Evolution 260 bio

AmB-loaded MCPs were fabricated using an emulsification method previously reported in the literature with some modifications [45 (link)]. Briefly, 10 mg of AmB was dissolved in 10 mL of an aqueous solution of alginate (5% wt/vol). Then, the alginate solution containing AmB was dispersed into a mixture of paraffin oil and span 80 (40 mL of paraffin oil and 2 mL of spam 80) at 1200 rpm for 30 minutes. Afterward, 20 mL of a 10% CaCl₂ solution was added and stirred at 800 rpm for an additional 2 hours. After this period of time, 20 mL of isopropanol was added and the mixture was allowed to react for 10 minutes. The final product was separated by centrifugation, washed three times with isopropanol, let dry at room temperature, and stored at −20^oC. AmB-loaded MCPs were characterized using different techniques: the hydrodynamic diameter and ζ-potential were determined using a particle size analyzer (Nanoplus HD, Particulate Systems, GA, USA), particle size and morphology were estimated using an optical microscope (Nikon, Nikon Instruments, NY, USA) and field-emission scanning electron microscopy (SEM; Raith150, Raith, Dortmund, Germany), and the FTIR spectrum and the absorption spectra were determined using an IR spectrophotometer (Spectrum Two, Perkin Elmer, CT, USA) and UV-Vis spectrophotometer (Evolution 260 Bio, Thermo Fisher Scientific, MA, USA), respectively.

Bacterial strains were cultivated in lysogeny broth (LB) and minimal medium M9 [48 (link)], supplemented (0.5% (w/v)) with beet molasses or yeast extract for four days in two different temperatures, i.e., 15 °C and 25 °C. Both bacterial strains were cultivated in an initial volume of 100 mL in triplicate. Every day, triplicates of each strain were divided into two equal parts. Part of the culture (i.e., 50 mL) was extracted and then measured spectrophotometrically using Evolution 260 Bio (Thermo Fisher Scientific, Waltham, MA, USA) to establish the maximum absorbance value. To determine the concentration of the crude carotenoid extract, the method of Liaaen-Jensen and Jensen was used [62 ]. The bacterial pellet was resuspended in 10 mL of acetone-methanol (7:2 v/v) solution, sonicated at ultrasonic cleaner for 5 min and filtered through Whatman filter paper (Whatman, Maidstone, United Kingdom). The absorbance of this extracted solution was measured spectrophotometrically at 453 nm and calculated according to Jensen’s equation [62 ]. The remaining half of bacterial cultures (i.e., 50 mL) was dried at 100 °C for 24 h and weighed to obtain the dry mass weight information.

All UV-Vis spectroscopic measurements were carried out using Thermo scientific Evolution 260 Bio double beam spectrophotometer (USA) with a 1 cm path length. Binding experiments were performed using a fixed concentration of HSA (1 μM) and 4-PBA in the range 1–20 μM. The UV-Vis spectral scan was recorded from 500–200 nm at 25 ± 0.2°C.

The DPPH (2,2-diphenyl-1-picrylhydrazyl) method was used to measure the free radical scavenging activity. The dilutions were prepared as followed: 2 mL of 0.1 mM DPPH in methanol was added to 2 mL of methanol containing different amounts of crude carotenoid extracts of ANT_H30 or ANT_H53B, i.e., to reach final concentrations of: 0.32, 0.64, 0.96, 1.92, 3.2, and 4 ug/mL of carotenoids. The absorbance at 517 nm was measured spectrophotometrically (Evolution 260 Bio (Thermo Fisher Scientific)) after 30 min. The scavenging of the DPPH radical (%) was calculated according to the formula ((A₀ − A₁)/A₀ × 100), where A₀ is the absorbance of the control reaction and A₁ is the absorbance of reactions containing crude carotenoid extract from ANT_H30 or ANT_H53B [64 (link)].

The ABTS test was carried out according to Kontek et al. [61 (link)]. The radical cations (ABTS+) were prepared by mixing an equal volume of 7 mM ABTS and 4.9 mM potassium persulfate. The working solution (Abs_734nm = 0.7 AU) was obtained by dilution with 50% MeOH. Concentrations of test extracts of TF (50–200 mg of material/mL), WSF (30–120 mg/mL) and DWSF (20–80 mg/mL), and Trolox (10−250 μg/mL) were prepared with 50% MeOH. A 30 μL of extract/standard was mixed with 1.5 mL of ABTS+ solution, and after 30 min, the Abs_734nm was measured using a UV-vis spectrophotometer (Evolution 260 Bio, Thermo Fisher). The absorbance inhibition (%) was calculated as follows: [(Abs_control–Abs_sample)/Abs_control] × 100. Results were determined from the linear curves (absorbance inhibition (%) vs. concentration (µg/mL)) of samples and are expressed as IC₅₀ values, defined as the concentration necessary to cause 50% radical inhibition.

The DPPH (2,2-diphenyl-1-picrylhydrazyl) method was used to measure the free radical scavenging activity. The dilutions were prepared as follows: 2 mL of 0.1 mM DPPH in methanol was added to 2 mL of methanol containing different amounts of melanin-like compound, i.e., to reach final concentrations of 1, 0.5, 0.25, 0.1, 0.01, and 0.001 mg/mL. The absorbance at 517 nm was measured spectrophotometrically (Evolution 260 Bio, Thermo Fisher Scientific) after 30 min. The scavenging of the DPPH radical (%) was calculated according to the formula ((A0 − A1)/A0 × 100) [82 (link)], where A0 is the absorbance of the control reaction and A1 is the absorbance of reactions containing melanin from the ANT_H4 strain. Each experiment was performed in triplicate.