Genesys 150

A UV–Vis spectrophotometer (Agilent Cary 5000 UV–Vis–NIR) was used to measure the optical properties of LNPs photonic crystal in the visible range. The reflectance spectrums of LNPs photonic crystal were recorded with the praying mantis diffuse reflectance accessory in the range of 350–800 nm with a specialized fluorine-based polymer as reference. And the beam spot size was 1.5 mm. A UV–Vis spectrophotometer (Thermoscientific Genesys 150) was used to measure the absorption spectra of lignin and LNPs. Lignin solution and LNPs dispersion were coated on a cover glass (borosilicate glass, 24 × 32 mm), respectively, and their absorption spectra were recorded. The spectrum of cover glass was recorded as a blank.

The extract was mixed with methanol and aluminum chloride, leaving the mixture in darkness for 10 min. Subsequently, the samples were read at 450 nm on a spectrophotometer (Genesys 150, Thermo Fisher Scientific, Waltham, MA, USA) using a quercetin calibration curve in the range from 0.2 to 1.2 mg/mL (R² of 0.97) [2 (link),21 (link)]. This analysis was performed on crude propolis, ethanolic propolis extracts, honey, and microencapsulates. Results were expressed on a dry basis as mg quercetin per g of sample.

Gallic acid was used for the calibration curve of phenolic compounds according to the Folin-Ciocalteu methodology. Methanolic extracts were prepared with 2 g of sample and 20 mL of 80% methanol, and they were left in the dark for 24 h at room temperature. A total of 3300 µL of methanolic extract, 150 µL of 20% Na₂CO₃, and 300 µL of 0.25 N Folin-Ciocalteu reagent were left to react for 15 min under dark conditions at room temperature; a blank was prepared under the same conditions using distilled water instead of the extract. Spectrophotometric readings were performed at 755 nm (Genesys 150, Thermo Fisher Scientific, Waltham, MA, USA), and the results were expressed on a dry basis as mg gallic acid equivalent (GAE)/g of sample [19 (link),25 (link),27 (link),29 (link)].

The methodology reported by Erpel et al. (2021) [17 (link)] was followed with some modifications. First, a 39.4 mg/L DPPH in a methanol solution was prepared and stored at 4 °C until use. Subsequently, five methanolic dilutions of the juice samples at different concentrations were prepared. Aliquots of 0.1 mL of each diluted sample were taken, and each was mixed with 3.9 mL of the DPPH solution. The samples were homogenized with a vortex shaker at medium speed and left to rest for 30 min at room temperature in dark conditions. Then, the absorbance of each sample was measured at 517 nm in a Genesys 150 UV–Vis spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The results were expressed in IC50 of mg gallic acid equivalents (GAE) per mL of juice, and the percentage of inhibition of DPPH radicals was calculated according to
$$\mathrm{\%}Inhibition=\left(1-\frac{Ab{s}_{blank}}{Ab{s}_{sample}}\right)·100$$
A 3.9 mL pure methanol sample was used as a blank, and 3.9 mL of the methanolic DPPH solution was used as a negative control.

The reducing power of the tea extract using the FRAP method was measured by the spectrophotometric method. FRAP reagent was made by mixing of 300 mM acetate buffer pH 3.6, 10 mM 2,4,6-tripyridyl-s-triazine (TPTZ) in 40 mM HCl and 20 mM FeCl₃.6H₂O with a 10: 1: 1 ratio of acetate buffer, TPTZ and FeCl₃.6H₂O (v/v). The reagent was then incubated at 37°C. Then, as much as 0.9 mL of reagent was reacted with 30 μl of tea extract sample and 30 μl of aquadest in the test tube for 4 minutes in a dark room. The absorbance was measured by a UV-Vis Spectrophotometer Thermoscientific GENESYS 150 at 593 nm [20 ]. The analysis uses the standard ascorbic acid standard curve (400, 420, 440, 460, and 480 μM). The result was expressed as mg ascorbic acid equivalent (mg AAE/g).

For extraction of γ-oryzanol, 1 g of (germinated) brown rice flour was suspended with 4 and 8 mL of 2-propanol in two centrifuge tubes and vortexed for 1 min. After centrifugation (centrifuge 5810 R; Eppendorf AG) at 700×g and 30 °C for 10 min, 1 mL of the obtained supernatant was added to a quartz cuvette containing 3 mL isopropanol. The absorbance was measured at 326 nm using UV-Vis spectrophotometer Genesys™ 150 (Thermo Fisher Scientific). Mass concentrations of γ-oryzanol extracted with V(2-propanol)=4 (γ₁) and 8 mL (γ₂) were determined using a linear regression (R²=0.999) constructed from standard γ-oryzanol solutions (γ=25-125 mg/L) and their corresponding absorbance values. Finally, γ-oryzanol mass fraction in mg per gram sample was calculated according to the following equation (1 (link), 14 (link)):

The biomass dry weight concentration was measured as changes in optical density (OD₇₅₀) using a spectrophotometer (Genesys 150, ThermoFisher Scientific, Waltham, MA, USA). Routines for cell dry weight determination and optical density correlation were recently described by Franke et al. [51 (link)].