# Production of Vegan Ice Cream: Enrichment with Fermented Hazelnut Cake
## 2 Materials and Methods
### Characterization of Ice Cream Products
The reaction was determined by measuring the change in absorbance value at 734 nm over 6 min between 0.1 mL of sample solution and 1.9 mL of 7 mmol/L ABTS radical solution (prepared in 2.5 mmol/L potassium persulfate). For the hydroxyl radical binding capacity, 100 \(\upmu\)L of sample and 3 mL of Smirnoff reagent were incubated at 37 \({}^{\circ}\)C for 30 min. Absorbance measurements were then performed at 510 nm. The results were expressed as trolox equivalents. Determination of iron chelating activity: The iron ion chelating capacities of the samples were determined spectrophotometrically ([PERSON] and [PERSON], 2007) [29]. Briefly, 2 mL of appropriately diluted sample solution was mixed with 0.1 mL of 1 mmol/L FeCl2.4H2O solution and incubated for 30 min. Then, 0.1 mL of 5 mmol/L ferrozine was added, mixed, and incubated for another 10 min. The resulting color was measured spectrophotometrically at 562 nm, and the results were expressed as EDTA equivalent. ACE inhibition: Briefly, 10 \(\upmu\)L of 0.25 units/mL ACE prepared in 0.01 mol/L saline phosphate buffer (pH: 7.0, NaCl concentration: 0.5 mol/L) was mixed with 10 \(\upmu\)L of sample solution. The mixture was incubated at 37 \({}^{\circ}\)C for 15 min, and then, the reaction was started by adding 150 \(\upmu\)L of 1.75 mmol/L FAPGG substrate solution (37 \({}^{\circ}\)C) prepared in saline phosphate buffer. The absorbance of the reaction was measured kinetically at 340 nm for 30 min at 37 \({}^{\circ}\)C. ACE activity was determined using the slope of the initial linear region of the absorbance-time curve, and captopril was used as a control ([PERSON] et al., 2014) [30]. Determination of acetylcholine esterase (AChE) inhibition activity: The inhibition activity of the samples was carried out as described in [PERSON] et al. (2007) [31]. The principle of inhibition activity is the measurement of the yellow anion TNB (5-thio-2-nitrobenzoate), which was released as a result of the reaction between thiocholine and DTNB (5,5'-dithio-2-nitrobenzoic acid) hydrolyzed by acetylcholinesterase, and at 412 nm absorbance, acetylthiocholine iodide (10.85 mg/5 mL phosphate buffer) was used as a substrate for activity determination. The solution containing various concentrations of sample or standard to be prepared for analysis was prepared with 250 \(\upmu\)L of 200 mM phosphate buffer at pH 7.7, 80 \(\upmu\)L DTNB, and 10 \(\upmu\)L enzyme (2 U/mL). The solution was incubated at 25 \({}^{\circ}\)C for 5 min for pre-incubation. Then, 15 \(\upmu\)L of substrate was added to the solution and incubated again for 5 min, and the yellow-colored compound was formed. Total phenolic content: Total phenolic content was determined using the Folin-Ciocalteu reagent ([PERSON] and [PERSON], 1965), and the results were expressed as gallic acid equivalents [32]. \(\upalpha\)-amylase/\(\upalpha\)-glucosidase inhibition: \(\upalpha\)-Glucosidase and \(\upalpha\)-amylase inhibition experiments were carried out according to the methods specified in [PERSON] et al. (2022) [33]. Briefly, 100 \(\upmu\)L of \(\upalpha\)-glucosidase enzyme (1 unit/mL) to be prepared in 0.1 mol/L sodium phosphate buffer (pH 6.8) was pre-incubated with 50 \(\upmu\)L of sample for 10 min at 37 \({}^{\circ}\)C. Then, 50 \(\upmu\)L of 10 \(\upmu\)M (4-Nitrophenyl \(\upbeta\)-D-Galactopyranoside) was added as a substrate to initiate the enzymatic reaction and the reaction, which continued at 37 \({}^{\circ}\)C for 30 min and was then terminated by adding 1 mL of 0.1 mol/L Na\({}_{2}\)CO\({}_{3}\). Enzyme activity was determined by measuring the formed p-nitrophenyl at 400 nm. For the other analysis, 100 \(\upmu\)L of \(\upalpha\)-amylase enzyme (1 unit/mL) prepared in 0.1 mol/L sodium phosphate buffer (pH 6.8) was incubated with 100 \(\upmu\)L of sample for 5 min at 37 \({}^{\circ}\)C. Then, 250 \(\upmu\)L of 1% (mg/mL) starch prepared in sodium phosphate buffer (pH 6.8) was added to start the reaction.