size of 0.45 µm was fixed between the donor and receptor compartment. The donor compartment was charged with 1 g of hydrogel. A 2 mL volume of sample was collected from the receptor compartment at predetermined time intervals, i.e., 0, 0.5, 1, 2, 4, 8, 12, 16, 20 and 24 h. The amount of Curcumin in the samples was analysed using a UV–Visible spectrophotometer (Shimadzu 1601, Kyoto, Japan) at a wavelength of 425 nm. The experiment was performed in triplicates and the results were averaged.