## 2 Materials and Methods
### Total Polyphenols Content and Antioxidant Activity
#### 2.6.7 Chelating Activity Fe\({}^{2+}\)  
The chelating ability for Fe\({}^{2+}\) was assessed using a ferrozine technique [35]. The metal ion chelation process significantly prevents reactive oxygen species formation [36]. A calibration curve was prepared by utilizing aqueous solutions of FeSO\({}_{4}\) to measure Fe\({}^{2+}\) ions. First, an initial FeSO\({}_{4}\) solution with a Fe\({}^{2+}\) concentration of 0.53 mmol/L was prepared. Next, a ferrozine initial solution (concentration of 3.2 mmol/L) was prepared. Then, 1000 \(\mu\)L of the initial FeSO\({}_{4}\) solution (final Fe concentrations of 3, 1.2, 0.6, and 0.3 mg/L) and 1000 \(\mu\)L of ferrozine were introduced into 10, 25, 50, and 100 mL volumetric flasks. The flasks were filled with distilled water, sealed snugly with a stopper, and then kept at room temperature for 10 min. The absorbance of the test solutions was measured (using the Thermo Scientific GENESYS 50 apparatus at the wavelength \(\lambda\) = 562 nm), obtaining a calibration curve for Fe\({}^{2+}\) ions y = 0.4888x + 0.0064, R\({}^{2}\) = 0.999.  
In the next stage, 1000 \(\mu\)L of the initial FeSO\({}_{4}\) solution, 100 \(\mu\)L of bio-ferment, and 1000 \(\mu\)L of ferrozine were introduced into 10 mL volumetric flasks. The flasks were filled with distilled water, sealed snugly with a stopper, and incubated at room temperature for 10 min. Then, the absorbance of the solutions was measured using a spectrophotometer at a wavelength of \(\lambda\) = 562 nm. Three separate tests were conducted.  
The assessment of the chelating activity for Fe\({}^{2+}\) ions was computed using the following formula:  
\[ChA=\frac{\left[\left(C_{Fe2+bs.}-C_{Fe2+t.s.}\right)\cdot V_{s}\right]}{V_{b}} \tag{2}\]  
where:  
ChA--chelating activity for Fe\({}^{2+}\) [mmol Fe\({}^{2+}\)/L];  
C\({}_{\rm Fe2+b.s.}\)--concentration of Fe\({}^{2+}\) ions in the blank sample [mmol/L];  
C\({}_{\rm Fe2+t.s.}\)--concentration of Fe\({}^{2+}\) ions in the tested sample [mmol/L];  
V\({}_{\rm s}\)--total volume of solution introduced into volumetric flasks [L];  
V\({}_{\rm b}\)--volume of bio-ferment introduced into volumetric flasks [L].  
Acidity testing [14] of appropriate bio-ferrments (B-P, B-E, B-O, and B-S) was carried out by titration with a standard sodium hydroxide solution and phenolphthalein as an indicator. In a ground conical flask, 10 mL of distilled water, 2 mL of the corresponding bio-ferrments (B-P, B-E, B-O, and B-S), and 3 drops of an ethanolic solution of phenolphthalein were introduced. The flask was closed with a stopper, and its contents were mixed and then titrated with NaOH solution until the solution turned slightly pink. Blank analyses were performed in parallel.  
The acidity (A) of the tested bio-ferrments was determined as the number of sodium hydroxides that are required to neutralize the test sample with the equivalent of carboxyl groups according to the following equation:  
\[A=\frac{(V-V_{0})\cdot N}{V_{b}} \tag{3}\]  
where:  
A--acidity [mmol COOH/L];  
V--volume of NaOH solution [L];  
V\({}_{\rm 0}\)--volume of NaOH solution used for titration of the blank [L];  
V\({}_{\rm b}\)--volume of NaOH solution used for titration of the sample of bio-ferment [L];  
N--normality of the NaOH solution used for the titration [0.1 N].