## 2 Materials and Methods
### Methods
#### 2.2.2 Total Polyphenols Content and Antioxidant Activity  
Total polyphenols content and antioxidant activity were determined in 80% methanol extracts (Sigma-Aldrich, Poznan, Poland). To sterile 50 mL Falcone tubes, on an analytical balance (AS 220/X, Radway, Radom, Poland) 1.0 g of the tested samples was weighed (with an accuracy of 0.001 g), 30 mL of 80% methanol solution was added and vortexed (Wizard Advanced IR Vortex Mixer, VELP Scientific Srl, Usmate, Italy) for 60 s (2000 rpm) and incubated in a shaking incubator (IKA KS 4000i Control, IKA(r) Poland Ltd., Warsaw, Poland) for 60 min (40 \({}^{\circ}\)C, 200 rpm). After incubation, the samples were vortexed again (60 s, 2000 rpm) and centrifuged (MPW-380 R, MPW Med. Instruments, Poland, Warsaw) for 15 min (3 \({}^{\circ}\)C, 10,000 rpm). In the obtained clear supernatants, the total content of polyphenols and antioxidant activity were determined.  
The total polyphenols content was determined by the colorimetric spectrophotometric method using Folin-Ciocalteu reagent (Sig-Ma-Aldrich, Poznan, Poland) according to the modified method of [PERSON] and [PERSON] (1965) [38]. The amounts of extracts (determined in a predetermined dilution scheme) were transferred to 50 mL volumetric flasks; then, 2.5 mL of Folin-Ciocalteu reagent and 5.0 mL of 20% sodium carbonate (Sigma-Aldrich, Poznan, Poland) were added, made up to the mark with distilled water and incubated  
Figure 2: Plant material used in the research: (**a**) WG—white garlic, (**b**) BG-C—black garlic cloves, (c) BG-S—black garlic spread and (**d**) BG-S—black garlic spread.  
for 60 min at room temperature (20 \({}^{\circ}\)C) protected from the light. After incubation, the absorbance was measured at a wavelength \(\lambda\) = 750 nm with a spectrophotometer (UV/Vis UV-6100A, Metash Instruments Co., Ltd., Shanghai, China). After taking into account the applied dilution schemes, based on the calibration curve (y = 2.231 + 0.1315, R\({}^{2}\) = 0.9995) for the standard substance, i.e., gallic acid (Sigma-Aldrich, Poznan, Poland), the results of total polyphenolic content were calculated and expressed as mg GAE (_Gallic Acid Equivalents_)--i.e., mg gallic acid per 1 g of dry matter (mg GAE/g d.m.).  
Antioxidant activity was determined by the colorimetric spectrophotometric method using ABTS\({}^{\star\bullet}\)(2,2'-azino-bis (3-ethylbenzothazoline-6-sulphonic) acid) (Sigma-Aldrich, Poznan, Poland) radical cation assay according to modified method of [PERSON] et al. (1999) [39]. The amounts (0.5-1.5 mL) of extracts (determined in a predetermined dilution scheme) were transferred into 10 mL glass test tubes; then, 3.0 mL of ABTS\({}^{\star\bullet}\) radical cations in PBS solution (Phosphate Buffer Solution, Sigma-Aldrich, Poznan, Poland) was added and incubated for 6 min at room temperature (20 \({}^{\circ}\)C). After incubation, the absorbance was measured at a wavelength \(\lambda\) = 734 nm with a spectrophotometer (UV/Vis UV-6100A, Metash Instruments Co., Ltd., Shanghai, China). After taking into account the applied dilution schemes, based on the calibration curve (y = -5.7012 + 0.7039, R\({}^{2}\) = 0.9998) for the standard substance, i.e., Trolox (Sigma-Aldrich, Poznan, Poland), the results of antioxidant activity were calculated and expressed as a \(\upmu\)mols TEAC (_Trolox Equivalent Antioxidant Capacity_)--i.e., \(\upmu\)M TEAC per 1 g of dry matter (\(\upmu\)M TEAC/1 g d.m.).  
The determinations of total polyphenolic content ant antioxidant activity were performed in six independent replications for each garlic sample.