*3.3. Saccharide Analysis*

The total oligosaccharides, mono- and disaccharides increased in all of the enzymatically hydrolyzed samples (Table 2). The quantity of oligosaccharides and mono- and disaccharides depended on the type and concentration of the enzyme as well as the duration of hydrolysis. The highest increase in total oligosaccharides after 1 h hydrolysis was obtained in the sample hydrolyzed with Viscozyme® L (3.6 times higher than control), while the lowest increase was determined in the sample hydrolyzed with Pectinex® Yieldmash Plus. Viscozyme® L is composed of β-glucanases, pectinases, hemicellulases, and xylanases. The enzymatic hydrolysis of β-glucan-type hemicellulose polymers in cranberry pomace increases the level of oligosaccharides [3].

Spadoni Andreani et al. [22] reported cranberry extracts after enzymatic hydrolysis with a significantly increased glucose level. The highest mono- and disaccharide content was observed in the sample hydrolyzed with Pectinex® Ultra Tropical. The lowest monoand disaccharide content was determined in the sample hydrolyzed with Celluclast® 1.5L (1.7 times lower than in the pomaces hydrolyzed with Pectinex® Ultra Tropical) compared with other enzymatically hydrolyzed pomaces. A longer enzymatic hydrolysis increased the mono- and disaccharide content, except for the pomace treated with Pectinex® Yieldmash Plus. However, a lower enzyme concentration was used for the longer hydrolysis. The greater increase in the saccharide content was determined using enzymes, which were mainly pectinases. Viscozyme® L used in this study (0.04 mL/g for 1 h hydrolysis) showed the highest level of total oligosaccharides, while the longer duration of hydrolysis and the higher enzyme concentration decreased the level of the oligosaccharides and increased the level of the mono- and disaccharides.

For these reasons, the determination of the further functional properties was evaluated in the enzymatically hydrolyzed samples for 1 h.

### *3.4. In Vitro Prebiotic Activity*

The growth of the probiotic bacteria in a medium supplemented with different carbohydrate sources is shown in Table 3. Both of the tested probiotic bacteria showed an ability to utilize the carbohydrates from cranberry water-soluble fractions as their carbon sources. The growth of the probiotic bacteria in the medium supplemented with carbohydrates was significantly higher than in the carbohydrate-free media. *L*. *acidophilus* DSM 20079 did not survive in the carbohydrate-free media after 48 h, while *B. animalis* DSM 20105 showed an ability to grow in this media. The cranberry water-soluble fraction after 24 h promoted the growth of *L. acidophilus* DSM 20079 better than glucose or inulin. However, with *L*. *acidophilus* DSM 20079 viability decreased in all of the samples after 48 h, except for the media supplemented with glucose when viability was compared after 24 h, whereas with *B. animalis* DSM 20105, growth increased in all of the samples after 48 h (Table 3). The highest increase was observed in the media supplemented with glucose and inulin. The cranberry water-soluble fraction obtained after treatment with Pectinex® Ultra Tropical promoted the growth of *L*. *acidophilus* DSM 20079 better than the other water-soluble fractions after 24 h. Furthermore, after 48 h, the *L*. *acidophilus* DSM 20079 in this medium showed a better cell viability than in the other media supplemented with cranberry extracts. However in most of the cases, no statistically significant differences were observed (*p* < 0.05). It may be due

to a higher concentration of mono- and disaccharides (Table 2) in this extract compared with the other cranberry extracts. The same proliferation effect of prebiotics with a higher sugar content was reported in other studies [23,24]. *L*. *acidophilus* could utilize a variety of carbohydrates, such as mono-, di-, and polysaccharides [25]. The *L. acidophilus* could use oligosaccharides as a carbon source and grow without sugar [26]. All of the cranberry water-soluble fractions had oligosaccharides which may have an impact on the faster proliferation of *L*. *acidophilus* DSM 20079, compared with glucose and inulin. However, the faster utilization of carbohydrates may lead to a decreased viability of the probiotic cells after 48 h.


**Table 3.** Growth of probiotics in medium supplemented with different carbohydrate source.


The extract obtained after treatment with Celluclast® 1.5L promoted the growth of *B. animalis* DSM 20105 after 24 h better than the other extracts, however, in most cases, no statistically significant differences were observed (*p* < 0.05). The *Bifidobacterium* shows a specific preference for prebiotic substrates within the genus, although most of the bacteria could utilize a range of different carbohydrates [27]. The molecular weight, degree of polymerization, and the type of linkage between the comprising units are known to influence the prebiotic activity of carbohydrates [23,28–30].

### *3.5. Dietary Fiber and TPC Content after Enzymatic Hydrolysis*

The dietary fiber content after enzymatic hydrolysis was evaluated in whole freezedried samples hydrolyzed for 1 h (Table 4). The IDF significantly decreased (*p* < 0.05) in all of the samples. The lowest content of IDF was observed in a sample hydrolyzed with Pectinex® Ultra Tropical (13.3% lower than the control). The pectinolytic enzymes increased the plant cell-wall breakdown of the pomace [31]. The content of the SDF decreased significantly (*p* < 0.05) in all of the samples, except for the sample hydrolyzed with Celluclast® 1.5L. The lowest SDF content was observed in the sample treated with Pectinex® Ultra Tropical (84.41% lower than the control) and the highest content was determined in the sample treated with Celluclast® 1.5 L, however, no significant differences were observed (*p* < 0.05) compared to the control. The results indicate that the SDF can be hydrolyzed to smaller fragments and does not precipitate with ethanol. Mrabet et al. [32] reported a similar decrease in the dietary fiber content treated with Viscozyme® L, while Yoon et al. [21] reported an increase in the alcohol-soluble dietary fiber after the enzymatic hydrolysis of carrot pomace. Spadoni Andreani et al. [33] reported an increased yield of oligosaccharides in cranberry carbohydrate extracts after enzymatic treatment with Viscozyme® L and Pectinex® Ultra SPL. The obtained results sugges<sup>t</sup> that Celluclast® 1.5L could be used for increasing the SDF, while Viscozyme® L and Pectinex® Ultra Tropical showed promising results for the production of oligosaccharides.


**Table 4.** Dietary fiber, TPC content and technological properties of enzymatically treated cranberry pomace.

Data values are expressed as means with the standard deviation of three replicates for SDF, IDF, ORC, WRC, WSC, solubility and of six replicates for TPC. Values in one column followed by the same letter are not significantly different (*p* < 0.05).

The TPC of the enzymatically treated cranberry pomace was evaluated (Table 4). The enzymatic hydrolysis increased the TPC in all of the samples, and varied in range from 7.04 to 7.96 mg GAE/g, however, no significant difference (*p* < 0.05) was determined between the control and the pomaces hydrolyzed with Celluclast® 1.5L and Pectinex® Yieldmash Plus. The highest amount of TPC was determined after hydrolysis with Pectinex® Ultra Tropical and Viscozyme® L (13.07 and 10.94% higher than the control, respectively). The results showed that decreasing the total dietary fiber content increases the TPC content. The antioxidants are usually stored in natural cell compartments, so they must be released during digestion to be absorbed in the gu<sup>t</sup> [34]. Gouw et al. [35] reported TPC in dried cranberry pomace of 7.58 mg GAE/g; other studies reported phenolic content of 6 mg/g [20], while Ross et al. [36] reported higher values of TPC in cranberry pomace (~13.55–15.17 mg GAE/g) depending on the drying conditions. The TPC content in the berries depends on many factors, such as cultivar peculiarities, cultivation technologies, region, weather conditions, ripeness, harvesting time, and storage conditions/time [37]. The higher content of TPC was reported in grape pomace (38.7 ± 0.36 mg GAE/g) [38] and blueberry pomace (~17.76–20.82 mg GAE/g) [36]. The phenolic compounds associated with soluble dietary fiber may present different structures, including soluble flavonoids and phenolic acids [34].
