Advanced Extraction Techniques for Bioactive Compounds from Berry Fruits: Enhancing Functional Food Applications

Abstract

The modern functional food market is developing dynamically, responding to the growing demand for products combining nutritional and health-promoting values. At the center of this evolution are natural bio-organic extracts, rich in bioactive compounds such as antioxidants, polyphenols, flavonoids, carotenoids, and vitamins, which can enrich traditional food products, including fruit juices, increasing their health-promoting values. The aim of the research was to compare the efficiency of extraction of bioactive compounds from various forms of plant raw material (dried, freeze-dried, frozen material) using innovative techniques: supercritical fluid extraction (SFE) and accelerated solvent extraction (ASE). The research showed that the ASE method demonstrated higher extraction efficiency, in some cases exceeding 40%, whereas SFE exhibited superior selectivity, achieving higher carotenoid contents (105.59 mg/100 g in sea buckthorn powder) and antioxidant activity (234.67 µmol TEAC/g in black elderberry fruit). The use of advanced extraction techniques is a modern approach to juice production, in line with current trends in functional food and healthy eating, which can contribute to the prevention of lifestyle diseases.

Full text

Foods 2024, 13, 4115 https://doi.org/10.3390/foods13244115
Article
Advanced Extraction Techniques for Bioactive Compounds
from Berry Fruits: Enhancing Functional Food Applications
Aneta Krakowska-Sieprawska
1,
*, Justyna Walczak-Skierska
1
, Paweł Pomastowski
1
, Róża Sobolewska
2
,
Jarosław Głogowski
2
, Cezary Bernat
2
and Katarzyna Rafińska
3,
*
1
Interdisciplinary Centre of Modern Technologies, Nicolaus Copernicus University, Wileńska 4 St.,
87-100 Torun, Poland; walczak-skierska@umk.pl (J.W.-S.); p.pomastowski@umk.pl (P.P.)
2
Fortuna Company, Tymienice 88, 98-220 Zduńska Wola, Poland; roza.sobolewska@fortuna.com.pl (R.S.);
jaroslaw.glogowski@fortuna.com.pl (J.G.); cezary.bernat@fortuna.com.pl (C.B.)
3
Department of Environmental Chemistry and Bioanalytics, Faculty of Chemistry, Nicolaus Copernicus
University, Gagarina 7 St., 87-100 Torun, Poland
* Correspondence: krakowska-sieprawska@umk.pl (A.K.-S.); katraf@umk.pl (K.R.);
Tel.: +48-888684482 (A.K.-S.); +48-665131825 (K.R.)
Abstract: The modern functional food market is developing dynamically, responding to
the growing demand for products combining nutritional and health-promoting values.
At the center of this evolution are natural bio-organic extracts, rich in bioactive com-
pounds such as antioxidants, polyphenols, flavonoids, carotenoids, and vitamins, which
can enrich traditional food products, including fruit juices, increasing their
health-promoting values. The aim of the research was to compare the efficiency of ex-
traction of bioactive compounds from various forms of plant raw material (dried,
freeze-dried, frozen material) using innovative techniques: supercritical fluid extraction
(SFE) and accelerated solvent extraction (ASE). The research showed that the ASE
method demonstrated higher extraction efficiency, in some cases exceeding 40%,
whereas SFE exhibited superior selectivity, achieving higher carotenoid contents (105.59
mg/100 g in sea buckthorn powder) and antioxidant activity (234.67 µmol TEAC/g in
black elderberry fruit). The use of advanced extraction techniques is a modern approach
to juice production, in line with current trends in functional food and healthy eating,
which can contribute to the prevention of lifestyle diseases.
Keywords: supercritical fluid extraction; accelerated solvent extraction; bioactive
compounds; antioxidant activity; plant material processing methods
1. Introduction
The contemporary functional food market is experiencing dynamic growth, re-
sponding to the increasing consumer demand for products that combine nutritional and
health-promoting properties. At the center of this evolution are plant-derived bioactive
extracts, rich in bioactive compounds such as antioxidants, polyphenols, flavonoids,
carotenoids, and vitamins, which can be used to enrich traditional food industry prod-
ucts, including fruit juices, thereby enhancing their health-promoting properties. This
approach aims to improve and enhance the characteristics of the input product, i.e.,
strengthening antioxidant properties, extending the shelf life of the product (natural
preservatives), and improving taste and color (natural dyes) [1,2]. The development of
Academic Editor: Isabel
Borrás-Linares
Received: 26 November 2024
Revised: 18 December 2024
Accepted: 19 December 2024
Published: 19 December 2024
Citation: Krakowska-Sieprawska,
A.; Walczak-Skierska, J.;
Pomastowski, P.; Sobolewska, R.;
Głogowski, J.; Bernat, C.; Rafińska,
K. Advanced Extraction Techniques
for Bioactive Compounds from
Berry Fruits: Enhancing Functional
Food Applications. Foods 2024, 13,
4115. https://doi.org/10.3390/
foods13244115
Copyright: © 2024 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license
(https://creativecommons.org/license
s/by/4.0/).

Foods 2024, 13, 4115 2 of 18
extraction techniques is an important task in the selective isolation of biologically active
compounds from plant material.
The use of advanced extraction techniques to obtain bioactive compounds from
plant materials represents a modern approach to juice production, fitting into current
development trends in the field of functional food [3]. This trend fits into the broader
context of growing consumer awareness of healthy eating. Although knowledge of the
term “functional food” among Polish consumers is still limited, research indicates an in-
creasing interest in products with health-promoting properties. The development of the
functional food market, including innovative juices enriched with natural extracts, re-
sponds to these changing consumer preferences and opens new perspectives for the food
industry. The increasing global demand for functional food products enriched with nat-
ural extracts is driven by rising consumer interest in health and wellness. This trend re-
flects growing health awareness, the desire for preventive healthcare, and lifestyle shifts
influenced by urbanization and aging populations. Key factors include consumer interest
in food products that not only satisfy basic nutritional needs but also offer additional
health benefits, such as improved immunity, better digestion, or enhanced cognitive
function [4]. While extensive research has been conducted on pomace, limited studies
directly compare the complementary advantages of accelerated solvent extraction (ASE)
and supercritical fluid extraction (SFE) in extracting diverse bioactive compounds, par-
ticularly for industrial scalability.
The extraction of bioactive compounds from various plant materials, such as sea
buckthorn, rowan, wild rose, or chokeberry, is a complex process that requires the ap-
plication of advanced techniques. The ultimate goal is to isolate the desired chemical
substances from plants using a solvent. Traditional extraction methods, such as macera-
tion and Soxhlet extraction, were not employed in this study due to their limitations, in-
cluding longer processing times, higher energy consumption, and extensive use of or-
ganic solvents, which pose environmental and safety concerns. Innovative extraction
techniques, such as SFE and ASE, open new possibilities for isolating valuable secondary
metabolites while preserving their original structure and biological properties [5,6]. The
ASE uses high pressure and temperature to achieve rapid extraction, while SFE operates
under supercritical conditions, enabling selective extraction of low-polarity compounds
with minimal solvent usage. Both techniques align with the principles of green chemistry
and enable the production of extracts with a high content of bioactive compounds while
minimizing the use of harmful organic solvents [6,7]. While numerous studies have fo-
cused on individual extraction techniques, there is limited comparative research evalu-
ating ASE and SFE for their complementary advantages in isolating bioactive compounds
from berry fruits. This study aims to bridge this gap by evaluating the efficiency, selec-
tivity, and antioxidant activity of extracts obtained using these methods.
The inspiration for using extracts from berry plants with health-promoting proper-
ties in the production of innovative juices comes from both scientific knowledge and folk
traditions, which are an integral part of our culture. This approach not only enhances the
nutritional value of products but also may contribute to the prevention of neurodegen-
erative and lifestyle-related diseases, including cancer, type 2 diabetes, obesity, and car-
diovascular diseases [8,9]. By employing two complementary extraction methods, it is
possible to obtain bioactive compounds with different physicochemical properties. The
use of innovative extraction techniques allows for the isolation of polyphenolic com-
pounds and carotenoids as health-promoting components of the juice. Their high content
in the extract may help mitigate reactive oxygen species in the body. Due to their unique
antioxidant properties, these extracts could serve as natural preservatives for a new
generation of juices, enabling a reduction in preservation parameters, including pas-
teurization [4]. Enriching juice with extracts from wild rose, similar to those from sea

Foods 2024, 13, 4115 3 of 18
buckthorn, will increase the level of vitamin C. Furthermore, literature data indicate that
wild rose extract inhibits browning and ensures color stability in juice [10]. An extract
obtained from rowan is unique due to its proven medicinal properties, such as its anti-
depressant effects, and its high content of vitamin A (retinol), beta-carotene, vitamin C,
and vitamin K, which collectively contribute to its beneficial effects on gastrointestinal
health [11]. Chokeberry, like black elderberry, is classified as a “superfruit” because it is a
unique source of antioxidants that inhibit aging processes and positively impact the car-
diovascular system [12]. In the case of plant materials, the ASE technique is aimed at
obtaining natural dyes. These dyes, due to their unique chemical structure containing
magnesium, can act as natural antioxidants and modulators of the nervous system, which
is significant in the context of functional food production [13].
The aim of the research was to compare the efficiency of extracting bioactive com-
pounds from various forms of plant raw material (dried, lyophilized, and frozen materi-
al) using complementary techniques: supercritical fluid extraction (SFE) and accelerated
solvent extraction (ASE). The raw materials (sea buckthorn, chokeberry, black elderberry,
rowan, wild blueberry, and wild rose) were selected based on ethnopharmacological
premises, which contribute to the development of innovative food products falling into
the category of functional food.
2. Materials and Methods
2.1. Plant Material
The plant materials used in the study included the following:
• Sea buckthorn (Hippophae rhamnoides L.)
• Chokeberry (Aronia melanocarpa (Michx.) Elliott)
• Black elderberry (Sambucus nigra L.)
• Rowan (Sorbus aucuparia L.)
• Wild blueberry (Vaccinium myrtillus L.)
• Wild rose (Rosa canina L.)
Table 1 presents the selected plant materials that were used in the studies on the
content of bioactive compounds.
Table 1. Selected plant materials.
Producer Plant Material
Form of Plant Materi-
al Processing
LIOGAM Foryś, Kot, Prześlak sp.J
Kielce Poland Black elderberry fruit Freeze-dried
Black elderberry fruit halves Freeze-dried
Black elderberry powder Freeze-dried
Black elderberry grits Freeze-dried
WPPH “ELENA”, Kalisz, Poland Chokeberry grits Freeze-dried
Chokeberry fruit halves Freeze-dried
Sea buckthorn fruit halves Freeze-dried
Black elderberry fruit halves Freeze-dried
PPHU “FROSTER”
,
Kielce, Poland Black elderberry fruit Freeze-dried
Chokeberry fruit Freeze-dried
Chokeberry powder Freeze-dried
Black elderberry powder Freeze-dried
Sea buckthorn powder Freeze-dried
Wild rose powder Freeze-dried
Wild rose fruit Freeze-dried

Foods 2024, 13, 4115 4 of 18
PPHU “AWB” Alina Becia
Łańcut, Poland Chokeberry fruit Dried
Black elderberry fruit Dried
Sea buckthorn fruit Dried
Wild rose fruit Dried
Wild rose fruit halves Dried
Rowan fruit Dried
FUNGOPOL, Sp. z o.o Sp.k. Brusy,
Poland Chokeberry fruit Frozen
Wild blueberry fruit Frozen
Rowan fruit Frozen
Wild rose fruit Frozen
Sea buckthorn fruit Frozen
Black elderberry fruit Frozen
2.2. Extraction Methods
Supercritical fluid extraction (SFE) was carried out using a MV-10 ASFE system
(Waters Corp., Milford, MA, USA) equipped with a 10 mL extraction cell. Approximately
1.5 g of plant material was loaded into the extraction cell, which was packed with glass
beads. Preliminary SFE experiments were conducted to determine the target compounds.
Extractions were performed at 60 °C and 200 bar, with a 30 min static time and a 30 min
dynamic mode (continuous flow), using scCO2 as the solvent and 96% EtOH as a
co-solvent. The flow rates were 10 mL/min for scCO2 and 0.4 mL/min for 96% EtOH. The
obtained extracts were stored in a refrigerator until further analysis.
Accelerated solvent extraction (ASE) was conducted using the Dionex ASE 350 sys-
tem (Thermo Scientific, Waltham, MA, USA). Approximately 1.5 g of plant material was
mixed with glass beads and loaded into the extraction cell. The extraction was performed
under the following conditions: 96% ethanol (EtOH) as the solvent, a temperature of
60°C, a pressure of 10 MPa, a static time of 15 min, and two static cycles. The obtained
extracts were stored in a refrigerator until further analysis.
The extraction parameters were selected based on preliminary optimization ex-
periments and established literature findings, ensuring the effective extraction of ther-
mally sensitive bioactive compounds, such as polyphenols and carotenoids, while
maximizing yield and maintaining stability.
2.3. Determination of Dry Matter Content (DM)
To determine the extraction efficiency of the extraction process, the dry mass of the
extracts was first obtained. To do this, 1 mL of SFE and ASE extract was measured into
Eppendorf tubes and evaporated. The procedure was carried out for each extract in three
repetitions. The extraction efficiency was calculated using the following relationship:
𝑌
(%) = 𝑚
𝑚
∙ 100 (1)
where Yextract is extraction yield expressed in %, mextract is the dry extract mass (g), and mfeed
is the feed mass (g).
2.4. Determination of Total Phenolic Content (TPC)
The total content of phenolic compounds in the obtained extracts was determined
using the Folin–Ciocalteu (FC) method. The procedure was based on the method of Sin-
gleton et al., with modifications [14]. To 12 µL of the extract, 188 µL of deionized water
and 12 µL of Folin–Ciocalteu reagent were added. The mixture was incubated in the dark

Foods 2024, 13, 4115 5 of 18
for 8 min, and then 38 µL of 20% sodium carbonate was added. After 30 min of incuba-
tion at 20 °C in the dark, the absorbance was measured at a wavelength of 765 nm using a
Varioskan™ LUX multimode microplate reader (Thermo Fisher Scientific, Waltham, MA,
USA). The measurement was performed against a prepared blank sample. The results
were expressed as gallic acid equivalents (GAE) in milligrams per gram of dry extract.
2.5. Determination of Total Carotenoids and β-Carotene Content
The determination of total carotenoids was carried out according to the Polish
Standard PN-90/A-75101/12 with some modifications [15]. Specifically, the sample was
prepared by combining 3 mL of the extract with 1 mL of ethyl ether. Carotenoids ex-
tracted from plant material should be obtained using a mixture of ethyl ether and ethanol
in a ratio of 5/10–15 (v/v). After extraction, the obtained extract was transferred to 96-well
plates in 250 µL aliquots. Then, a spectrophotometric measurement was performed,
measuring the absorbance of the extract at a wavelength of 467 nm, which corresponds to
the absorption maximum of β-carotene. The calculation of carotenoid concentration, ex-
pressed as β-carotene, was performed using the equation:
A = 0.46605n + 0.0332 [µg/mL] (2)
where A represents the concentration of β-carotene in 1 mL of the solution [µg/mL], and
n is the absorbance at 467 nm.
Then, the total carotenoid content in the sample was calculated according to the
formula:
X = (A × V × 100)/(G × 100) [mg/100g] (3)
where A is the concentration of β-carotene in 1 mL of the solution [µg/mL], V is the total
volume of the extract in mL, and G is the sample mass in grams.
2.6. DPPH Method
The study assessed the free radical scavenging activity of the obtained extracts using
the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging method. The procedure
was based on the method described by Espín et al., with some modifications [16]. To 200
µL of a 0.1 mM ethanol solution of DPPH (initial absorbance of DPPH = 0.9), 50 µL of the
extracts were added and incubated in the dark for 30 min. Absorbance was measured at a
wavelength of 517 nm using a Varioskan™ LUX Multimode microplate reader (Thermo
Fisher Scientific, Waltham, MA, USA). The results were expressed as micromoles of
Trolox Equivalent Antioxidant Capacity (TEAC) per gram of dry extract.
2.7. Determination of Phenolic Compounds Using HPLC-ESI-MS/MS Analysis
Phenolic compounds were identified using a method developed by Krakowska et al.
[6]. For the analysis, 1 mL of the extracted sample was evaporated, and the dry residue
was reconstituted in 1 mL of methanol before filtration. The analysis was conducted us-
ing a Shimadzu LC-MS 8050 triple quadrupole mass spectrometer (Kyoto, Japan), which
included a binary solvent delivery system (LC-30 CE), a controller (CBM 20 A), an au-
tosampler (SIL-30 A), and a column thermostat (CTO-20 AC). Data analysis was per-
formed with the LabSolution 5.8 software. Separation of phenolic compounds was
achieved on a Kinetex F5 column (100 × 2.1 mm, 2.6 µm; Phenomenex, Torrance, CA,
USA), using 0.1% formic acid in water as mobile phase A and acetonitrile as mobile phase
B. The gradient program was as follows: 0–7 min, 0–80% B; 7–8 min, 80% B; AND 8–10
min, 80–0% B. The flow rate was set at 0.4 mL/min, with an injection volume of 10 µL.
The MS/MS analysis was performed in both positive and negative ionization modes.
Multiple reaction monitoring (MRM) was employed for qualitative and quantitative

Foods 2024, 13, 4115 6 of 18
analysis. Key electrospray ionization (ESI) settings were as follows: nebulizing gas flow
at 3 L/min, heating gas flow at 10 L/min, drying gas temperature at 400 °C, desolvation
line (DL) temperature at 250 °C, and interface temperature at 300 °C. Detailed MRM
transitions for all identified compounds were reported by Krakowska et al. [6].
2.8. MALDI-TOF-MS Analysis
For the MALDI-TOF-MS analysis, 1 µL of ethanol extract obtained through ASE and
SFE methods was deposited onto a ground steel MALDI target. The analysis was per-
formed using an UltrafleXtreme II MALDI-TOF/TOF mass spectrometer (Bruker Dal-
tonics, Bremen, Germany), equipped with a modified neodymium-doped yttrium alu-
minum garnet (Nd) laser (smartbeam II) operating at a frequency of 2 kHz and a wave-
length of 355 nm. Spectra were recorded in reflector positive mode, with an acceleration
voltage of 25 kV, covering an m/z range of 100–4500 Da. All acquired mass spectra were
processed using the flexControl and flexAnalysis 3.4 software (Bruker Daltonik). Addi-
tionally, cluster analysis was conducted using ClinProTools 3.4 software (Bruker Dal-
tonik) to further process the MALDI-TOF/TOF MS data.
3. Results and Discussion
Research on the extraction of bioactive compounds from plants focuses on the effi-
ciency of various methods, which is crucial for the food industry. In particular, SFE and
ASE are two innovative techniques that differ in terms of efficiency, quality of obtained
extracts, and the type of bioactive compounds recovered. The present study focused on
comparing these two methods in the context of phenolic content, carotenoid content, and
antioxidant activity in various plant samples.
3.1. Comparison of Extraction Methods
The results indicate significant differences in the efficiency of both methods:
• Yields of Extraction
The extraction efficiency was significantly higher for the ASE method, both for
freeze-dried, dried, and frozen materials. The highest values, exceeding 40%, were ob-
tained for sea buckthorn fruit halves and the powder and grits from chokeberry. The SFE
extraction method demonstrated a considerably lower extraction efficiency, which ex-
ceeded 10% only for freeze-dried sea buckthorn halves and frozen wild berry fruits. Such
differences indicate a greater effectiveness of ASE in obtaining bioactive compounds,
which can be advantageous in an industrial context where efficiency is a key factor.
However, as noted by Patra et al. [17], higher efficiency does not always translate to ex-
tract quality, suggesting that some bioactive compounds may be better preserved during
the SFE process. Previous studies using MALDI-TOF-MS have shown that SFE is a more
selective extraction method than ASE, as ASE extracts contain a significant amount of
interfering compounds, such as peptides and small proteins, as well as fragments of
polysaccharides [7]. A high content of interfering compounds in the extract can nega-
tively affect the stability and turbidity of juices, ultimately reducing their final quality. It
is also worth noting that differences in efficiency may result from various extraction pa-
rameters such as time, temperature, and the ratio of raw material to solvent, which are
crucial for the final quality of the extracts as well as the form of material preparation [18].
The observed variations in extraction yield have significant implications for indus-
trial scalability and cost-effectiveness. Higher yields, as demonstrated by the ASE
method, are advantageous for industrial applications where maximizing output is critical
to meeting demand and ensuring cost efficiency. Specifically, the ASE method achieved
extraction yields exceeding 40% for certain materials, such as sea buckthorn fruit halves

Foods 2024, 13, 4115 7 of 18
and chokeberry grits, compared to the lower yields (<10%) of SFE. This difference trans-
lates to lower raw material requirements and reduced processing time for ASE, directly
reducing operational costs. However, the lower yields associated with SFE may be offset
by the higher purity and stability of the extracts, which could justify the cost of
high-value products like nutraceuticals. A more detailed economic analysis could evalu-
ate whether the higher capital costs of ASE or SFE are mitigated by their respective effi-
ciencies and product quality (Figure 1).
Figure 1. Extraction efficiency in extracts obtained by SFE and ASE.
• Total phenolic content
The ASE method showed higher phenolic content values in most of the analyzed
samples. The highest values were recorded for extracts from freeze-dried chokeberries
(31.95 mg GAE/g of dry extract) and frozen fruits from wild rose (30.31 mg GAE/g of dry
extract) and black elderberry (30.56 mg GAE/g of dry extract) (Figure 2). In the case of the
SFE method, the highest phenolic values were obtained for frozen black elderberry fruits
(31.23 mg GAE/g of dry extract) and freeze-dried powder from wild rose (22.25 mg
GAE/g of dry extract) (Figure 2). To provide additional context, our findings were com-
pared to the study by Patra et al. [17], where ASE achieved a 30–40% higher total phenolic
content compared to traditional methods. In our study, the ASE method produced the
highest phenolic content (e.g., 31.95 mg GAE/g of dry extract for freeze-dried chokeber-
ries), which is approximately 20% higher than the SFE results for the same material.
Similarly, the ASE extracts from frozen wild rose fruits (30.31 mg GAE/g) exceeded the
phenolic content reported for Soxhlet extraction by Mustafa and Turner [19] by ap-
proximately 35%. These comparisons highlight the significant advantages of advanced
extraction methods over conventional ones and validate their applicability in producing
phenolic-rich extracts for the food industry.

Foods 2024, 13, 4115 8 of 18
Figure 2. Total phenolic content in the obtained extracts using SFE and ASE.
• Carotenoid Content
Carotenoid stability is a critical factor influencing the effectiveness of extraction
processes. Carotenoids are highly susceptible to oxidation and isomerization under heat,
light, and oxygen exposure. The SFE method proved to be superior for carotenoid ex-
traction, achieving significantly higher carotenoid content (e.g., 105.59 mg/100 g for
freeze-dried sea buckthorn powder) compared to ASE (6.04 mg/100 g) (Figure 3). This can
be attributed to the supercritical CO2 environment, which minimizes exposure to oxygen
and operates at lower temperatures, preserving the bioactive compounds’ trans-isomeric
forms. In contrast, the higher temperatures and pressures used in ASE might lead to
partial degradation or isomerization of carotenoids. These findings underscore the im-
portance of method selection in maintaining the structural integrity and bioactivity of
carotenoids throughout the extraction process. These differences highlight the im-
portance of selecting the extraction method based on the type of bioactive compounds we
wish to obtain. The advantage of SFE arises from its ability to selectively extract
low-polarity compounds, such as carotenoids, under supercritical conditions. Super-
critical fluid extraction (SFE) prevents the oxidation of bioactive compounds during the
extraction process, thereby helping to preserve the antioxidant activity of carotenoids by
optimizing pressure and temperature, as well as improving the extraction of a more sta-
ble (trans) form of these compounds [20]. According to research by Mattea et al. [21],
carotenoid extraction, particularly β-carotene, lutein, and lycopene, can be effectively
carried out using supercritical carbon dioxide (CO2) technology. This extraction method
stands out among other techniques because it provides better mass transfer. This is due
to the low viscosity, surface tension, and density of supercritical CO2, which, combined
with higher diffusion compared to conventional solvents, significantly enhances the effi-
ciency of the extraction process [5,6].

Foods 2024, 13, 4115 9 of 18
Figure 3. Total carotenoid content in extracts obtained using SFE and ASE.
To ensure the consistency of the results throughout the manuscript, we have added
Table 2, which presents the total carotenoid content expressed per gram of dry mass ex-
tract. Additionally, these results confirm the superiority of the SFE method, as demon-
strated by its significantly higher total carotenoid content compared to ASE across vari-
ous plant materials.
Table 2. Total carotenoid content in extracts obtained using SFE and ASE calculated per gram of
dry mass of the extract.
Total Carotenoids Content
[mg/g DM Extract]
Plant Material Form of Plant Material
Processing SFE ASE
Black elderberry fruit Freeze-dried 21.09 ± 1.24 0.21 ± 0.02
Black elderberry fruit halves Freeze-dried 23.25 ± 1.29 0.32 ± 0.01
Black elderberry powder Freeze-dried 11.01 ± 0.42 0.28 ± 0.02
Black elderberry grits Freeze-dried 20.64 ± 0.96 0.51 ± 0.04
Chokeberry grits Freeze-dried 15.74 ± 1.78 0.33 ± 0.03
Chokeberry fruit halves Freeze-dried 12.06 ± 0.73 0.23 ± 0.03
Sea buckthorn fruit halves Freeze-dried 6.56 ± 0.93 0.15 ± 0.02
Black elderberry fruit halves Freeze-dried 12.19 ± 1.20 0.57 ± 0.05
Black elderberry fruit Freeze-dried 44.45 ± 3.03 0.37 ± 0.08
Chokeberry fruit Freeze-dried 10.78 ± 0.10 0.31 ± 0.01
Chokeberry powder Freeze-dried 5.95 ± 0.53 0.27 ± 0.05
Black elderberry powder Freeze-dried 9.05 ± 1.01 0.26 ± 0.01
Sea buckthorn powder Freeze-dried 15.55 ± 1.38 0.16 ± 0.01
Wild rose powder Freeze-dried 15.05 ± 0.86 0.32 ± 0.01

Foods 2024, 13, 4115 10 of 18
Wild rose fruit Freeze-dried 62.39 ± 4.13 0.56 ± 0.02
Chokeberry fruit Dried 25.27 ± 1.32 0.23 ± 0.01
Black elderberry fruit Dried 11.11 ± 1.83 0.18 ± 0.01
Sea buckthorn fruit Dried 3.71 ± 0.96 0.27 ± 0.03
Wild rose fruit Dried 49.20 ± 3.32 1.08 ± 0.14
Wild rose fruit halves Dried 33.88 ± 2.13 0.57 ± 0.04
Rowan fruit Dried 7.19 ± 1.45 0.10 ± 0.01
Chokeberry fruit Frozen 15.60 ± 0.24 0.21 ± 0.01
Wild blueberry fruit Frozen 46.24 ± 3.06 0.34 ± 0.05
Rowan fruit Frozen 15.47 ± 1.53 0.27 ± 0.01
Wild rose fruit Frozen 34.23 ± 2.26 2.05 ± 0.15
Sea buckthorn fruit Frozen 2.11 ± 0.07 0.25 ± 0.02
Black elderberry fruit Frozen 64.76 ± 3.61 0.37 ± 0.01
• Antioxidant Activity (DPPH)
The results of the DPPH test demonstrated varied antioxidant activity depending on
the extraction method and type of fruit. The observed fluctuations in antioxidant activity
across the 27 samples were analyzed using one-way ANOVA to determine the statistical
significance between the two groups (ASE and SFE). It was determined that there were
no statistically significant differences between the ASE and SFE methods for the extract
from dried wild rose fruit. For all other samples, the differences in antioxidant activity
between the two methods were found to be statistically significant. The highest DPPH
values were obtained for extracts from frozen black elderberry fruits (234.67 µmol
TEAC/g of dry extract) and freeze-dried wild rose powder (205.36 µmol TEAC/g of dry
extract) using the SFE method (Figure 4). The ASE method showed lower values in the
analyzed samples (Figure 4). These differences may arise from the varying abilities of the
extraction methods to preserve and concentrate antioxidants. Antioxidant activity is
closely related to the presence of phenols and carotenoids, suggesting that extraction
methods should be tailored to specific goals to maximize health benefits. Significant dif-
ferences in antioxidant potential may also result from the selectivity of the extraction
processes. Paes et al. [22] also emphasize that while the SFE method can yield similar
amounts of phenolic compounds as ASE, it produces extracts with higher antioxidant
activity. The higher selectivity of SFE allows for better preservation of the bioactivity of
phenols, which is particularly important given their role as antioxidants. SFE enables
precise extraction of low-polarity compounds, which is advantageous for obtaining
high-quality extracts. The results obtained may indicate a high level of interfering com-
pounds in ASE extracts that do not exhibit biological activity. Shahid et al. [23] also note
that while ASE is more efficient, it may lead to the extraction of interfering compounds
that can diminish the quality and stability of final products. In contrast, extracts obtained
using SFE, despite lower yields, exhibited high antioxidant potential, indicating greater
selectivity of this technique. Therefore, extracts obtained by SFE may be particularly
valuable for the food industry. SFE, due to supercritical conditions, allows for selective
extraction of lipophilic compounds such as carotenoids, as confirmed by results obtained
by Yaqoob et al. [24]. Carotenoids play a crucial role in antioxidant mechanisms, and
their effective extraction is essential for achieving high antioxidant activity in industrial
extracts. An additional advantage of this method is its ability to perform extraction
without the addition of a co-solvent, making the final product ready for direct applica-
tion in production. In the case of ASE extraction, evaporating the solvent can be prob-
lematic, requiring an additional step in the production process that involves investment
in appropriate equipment as well as costs related to operation and electricity. In this

Foods 2024, 13, 4115 11 of 18
study, the ASE method demonstrated lower antioxidant activity compared to SFE, which
may be associated with the presence of these interfering compounds. These compounds
can negatively affect the stability, clarity, and overall quality of extracts, representing a
significant limitation of this technique.
Figure 4. Antioxidant activity measured using the DPPH method in the extracts obtained using SFE
and ASE.
3.2. Comparison of Different Methods of Processing Plant Material
Table 1 presents various forms of processed plant material selected for their
health-promoting properties and potential in functional food production. The choice of
raw materials was based on their ethnopharmacological and organoleptic characteristics,
as well as their popularity among consumers, making them suitable candidates for fur-
ther research on their application in innovative food products. The selection of pro-
cessing methods for fruits is crucial for preserving their nutritional value and bioactive
properties. Freeze-drying has proven to be the most effective method for maintaining
bioactive compounds, while hot air drying often leads to significant losses of these
compounds [25]. Freezing, although effective in maintaining quality, poses challenges in
terms of storage. Understanding the impact of plant raw material preparation methods
on the content of bioactive compounds, as well as potential issues related to their pro-
cessing, is essential for the food industry striving to produce healthy and high-quality
products. This knowledge allows for the optimization of technological processes to
maximize the content of desired bioactive components in final products. Furthermore,
identifying and addressing technological challenges associated with processing raw
materials rich in bioactive compounds enables the development of efficient and effective
methods for their extraction and stabilization. The aim of the study was to understand
which of the most popular methods of preparing plant raw material—namely drying,
freeze-drying, or freezing—best preserves the beneficial bioactive properties of the se-
lected plant material.

Foods 2024, 13, 4115 12 of 18
• Freeze-drying
Freeze-drying demonstrated the highest efficiency in preserving bioactive com-
pounds, as shown by quantitative data from this study. For example, extracts from
freeze-dried chokeberry powder contained the highest total phenolic content (31.95 mg
GAE/g using ASE), while for dried chokeberry powder was 13.93 mg GAE/g. In the case
of SFE extracts, the highest levels of carotenoids were obtained for the extract from
freeze-dried sea buckthorn powder (105.59 mg/100 g). Moreover, for the extract obtained
by SFE from freeze-dried sea buckthorn fruit halves, the total carotenoid content was
almost 10 times higher (77.39 mg/100 g) compared to the extracts from dried and frozen
sea buckthorn fruit (7.86 mg/100 g). These differences can be attributed to the low tem-
peratures used in freeze-drying, which minimize oxidative and thermal degradation. The
results for black elderberry and sea buckthorn extracts in the form of freeze-dried pow-
ders are consistent with previous studies suggesting that freeze-drying can preserve up
to 90% of phenols and antioxidant activity compared to fresh fruits. The obtained results
confirm earlier literature findings that identify freeze-drying as the most effective
method for maintaining bioactive compounds in fruits [26,27].
• Drying
Drying is currently one of the most common methods for preserving nutrients and
bioactive compounds during food production [22]. Extracts obtained from dried berry
fruits exhibited lower levels of bioactive compounds (Figure 1) and total carotenoid
content (Figure 3). An exception was noted for extracts from whole wild rose fruits,
where the level of phenolic compounds was higher than that in freeze-dried fruits. This
difference may result from structural changes occurring during the drying process,
which facilitate better mass transfer in larger fruit materials (1–2 cm). In the case of
powders, tissue fragments are small in both dried and freeze-dried fruits, leading to ef-
fective mass transport in both forms. Therefore, additional structural changes that occur
during drying do not significantly affect mass transfer [7,28].
• Frozen
Freezing demonstrated variable efficiency depending on the type of fruit, with fro-
zen wild berries achieving results comparable to freeze-dried samples. Extracts from
frozen fruits exhibited high levels of phenolic compounds. This is likely due to two fac-
tors: (i) the good preservation of phenolic acid structures and (ii) the structural changes
that occur during freezing, which positively affect mass transfer. Numerous studies have
shown that freezing typically preserves more bioactive compounds than traditional
drying methods. For example, a study on strawberries found that freezing retained 90%
of the vitamin C content compared to only 50% in air-dried samples [29]. It is important
to note that extracting from frozen plant raw materials was problematic due to specific
storage conditions, difficulties in filling extraction systems, and the presence of water in
the resulting extract, which increases the risk of microbial contamination. Therefore,
freeze-drying proved to be more advantageous than freezing in terms of preserving bi-
oactive compounds in certain fruits and its suitability for use in the food processing in-
dustry.
3.3. Chemical Analysis of Obtained Extracts by HPLC-ESI-MS/MS
Analysis of HPLC conducted on extracts obtained through SFE and ASE methods
revealed significant differences in the content of key bioactive compounds, such as fla-
vone, hesperidin, and chlorogenic acid, depending on the extraction method used and
the form of plant material processing (Table 3).

Foods 2024, 13, 4115 13 of 18
Table 3. Flavone, hesperidin, and chlorogenic acid content in extracts measured using HPLC-MS.
SFE
ASE
Plant Material
Form of Plant
Material
Processing
Flavone
[µg/100 g] SD Hesperidin
[µg/100 g] SD
Chlorogenic
Acid
[µg/100 g]
SD Flavone
[µg/100 g] SD Hesperidin
[µg/100 g] SD
Chlorogenic
Acid
[µg/100 g]
SD
Black elderberry
fruit Freeze-dried 12.1 2.3 3.5 0.7 236.7 35.8 5.1 4.8 8.3 4.5 20.3 0.6
Black elderberry
fruit halves Freeze-dried 6.1 0.3 3.6 0.4 111.5 0.0 5 0.2 10.6 1.2 16.6 1.2
Black elderberry
powder Freeze-dried 2.4 0.3 1.2 0.1 ND ND 3.8 0.3 6.3 0.2 5.5 0.2
Black elderberry
grits Freeze-dried 8.9 0.7 4.6 0.8 222.3 17.4 4.4 0.2 7.5 0.5 24.4 0.3
Chokeberry grits Freeze-dried 7.8 0.3 7.4 0.7 249.0 35.4 3.2 0.0 13.6 0.1 33.1 0.4
Chokeberry fruit
halves Freeze-dried 22.5 2.1 5.1 0.1 ND ND 6.2 0.1 29.9 0.3 41.4 0.0
Sea buckthorn
fruit halves Freeze-dried 23.6 1.1 11.0 0.2 ND ND 4.1 0.8 9.3 0.2 0.1 0.0
Black elderberry
fruit halves Freeze-dried 13.2 3.8 3.4 0.1 202.4 1.0 8.8 0.2 16.7 0.4 48.7 0.0
Black elderberry
fruit Freeze-dried 32.7 1.1 3.4 0.5 ND ND 8.4 0.3 14.3 0.7 15.6 0.0
Chokeberry fruit Freeze-dried 25.1 0.3 6.6 0.1 98.0 4.0 14 0.7 18.9 1.6 84 0.8
Chokeberry
powder Freeze-dried 21.4 0.3 9.5 0.0 813.3 30.0 3.5 0.0 31.1 0.8 61.8 1.8
Black elderberry
powder Freeze-dried 15.7 0.0 1.9 0.0 63.8 7.6 3.6 0.3 77.1 0.0 11.6 0.8
Sea buckthorn
powder Freeze-dried 30.4 2.1 1 0.0 32.1 0.4 3.3 0.0 0.2 0.0 0.2 0.0
Wild rose
powder Freeze-dried 21.8 1.1 ND ND 86.0 3.1 6.9 0.5 8.9 0.1 0.8 0.0
Wild rose fruit Freeze-dried 275.6 32.6 ND ND ND ND 33.6 0.2 14.4 0.6 6.2 0.1
Chokeberry fruit Dried 61.5 2.7 17.8 0.6 ND ND 22.8 0.0 3.4 0.6 4.0 0.0
Black elderberry
fruit Dried 50.0 1.4 43.3 1.7 ND ND 14.7 0.2 2.7 0.0 ND ND
Sea buckthorn
fruit Dried 22.7 1.8 26.9 2.0 0.6 0.0 7.1 0.1 1.3 0.0 1.5 0.0
Wild rose fruit Dried 38.6 0.4 19.3 0.5 ND ND 129.1 13.
0 22.8 0.8 ND ND
Wild rose fruit
halves Dried 40.9 1.6 67.3 0.8 ND ND 61.1 3.2 26.0 0.5 ND ND
Rowan fruit Dried 29.2 0.9 11.0 0.3 10.2 4.7 19.4 2.2 21.3 0.0 1.5 0.1
Chokeberry fruit Frozen 29.4 4.7 ND ND 596.0 35.7 10.3 1.2 2.1 0.0 23.5 1.1
Wild blueberry
fruit Frozen 0.9 0.0 14.7 2.0 30.1 1.0 5.2 0.0 62.1 4.6 138.4 0.5
Rowan fruit Frozen 35.3 3.0 14.3 0.6 ND ND 13.2 1.1 25.4 1.1 ND ND
Wild rose fruit Frozen 32.3 3.0 21.3 2.6 6.6 0.1 117.1 9.8 199.7
15.
1 149.8 5.3
Sea buckthorn
fruit Frozen 22.7 1.1 21.4 1.1 ND ND 6.2 0.4 2.8 0.1 1.0 0.1
Black elderberry
fruit Frozen 81.1 7.9 65.1 5.2 ND ND 24.6 1.5 12.4 1.4 ND ND
ND—not detected; SD—standard deviation.

Foods 2024, 13, 4115 14 of 18
• Flavone
In the case of flavone, the SFE method demonstrated significantly greater efficiency
for freeze-dried wild rose fruits, where the content of these compounds reached 275.6
µg/100 g. In comparison, the ASE method yielded only 129.1 µg/100 g of flavone from
dried fruits of the same species. These results may indicate that supercritical CO2, par-
ticularly in combination with modifiers, better dissolves flavones due to its lower polar-
ity and specific extraction properties.
• Hesperidin
Hesperidin, which is a more polar compound, was better extracted using ASE. The
highest concentration (199.7 µg/100 g) achieved for frozen wild rose fruits may be asso-
ciated with the better solubility of hesperidin in more polar solvents used in this method.
In the SFE method, although relatively high values were obtained in some cases, such as
for dried wild rose fruit halves (67.3 µg/100 g), overall extraction of hesperidin was less
efficient, which may result from the limited ability of supercritical CO2 to solvate more
polar compounds without appropriate modifiers.
• Chlorogenic Acid
The highest content of chlorogenic acid was obtained using SFE from freeze-dried
chokeberry powder, reaching a value of 813.3 µg/100 g. Such a high concentration may
indicate a strong selectivity of the SFE method for extracting this compound, especially
when ethanol is used as a modifier. In comparison, the ASE method achieved the highest
values of chlorogenic acid for frozen wild rose fruits (149.8 µg/100 g), which is signifi-
cantly lower. This suggests that SFE may be a more effective extraction method for me-
dium-polarity compounds like chlorogenic acid while preserving their chemical integri-
ty.
Different forms of processing had a significant impact on extraction efficiency.
Freeze-drying proved to be the best method for preserving bioactive compounds, which
can be attributed to minimizing losses due to thermal and oxidative degradation. An
example is freeze-dried chokeberry powder, which showed the highest concentration of
chlorogenic acid (813.3 µg/100 g) using SFE, significantly surpassing other processing
forms. Freezing also demonstrated high efficacy in preserving phenolic compounds;
however, challenges related to storage and potential microbial contamination may limit
its application in industry.
3.4. Selectivity of Extraction Methods
In this study, two extraction techniques—ASE and SFE—were compared in terms of
their impact on the mass profile of the analyzed using MALDI-TOF-MS. The results in-
dicate significant differences in the distribution of mass-to-charge ratios (m/z) between
the extracts obtained using both methods (Figures 5 and 6).
Figure 5 illustrates the percentage distribution of m/z (mass-to-charge ratio) values
below and above 800 in extracts obtained from various plant materials using two extrac-
tion methods, ASE and SFE. The interpretation of this data focuses on comparing the ef-
ficiency and selectivity of these two extraction techniques in isolating compounds of
different molecular weights. Firstly, the distinction between ASE and SFE methods may
influence their efficiency in extracting molecules of different m/z ranges. SFE shows a
higher percentage of m/z values below 800, which indicates that this method is more ef-
fective in isolating smaller molecules from plant materials. Conversely, ASE shows a
greater proportion of m/z values above 800, which suggests that this method may extract
larger compounds. Molecules with m/z values below 800 often correspond to smaller
chemical molecules such as flavonoids or alkaloids, while those with m/z values above
800 may represent more complex structures, including interfering molecules like lipids or

Foods 2024, 13, 4115 15 of 18
proteins. These findings are consistent with previous reports indicating that SFE is more
effective in extracting small-molecule secondary metabolites, while ASE prefers the ex-
traction of compounds with more complex molecular structures [7].
Figure 5. Percentage distribution of m/z values below and above 800 in plant extracts obtained us-
ing two extraction methods: accelerated solvent extraction (ASE) and supercritical fluid extraction
(SFE).
Additionally, the gel view presented in Figure 6 provides further insights into the
differences in the chemical composition of the obtained extracts. The spectra obtained for
samples from SFE and ASE show differences in signal intensity and distribution, sug-
gesting that extraction methods influence the final chemical composition of the extracts.
SFE exhibits a more uniform spectral profile for all tested extracts, with almost all m/z
values below 1000, which may result from the greater selectivity of this method. In con-
trast, ASE, due to the use of higher temperatures and pressures, appears to facilitate the
release of a more diverse range of higher molecular weight compounds, especially for
freeze-dried and dried elderberry. Such differences may have significant implications for
the applications of extracts in pharmacology and biotechnology, where specific classes of
chemical compounds are preferred. From a practical standpoint, the choice of extraction
method should depend on the intended goal of the study. If the aim is to obtain low
molecular weight compounds such as terpenes or phenols, SFE seems to be the more
appropriate technique. Conversely, ASE may be a better choice when higher molecular
weight compounds such as alkaloids, polysaccharides, or proteins are desired. This in-
dicates a potential complementarity between both techniques in studies on plant con-
stituents.

Foods 2024, 13, 4115 16 of 18
Figure 6. Gel view of MALDI-TOF-MS spectra in plant extracts (1–27) obtained using supercritical
fluid extraction (SFE) and accelerated solvent extraction (ASE): 1. Black elderberry fruit
freeze-dried, 2. Black elderberry fruit halves freeze-dried, 3. Black elderberry powder freeze-dried,
4. Black elderberry grits freeze-dried, 5. Chokeberry grits freeze-dried, 6. Chokeberry fruit halves
freeze-dried, 7. Sea buckthorn fruit halves freeze-dried, 8. Black elderberry fruit halves
freeze-dried, 9. Black elderberry fruit freeze-dried, 10. Chokeberry fruit freeze-dried, 11. Choke-
berry powder freeze-dried, 12. Black elderberry powder freeze-dried, 13. Sea buckthorn powder
freeze-dried, 14. Wild rose powder freeze-dried, 15. Wild rose fruit freeze-dried, 16. Chokeberry
fruit dried, 17. Black elderberry fruit dried, 18. Sea buckthorn fruit dried, 19. Wild rose fruit dried,
20. Wild rose fruit halves dried, 21. Rowan fruit dried, 22. Chokeberry fruit frozen, 23. Wild blue-
berry fruit frozen, 24. Rowan fruit frozen, 25. Wild rose fruit frozen, 26. Sea buckthorn fruit frozen,
27. Black elderberry fruit frozen.
4. Conclusions
The results of this study clearly demonstrate that ASE and SFE methods offer dis-
tinct advantages for extracting bioactive compounds. ASE is highly efficient, especially
for phenolic compounds and antioxidants, making it ideal for maximizing yield. How-
ever, its efficiency may introduce interfering non-bioactive compounds, potentially re-
ducing product quality. In contrast, SFE provides higher selectivity and better preserva-
tion of carotenoids and lipophilic compounds, producing purer extracts with minimal
interference. Combining SFE with freeze-drying effectively preserves bioactive proper-
ties, as shown in studies on black elderberry and sea buckthorn, maintaining high phenol
and carotenoid concentrations. This approach is particularly promising for high-quality
berry fruit extracts, supporting innovations in functional foods.
Freezing and drying methods, however, lead to greater bioactive compound losses,
limiting their application. Future research should optimize ASE and SFE parameters to
improve efficiency, scalability, and compatibility with diverse plant materials. This in-
cludes exploring novel SFE co-solvents for polar compounds, refining ASE to protect
heat-sensitive substances, and assessing the economic and environmental feasibility of
industrial integration. Hybrid systems combining ASE and SFE could enhance yields and
selectivity, unlocking new opportunities in functional foods and nutraceuticals.

Foods 2024, 13, 4115 17 of 18
Author Contributions: Conceptualization: A.K.-S., P.P., J.G., C.B., and K.R.; methodology: A.K.-S.,
J.W.-S., R.S., and K.R.; validation: J.W.-S.; writing—original draft preparation: A.K.-S. and K.R.;
writing—review and editing: A.K.-S., P.P., and K.R.; visualization: A.K.-S.; supervision: A.K.-S.,
P.P., and K.R.; project administration: J.G. and C.B; funding acquisition: C.B. All authors have read
and agreed to the published version of the manuscript.
Funding: This research was funded by NUTRITECH project I-000W/22, funded by the National
Centre for Research and Development, titled “Development of an improved technology for ob-
taining juice based on lithium concentrates, biologically active fruit extracts, and extended shelf
life”. We express our heartfelt thanks for the support and the opportunity to conduct research that
contributed to the development of technology and the improvement of food product quality.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The original contributions presented in the study are included in the
article; further inquiries can be directed to the corresponding author.
Acknowledgments: Justyna Walczak-Skierska and Paweł Pomastowski are members of Toruń
Center of Excellence “Towards Personalized Medicine” operating under Excellence Initia-
tive-Research University.
Conflicts of Interest: The authors declare no conflicts of interest. Author Jarosław Głogowski,
Cezary Bernat, Róża Sobolewska, were employed by the company Fortuna Company, Tymienice
88, 98-220 Zduńska Wola. The remaining authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be construed as a potential conflict
of interest. The authors declare that this study received funding from NUTRITECH project
I-000W/22 funded by the National Centre for Research and Development. The funder was not in-
volved in the study design, collection, analysis, interpretation of data, the writing of this article or
the decision to submit it for publication.
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