Uv 1650

Anthocyanins content was determined as reported by Ribereau Gayon and Stonestreet [32 ]. One milliliter of sample was mixed with 1 mL of HCl/EtOH solution (0.1% v/v) and 20 mL of HCl/H₂O solution (2% v/v). Then, 2.5 mL of sample mixture was added with 1 mL of deionized H₂O and other 2.5 mL of sample mixture with 1 mL of potassium bisulphite solution (20% w/v). After 15 min of reaction, the absorbance of each solution was measured at 520 nm using an UV-Vis spectrophotometer (Shimadzu UV 1650, Milano, Italy), using distilled water as a control. The anthocyanin content, expressed as milligrams of malvidin-3-glucoside equivalent per liter (mg/L), was calculated considering a calibration curve, obtained with different solutions of malvidin-3-glucoside as standard. All the determinations were performed in triplicate.

UV-visible spectra were measured on UV-1650 Shimadzu spectrophotometer in the range of 220 to 320 nm. CD spectra were recorded on JASCO J-815 spectrophotometer from 220 to 320 nm range, with the average of three scans. The DNA melting studies were done on CARY 100 spectrophotometer (Varian America) equipped with temperature controller.

UV–visible optical transmission spectroscopy (UV–1650, Shimadzu) (Kyoto, Japan) was performed on the PI films in the wavelength range of 200–800 nm with a resolution of 0.5 nm and a scanning rate of 300 nm per min⁻¹.

To cultivate R. toruloides the medium used was minimal media (MM; glucose 5 g/l; Na₂HPO₄ 6 g/l; NaCl 5 g/l; KH₂PO₄ 3 g/l; NH₄Cl 2 g/l; MgSO₄ 0.1 g/l; yeast extract 2 g/l) solidified with 1.5% agar. The culture was maintained at RT (~25–30°C) and shaking speed was 120 rpm. Growth was measured using optical density at 600 nm (Shimadzu UV-1650) and by quantitating biomass. A 48 h old culture at 10% (v/v) was used as inoculum for all studies. To obtain biomass, cells were harvested by centrifugation, washed with de-ionized water and dried at 85 ± 3°C and expressed as g/L. The effect of NaCl supplementation was studied with 1, 5, and 10% NaCl (w/v); the control media was devoid of NaCl. The residual amount of reducing sugar was estimated using 3,5 dinitrosalysilic acid (DNSA) method (Miller, 1959 (link)) in the fermentation media.

To increase the dissolution rate and solubility of curcumin, PEGylated curcumin complex (PEG-CUR) was prepared by fusion method as already reported by Nguyen et al., [10 ]. Briefly; on the basis of results of phase solubility diagram polyethylene glycol (PEG) 6000 (3.75g) was melted in a water bath at 70°C and CUR (1.25g) was dispersed into molten mixture with continuous mechanical stirring until uniform. The resulted mixture was placed in refrigerator for 4–5 min to cool, solidify and dried at room temperature. Resulted PEG-CUR complex was grinded in pestle mortar, screened from sieve (mesh #40), preserved in aluminum foil, and kept in a dry place. Moreover, confirmation of PEG-CUR complex was done by FTIR spectra as shown in Fig 4 and solubility studies. For solubility analysis through shake flask method about 20mg prepared PEG-CUR complex was dispersed in 10 ml of distilled water at 37°C at 50 rpm for 24h. After shaking for 24h samples were filtered and analyzed at 425 nm using UV-visible spectrophotometer (Shimadzu UV-1650, Tokyo, Japan) (Fig 1).