Genesys 50

A cell suspension of the tested strains was prepared by inoculating 10 mL MRS broth with a single colony and incubating at 30 °C for 24 h. From this pre-inoculum, 100 µL were sub-cultured into 10 mL fresh MRS broth and incubated for further 24 h. The concentration of colony forming units (CFU) was measured by optical density at a wavelength of 600 nm using a UV-VIS spectrophotometer (Genesys 50, Thermo Scientific, Waltham, MA, USA). The amount of inoculum necessary to reach 7 log CFU per g lentil emulsion was centrifuged at 4500 rpm for 5 min, the pellet was washed in 10 mL sterile tap water and centrifuged again. The resulting pellet was suspended in fructose-containing lentil emulsion heated to 30 °C, shaken manually for 5 min, transferred to sterile containers and fermented for 12 h at 30 °C. For the control, the necessary amount of frozen Yoflex culture to reach 7 log CFU per g lentil emulsion was added to sucrose-containing lentil emulsion heated to 42 °C, shaken manually for 5 min, transferred to sterile containers and fermented for 12 h at 42 °C. The YA were stored at 6 °C for 12–16 h prior to further analyses. Each fermentation was performed in three biological replicates.

The total concentrations of chlorophylls (CHL) and carotenoids (TCA) were determined in green juice fractions and ethanol extracts by spectrophotometry. The absorbance was measured at 470 nm, 649 nm, and 665 nm using a UV-visible spectrophotometer (Genesys 50, Thermo Scientific, Waltham, CA, USA) with a quartz cuvette. Turbidity at 750 nm was considered. Calculations were carried out using the formulas described by Lichtenthaler and Wellburn [102 (link)]:

Total flavonoid content was determined by aluminum chloride colorimetric assay (Zhishen et al., 1999 (link)). Total flavonoid content was extracted from 300 mg of frozen ground basil and 1.5 ml of methanol/H₂O_/acetone (60:30:10 v/v/v) in an ultrasonic bath (Branson 2800, Richmond, VA, United States) for 15 min. Samples were centrifuged at 15,000 RCF (Sorvall Legend Micro 21R, Thermo Fisher Scientific, Waltham, MA, United States) at 4°C for 10 min, and the supernatant was collected. Catechin was used as a quantifying standard. In a 3-ml cuvette, 50 μL of the extracted sample was mixed with 1.95 ml water and 75 μL of 5% NaNO₂. After 6 min 150 μL of 10% AlCl₃ was added and after another 5 min, 500 μL of 1 M NaOH was added. The absorbance was measured at a wavelength of λ = 250 nm in a spectrophotometer (Genesys 50, Thermo Fisher Scientific, Waltham, MA, United States) against a blank. Data was expressed on the base of dry weight as mg/g DW.

Hydrogen peroxide (H₂O₂) was determined according to Junglee et al. (2014) (link) with some modifications. H₂O₂ was extracted from 0.1 g of frozen ground basil leaves with 0.4 ml of 0.1% TCA, 0.4 ml of potassium phosphate buffer (pH 7.6), and 0.8 ml of potassium iodide. After incubation for 10 min at 4°C, the samples were centrifuged at 15,000 RCF (Sorvall Legend Micro 21R, Thermo Fisher Scientific, Waltham, MA, United States) at 4°C for 10 min, and the supernatant was collected. Samples were measured in UV-cuvettes at a wavelength of λ = 350 nm in a spectrophotometer (Genesys 50, Thermo Fisher Scientific, Waltham, MA, United States) against the blank. For quantification, a calibration curve was prepared with H₂O₂ solutions with concentrations from 10 to 400 μmol/L. For each sample, three technical replicates were prepared. Data were expressed on the base of dry weight as mg/g DW.

Acetone and methanol extracts were dried under nitrogen and resuspended in ethanol. The absorbance (Ax) was measured at 445, 663 and 750 nm by a UV–visible spectrophotometer (Genesys 50, Thermo Scientific). Fucoxanthin concentrations, expressed as mg g⁻¹ dry weight (DW), was estimated using the following equation [79 (link)]:
where:
W = sample weight (g of dry weight)
V = ethanol volume (L)
A455, A663, A750 = absorbance at x nm

The polarity of the prepared NADES was determined using solvatochromic method with Nile red, described previously in Fernandes et al. [11 (link)]. The absorbance of the samples was obtained using a UV-spectrophotometer (GENESYS 50, Thermo Scientific, Waltham, MA, USA) wavelength range of 400–800 nm. Measurements were conducted in triplicates. Polarity of BE:Gly was determined previously [12 (link)].