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Citation: Vlad, C.C.;
P˘acularu-Burada, B.; Vasile, A.M.;
Milea, S
,.A.; Bahrim, G.-E.; Râpeanu,
G.; St˘anciuc, N. Upgrading the
Functional Potential of Apple
Pomace in Value-Added Ingredients
with Probiotics. Antioxidants 2022,11,
2028. https://doi.org/10.3390/
antiox11102028
Academic Editor: Rosalba Siracusa
Received: 1 September 2022
Accepted: 12 October 2022
Published: 14 October 2022
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antioxidants
Article
Upgrading the Functional Potential of Apple Pomace in
Value-Added Ingredients with Probiotics
Camelia Cristina Vlad, Bogdan Păcularu-Burada , Aida Mihaela Vasile, S
,tefania Adelina Milea ,
Gabriela-Elena Bahrim , Gabriela Râpeanu and Nicoleta Stănciuc *
Faculty of Food Science and Engineering, Dunărea de Jos University of Galati, DomneascăStreet 111,
800201 Galati, Romania
*Correspondence: nsava@ugal.ro
Abstract:
Emerging customized designs to upgrade the functional potential of freeze-dried apple
pomace was used in this study, in order to transform the industrial by-products into ingredients
containing probiotics, for a better and healthier food composition. The freeze-dried apple pomace
was analyzed for free and bounded phenolic contents, highlighting a significant level of caffeic acid
(4978.00
±
900.00 mg/100 g dry matter (DM)), trans-cinnamic acid (2144.20
±
37.60 mg/
100 g DM
)
and quercetin 3-
β
-D-glucoside (236.60
±
3.12 mg/100 g DM). The pectin extraction yield was ap-
proximatively 24%, with a degree of esterification of 37.68
±
1.74%, and a methoxyl content of
5.58 ±0.88%
. The freeze-dried apple pomace was added in a different ratio as a supplement to cul-
tural medium of Loigolactobacillus bifermentans MIUG BL 16, suggesting a significant prebiotic effect
(
p< 0.05
) at concentration between 1% and 2%. The apple pomace was used to design three freeze-
dried ingredients containing probiotic, with a high level of polyphenolic content (
6.38 ±0.14 mg
gallic acid equivalents/g DM) and antioxidant activity (42.25
±
4.58 mMol Trolox/g DM) for the
powder containing apple pomace ethanolic extract. When inulin was used as a prebiotic adjuvant, the
obtained powder showed a 6 log/g DM viable cell count. The ingredients were added to fermented
vegetable soy milk-based products, allowing us to improve the polyphenolic content, antioxidant
activity and viable cell counts. The approach designed in this study allowed us to obtain ingredients
suitable to add value to food, whereas premises to align with the current circular economy premises,
by reintegrating the industrial waste as sources of high added value compounds, are also provided.
Keywords:
apple pomace; probiotics; prebiotics; pectin; value-added; polyphenols; circular economy
1. Introduction
The actual projection indicates a continuous population growth, which will probably
reach the level of about 10 billion in 2050; this imposes a high pressure on the agri-food
market to offer alternative sources of food, which should meet the nutritional needs and
also to the current trend of healthy and sustainable foods consumption [
1
,
2
]. However, huge
quantities of food wastes are generated by the agro-food industries, resulting in a negative
impact on environment, due to their high moisture content and instability, thus favoring
its microbial decomposition with a concomitant production of greenhouse gas emissions
and nitrogen contamination of soil and water [
3
]. An estimation of agro-food waste
during production and processing of fruits and vegetable reaches approximately 14.8%,
accounting for the largest source of food loss and wastes globally [
4
], involving significant
economic and environmental issues. Commonly, these agri-food wastes are used as animal
feed, landfilling, composting, anaerobic digestion, carbonization or thermal treatment [
5
].
Recent strategies for reducing or recycling agri-food waste have reconsidered integrated
approaches for the production of bioenergy, biochemicals, and value-added products,
driven by diversity and high concentration of compounds and secondary metabolites, with
multiple biological and technological functions [
6
]. Fruit and vegetable wastes are rich in
Antioxidants 2022,11, 2028. https://doi.org/10.3390/antiox11102028 https://www.mdpi.com/journal/antioxidants
Antioxidants 2022,11, 2028 2 of 15
non-digestible carbohydrates (resistant starch, inulin, cellulose, hemicellulose, pectin and
alginates), polyphenols, carotenoids, vitamins, etc. [6].
The apple (Malus domestica) is one of the most consumed fruits worldwide, both as
fresh and processed products. In order to meet the global demands for juices, juice concen-
trates and cider, 11.6 million tons of apples are processed [
7
], resulting in 30% of the product
becoming wastes. Globally, these wastes may represent up to 4 million tons per year of
apple pomace [
6
], consisting of pulp, skins, seeds and stalks of the fruit [
8
]. The apple
pomace is rich in minerals, dietary fiber, polyphenols, and pectin [
9
,
10
]. Two important
recycling strategies for fruit and vegetable wastes were reported, divided into different
technologies, such as composting, processing to flour or conversion into water, and ex-
tractions [11]. Therefore, the application of apple pomace could be divided into two main
ways, respectively, conventional (such as an additive in animal feed, fermentation conver-
sion to compost, or produce nutrition enhancement) and high-value products (functional
ingredients exploring the carbohydrates, phenolic compounds and pentacyclic triterpenes
content, and extraction or fermentation conversion to produce enzymes, organic acid,
pigment, biofuels) [
12
]. The application of selected extraction to obtain high-value-added
ingredients may be considered as an economically and environmental efficient strategy,
since novel extraction technologies of a low-cost, easily available material guarantee a
high extraction rate and yield, while reducing the use of organic solvents [
11
]. However,
when designing a technology to extract selected bioactives based on affordable, sustainable
and profitable technologies at an industrial level [
13
], several cost-related issues should be
considered such as the costs of initial investment, the industrial application, the disposal of
the relatively high amounts of residual waste, etc. [13].
The strain-specific probiotic bioactivities are extensively studied, especially
in vitro
,
whereas very few have tested probiotic efficacy in an animal or human model [
5
]. The
health-associated benefits of probiotics are related to increasing the food’s nutritional
value, intestinal infections regulation, lactase biosynthesis to improve lactose digestion,
anti-cancer property and antibiotic therapy, and reducing diarrhea incidence and blood
cholesterol [14,15]. The survivability of probiotics is significantly questioned, since the ex-
trinsic and native features such as the production of hydrogen peroxide, post-acidification,
oxygen, pH, storage temperature, and processing conditions can greatly reduce their
activity [
16
,
17
]. In order to improve the probiotics viability in different environments,
different prebiotics, such as oligosaccharides, mannan oligosaccharide, galactooligosac-
charide, arabinoxylan-oligosaccharide, inulin, and
β
-glucan, have been well studied [
5
].
The prebiotic mechanism is explained by their slow fermentation from complex structures,
thus providing fermentable carbohydrates for bacteria in the distal colon, allowing us to
regulate the dysbiosis of gut microbiota, while producing metabolic butyrate to modulate
the gut barrier function and anti-inflammatory effect [
18
]. The prebiotics targeted for the
gut should be resistant to gastrointestinal digestion, including the low pH of the stomach,
hydrolysis by intestinal enzymes and gastrointestinal absorption [
19
]. It has been suggested
that approximately 85–90% of the ingested pectin reached the terminal ileum [
20
], being
available for microbial fermentation at the colon.
The vital role of the lactic acid bacteria (LAB) in human health [
21
] is well studied,
with a significant dimension of industrial applications, both in the health and agri-food
industries to enhance food quality and human well-being [
22
]. Recently, the LAB taxonomy
included 261 species, divided into 26 genera based on their whole genome sequences [
23
].
Significant scientific data support the benefits of lactic acid bacteria as probiotics, modu-
lating the gut microbiota due to their ability to compete with pathogens, as well as their
immunomodulatory, anti-obesity, anti-diabetic, and anti-cancer activities [
14
]. Loigolac-
tobacillus bifermentans (Lo. bifermentans) is a facultatively heterofermentative lactic acid
bacterium, generally isolated from cheeses, with a potential for fermentation of lactic acid
into acetic acid, ethanol, traces of propionic acid, carbon dioxide, and hydrogen [24].
Therefore, the aim of our study was to test the hypothesis that apple pomace is suitable
as a potential valuable resource for full-components utilization, in terms of polyphenols
Antioxidants 2022,11, 2028 3 of 15
and prebiotic fibers, in order to design novel formulations based on apple pomace and
probiotics [
6
]. In this context, the pomace resulted from apple juice (Idared variety) was
selected as an emerging source of polyphenols, and pectin as new prebiotics for the selected
LAB strain. The apples pomace was freeze-dried and used to advance the phytochemical
content, in terms of polyphenols and pectin. Further, in order to customize the technological
design, the freeze-dried apple pomace was tested for prebiotic potential for Loigolactobacillus
bifermentans MIUG BL 16 (Lo. bifermentans MIUG BL 16). The strain was isolated from
cheese and conserved with the indicative of MIUG 16 in the Microorganisms Collection
of University Dunarea de Jos of Galati, Romania. Three customized ingredients were
developed based on apple pomace and Lo. bifermentans MIUG BL 16, with different
adjuvants (inulin and soy protein isolates), in order to maintain unique attributes such as
shelf-life and probiotic cell viability to fulfill the specific needs of dosage. The resulting
powders were tested for phytochemical content, in terms of polyphenols and flavonoids
content, antioxidant activity, and viable cell counts, whereas the value-added functional
properties were analyzed by adding to vegetable fermented foods.
2. Materials and Methods
2.1. Chemicals
The HPLC analytical-grade hexane, acetone, acetonitrile, ethyl acetate, methanol and
analytical grade 2,2-diphenyl-1-picrylhydrazyl (DPPH), 6-Hydroxy-2,5,7,8-
tetramethylchromane-2-carboxylic acid (Trolox), hydrochloric acid, citric acid, sodium
hydroxide, sodium chloride, aluminum chloride, ethanol, Folin–Ciocâlteu reagent, gal-
lic acid, inulin were purchased by Sigma Aldrich (Darmstadt, Germany). For cromato-
graphic analysis the following reagents were used: HCl ACS reagent (37%), acetic acid,
methanol, ethyl acetate, acetonitrile, theaflavin, cafestol, procyanidin A1, procyanidin B1,
(
−
)-epigallocatechin, catechin, caffeine, caffeic acid, ellagic acid, gallic acid, protocatechuic
acid, trans-cinnamic acid, quercetin 3-glucoside, quercetin 3-D-galactoside, quercetin 3-
β
-
D-glucoside, naringin, hesperidin, myricetin, apigenin, kaempferol, luteolin, and isorham-
netin (HPLC-grade), purchased from Sigma-Aldrich (Darmstadt, Germany). Other reagents
such as sodium bicarbonate were purchased from Honeywell, Fluka (Seelze, Germany).
The Lo. bifermentans MIUG BL 16 strain was from Microorganism Collection of Dunarea de
Jos University (acronym MIUG, Galati, Romania). de Man, Rogosa and Sharpe agar (MRS
agar) was purchased from Merck (Darmstadt, Germany).
2.2. Fruits Processing
Apples from Idared variety were purchased from a local market (Galati, Romania) in
October 2021 and washed. The seeds and stems were removed manually, and the apples
were portioned into small slices (approximatively 5 cm). Further, in order to limit the
oxidative processes, the slices were immersed in a solution consisting of lemon juice and
honey (1000 mL of distillated water, 10 mL of lemon juice and 10 g of honey) for 4 h.
The composition of the apple’s immersion solution took into account the use of natural
sources of ascorbic acid and sugars, such as lemon juice and honey. Ascorbic acid is a
common antioxidant that can rapidly reduce quinones and inhibit enzymatic browning [
25
].
The apple slices were squeezed, using a fruits juicer (Stainless Steel Fruit Vegetable Juice
Extractor Juicer Squeezer, Guangdong, China). The resulting pomace was immediately
frozen (
−
18
◦
C) and subjected to freeze-drying (CHRIST Alpha 1-4 LD plus, Osterode
am Harz, Germany) at
−
42
◦
C under a pressure of 0.10 mBar for 48 h. Afterwards, the
freeze-dried pomace was collected and packed in metallized bags and stored at 4
◦
C until
further analysis.
2.3. Conventional Solid–Liquid Solvent Ultrasound Assisted Extraction of Polyphenols from
Freeze-Dried Apple Pomace
In order to have an overall view of the global polyphenols content, a conventional
solid–liquid solvent ultrasound assisted method was applied. Therefore, an amount of
Antioxidants 2022,11, 2028 4 of 15
1 g of freeze-dried apple pomace was homogenized with 25 mL of 70% ethanol solution
and extracted in an ultrasonic bath, at 35
◦
C, for 30 min. Afterwards, the mixture was
centrifuged at 3420
×
gfor 10 min, at 10
◦
C (Hettich Universal 320R, Tuttlingen, Germany),
the supernatant was collected, and the extraction was repeated three times. The collected
supernatants were subjected to concentration, at the temperature of 40
◦
C, under reduced
pressure to dryness (AVC 2-18 concentrator, CHRIST, Osterode am Harz, Germany). The
obtained extract was used for the spectrophotometric analysis of total polyphenolic (TPC)
and flavonoids contents (TFC).
2.4. Spectrophotometric Analysis of Total Polyphenolic and Flavonoids Contents from Freeze-Dried
Apple Pomace
To measure the content of TPC and TFC, the colorimetric method were used. For TFC
content analysis, the aluminum chloride method was used, involving the mixing of
0.25 mL
of a solution consisting of 10 mg of concentrated extract dissolved in 5 mL of ultrapure
water, with 1.25 mL of distilled water and 0.075 mL of 5% sodium nitrite solution. After
5 min of reaction, at room temperature, a volume of 0.150 mL of 10% aluminum chloride
solution was added and allowed to react for 6 min [
26
]. Further, a volume of 0.5 mL of 1M
sodium hydroxide and 0.750 mL of distilled water were added and the absorbance of the
mixtures was immediately measured at 510 nm (Jenway 6505 UV-Vis spectrophotometer,
Loughborough, UK). The TFC content was expressed in mg catechin equivalents/g dry
matter (mg CE/g DM), based on catechin standard curve.
The Folin–Ciocâlteu method was used to determine the TPC content, by using a
volume of 0.2 mL extract dissolved in ultrapure water, mixed with 15.8 mL of distilled
water and 1 mL of Folin–Ciocâlteu reagent [
26
]. The mixture was allowed to react for
10 min
,
at room temperature, followed by addition of 3 mL of 20% sodium carbonate solution.
The absorbance was measured after 60 min of reaction in the dark at a wavelength of
765 nm
(Jenway 6505 UV-Vis spectrophotometer, Loughborough, UK). The TPC content was
expressed as mg gallic acid equivalents (GAE)/g DM, based on gallic acid
standard curve
.
2.5. Extraction of Free and Bounded Polyphenols from Freeze-Dried Apple Pomace
The free and bounded bioactive compounds’ extraction and separation was performed
as described by Xiong et al. [
27
], slightly modified as Zhang et al. [
28
]. Briefly, the freeze-
dried apple pomace’s free bioactives were extracted with methanol (80% v/v), at 25
◦
C
and 150 rpm, for 2 h (Lab Companion SI-300, GMI, Washington, DC, USA), the super-
natant being concentrated afterwards. For the bounded bioactive compounds from the
analyzed sample, the pellet resulted after free bioactives’ extraction was mixed with 2M
HCl and hydrolyzed at 100
◦
C for 1 h. Thereafter, the bounded bioactive compounds were
extracted with ethyl acetate and then concentrated. The obtained fractions were used for
chromatographic analysis of the polyphenols.
2.6. Cromatographic Analysis of Polyphenols from Freeze-Dried Apple Pomace
The separation and identification of the bioactive compounds from freeze-dried apple
pomace was performed by an Agilent 1200 HPLC system equipped with autosampler,
degasser, quaternary pump system, multi-wavelength detector (MWD) and column ther-
mostat (Agilent Technologies, Santa Clara, CA, USA). A Synergi Max-RP-80 Å column
(250
×
4.6 mm, 4
µ
m particle size, Phenomenex, Torrance, CA, USA) was used for the free
and bounded polyphenols analysis. The concentrated samples, free and bounded fraction,
respectively, were dissolved in methanol and subjected to the HPLC separation, at 30
◦
C,
using 10
µ
L injection volume, with solvent A (1% v/v acetic acid in ultrapure water) and
solvent B (acetonitrile) using a flow rate of 0.65 mL/min. The method runtime was 80 min,
the compounds of interest were detected at 280 nm and 320 nm, using a
10 nm
bandwidth
for each detection wavelength to increase compounds’ selective separation. The identifica-
tion of the bioactive compounds from apple pomace was made by comparing the retention
times of the peaks with those obtained for standard solutions of bioactives. The identified
Antioxidants 2022,11, 2028 5 of 15
compounds were quantified by external calibration curves using the peak area. Data ac-
quisition was performed by Chemstation software, version B.04.03 (Agilent Technologies,
Santa Clara, CA, USA). Results were expressed in mg/100 g DW freeze-dried pomace.
2.7. Pectin Analysis
The citric acid method proposed by Spinei and Oroian [
29
] was used for the extraction
of pectin from freeze-dried apple pomace. Briefly, the freeze-dried apple pomace was
mixed with ultrapure water (1:10 w/v), the pH of this mixture being adjusted to 2.0, and
then kept, at 90
◦
C, for 3 h in a water bath (Julabo, Seelbach, Germany). Thereafter, the
supernatant was separated by centrifugation (2320
×
g, 22
◦
C, 35 min) followed by an equal
amount of ethanol (96% v/v) addition (1:1 v/v). The precipitated pectin was separated after
12 h of storage, at 4
◦
C, and centrifugation in the same conditions. Finally, the extracted
pectin was washed with ethanol (96% v/v) and dried at 50
◦
C. The extraction yield was
expressed using Equation (1):
Pectin yield, % =m0
m×100 (1)
where m0is the weight of dried pectin (g), and mis the weight of dried pomace (g).
The degree of esterification for the extracted pectin was assessed by the titrimetric
method using NaOH 0.10 N and phenolphthalein [
30
]. Results were expressed as described
in Equation (2):
De gree o f esteri f i catio n, % =V2
V1+V2
×100 (2)
where V
1
is the volume of NaOH 0.10 N used for the first titration (mL), and V
2
is the
volume of NaOH 0.10 N used for the second titration (mL).
Furthermore, the equivalent weight (EW) for the pectin extracted from the freeze-dried
apple pomace was determined. Firstly, for the EW estimation, 0.25 g pectin was dissolved
in 100 mL ultrapure water and continuously mixed (300 rpm, 1 h, 25
◦
C). The EW was
evaluated by the titrimetric method with NaOH 0.10 N, and prior to the titration, 5 g of
NaCl and phenolphthalein were added in the pectin solution. Results were calculated
using Equation (3) [
30
]. This resulting solution was further used to determine the methoxyl
content by titration with NaOH 0.10 N as Dranca et al. [
31
] reported. Methoxyl content was
calculated by Equation (4).
EW,g/mol =1000 ×m
V×N(3)
where mis the sample weight (g), Vis the volume of NaOH 0.10 N used for titration (mL),
and Nis the normality of NaOH solution.
Methoxyl content, % =V×N×3.10
m(4)
where Vis the volume of NaOH 0.10 N used for titration (mL), Nis the normality of NaOH
solution, and mis the sample weight (g).
2.8. Antiradical Scavenging Activity
The antiradical activity of the extract on 2,2-diphenyl-1-picrylhydrazyl (DPPH) was
used to evaluate the antioxidant activity of the apple pomace. The protocol involved
mixing a volume of 3.9 mL of DPPH solution (0.1 M in methanol) with 0.1 mL of extract
solution dissolved in ultrapure water. The mixture was allowed to react for 90 min, at room
temperature, in the dark, followed by absorbance reading at 515 nm (Jenway 6505 UV-Vis
spectrophotometer). The antiradical activity was expressed as mMol Trolox/g DM, based
on 6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox) standard curve [
26
].
Antioxidants 2022,11, 2028 6 of 15
2.9. Lo. bifermentans MIUG BL 16 Reactivation and Inoculum Preparation
Prior to each testing and formulation, all the growth media were autoclaved, at 121
◦
C,
for 15 min (ES-315, TOMY Digital Biology Co., Ltd., Tokyo, Japan), whereas the freeze-dried
apple pomace was sterilized using a UV lamp (Faster SafeFast Elite, Cornaredo, Italy).
The bacterial Lo. Bifermentans MIUG BL 16 strain stored in MRS broth medium with 40%
glycerol, at −80 ◦C, in MIUG Collection was reactivated in MRS broth medium overnight
and incubated for 48 h, at 37
◦
C, followed by being spread plated in the polystyrene Petri
dishes plates containing the MRS agar surface. The inoculum was obtained from a single
colony harvested from MRS agar, transferred to 50 mL MRS broth medium, and cultured
for 48 h, at 37
◦
C. The optical density of the inoculum was 2.0 at a wavelength of 600 nm,
using a Jenway 6505 UV-Vis spectrophotometer.
2.10. The Prebiotic Effect of Freeze-Dried Apple Pomace
In order to test the prebiotic effect, different ratios of the freeze-dried apple pomace
(0.5%, 1.0% and 2.0%, w/v) were added to 98 mL of liquid MRS medium, followed by
addition of 2% (v/v) inoculum. The control sample obtained without the addition of
apple pomace was prepared using the same conditions. The samples were fermented in a
stationary system at, 37
◦
C, for 48 h. After fermentation, the samples were stored, at 4
◦
C,
for 14 days. The prebiotic effect was calculated based on Equation (5):
Prebi otic e f f ect =
logCFU
mL sample
logCFU
mL control
(5)
where log (CFU)/mL
sample
represents the logarithm of the colony-forming units of the sample
with the addition of freeze-dried apple pomace, and log (CFU)/mL
control
represents the
logarithm of the colony-forming units of the control sample (without the addition of
apple pomace).
2.11. Viable Cell Counts
In order to determine the viable cell counts, the method described by Vasile et al. [
32
]
was used by 10-fold serial dilutions in a sterile physiological serum (0.9 g NaCl%, w/v),
by the pour plate technique. The viable cell number was determined by estimating the
number of colony-forming units (CFU) on the MRS agar plates (medium at pH 5.7) after
72 h of aerobic incubation, at 37 ◦C. The counts were expressed as CFU/g DM.
2.12. Customised Carriers for Inoculation of Freeze-Dried Apple Pomace with Lo. bifermentans
MIUG BL 16
Three experimental variants were prepared based on freeze dried apple pomace and
1% L. bifermentans MIUG BL 16 inoculum, as follows: Variant 1 (V1) was obtained by using
a single combination of freeze-dried apple pomace dissolved in sterile distillated water
(20%) and probiotic culture, Variant 2 (V2) combined freeze-dried apple pomace dissolved
in sterile distillated water (20%) with 2% inulin and probiotic culture, whereas Variant 3
(V3) used an amount of 5 g of the concentrated extract obtained from freeze-dried apple
pomace, dissolved in biopolymeric solution of soy protein isolate (2%) and inulin (1%)
and probiotic culture. All the samples were immediately frozen (
−
18
◦
C) and subjected
to freeze-drying (CHRIST Alpha 1-4 LD plus, Osterode am Harz, Germany), at
−
42
◦
C,
under a pressure of 0.10 mBar for 48 h. Afterwards, the carriers were collected and packed
in metallized bags and stored, at 4 ◦C, until further analysis.
2.13. Global Characterization of Powders
The powders were extracted for phytochemicals, and characterized as TPC, TFC, antioxi-
dant activity, and viable cell counts as described according to the protocols abovementioned.
Antioxidants 2022,11, 2028 7 of 15
2.14. Testing the Powders in Food Matrices
The powders were tested for value-added potential by adding into a fermented soya
milk product in a ratio of 2%. The samples were coded as C (control), S1 (fermented soya
milk products with 2% addition of V1), S1 (fermented soya milk products with 2% addition
of V2), and S3 (fermented soya milk products with 2% addition of V3). After addition,
the resulting samples were kept for 24 h under refrigeration conditions, for uniformity
and homogenization. The samples were characterized for TPC, TFC, antioxidant activity
and viable cell counts. In order to assess the cells viability during storage, the foods were
analyzed after 7 and 14 days of storage in refrigerated conditions.
2.15. Statistical Analysis
Reported results are average values for triplicates (n= 3) and duplicate measurements
(n= 2) in case of HPLC data, followed by standard deviations. Significant differences were
determined by ANOVA and Tukey test for 95% confidence interval (p< 0.05) using Minitab
19 software (Minitab Inc., State College, PA, USA).
3. Results and Discussion
3.1. Phytochemical Content of the Freeze-Dried Apple Pomace
Identification of phenolics in the extracts obtained from freeze-dried apple pomace was
achieved by comparing their spectra and retention times with those of externally injected
standards. Several compounds for which standards were not available, and identification
was made using references. Figure 1shows the detected free phenolics analyzed in freeze-
dried apple pomace, at different wavelength (280 nm and 320 nm). A total of 52 and 44
phenolic substances were separated through retention time (Rt), respectively (Figure 1a,b).
Six classes of free compounds were identified and quantified, based on external
calibration curves (Table 1). The total free polyphenolic content, estimated based on
quantity of identified compounds were 134.96 mg/100 g DM pomace at 280 nm and
137.52 mg
/
100 g DM
pomace. When measuring at 280 nm, the flavan-3-ols were identi-
fied as the major polyphenolic class (79.12%), followed by flavanones (10%) and phenolic
acids (6.93%). When measuring at 280 nm, fourteen free polyphenols were identified and
quantified, according to the reference standards, as follows: caffeic acid, ellagic acid, proto-
catechuic acid, theaflavin, catechin, cafestol, procyanidins A1 and B1, (
−
)-epigallocatechin,
caffeine, quercetin 3-glucoside, hesperidin and naringin. However, at 320 nm, gallic acid,
caffeic acid, myricetin, quercetin 3-glucoside and hesperidin were found (Table 1). The
major free bioactive were represented by gallic acid (86.42
±
2.26 mg/100 g DM), theaflavin
(71.73 ±0.72 mg/100 g DM) and hesperidin (42.83 ±0.27 mg/100 g DM).
Antioxidants 2022,11, 2028 8 of 15
Antioxidants 2022, 11, x FOR PEER REVIEW 7 of 15
2.13. Global Characterization of Powders
The powders were extracted for phytochemicals, and characterized as TPC, TFC, antiox-
idant activity, and viable cell counts as described according to the protocols abovementioned.
2.14. Testing the Powders in Food Matrices
The powders were tested for value-added potential by adding into a fermented soya
milk product in a ratio of 2%. The samples were coded as C (control), S1 (fermented soya
milk products with 2% addition of V1), S1 (fermented soya milk products with 2% addi-
tion of V2), and S3 (fermented soya milk products with 2% addition of V3). After addition,
the resulting samples were kept for 24 h under refrigeration conditions, for uniformity
and homogenization. The samples were characterized for TPC, TFC, antioxidant activity
and viable cell counts. In order to assess the cells viability during storage, the foods were
analyzed after 7 and 14 days of storage in refrigerated conditions.
2.15. Statistical Analysis
Reported results are average values for triplicates (n = 3) and duplicate measure-
ments (n = 2) in case of HPLC data, followed by standard deviations. Significant differ-
ences were determined by ANOVA and Tukey test for 95% confidence interval (p < 0.05)
using Minitab 19 software (Minitab Inc., Pennsylvania, USA).
3. Results and Discussion
3.1. Phytochemical Content of the Freeze-Dried Apple Pomace
Identification of phenolics in the extracts obtained from freeze-dried apple pomace was
achieved by comparing their spectra and retention times with those of externally injected
standards. Several compounds for which standards were not available, and identification
was made using references. Figure 1 shows the detected free phenolics analyzed in freeze-
dried apple pomace, at different wavelength (280 nm and 320 nm). A total of 52 and 44
phenolic substances were separated through retention time (Rt), respectively (Figure 1a,b).
(a)
Antioxidants 2022, 11, x FOR PEER REVIEW 8 of 15
(b)
Figure 1. HPLC analysis for free polyphenolic compounds in freeze-dried apple pomace extract at
280 nm (a) and 320 nm (b). Peaks’ identification: (a): 4—theaflavin, 5—cafestol, 16—protocatechuic
acid, 17—procyanidin B1, 19—(−)-epigallocatechin, 22—catechin, 24—caffeine, 26—caffeic acid,
31—procyanidin A1, 36—ellagic acid, 38—quercetin 3-glucoside, 41—naringin, 43—hesperidin, 1–
3, 6–15, 18, 20, 21, 23, 25, 27–30, 32–35, 37, 39, 40, 42, 44–52—unidentified compounds; (b): 2–theafla-
vin, 3—cafestol, 5—gallic acid, 9—procyanidin B1, 14—caffeine, 16—caffeic acid, 19—procyanidin
A1, 23—ellagic acid, 24—quercetin 3—D-galactoside, 25—quercetin 3—β-D-glucoside, 27—nar-
ingin, 28—hesperidin, 36—trans-cinnamic acid, 39—apigenin, 40—kaempferol, 1, 4, 6–8, 10–13, 15,
17, 18, 20–22, 26, 29–35, 37, 38, 41–44—unidentified peaks.
Six classes of free compounds were identified and quantified, based on external cali-
bration curves (Table 1). The total free polyphenolic content, estimated based on quantity
of identified compounds were 134.96 mg/100 g DM pomace at 280 nm and 137.52 mg/100
g DM pomace. When measuring at 280 nm, the flavan-3-ols were identified as the major
polyphenolic class (79.12%), followed by flavanones (10%) and phenolic acids (6.93%).
When measuring at 280 nm, fourteen free polyphenols were identified and quantified,
according to the reference standards, as follows: caffeic acid, ellagic acid, protocatechuic
acid, theaflavin, catechin, cafestol, procyanidins A1 and B1, (−)-epigallocatechin, caffeine,
quercetin 3-glucoside, hesperidin and naringin. However, at 320 nm, gallic acid, caffeic
acid, myricetin, quercetin 3-glucoside and hesperidin were found (Table 1). The major free
bioactive were represented by gallic acid (86.42 ± 2.26 mg/100 g DM), theaflavin (71.73 ±
0.72 mg/100 g DM) and hesperidin (42.83 ± 0.27 mg/100 g DM).
Table 1. The free and bounded phenolic compounds in freeze-dried apple pomace.
Bioactive Compound
Concentration (mg/100 g DM Freeze-Dried
Pomace)
Free Fraction
Bounded Fraction
Phenolic acids
Gallic acid
86.42 ± 2.26 a
8.30 ± 0.26 b
Caffeic acid
3.62 ± 0.80 b
4978.00 ± 900.00 a
Ellagic acid
2.90 ± 0.02 b
50.24 ± 0.34 a
Protocatechuic acid
2.88 ± 0.02 b
2144.20 ± 37.60 a
Flavan-3-ols
Theaflavin
71.73 ± 0.72 a
10.23 ± 0.21 b
Catechin
9.98 ± 0.00 a
N.D.
Cafestol
12.96 ± 0.22 a
4.17 ± 0.81 b
Procyanidin A1
5.27 ± 0.00 b
5.77 ± 0.02 a
Procyanidin B1
4.28 ± 0.01 a
1.20 ± 0.17 b
(−)-Epigallocatechin
2.46 ± 0.57 a
N.D.
Xanthine
Figure 1.
HPLC analysis for free polyphenolic compounds in freeze-dried apple pomace extract at
280 nm (
a
) and 320 nm (
b
). Peaks’ identification: (
a
): 4—theaflavin, 5—cafestol, 16—protocatechuic
acid, 17—procyanidin B1, 19—(
−
)-epigallocatechin, 22—catechin, 24—caffeine, 26—caffeic acid,
31—procyanidin A1, 36—ellagic acid, 38—quercetin 3-glucoside, 41—naringin, 43—hesperidin, 1–3,
6–15, 18, 20, 21, 23, 25, 27–30, 32–35, 37, 39, 40, 42, 44–52—unidentified compounds; (
b
): 2–theaflavin,
3—cafestol, 5—gallic acid, 9—procyanidin B1, 14—caffeine, 16—caffeic acid, 19—procyanidin A1,
23—ellagic acid, 24—quercetin 3—D-galactoside, 25—quercetin 3—
β
-D-glucoside, 27—naringin,
28—hesperidin, 36—trans-cinnamic acid, 39—apigenin, 40—kaempferol, 1, 4, 6–8, 10–13, 15, 17, 18,
20–22, 26, 29–35, 37, 38, 41–44—unidentified peaks.
Antioxidants 2022,11, 2028 9 of 15
Table 1. The free and bounded phenolic compounds in freeze-dried apple pomace.
Bioactive Compound Concentration (mg/100 g DM Freeze-Dried Pomace)
Free Fraction Bounded Fraction
Phenolic acids
Gallic acid 86.42 ±2.26 a8.30 ±0.26 b
Caffeic acid 3.62 ±0.80 b4978.00 ±900.00 a
Ellagic acid 2.90 ±0.02 b50.24 ±0.34 a
Protocatechuic acid 2.88 ±0.02 b2144.20 ±37.60 a
Flavan-3-ols
Theaflavin 71.73 ±0.72 a10.23 ±0.21 b
Catechin 9.98 ±0.00 aN.D.
Cafestol 12.96 ±0.22 a4.17 ±0.81 b
Procyanidin A1 5.27 ±0.00 b5.77 ±0.02 a
Procyanidin B1 4.28 ±0.01 a1.20 ±0.17 b
(−)-Epigallocatechin 2.46 ±0.57 aN.D.
Xanthine
Caffeine 4.89 ±0.07 a1.93 ±0.03 b
Flavonols
Myricetin 4.84 ±0.01 aN.D.
Quercetin 3-glucoside 0.40 ±0.00 aN.D.
Quercetin 3-D-galactoside N.D. 22.18 ±0.10 a
Quercetin 3-β-D-glucoside N.D. 236.60 ±3.12 a
Isorhamnetin N.D. 17.06 ±0.83 a
Flavanones
Hesperidin 42.83 ±0.27 aN.D.
Naringin 2.64 ±0.24 aN.D.
Apigenin N.D. 3.18 ±0.12 a
Kaempferol N.D. 27.80 ±1.36 a
Luteolin N.D. 0.86 ±0.05 a
Values in the same row that do not share a letter (
a, b
) are statistically different (p< 0.05) according to the Tukey
test (95% confidence level). N.D.—not determined.
In case of bounded phenolics, from Figure 2a,b, it can be observed that 44 and 24
phenolic substances were separated through retention time (Rt), at 280 nm and
320 nm
,
respectively. The total concentration for the bounded phenolics measured at 280 nm
was of
5362.71 mg
/100 g DM apple pomace, whereas at 320 nm, a concentration of
2299.21 mg
/
100 g DM
apple pomace was found. From Table 1, it can be seen that caffeic
acid prevails, with the highest concentration of approximatively 50 mg/g DM
(
49.78 ±9.00 mg/g DM
), followed by trans-cinnamic acid, with 21.44
±
0.37 mg/g DM
and quercetin 3-β-D-glucoside, with 2.36 ±0.03 mg/g DM.
The phenolic acids were the main bounded compounds, representing 94% from the
total polyphenolics. Different studies reported that in fresh apple pomace, phenolic com-
pounds are dominated by chlorogenic acid, caffeic acid, (+)-catechin, (
−
)-epicatechin, rutin,
and quercetin glycosides [
33
], whereas after freeze-drying, phlorizin (phloretin-2
0
-
β
-D-
glucopyranoside) is the most prominent polyphenol [34].
Additionally, Górna´s et al. [
35
] suggested that di-hydrochalcones (such as phlorizin)
are the main polyphenols found in apple seeds and stems, whereas the flesh part consists
mainly of chlorogenic acid and flavonol glycosides. However, in this study, the seeds and
stems were removed in the apples processing steps, and therefore, it is not expected to
identify and quantify these compounds.
Antioxidants 2022,11, 2028 10 of 15
Antioxidants 2022, 11, x FOR PEER REVIEW 9 of 15
Caffeine
4.89 ± 0.07 a
1.93 ± 0.03 b
Flavonols
Myricetin
4.84 ± 0.01 a
N.D.
Quercetin 3-glucoside
0.40 ± 0.00 a
N.D.
Quercetin 3-D-galactoside
N.D.
22.18 ± 0.10 a
Quercetin 3-β-D-glucoside
N.D.
236.60 ± 3.12 a
Isorhamnetin
N.D.
17.06 ± 0.83 a
Flavanones
Hesperidin
42.83 ± 0.27 a
N.D.
Naringin
2.64 ± 0.24 a
N.D.
Apigenin
N.D.
3.18 ± 0.12 a
Kaempferol
N.D.
27.80 ± 1.36 a
Luteolin
N.D.
0.86 ± 0.05 a
Values in the same row that do not share a letter (a, b) are statistically different (p < 0.05) according
to the Tukey test (95% confidence level). N.D.—not determined.
In case of bounded phenolics, from Figure 2a,b, it can be observed that 44 and 24
phenolic substances were separated through retention time (Rt), at 280 nm and 320 nm,
respectively. The total concentration for the bounded phenolics measured at 280 nm was
of 5362.71 mg/100 g DM apple pomace, whereas at 320 nm, a concentration of 2299.21
mg/100 g DM apple pomace was found. From Table 1, it can be seen that caffeic acid pre-
vails, with the highest concentration of approximatively 50 mg/g DM (49.78 ± 9.00 mg/g
DM), followed by trans-cinnamic acid, with 21.44 ± 0.37 mg/g DM and quercetin 3-β-D-
glucoside, with 2.36 ± 0.03 mg/g DM.
The phenolic acids were the main bounded compounds, representing 94% from the
total polyphenolics. Different studies reported that in fresh apple pomace, phenolic com-
pounds are dominated by chlorogenic acid, caffeic acid, (+)-catechin, (−)-epicatechin, ru-
tin, and quercetin glycosides [33], whereas after freeze-drying, phlorizin (phloretin-2′-β-
D-glucopyranoside) is the most prominent polyphenol [34].
(a)
Antioxidants 2022, 11, x FOR PEER REVIEW 10 of 15
(b)
Figure 2. HPLC analysis for bounded polyphenolic compounds in freeze-dried apple pomace ex-
tract at 280 nm (a) and 320 nm (b). Peaks’ identification: (a): 5—gallic acid, 21—caffeic acid, 30—
quercetin 3—glucoside, 36—hesperidin, 40—myricetin, 1–4, 6–20, 22–29, 31–35, 37–39, 41–44—uni-
dentified peaks; (b): 4—caffeic acid, 9—quercetin 3—D-galactoside, 12—hesperidin, 19—luteolin,
20—trans-cinnamic acid, 22—kaempferol; 23—isorhamnetin, 1–3, 5–8, 10, 11, 13–18, 21, 24–28—un-
identified peaks.
Additionally, Górnaś et al. [35] suggested that di-hydrochalcones (such as phlorizin)
are the main polyphenols found in apple seeds and stems, whereas the flesh part consists
mainly of chlorogenic acid and flavonol glycosides. However, in this study, the seeds and
stems were removed in the apples processing steps, and therefore, it is not expected to
identify and quantify these compounds.
3.2. Pectin Quantification and Characterization from the Freeze-Dried Apple Pomace
The citric acid method used in our study allowed us to obtain a pectin yield of 23.62
± 0.70%, with a degree of esterification of 37.68 ± 1.74%, EW of 149.26 ± 1.89, and a meth-
oxyl content of 5.58 ± 0.88%. Our results are in good agreement with Colodel & Petkowicz
[36], who reported a pectin yield of 27.40% after 1 h of extraction with citric acid. It has
been suggested that the pectin yield may be both positively and negatively affected by the
preliminary treatments applied to the pomace, as well as by the pH and type of solvent,
extraction time and temperature [37]. Pectin yields ranging 21.49–22.17% were deter-
mined after apple pomace’s treatment, at 80 °C, for 1 or 2 h [38].
Zheng et al. [39] reported a degree of esterification of 34.60% for the pectin extracted
from apple pomace, which is in fair agreement with data reported in this study (37.68 ±
1.74%). However, it has been suggested that a degree of esterification higher than 50% can
be achieved by a statistical optimization of the pectin’s extraction process. The methoxyl
content (5.58 ± 0.88%) and EW values (149.26 g/mol) can be correlated with extraction pa-
rameters, such as pH and temperature, whereas some other parameters (such as citric acid
and extraction time) affect the monosaccharides’ structures of pectin, leading to a low
value for EW [31]. Spinei and Oroian [29] reported methoxyl content ranging from 3.98 to
6.18% for the pectin extracted from grape pomace.
3.3. Potential Prebiotic Effect of Freeze-Dried Apple Pomace Supplementation of Broth Culture
Medium
Different studies showed that the chemical compounds of apple pomace, in terms of
pectin and polyphenols, make this by-product suitable for valorization as a potential
prebiotic [5]. For example, different studies suggested that apple pectin is able to interact
both with the intestinal microbiota and the intestinal immune cells, thus acting as a prebi-
otic substrate capable of promoting the intestinal immune barrier [40]. In this study, the
MRS broth was supplemented with different ratios of freeze-dried apple pomace (0–2%,
w/v), followed by inoculation with Lo. bifermentans MIUG BL 16 (2%) and fermentation
Figure 2.
HPLC analysis for bounded polyphenolic compounds in freeze-dried apple pomace
extract at 280 nm (
a
) and 320 nm (
b
). Peaks’ identification: (
a
): 5—gallic acid, 21—caffeic acid,
30—quercetin 3—glucoside, 36—hesperidin, 40—myricetin, 1–4, 6–20, 22–29, 31–35, 37–39, 41–
44—unidentified peaks; (
b
): 4—caffeic acid, 9—quercetin 3—D-galactoside, 12—hesperidin, 19—
luteolin, 20—trans-cinnamic acid, 22—kaempferol; 23—isorhamnetin, 1–3, 5–8, 10, 11, 13–18, 21,
24–28—unidentified peaks.
3.2. Pectin Quantification and Characterization from the Freeze-Dried Apple Pomace
The citric acid method used in our study allowed us to obtain a pectin yield of
23.62 ±0.70%
, with a degree of esterification of 37.68
±
1.74%, EW of 149.26
±
1.89, and
a methoxyl content of 5.58
±
0.88%. Our results are in good agreement with Colodel &
Petkowicz [
36
], who reported a pectin yield of 27.40% after 1 h of extraction with citric acid.
It has been suggested that the pectin yield may be both positively and negatively affected
by the preliminary treatments applied to the pomace, as well as by the pH and type of
solvent, extraction time and temperature [
37
]. Pectin yields ranging 21.49–22.17% were
determined after apple pomace’s treatment, at 80 ◦C,for1or2h[38].
Zheng et al. [
39
] reported a degree of esterification of 34.60% for the pectin ex-
tracted from apple pomace, which is in fair agreement with data reported in this study
(
37.68 ±1.74%
). However, it has been suggested that a degree of esterification higher than
50% can be achieved by a statistical optimization of the pectin’s extraction process. The
methoxyl content (5.58
±
0.88%) and EW values (149.26 g/mol) can be correlated with
extraction parameters, such as pH and temperature, whereas some other parameters (such
as citric acid and extraction time) affect the monosaccharides’ structures of pectin, leading
to a low value for EW [
31
]. Spinei and Oroian [
29
] reported methoxyl content ranging from
3.98 to 6.18% for the pectin extracted from grape pomace.
Antioxidants 2022,11, 2028 11 of 15
3.3. Potential Prebiotic Effect of Freeze-Dried Apple Pomace Supplementation of Broth
Culture Medium
Different studies showed that the chemical compounds of apple pomace, in terms
of pectin and polyphenols, make this by-product suitable for valorization as a potential
prebiotic [
5
]. For example, different studies suggested that apple pectin is able to interact
both with the intestinal microbiota and the intestinal immune cells, thus acting as a prebiotic
substrate capable of promoting the intestinal immune barrier [
40
]. In this study, the MRS
broth was supplemented with different ratios of freeze-dried apple pomace (0–2%, w/v),
followed by inoculation with Lo. bifermentans MIUG BL 16 (2%) and fermentation under
controlled stationary system (37
◦
C for 48 h). In order to estimate the potential prebiotic
effect, the viable cell counts were also determined after a 21 days storage, at 4
◦
C. The
prebiotic effect of the supplementation is provided in Table 2.
Table 2.
Potential prebiotic effect of freeze-dried apple pomace supplementation of MRS broth for
Loigolactobacillus bifermentans MIUG BL 16.
Prebiotic Effect
Storage (Days) Supplementation Ratio, % (w/v)
0.5 1 2
0 0 0 0
7 1.50 ±0.02 a1.53 ±0.07 a1.54 ±0.08 a
14 3.22 ±0.12 a3.63 ±0.19 b3.87 ±0.22 ab
21 3.99 ±0.56 a4.58 ±0.41 b4.74 ±0.21 c
Data are showed as mean
±
SD (n = 3). Values in the same row that do not share a letter (
a, b, c
) are statistically
different (p< 0.05) according to the Tukey test (95% confidence level).
From Table 2it can be observed that after 7 days of storage, no significant differences
(p> 0.05) were found in potential prebiotic effect of apple pomace supplementation of
culture medium, whereas an increase was observed after 14 and 21 days, respectively. The
potential prebiotic effect is much more obvious after 21 days of storage, at 4
◦
C, when a
significant increase (p< 0.05) was observed at added concentration of 1 and 2% (Table 3).
Table 3.
Global phytochemical content, antioxidant activity and viable cell counts of the ingredients.
Compound (/g DM) Variant 1 Variant 2 Variant 3
Total polyphenolic
content (mg GAE) 5.32 ±0.06 c5.64 ±0.09 b6.38 ±0.14 a
Total flavonoids
content (mg CE) 4.02 ±0.09 b5.21 ±0.35 a5.59 ±0.22 a
Antioxidant activity
(mMol Trolox) 22.02 ±2.11 b23.01 ±1.37 b42.25 ±4.58 a
Viable cells (CFU) 3.0 ×105 c 2.05 ×106 a 1.10 ×106 b
Variant 1—freeze-dried apple pomace dissolved in sterile distillated water (20%) and 1% probiotic culture; Variant
2—freeze-dried apple pomace dissolved in sterile distillated water (20%) with 2% inulin and 1% probiotic culture;
Variant 3—freeze-dried powder containing concentrated extract in biopolymeric solution of soy protein isolate
(2%), inulin (1%) and 1% probiotic culture. Values in the same row that do not share a letter (
a, b, c
) are statistically
different (p< 0.05) according to the Tukey test (95% confidence level).
The potential prebiotic effect of freeze-dried apple pomace could be explained by a
synergic effect of both polysaccharides and polyphenolics. The prebiotic effect of polysac-
charides is related to probiotics ability to degrade pectins, and/or utilize them and other
metabolites, by cross-feeding interactions [
41
]. Larsen et al. [
41
] suggested that one of
the most important factors in prebiotic effect of pectin is the degree of esterification of
polygalacturonic acid, with an increase in microbiota composition for the low methoxyl
pectins. Therefore, the significant prebiotic effect of freeze-dried apple pomace could be ex-
plained by the low methoxyl content for the pectin determined in this study (
5.58 ±0.88%
).
Antioxidants 2022,11, 2028 12 of 15
However, these authors highlighted the possibility of differential stimulation of bacterial
populations using pectins with different sugar content [41].
Besides the well-recognized resistant oligosaccharides prebiotics (inulin, fructo-
oligosaccharides and galacto-oligosaccharides) [
42
], recent studies have shown the in-
teraction between polyphenols and the gut microbiota, suggesting them as candidate
compounds to prebiotics [
43
,
44
]. For example, de Araùjo Chagas Vergara et al. [
45
] sug-
gested the prebiotic effect of the fermented cashew apple juice containing oligosaccharides
on Leuconostoc mesenteroides and Lactobacillus johnsonii growth.
Regarding the prebiotic potential of polyphenols, a bidirectional interaction was sug-
gested, involving a modulation effect of polyphenols on gut microbiota and, conversely,
microorganisms can modulate the activity of the phenolic compounds [
46
]. This bidirec-
tional relationship can regulate the metabolism and the bioavailability of polyphenols,
converting them into metabolites, which may have different effects on the host health, as
explained by Singh et al. [44].
Although the prebiotic effect of apple pomace was clearly evidenced in this study, the
potential role of polyphenols, sugars and pectin as modulators of gut microbiota should be
further tested [
47
]. This is based on the hypothesis that the structure and function of each
individual polyphenol can be influenced by the food matrix and individual composition of
the human microbiota [48].
3.4. Customised Designs for Ingredients Based on Apple Pomace and Lactic Acid Bacteria
Three experimental variants of freeze-dried powders based on freeze-dried apple
pomace and the corresponding extract and Lo. bifermentans MIUG BL 16 were obtained,
with and without adjuvants. The powders were tested for global phytochemical content
and viable cells (Table 3).
As expected, variant V3 containing apple pomace extract showed a higher TPC
content of 6.38
±
0.14 mg GAE/g DM powder, whereas the samples with apple po-
mace displayed comparable TPC values of 5.32
±
0.06 mg GAE/g DM powder (V1) and
5.64 ±0.09 mg GAE
/g DM powder (V2). The same trend was observed in TFC, with lower
value in V1 (
4.02 ±0.09 mg CE/g DM
powder), followed by V21 with
5.21 ±0.35 mg
CE/g
DM powder and V3 with
5.59 ±0.22 mg
CE/g DM powder. The higher TPC content in
V3 had a significant impact on DPPH antiradical scavenging activity, yielding a value
of
42.25 ±4.58 mMol
Trolox/g DM powder. The significant difference between variants
could be explained by the use of the extract from apple pomace in designing V3, which
concentrates various bioactive compounds, thus impacting the antioxidant activity. How-
ever, the customized design significantly impacted the viable cell counts (Table 3), with V2
and V3 reaching 6.30 log and 6.04 log, respectively, when compared with 5.40 log in V1.
These results highlighted the cumulative effect of pectin, inulin and polyphenols in V2.
3.5. Testing the Value-Added Potential of the Ingredients in Food Matrice
In order to test the potential value-added functionality of the customized variants, a
fermented commercial product obtained from soy milk was selected, from the perspective
to improve the nutritional and biological profile of plant-based food products. The variants
were added in a ratio of 2% and characterized for phytochemical content, antioxidant
activity and viable cell counts (Table 4).
An increase in TPC and TFC contents was observed for all the sample, as compared
with control (C), according to the profile of the powders. The value-added functional
properties were highlighted in terms of both antioxidant activity and viable cell counts.
The antioxidant activity values were significantly higher (p< 0.05) in variants S2 and
S3 (
37.95 ±1.31 mMol
Trolox/100 g DM product and 40.23
±
1.27 mMol Trolox/100 g
DM product, respectively), while viable bacterial cells showed higher values with 1 log,
when compared with control (Table 4). Wang et al. [
15
] suggested polyphenol contents of
56.2 ±1.8 µg
GAE/g, 66.4
±
3.4
µ
g GAE/g and 74.9
±
2.6
µ
g EAG/g in yogurt samples
fortified with 1%, 2% and 3% freeze-dried apple pomace, respectively.
Antioxidants 2022,11, 2028 13 of 15
Table 4.
Global phytochemical content, antioxidant activity and viable cell counts of value-added
fermented soya milk.
Compound (/100 g DM) C S1 S2 S3
Total polyphenolic content (mg GAE)
49.96 ±1.92 b53.01 ±1.63 ab 51.17 ±0.33 ab 56.49 ±2.00 a
Total flavonoids content (mg CE) 42.84 ±2.44 b47.05 ±2.44 b46.54 ±1.19 b55.31 ±1.38 a
Antioxidant activity (mMol Trolox) 22.08 ±3.23 c30.06 ±3.71 bc 37.95 ±1.31 ab 40.23 ±1.27 a
Viable cells (CFU)/g DM 5.52 ×106 d 1.58 ×107 c 4.88 ×107 a 3.82 ×107 b
C—fermented soya milk without powder addition; S1—fermented soya milk with addition of 2% from powder
V1; S2—fermented soya milk with addition of 2% from powder V2; S3—fermented soya milk with addition of
2% from powder V3. Values in the same row that do not share a letter (
a, b, c, d
) are statistically different (p< 0.05)
according to the Tukey test (95% confidence level).
4. Conclusions
The research carried out in this study is an attempt to answer the current demands
to add value in food by the incorporation of natural ingredients. As apple is one of
the most processed fruits worldwide, the immense amount of apple pomace generated
is considered an important and cheap source of bioactives, with important benefits for
health. Therefore, in our study, different customized designs to valorize apple pomace
into ingredients were studied, as a valuable strategy to expand the food chain with less
impact on the environment, while providing nutritional and biological added value in
terms of bioactives, prebiotics and probiotics in single formulas. The apple pomace was
used in a freeze-dried form to analyze the polyphenolic and pectin content. The preliminary
chromatographic analysis evidenced the presence of both free and bonded polyphenolic
compounds, allowing us to quantify a total amount of around 135 mg/100 g DM and
5363 mg/100 g DM freeze-dried apple pomace, respectively. Gallic acid, theaflavin and
hesperidin were the major free compounds separated from the methanolic fraction, whereas
caffeic acid showed the highest concentration of approximatively 50 mg/g DM, followed by
trans-cinnamic acid, and quercetin 3-
β
-D-glucoside, these compounds being identified from
the bounded fraction. However, an in-depth bioactives’ characterization along with their
interactions and mechanisms of action must be considered to emphasize and understand
the functionality of the designed products with apple pomace. The pectin yield was of
approximatively 24%, with a degree of esterification of 38%. The prebiotic effect was
tested for a probiotic strain (Loigolactobacillus bifermentans MIUG BL 16) in different ratios,
varying from 0 to 2%, suggesting a cumulative effect of both pectin’s and polyphenols on
cell survivability. Three customized technological designs were developed, incorporating
freeze-dried apple pomace and inulin, whereas the extract was microencapsulated in
a biopolymeric matrix based on soy protein isolates and inulin. The ingredients were
analyzed for the phytochemical content and viable cell count of at least 6 log/g. The
powders were added as ingredients in a fermented soy milk, allowing us to obtain an
increase in antioxidant activity and viable cell counts. The results obtained in this study
unveil a readily available method for straightforward processes in order to upgrade the
great potential of fruit by-products by transforming them into value-added ingredients for
different applications.
Author Contributions:
Conceptualization, N.S.; methodology, N.S.,
S
,
.A.M. and B.P.-B.; software,
C.C.V., B.P.-B., and
S
,
.A.M.; validation, G.-E.B., A.M.V., G.R., and N.S.; formal analysis, C.C.V., B.P.-B.,
A.M.V., and
S
,
.A.M.; investigation, C.C.V., A.M.V., B.P.-B., and
S
,
.A.M.; resources, G.-E.B., G.R. and
N.S.; writing—original draft preparation, C.C.V., B.P.-B.,
S
,
.A.M., A.M.V., and N.S.; writing—review
and editing, G.-E.B., G.R. and N.S.; visualization, A.M.V., G.R. and N.S.; supervision, N.S.; project
administration, N.S.; funding acquisition, G.-E.B. and G.R. All authors have read and agreed to the
published version of the manuscript.
Funding:
This work was supported by the Internal Grant financed by Dunarea de Jos University of
Galati, Romania, Contract no. 3637/30.09.2021.
Institutional Review Board Statement: Not applicable.
Antioxidants 2022,11, 2028 14 of 15
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments:
The Integrated Center for Research, Expertise and Technological Transfer in
Food Industry is acknowledged for providing technical support.
Conflicts of Interest: The authors declare no conflict of interest.
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