Genesys 10

Complexation of freshly precipitated Fe (10⁻⁵ M FeCl₃) by bacillibactin (10⁻⁵ M) in water was monitored over time by UV-visible (UV-Vis) spectrometry (Genesys 10S; Thermo Scientific). Experiments on Fe complexation by a catechol siderophore in the presence of biofilm were performed at a high concentration of ligand (10⁻⁴ M) to overcome the spectral interferences of the biofilm matrix. Due to the limited commercial availability of bacillibactin, we used azotochelin, a biscatechol from Azotobacter vinelandii, as a model catechol siderophore. Azotochelin was synthetized as described previously (37 (link), 38 ). The B. subtilisdhbA-F mutant (deficient in siderophore production) was grown in the presence of 10⁻⁴ M FeCl₃ and 10⁻⁵ M desferrioxamine E (DefE; Cedarlane, Burlington, Ontario, Canada) to obtain a catechol siderophore-free biofilm. After biofilm establishment (22 h after inoculation), 10⁻⁴ M azotochelin was added to the medium, and the formation of Fe-azotochelin complex was monitored by UV-Vis spectrometry (Genesys10s; Thermo Scientific).

The physicochemical properties were done as follows: the titratable acidity of verjuice was calculated as the percentage of tartaric acid since it is the most abundant organic acid in verjuice [14 , 15 (link)]. A pH/temperature microprocessor-based bench meter, equipped with digital probe 230 VAC, was used to measure the pH. A handheld refractometer (Model REF 107, 0-90% Brix) was used to measure the soluble solid content of verjuice [15 (link)]. Color intensity of all verjuice samples was measured spectrophotometrically (spectrophotometer, Thermo, model Genesys 10-S) based on the color intensity measurement of wine (I = (A at 420 nm) + (A at 520 nm), where I is the intensity and A is the absorbance) [16 ]. The Folin-Ciocalteu method for wines was adopted to find the total quantity of polyphenols in verjuice [17 (link)]. The antioxidant activity of the polyphenols found in verjuice was tested by the ability of these polyphenols to scavenge hydrogen peroxide [18 (link), 19 (link)]. Suspended solids were determined using Method 160.2 in Methods for Chemical Analysis of Water and Wastes (USEPA, 1983). Moisture percentage was measured using a moisture analyzer (Redwag MA 210.R, 563610, Tmax 160°C). A UV-visible spectrophotometer (spectrophotometer, Thermo, model Genesys 10-S) was used for measuring the browning index [20 (link)].

The DPPH radical-scavenging activity was assessed following the method of Phongthai et al. [9 (link)]. The digested and non-digested proteins (1 mg/mL, 0.5 mL) were mixed with 0.1 mM DPPH (2 mL) and then incubated for 30 min (dark condition). Distilled water was used as a control. The absorbance was read at 517 nm (Genesys™ 10S, Thermo Scientific, Waltham, MA, USA). The percentage of inhibition activity was calculated using the following equation:
where Abs_control and Abs_sample are the absorbances of the control and sample, respectively.
The ABTS radical-scavenging activity was assessed as described in Phongthai et al. [9 (link)]. Potassium persulfate (2.6 mM) and 7.4 mM ABTS solutions were mixed (1:1) and incubated for 12 h. The working solution was prepared by diluting the stock solution in DI water at a ratio of 1:60. The digested and non-digested protein solution (150 µL) was mixed with 28.5 mL of the working solution and incubated for 2 h in the dark. The absorbance was measured at 734 nm (Genesys™ 10S, Thermo Scientific, Waltham, MA, USA). The percentage of inhibition activity was calculated using the following equation:
where Abs_control and Abs_sample are the absorbances of the control and sample, respectively.