foods

Article
Technological Properties and Composition of Enzymatically
Modified Cranberry Pomace
Jolita Jagelaviciute 1 , Loreta Basinskiene 1, *, Dalia Cizeikiene 1 and Michail Syrpas 1,2

1 Department of Food Science and Technology, Kaunas University of Technology, Radvilenu Rd. 19,
LT-50254 Kaunas, Lithuania; jolita.jagelaviciute@ktu.lt (J.J.); dalia.cizeikiene@ktu.lt (D.C.);
michail.syrpas@ktu.lt (M.S.)
2 Bioprocess Research Centre, Kaunas University of Technology, Radvilenu Rd. 19, LT-50254 Kaunas, Lithuania
* Correspondence: loreta.basinskiene@ktu.lt

Abstract: Cranberry pomace obtained after juice production is a good source of dietary fiber and
other bioactive compounds. In this study, cranberry pomace was hydrolyzed with Viscozyme® L,
Pectinex® Ultra Tropical, Pectinex® Yieldmash Plus, and Celluclast® 1.5L (Novozyme A/S, Den-
mark). The soluble and insoluble dietary fiber was determined using the Megazyme kit, while the
changes in mono-, disaccharide and oligosaccharides’ contents were determined using HPLC-RI;
the total phenolic contents were determined by Folin−Ciocalteu’s Assay. Prebiotic activity, using
two probiotic strains Lactobacillus acidophilus DSM 20079 and Bifidobacterium animalis DSM 20105,
was investigated. The technological properties, such as hydration and oil retention capacity, were
evaluated. The enzymatic treatment increased the yield of short-chain soluble saccharides. The
highest oligosaccharide content was obtained using Viscozyme® L and Pectinex® Ultra Tropical. All
of the tested extracts of cranberry pomace showed the ability to promote growth of selected probiotic
bacteria. The insoluble dietary fiber content decreased in all of the samples, while the soluble dietary
fiber increased just in samples hydrolyzed with Celluclast® 1.5L. The highest content of total phenolic
Citation: Jagelaviciute, J.; compounds was obtained using Viscozyme® L and Pectinex® Ultra Tropical (10.9% and 13.1% higher
Basinskiene, L.; Cizeikiene, D.; than control, respectively). The enzymatically treated cranberry pomace exhibited lower oil and
Syrpas, M. Technological Properties water retention capacities in most cases. In contrast, water swelling capacity increased by 23% and
and Composition of Enzymatically 70% in samples treated with Viscozyme® L and Celluclast® 1.5L, respectively. Enzymatically treated
Modified Cranberry Pomace. Foods cranberry pomace has a different composition and technological properties depending on the enzyme
2022, 11, 2321. https://doi.org/ used for hydrolysis and can be used in various novel food products.
10.3390/foods11152321

Academic Editors: Marco Poiana, Keywords: cranberry pomace; enzymatic hydrolysis; dietary fiber; technological properties
Francesco Caponio and Antonio Piga

Received: 14 July 2022

Accepted: 1 August 2022
1. Introduction
Published: 3 August 2022
Agricultural by-products form a large part of waste, with approximately 88 metric
Publisher’s Note: MDPI stays neutral
tons per year generated in Europe alone [1]. However, these by-products are considered
with regard to jurisdictional claims in
to be potential sources of various high added-value, bioactive compounds (dietary fibers,
published maps and institutional affil-
pigments, essential minerals, fatty acids, antioxidant polyphenolic compounds, etc.) [2].
iations.
Berry pomace (or press cake) obtained after juice pressing usually contains skin, stems, and
seed parts [1]. Various fruit and berry products are substantially composed of structural
polysaccharides and their related oligosaccharides and these compounds are commonly
Copyright: © 2022 by the authors.
described as insoluble (IDF) and soluble (SDF) dietary fibers [3]. The dietary fiber in berry
Licensee MDPI, Basel, Switzerland. pomace is usually composed of pectin, lignin, cellulose, hemicellulose, and inulin [1]. These
This article is an open access article types of compounds have a potentially significant impact on human health [3], such as a
distributed under the terms and decrease in the risk of hypertension, obesity, diabetes, coronary heart disease, stroke, and
conditions of the Creative Commons certain gastrointestinal disorders [1]. The Food and Drug Administration recommendation
Attribution (CC BY) license (https:// for the use of dietary fiber in the diet is approximately 25–35 g per day, and 6 g of it should
creativecommons.org/licenses/by/ be SDF. Furthermore, some studies show that 10 g of extra dietary fiber in a diet could help
4.0/). decrease the risk of death caused by coronary heart disease by up to 35% [1,4].

Foods 2022, 11, 2321. https://doi.org/10.3390/foods11152321 https://www.mdpi.com/journal/foods

Foods 2022, 11, 2321 2 of 14

Polyphenols and cell-wall polysaccharides can also positively modulate the profile
of the gut microbiota, which may have implications in the prevention against metabolic
diseases [5]. Other studies indicate that SDF can be used as a prebiotic [1]. Islam et al. [6]
reported that the incorporation of cranberry pomace in the food not only improved the
short-term iron and cholesterol levels in the blood serum of broilers, but also increased
the levels of beneficial bacterial genera and decreased the undesirable ones. Other studies
indicated that the fractions of cranberry oligosaccharide and the xyloglucan and purified
cranberry xyloglucan compounds have shown a distinct lack of antimicrobial properties
or cytotoxic effects in numerous bacterial and human cell lines [3]. The oligosaccharides
extracted from cranberries even shown the ability to modify the biofilm formation of some
E. coli strains [7]. Liu et al. [8] reported that cranberry oligosaccharides can be used as a
carbon source for Lactobacillus, however the oligosaccharide metabolization is strain specific.
The supplementation of fermented meat with cranberry pomace significantly inactivates
Salmonella and increases the growth of lactic acid bacteria [9].
An increase in the level of dietary fiber in food products usually negatively influences
the products’ texture and color, depending on the properties and level of fiber. However,
some studies indicated that enzymatic treatment is practical and a potential modification
method to obtain functional food materials with better technological properties from
agriculture by-products [10,11]. For example, the enzymatically treated tomato peels have
higher contents of lycopene and dietary fiber [10]. The enzymatic treatment can effectively
increase the release of antioxidants and phenolic compounds and the fiber composition.
However, this is not the case for all of the fruit and berry matrixes; in some cases, enzymatic
treatment can reduce the fiber content and increase the free monosaccharides [11]. The
treatment of fruit fiber with enzymes of different activities has resulted in structural changes
that have altered the technological properties, such as the water-holding and swelling
capacities [12].
In this study, cranberry pomace was enzymatically treated with several commercially
available enzymes to modify their technological properties and the composition of the
dietary fiber. The technological properties were determined, such as water-holding, oil-
holding, swelling, emulsion capacities, dietary fiber composition, and prebiotic potential.
Most previously published studies indicated cranberry by-products as a good source for
isolating bioactive compounds. However, there is a lack of information regarding the effect
of enzymatic treatments on cranberry pomace properties. This study provides experimental
support for the whole cranberry pomace as a source of dietary fiber and for developing
functional and innovative food products.

2. Materials and Methods

2.1. Cranberry Pomace
The cranberry pomace was kindly donated by the company “I˛vairios sultys” (Kėdainiai,
Lithuania). The moisture content was 3.96%. The pomace was ground to a particle size of
0.5 mm and stored at 4 ◦ C.

2.2. Proximate Composition Analysis of Cranberry Pomace

The moisture content was determined by drying 1 g of cranberry pomace in an
oven at 105 ◦ C to a constant weight, according to the Association of Official Analytical
Collaboration (AOAC) Method 925.10-1925. After drying, the final weight of the pomace
was subtracted from the initial weight divided by the weight of the sample multiplied
by 100 and expressed as g/100 g. The proteins were determined on 1 g of pomace by
the Kjeldahl method (N × 6.25), according to the AOAC Method 978.04. The results were
expressed as g/100 g of the dry weight sample. The lipids were determined on 1 g of
pomace by drying the sample and subsequently the Soxhlet extraction with hexane was
performed for 5 h, according to the AOAC Method 948.22. After the extraction, the residues
were dried in an oven at 105 ◦ C to a constant weight. The lipid content was calculated from
the initial dry weight of pomace by subtracting the final weight of the residues divided by
Foods 2022, 11, 2321 3 of 14

the weight of the dry sample multiplied by 100 and expressed as g/100 g of the dry weight
sample. The ash content was determined by charring 2 g of pomace for 30 min, followed by
incineration in a muffle furnace at 525 ◦ C for 4 h, according to the AOAC Method 930.05.
The residues obtained after incineration were recorded and divided by the weight of the
sample weight and multiplied by 100 and expressed as g/100 g of the dry weight sample.
The total carbohydrate content was calculated by subtracting the value of the protein, ash,
and lipid contents from 100% of the dry weight. The soluble and insoluble dietary fiber were
quantified using a Megazyme kit, based on the American Association of Cereal Chemists
(AACC) Method 32-07.01 and the AOAC Method 991.43 [13]. One g of pomace (duplicate)
was mixed with 40 mL MES-Tris buffer (0.05 M pH 8.2) and hydrolyzed with 50 µL of
α-amylase (Megazyme E-BLAAM) at 95 ◦ C for 30 min under constant stirring (120 rpm).
After hydrolysis, the temperature was cooled to 60 ◦ C and the sample was hydrolyzed with
100 µL of protease (Megazyme E-BSPRT) at 60 ◦ C for 30 min under constant stirring (120
rpm). Then, the pH was adjusted to 4.1–4.8 with 0.561 N HCl and further hydrolysis was
performed with 200 µL of amyloglucosidase (Megazyme E-AMGDF) at 60 ◦ C for 30 min
under constant stirring (120 rpm). The suspension was vacuum-filtered in the Fibertec
1023 E equipment (Foss System, Hilleroed, Denmark) through a celite (Megazyme cat. No.
G-CELITE) in a bed crucible. The residues were washed with hot distilled water (70 ◦ C),
95% ethanol and acetone, dried and recorded as IDF. The obtained supernatant was mixed
with four volumes of 95% ethanol (60 ◦ C) and left to stand at room temperature for 1 h;
the SDF was recorded by filtration of the ethanolic suspension in the same conditions as
the IDF and washed with 78% ethanol, 95% ethanol, and acetone. The IDF and SDF were
calculated by subtracting the protein and ash contents determined in the obtained residues
and expressed as g/100 g of the dry weight sample.

2.3. Enzymatic Hydrolysis

The enzymatic hydrolysis of the cranberry pomace was produced using commer-
cially available Novozyme A/S enzymes (Novozyme, Denmark), such as Viscozyme® L
(β-glucanases, pectinases, hemicellulases, and xylanases), Pectinex® Ultra Tropical (pecti-
nases, 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 calcula-
tion, 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
Foods 2022, 11, 2321 4 of 14

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:
PI = average LogA − average LogB (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 reagent
(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:

WRC (g/g) = (Mb − Ma)/Ma (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 mod-
ifications. The cranberry pomace (0.2 g) was weighted in graduated test tubes and hydrated
Foods 2022, 11, 2321 5 of 14

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:

WSC (mL/g) = (V1 − V0)/W (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:
ORC (g/g) = (W1 − W0)/W0 (4)
where W1 is the weight of the pellet (g) and W0 is the weight of the dry sample (g).

2.10. Solubility
The yield of soluble material in the pomace was determined, using the supernatant
from WRC analysis. The supernatant, after decantation, was dried at 105 ◦ C to a constant
weight. The solubility was calculated by the following equation:

Solubility (%) = (M/M0) × 100 (5)

where M is the weight of dried soluble material (g); M0 is the weight of the dry pomace
used for analysis (g).

2.11. Emulsion Stability

The emulsion was prepared by mixing the pomace (0.16 g) with sodium phosphate
(0.02 M) and citric acid (0.1 M) buffer pH 4, 6, 8 or distilled water (8 g), then the sunflower
oil was added (8 g) and emulsified using a rotor–stator homogenizer (IKA® T-25 digital,
Ultra-Turrax, Staufen, Germany) for 5 min at 10,000 rpm. The emulsions were transferred
into graduated tubes and stored for 3 weeks at 4 ◦ C. The emulsion stability was recorded
in terms of phase separation and expressed by the following equation:

Emulsion stability = (VE/VT) × 100 (6)

where VE is the volume of emulsion; VT is the total volume.

To evaluate the thermal stability of emulsions, the tubes were heated in an 80 ◦ C
water bath for 30 min. Then, the tubes were cooled to room temperature and stored for
3 weeks at 4 ◦ C. The thermal stability of the emulsion was recorded and expressed as
previously described.

2.12. Statistical Analyses

The mean values and standard deviations were calculated using MS Excel 2019 (Mi-
crosoft Corp, Albuquerque, NM, USA). The statistical analysis was performed using the
Statgraphics Centurion 19 statistical package. One-way analysis of variation (ANOVA),
followed by Tukey’s honest significant difference (HSD) test were performed to determine
significant differences (p < 0.05).

3. Results and Discussion

3.1. Chemical Composition
The chemical composition of the cranberry pomace is shown in Table 1. The major
component of the pomace is carbohydrates, in which dietary fibers represent 72.67 g/100 g
Foods 2022, 11, 2321 6 of 14

of dry weight (IDF—59.93 g/100 g and SDF—12.74 g/100 g). The dietary fibers obtained
from the various berries mainly include: lignin, pectin, cellulose, and hemicellulose [1].
Islam et al. [6] reported a similar amount of SDF (11–12.3 g/100 g) in cranberry pomace,
while the protein content was reported as lower (5.75 g/100 g). White et al. [20] reported a
similar carbohydrate content in cranberry pomace, but a lower SDF and protein content and
a higher IDF and fat content. The chemical composition of the cranberry pomaces varies and
is influenced by several conditions, such as the cultivar, ripeness, or processing conditions.

Table 1. Chemical composition of cranberry pomace.

Parameter Content (g/100 g)

Moisture 5.57 ± 0.11
Total fat a 9.83 ± 0.46
Ash a 0.96 ± 0.04
Protein a 7.40 ± 0.06
Carbohydrate b 81.81
Insoluble dietary fiber a 59.93 ± 1.46
Soluble dietary fiber a 12.74 ± 0.09
a Expressed as g/100 g of dry weight; b Carbohydrate content was determined by subtracting the value of protein,
ash, and lipid content from 100% of dry weight. Data values are expressed as means with the standard deviation
(n = 3).

3.2. Yield of Water-Soluble Fraction

The enzymatic hydrolysis of the cranberry pomace was performed using four different
commercial enzymes. The different times for hydrolysis and different concentrations of
enzymes were evaluated (rationale). The yield of the water-soluble fraction obtained
after enzymatic hydrolysis is presented in Table 2. As could be expected, the enzymatic
hydrolysis increased the water-soluble material content in all of the samples. The extraction
yield depended on the composition and concentration of enzymes, and the duration
of the hydrolysis. The highest yield of the water-soluble fraction was obtained using
Viscozyme® L and Pectinex® Ultra Tropical enzymes. Compared with the yield of the
control sample after 1 h of incubation, the lowest increase in the water-soluble fraction was
observed with Celluclast® 1.5L (increased by 19.68%), while the highest increase was in the
sample hydrolyzed with Pectinex® Ultra Tropical (increased by 93.34%). Previous studies
have reported that enzymatic treatment increases the water-soluble fraction yield [10].
Yoon et al. [21] reported an increase in the alcohol-insoluble dietary fiber by prolonging the
time of enzyme hydrolysis, while the alcohol-soluble dietary fiber yield did not change
significantly after 24 h of enzymatic hydrolysis. However, increasing the enzyme level
can reduce hydrolysis time (Table 2). In most cases, there were no significant differences
(p < 0.05) between the yields of water-soluble fraction obtained using the same enzyme by
increasing the enzyme level and decreasing enzymatic hydrolysis time.

Table 2. Water-soluble fraction yield and saccharide composition of cranberry pomace after enzymatic
hydrolysis.

Time E/S Ratio Water-Soluble Total Oligosaccharides, Total Mono- and

Enzyme
(h) (mL/g) Fraction Yield, % mg/g DW Disaccharides, mg/g DW
1 0.04 Viscozyme® L 25.56 ± 1.33 e 71.6 ± 4.4 b 178.5 ± 8.9 abcd
5.5 0.1 Viscozyme® L 27.98 ± 0.20 e 61.3 ± 2.1 b 222.9 ± 4.3 d
1 0.1 Pectinex® Yieldmash Plus 21.50 ± 0.78 d 21.2 ± 0.6 a 197.0 ± 0.3 bcd
7 0.02 Pectinex® Yieldmash Plus 19.95 ± 0.23 cd 16.3 ± 3.9 a 173.7 ± 11.4 abcd
1 0.08 Pectinex® Ultra Tropical 27.34 ± 1.56 e 59.0 ± 6.0 b 211.9 ± 24.3 cd
4 0.06 Pectinex® Ultra Tropical 27.41 ± 0.71 e 56.4 ± 4.3 b 216.3 ± 12.7 cd
1 0.02 Celluclast® 1.5L 18.50 ± 1.35 bc 32.2 ± 3.3 a 123.3 ± 14.8 ab
7 0.1 Celluclast® 1.5L 21.74 ± 0.71 d 56.5 ± 3.1 b 150.2 ± 9.0 abcd
1 - - 14.12 ± 0.34 a 19.9 ± 2.2 a 108.9 ± 2.5 a
Foods 2022, 11, 2321 7 of 14

Table 2. Cont.

Time E/S Ratio Water-Soluble Total Oligosaccharides, Total Mono- and

Enzyme
(h) (mL/g) Fraction Yield, % mg/g DW Disaccharides, mg/g DW
4 - - 16.14 ± 0.14 ab 18.9 ± 2.5 a 118.9 ± 7.5 a
5.5 - - 16.56 ± 0.68 abc 20.9 ± 2.9 a 140.4 ± 20.9 abc
7 - - 17.08 ± 0.17 abc 22.0 ± 2.9 a 143.3 ± 21.9 abc
E/S—enzyme/substrate (pomace) ratio. - without enzyme (control samples). Data values are expressed as means
with the standard deviation (n = 3). Values in one column followed by the same letter are not significantly different
(p < 0.05).

3.3. Saccharide Analysis

The total oligosaccharides, mono- and disaccharides increased in all of the enzymat-
ically 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 du-
ration 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® Yield-
mash 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 mono-
and 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 carbo-
hydrate 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
Foods 2022, 11, 2321 8 of 14

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 pro-
liferation 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.

Lactobacillus acidophilus DSM 20079 Bifidobacterium animalis DSM 20105

Prebiotics 0h 24 h 48 h 0h 24 h 48 h
Log CFU/g PI Log CFU/g PI Log CFU/g PI Log CFU/g PI
Carbohydrate-free a a a a a a
5.03 ± 0.07 4.27 ± 0.13 −0.66 - −5.03 5.03 ± 0.05 6.64 ± 0.09 1.61 6.45 ± 0.03 1.42
MRS
Glucose 4.78 ± 0.20 a 7.31 ± 0.22 bc 2.53 7.62 ± 0.08 e 2.84 5.28 ± 0.05 b 7.99 ± 0.22 b 2.71 9.00 ± 0.41 cd 3.72
Inulin 4.81 ± 0.23 a 7.11 ± 0.25 b 2.29 5.52 ± 0.57 b 0.71 5.28 ± 0.08 b 8.36 ± 0.25 bc 3.08 9.07 ± 0.12 d 3.78
Control 4.77 ± 0.06 a 7.72 ± 0.10 de 2.95 5.86 ± 0.06 bc 1.09 5.52 ± 0.09 c 8.03 ± 0.52 b 2.51 8.83 ± 0.12 cd 3.30
Viscozyme® L 4.79 ± 0.09 a 7.69 ± 0.11 cde 2.90 6.21 ± 0.04 cd 1.41 5.60 ± 0.06 c 8.37 ± 0.13 bc 2.77 8.37 ± 0.05 b 2.77
Pectinex® Ultra
4.72 ± 0.07 a 7.84 ± 0.23 e 3.13 6.45 ± 0.14 d 1.73 5.58 ± 0.05 c 8.40 ± 0.02 bc 2.81 8.63 ± 0.05 bc 3.04
Tropical
Pectinex®
5.00 ± 0.01 a 7.53 ± 0.14 cde 2.53 5.81 ± 0.19 bc 0.82 5.52 ± 0.09 ab 8.03 ± 0.52 bc 2.51 8.77 ± 0.19 bcd 3.24
Yieldmash Plus
a c c
Celluclast® 1.5L 4.74 ± 0.06 7.44 ± 0.15 cd 2.70 5.69 ± 0.09 b 0.95 5.62 ± 0.09 8.66 ± 0.03 3.04 8.93 ± 0.07 cd 3.31
- No growth; data values are expressed as means with the standard deviation (n = 4). Values in one column
followed by the same letter are not significantly different (p < 0.05).

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 poly-
merization, 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 freeze-
dried 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 suggest 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.
Foods 2022, 11, 2321 9 of 14

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

E/S
Time TPC, mg of
Sample Ratio IDF, g/100 g SDF, g/100 g ORC, g/g WSC, mL/g WRC, g/g Solubility, %
(h) GAE/g
(mL/g)
Control 1 - 62.48 ± 0.46 d 11.23 ± 0.84 c 6.50 ± 0.08 a 1.80 ± 0.3 a 10.59 ± 0.20 a 15.4 ± 0.9 a 7.04 ± 0.41 a
Viscozyme® L 1 0.04 56.46 ± 0.15 ab 2.64 ± 0.26 a 4.37 ± 0.25 b 2.22 ± 0.01 b 8.58 ± 0.24 b 27.5 ± 1.3 b 7.81 ± 0.36 b
Pectinex®
Yieldmash 1 0.1 58.48 ± 0.44 bc 5.44 ± 0.30 b 3.99 ± 0.13 b 1.65 ± 0.01 a 7.25 ± 0.22 c 24.0 ± 0.6 c 7.42 ± 0.24 ab
Plus
Pectinex®
1 0.08 54.17 ± 1.38 a 1.75 ± 0.29 a 4.11 ± 0.16 b 1.57 ± 0.11 a 8.10 ± 0.43 b 29.6 ± 1.2 d 7.96 ± 0.29 b
Ultra Tropical
Celluclast®
1 0.02 60.52 ± 0.17 cd 11.90 ± 0.53 c 5.80 ± 0.37 c 3.06 ± 0.16 c 11.02 ± 0.24 a 20.3 ± 1.0 e 7.06 ± 0.28 a
1.5 L
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® Yield-
mash 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 con-
tent. The antioxidants are usually stored in natural cell compartments, so they must be
released during digestion to be absorbed in the gut [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].

3.6. Technological Properties

Fiber-rich by-products can be included in food products as low-cost and non-caloric
bulking agents to partially replace flour, fat or sugar, as water and oil retention agents, and
to improve the stability of emulsions [39]. The technological properties were evaluated of
samples hydrolyzed for 1 h (Table 4). The ORC after enzymatic hydrolysis significantly
decreased (p < 0.05) in all of the samples compared to the control. The lowest ORC was
observed in a sample hydrolyzed with Pectinex® Yieldmash Plus; however, a significant
difference (p < 0.05) between the samples treated with enzyme preparations with pectinases
was not observed. Zheng et al. [40] reported a decrease in the coconut cake dietary fiber
ORC after enzymatic hydrolysis with cellulase. Oil retention is related mainly to the surface
properties, overall charge density, and the constituents’ hydrophilic nature [39]. Enzymatic
hydrolysis decreased the total dietary fiber ratio and quantity, which may influence the
surface properties of the enzymatically treated pomace. Despite the decreased ORC,
all of the pomaces showed good ORC properties compared to the other berry pomaces.
Gouw et al. [35] reported a lower ORC of cranberry pomaces of 1.97 g/g; other authors
also reported lower ORC values of berry pomaces (blackcurrant, redcurrant, chokeberry,
rowanberry, and gooseberry) that ranged from 1.91 to 2.27 g/g [41]. Dietary fiber with a
good ability to retain oil can be used for the stabilization of emulsions and high-fat food
products [39].
The WSC increased in the samples hydrolyzed with Viscozyme® L and Celluclast®
1.5L (23.33% and 70%, respectively) compared to the control, while treatment with other
Foods 2022, 11, 2321 10 of 14

enzymes decreased the WSC. The enzymatic hydrolysis of cellulose and hemicellulose leads
to the exposure of more hydrogen bonds, which influence a higher WSC [40]. However, a
high IDF and a low SDF content (especially pectin) have an adverse effect on the WSC [42].
The particle sizes also affect the WSC, and, in most cases, the decrease in the particle size
increases the WSC; however, it can also be reduced due to the destruction of the dietary fiber
matrix and the links between polysaccharides [43]. Reißner et al. [41] reported higher WSC
(5.50–7.09 mL/g) of other berry pomaces (blackcurrant, redcurrant, gooseberry, rowanberry,
and chokeberry). Gouw et al. [35] reported a higher WSC of cranberry pomace (5.87 mL/g).
A previous study showed that the WSC in most cases increased after enzymatic treatments.
However, it depends not only on the kind of fiber or enzyme, but on other conditions used
in the treatment [12].
The WRC decreased significantly after enzymatic treatment with most of the enzyme
preparations, while enzymatic treatment with Celluclast® 1.5L increased the WRC, however,
no significant differences were observed (p < 0.05) compared with the control. The fibers
consisting mainly of primary cell walls generally have higher water hydration values than
the fibers consisting mainly of secondary cell walls [44]. The properties of water hydration
decreased after enzymatic hydrolysis with enzymes, the main declared activities being
pectinases. Spadoni Andreani et al. [33] indicated that the cranberry pomace cell wall is rich
in pectic polysaccharides. The hydration properties of dietary fibers are strongly related to
the source of the dietary fiber [39,41].
The stability of the emulsion is the ability to maintain the emulsion and its rupture
resistance [45]. Kalla-Bertholdt et al. [46] reported that an emulsion prepared with high
amounts of SDF has a micelle-like network, leading to a faster initial fat digestion. The
emulsion stability of the enzymatically hydrolyzed cranberry pomaces was determined
(Figure 1). The stability of the emulsion depended not only on the enzymatically treated
pomace, but also on the pH value. This also confirms other studies [47]. The stability of
the emulsion decreased during storage in all of the samples, however, in most cases, the
stability of the emulsion did not change significantly (p < 0.05) during 168 and 504 h of
storage (Figure 1). The pomaces hydrolyzed with Pectinex® Yieldmash Plus showed the
lowest emulsion stability compared with the other samples. The pomaces hydrolyzed with
Pectinex® Ultra Tropical and Celluclact® 1.5 L showed the highest emulsion stability in
water (pH 3.27) and pH 4 buffer solution, while, at pH 6, better emulsion stability was
shown in the pomaces hydrolyzed with Viscozyme® L and Celluclast® 1.5L. The control
sample and the sample hydrolyzed with Celluclast® 1.5L showed a higher stability of the
emulsion in a pH 8 buffer compared to the other enzymatically treated pomaces. The
thermal stability of the emulsions was lower (Figure 2). The lowest thermal stability in
all of the cases was observed using pomaces hydrolyzed with Pectinex® Yieldmash Plus.
The highest stability was obtained in most of the cases using pomace hydrolyzed with
Celluclast® 1.5L. Huc-Mathis et al. [48] reported that IDF helps maintain the stability
of the emulsions through the Pickering mechanism and/or network formation in the
continuous phase, probably favored by the stabilization of the proteins and the pectins in
the soluble fraction.
 the cases was observed using pomaces hydrolyzed with Pectinex®® Yieldmash Plus. The 
the cases was observed using pomaces hydrolyzed with Pectinex  Yieldmash Plus. The 
highest stability was obtained in most of the cases using pomace hydrolyzed with Cellu‐
highest stability was obtained in most of the cases using pomace hydrolyzed with Cellu‐
clast®®   1.5L. 
clast 1.5L.  Huc‐Mathis 
Huc‐Mathis  et 
et  al. 
al.  [48] 
[48]  reported 
reported  that 
that  IDF 
IDF  helps 
helps  maintain 
maintain  the 
the  stability 
stability  of 
of  the 
the 
emulsions through the Pickering mechanism and/or network formation in the continuous 
emulsions through the Pickering mechanism and/or network formation in the continuous 
Foods 2022, 11, 2321 phase, probably favored by the stabilization of the proteins and the pectins in the soluble 
11 of 14
phase, probably favored by the stabilization of the proteins and the pectins in the soluble 
fraction. 
fraction. 

  
Figure 1. Emulsion stability of enzymatically hydrolyzed cranberry pomaces at pH 3.27 (water) (a);
Figure 1. Emulsion stability of enzymatically hydrolyzed cranberry pomaces at pH 3.27 (water) (a); 
Figure 1. Emulsion stability of enzymatically hydrolyzed cranberry pomaces at pH 3.27 (water) (a); 
4 (b); 6 (c); 8 (d). Data values are expressed as means with the standard deviation (n = 3).
4 (b); 6 (c); 8 (d). Data values are expressed as means with the standard deviation (n = 3). 
4 (b); 6 (c); 8 (d). Data values are expressed as means with the standard deviation (n = 3). 

  
Figure  2.  Thermal 
Figure emulsion stability 
Thermalemulsion stability  of 
emulsion stability ofof  enzymatically 
enzymatically hydrolyzed cranberry 
hydrolyzed pomaces 
cranberry pomaces at  pH  3.27 
at pH at 
3.27
Figure  2. 2.Thermal  enzymatically  hydrolyzed cranberry  pomaces  pH  3.27 
(water) (a); 4 (b); 6 (c); 8 (d). Data values are expressed as means with the standard deviation (n = 3). 
(water) (a); 4 (b); 6 (c); 8 (d). Data values are expressed as means with the standard deviation (n = 3).
(water) (a); 4 (b); 6 (c); 8 (d). Data values are expressed as means with the standard deviation (n = 3). 

  
Foods 2022, 11, 2321 12 of 14

4. Conclusions
Cranberry pomace is a good source of dietary fiber, containing 59.93 g/100 g (dry
weight) of IDF and 12.74 g/100 g of SDF. The enzymatic hydrolysis changed the tech-
nological properties of cranberry pomace, the ratio of SDF and IDF, and their quantities.
The pomace treated with Celluclast® 1.5L resulted in the highest SDF content and an
increase in the WSC and WRC. The highest amount of oligosaccharides was obtained
with Viscozyme® L, while hydrolysis with Pectinex® Ultra Tropical resulted in the highest
amount of mono- and disaccharides and TPC content. The pomace treated with these
enzymes can be used to enhance products with oligosaccharides and phenolic compounds.
All of the tested water-soluble fractions showed prebiotic activity and enhanced the growth
of Lactobacillus acidophilus DSM 20079 and Bifidobacterium animalis DSM 20105 after 24 h
of fermentation, while viability after 48 h of fermentation was strain and carbon source-
dependent. The ORC after the enzymatic hydrolysis significantly decreased (p < 0.05) in
all of the samples. However, all of the pomaces showed good ORC properties and could
be used to stabilize high-fat foods. The highest emulsion stability was obtained in most
cases using the pomace hydrolyzed with Celluclast® 1.5L. The enzymatic hydrolysis using
different enzymes gave cranberry pomaces with different compositions and technological
properties, which could be used in developing different novel food products. Therefore,
enzymatic hydrolysis can open new market perspectives for the application of cranberry
pomace as a new, cheap and valuable ingredient to improve the nutritional values and
technological properties of food products.

Author Contributions: Conceptualization, L.B. and D.C.; methodology, D.C.; formal analysis, J.J.;
investigation, J.J., D.C. and M.S; resources, L.B.; data curation, L.B. and D.C; writing—original draft
preparation, J.J.; writing—review and editing, L.B., D.C. and M.S.; visualization, J.J.; supervision, L.B.
and D.C. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available in the article.
Acknowledgments: The authors acknowledge Novozymes A/S, Denmark, for kindly providing the
enzymes required for this research.
Conflicts of Interest: The authors declare no conflict of interest.

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