*2.3. Enzymatic Hydrolysis*

The enzymatic hydrolysis of the cranberry pomace was produced using commercially available Novozyme A/S enzymes (Novozyme, Denmark), such as Viscozyme® L (β-glucanases, pectinases, hemicellulases, and xylanases), Pectinex® Ultra Tropical (pectinases, cellulases, hemicellulases, and β-glucanases), Pectinex® Yieldmash Plus (pectinases), and Celluclast® 1.5L (cellulases). The cranberry pomace was mixed with distilled water in a 1:10 ration (2.5 g of pomace with 25 mL of distilled water) and the enzyme was added at various concentration combinations in a range from 0.02 to 0.1 mL/g (control without enzymes). The mixture was incubated at 50 ◦C with shaking (200 rpm), and various time combinations from 1 to 7 h were used. The independent variable concentration of enzymes and duration of hydrolysis were chosen on the manufacturer's recommendations, and literature data [14,15]. After the hydrolysis, the enzyme activity was terminated by heating the sample in a 95 ◦C water bath for 20 min. For the water-soluble fraction yield calculation, the prebiotic activity and the mono-, disaccharides and oligosaccharides analysis, the mixture was cooled at room temperature (20 ◦C) and centrifugated (8000 rpm, 20 min). The water-soluble part (the resulting supernatant) was collected and freeze-dried (Harvest Right, North Salt Lake, UT, USA). The yield of the water-soluble fraction was determined gravimetrically after freeze-drying. The hydrolyzed cranberry pomace samples used for the determination of the functional properties were collected whole and freeze-dried. All of the freeze-dried samples were kept in a dark place at room temperature.

### *2.4. Mono-, Disaccharides' and Oligosaccharides' Analysis by High-Pressure Liquid Chromatography with Refractive Index Detector (HPLC-RI)*

For the saccharide analysis, 10 mg of the freeze-dried water-soluble fraction, was dissolved in 1 mL of Millipore water (10 mg/mL). The saccharide analysis was performed as previously described by Syrpas et al. [16]. The total oligosaccharides were recorded as the sum of all of the detected and quantified fractions of oligosaccharides with a degree of polymerization (DP) DP 7–10, DP 5–6, DP4, DP3, while the total mono- and disaccharides were recorded as a sum of the sucrose, glucose, fructose, sugar alcohols, and galacturonic

acids. According to the obtained results, the samples enzymatically hydrolyzed for 1 h were selected for further analysis.

### *2.5. In Vitro Assessment of the Prebiotic Activity*

The in vitro prebiotic activity of the water-soluble fraction was assessed by evaluating the probiotic bacteria growth stimulation, as described by Moreno-Vilet et al. [17] with some modifications. Two probiotic strains, *Lactobacillus acidophilus* DSM 20079 and *Bifidobacterium animalis* DSM 20105, were used. The probiotic strains were grown in De Man Rogosa and Sharpe (MRS) medium (Biolife, Milan, Italy), supplemented with 0.05% L-cysteine (*w*/*v*) under anaerobic conditions at 37 ◦C for 18 h. The bacterial inoculants were prepared by centrifugation (5000 g, 10 min, 4 ◦C) and washing twice with saline solution. After that, the bacterial cell pellets were collected and re-suspended in saline solution, using McFarland standard No. 0.5 (Liofilchem, Waltham, MA, USA) to standardize the bacterial concentrations. The growth of probiotics was evaluated using carbohydrate-free culture media with the same composition as the MRS supplemented with 1% ( *w*/*v*) from a different carbohydrate source. The water-soluble fraction obtained after 1 h hydrolysis was used as the carbohydrate source. Glucose was used as the negative control; commercial inulin was used as the positive control; and carbohydrate-free media was used as the blank control. Each tube was inoculated with 1% of the probiotic suspension and incubated for 48 h at 37 ◦C under anaerobic conditions. The probiotic bacteria counts were determined after 24 and 48 h. The probiotic growth was performed in quadruplet and measured by serial dilutions using the standard plate count technique following the calculation of the number of cells on the agar plate after 72 h incubation at optimal temperature and was expressed as log10 value of CFU/g. The proliferative index (PI) was calculated by the following equation:

$$\text{PI} = \text{average LogA} - \text{average LogB} \tag{1}$$

where B is the bacterial count at 0 h (CFU/g) and A is the bacterial count at 24 or 48 h (CFU/g).

### *2.6. Total Phenolic Content (TPC) by Folin* −*Ciocalteu's Assay*

The TPC of the enzymatically modified cranberry pomace was determined by Folin − Ciocalteu's Assay according to Singleton et al. [18] with some modifications. The extracts were obtained by mixing 1 g of enzymatically hydrolyzed cranberry pomace with 10 mL of methanol solution (0.1% HCl in methanol (*v*/*v*)) and left overnight in the dark at 4 ◦C. One ml of the extract solution in methanol was mixed with 5 mL of Folin −Ciocalteu's reagen<sup>t</sup> (1:9 *v*/*v*) and 4 mL sodium carbonate solution (7.5%). The samples were kept in the dark for 30 min and absorption was measured at 765 nm. The TPC was expressed in mg of gallic acid equivalents (GAE)/g of dry weight of cranberry pomace.

### *2.7. Water Retention Capacity (WRC)*

The water retention capacity was performed according to Yu et al. [19] with some modifications. The cranberry pomace (0.2 g) was mixed with distilled water (6 mL) at room temperature and left for 18 h. After that, the sample was centrifugated at 5000 rpm for 20 min. The resulting residues were weighted before and after drying at 105 ◦C. The WRC was calculated by the following equation:

$$\text{WRC} \left( \text{g/g} \right) = \left( \text{Mb} - \text{Ma} \right) / \text{Ma} \tag{2}$$

where Mb is the weight of residues before drying (g) and Ma is the weight of residues after drying (g).

### *2.8. Water Swelling Capacity (WSC)*

The water-swelling capacity was determined according to Yu et al. [19] with some modifications. The cranberry pomace (0.2 g) was weighted in graduated test tubes and hydrated

in 6 mL of distilled water at room temperature for 18 h. The volume of the pomace was recorded before and after hydration; the WSC was calculated by the following equation:

$$\text{WSC } (\text{mL/g}) = (\text{V1} - \text{V0}) / \text{W} \tag{3}$$

where V1 is the volume of pomace after hydration (mL); V0 is the volume of pomace before the hydration; W is the weight of the dry pomace prior to hydration (g).

### *2.9. Oil Retention Capacity (ORC)*

The ORC was performed according to Yu et al. [19] with some modifications. The dried pomace (0.2 g) was mixed with 2 g of sunflower oil and left for 1 h at room temperature. After that, the mixture was centrifugated at 300 rpm for 10 min and the supernatant was carefully decanted, and the pellet recovered was weighed. ORC was calculated by the following equation:

$$\text{ORC } (\text{g/g}) = (\text{W1} - \text{W0}) / \text{W0} \tag{4}$$

where W1 is the weight of the pellet (g) and W0 is the weight of the dry sample (g).
