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Received: 24 February 2020    Revised: 13 June 2020    Accepted: 19 June 2020

DOI: 10.1111/jfpp.14744

ORIGINAL ARTICLE

Valorization of red raspberry (Rubus idaeus L.) seeds as a

source of health beneficial compounds: Extraction by different
methods

Boško Marić1,2 | Biljana Abramović2 | Nebojša Ilić1 | Jelena Krulj1 | Jovana Kojić1 |

Jelena Perović1 | Marija Bodroža-Solarov1  | Nemanja Teslić1

1
University of Novi Sad Institute of Food
Technology, Novi Sad, Serbia
2
Department of Chemistry, Biochemistry
and Environmental Protection, University
of Novi Sad Faculty of Science, Novi Sad,
Serbia
Correspondence
Nemanja Teslić University of Novi Sad
Institute of Food Technology, Bulevar cara
Lazara 1, Novi Sad 21000, Serbia.
Email: nemanja.teslic@fins.uns.ac.rs
Funding information
Ministarstvo Prosvete, Nauke i Tehnološkog
Razvoja, Grant/Award Number: 451-03-
68/2020-14/200222; Ministry of Education,
Grant/Award Number: 451-03-68, 2020-14
and 200222
Abstract
Fruit processing by-product, raspberry (Rubus idaeus L.) seeds were utilized to
obtain health beneficial oils. Cold pressing (CP) and supercritical CO2 extraction
(SFE) were applied to separate oil from seeds. Raspberry seed oils were analyzed
in terms of fatty acids (FA) content, tocopherols, and functional quality indices.
The residues after extractions were analyzed in terms of free and total ellagic acid
content (TEAC). CP provided higher extraction yield than the SFE method. Oils
isolated by both methods have a significant amount of ω-3 FA, even though oil
obtained by SFE had slightly higher ω-3 FA content. Furthermore, oils obtained
with both extraction methods exhibited high values of tocopherol content. In
terms of ellagic acid content, residues after SFE had higher values of free and
TEAC than residues after CP.
Practical applications
Raspberry seeds are currently underutilized by-product of the fruit processing in-
dustry. These seeds could be further processed to obtain oil with high quantity of
essential ω-3 fatty acid and polyphenol extracts with high content of ellagic acid.
Isolated oil and phenolic extracts could be used in various food, cosmetic, and
nutraceutical products to increase its nutritive composition and functional proper-
ties. Utilization of cold pressing (conventional extraction technique) and supercriti-
cal CO2 extraction (novel and green extraction technique) for oil recovery in the
same study enables a direct comparison of these techniques and reveals the ad-
vantages and disadvantages of supercritical CO2 extraction for potential industrial
application.

1 |  I NTRO D U C TI O N
World population expansion leads to an epidemic of undernutrition
resulting in a need for increased food availability. Since functional
foods can decrease this problem, there is a need to create more
sustainable systems to create more functional foods and provide
food security for the world population. The use of food processing
by-products can help to create more functional foods, which can
decrease the problem of undernutrition and world hunger (Tripathi,
Mishra, Maurya, Singh, & Wilson, 2019).
Serbia is known as a significant producer of berries, primar-
ily raspberries with the production of 109.742 tons of fresh

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https://doi.org/10.1111/jfpp.14744
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raspberries in 2017 (FAOSTAT, 2019). In the processing of berries
for the production of berry-based juices, concentrates, jams, and
jellies, the berry seeds become a by-product, which is under-ex-
ploited or wasted (Rodríguez Couto, 2008). The berry seed ma-
terials removed as waste by-products contain health-beneficial,
bioactive compounds, which are known as natural antioxidants
(Nile & Park, 2014).
Previous research shows a relatively high content of ω-3 FA
in raspberry seeds (Mazurek, Chmiel, & Górecka, 2017; Parry
et al., 2005). The seed oil is rich in ω-3 FA as it contains about 30%
α-linolenic acid (Parry & Yu,  2004). Johansson, Laakso, and Kallio
(1997) found that linoleic, α-linolenic, oleic, and palmitic acids were
typically the most abundant FA from the seed oil of 22 common
edible berries including raspberry and the major ω-3 FA in plants
is α-linolenic acid (LNA; 18:3, ω-3). α-linolenic acid is an essential
FA that humans can not synthesize in the body and have to obtain
through the diet. Furthermore, α-linolenic acid is the only ω-3 FA
that may be present in botanical materials, including oilseeds (Parry
& Yu, 2004). Previous studies show that organic solvents, for exam-
ple, hexane (Oomah, Ladet, Godfrey, Liang, & Girard, 2000) or cold
pressing extraction can be used for extraction of oil from raspberry
seeds to gain a significant amount of oil. Therefore, raspberry seed
oil can be used as an alternative source of ω-3 FA.
In diseases treating, the optimal ratio of ω-6/ω-3 varies from 1 to
4 depending on the disease under consideration (Simopoulos, 2002).
Raspberry seed oil exhibited good effects on the activities of the in-
tracellular antioxidant enzymes and seems to protect the liver from
oxidative stress through the inhibition of mitogen-activated protein
kinases (Teng et al., 2017). Therefore, the extraction of raspberry
seed oil from this waste material could produce a beneficial food
ingredient, help to solve environmental problems, and expand the
raspberry market.
Raspberry seeds also contain a lot of antioxidants such as phe-
nols, flavonoids, anthocyanins, vitamins, etc. (De Souza et al., 2014).
Godjevac, Tešević, Vajs, Milosavljević, and Stanković (2009) reported
that raspberry seeds contain a lot of polyphenolic compounds such
as gallic acid, procyanidin dimer, sanguiin, roshenin, ellagic acid, etc.
Raspberry seeds contain 87.8% of the total content of ellagic acid in
raspberries according to Daniel et al. (1989).
Studies show that ellagic acid exerts biological properties
(Puupponen-Pimia et al., 2001). One of the most important effects
of ellagic acid is the anticancer effect and one of the main reasons
for the anticancer activity is the modulation of the toxin metabo-
lism and prevention of carcinogenesis induced by these compounds
(Seeram et al., 2006). Further, polyphenolic compounds, such as el-
lagic acid, have a strong effect on the inhibition of the α-amylase
and pancreatic lipase, which can be helpful in solving diabetic and
obesity problems (Abdin et  al.,  2019). Ellagic acid also inhibits the
activity of aflatoxin and in that way exerts its anti-microbiological
properties (Soni, Lahiri, Chackradeo, Bhide, & Kuttan, 1997). Besides
raspberries, previous research reported that pomegranates, black
raspberries, strawberries, walnuts, and almonds in their chemical
composition have ellagic acid (Landete, 2011).
The main objective of the present research was to investigate the
potential of raspberry seeds valorization for bioactive compounds
beneficial to human health. Two different extraction techniques
were selected for oil recovery from raspberry seeds. Obtained oil
extracts were analyzed in terms of total extraction yield, FA pro-
file, and tocopherols content. Isolated oils were also characterized
in terms of indices (ratio between hypocholesterolemic and hyper-
cholesterolemic FA, polyunsaturated and saturated FA ratio, athero-
genicity index, and thrombogenicity index) addressing functional oil
quality and suitability for human diet. Additionally, total and free
ellagic acid content was determined in residues after oil extraction.
2 | M ATE R I A L S A N D M E TH O DS
2.1 | Chemicals
Hydrochloric acid was purchased from Lach-Ner (Brno, Czech
Republic). Methanol was purchased from Macron (Avantor, Gliwice,
Poland). Tetrahydrofuran was purchased from Carlo Erba (Chaussée
du Vexin, France). All other chemicals were purchased from Sigma-
Aldrich (St. Louis, MO, USA).
2.2 | Plant material and methods
Plant material was fruit processing by-product raspberry seeds
(Willamette cultivar) which were kindly donated by Mondi Lamex
d.o.o. (Kraljevo, Serbia). Different quantities of seeds needed for
analyses were milled in a laboratory mill (Glen Mills, Clifton, NJ,
USA). The moisture content of the grains was determined using
gravimetrical AOAC Method 950.46/1990 also known as the “ov-
endry” method. Dietary fibers were determined by Megazyme
AOAC Method 991.43/2012. The ash content was determined by
the AOAC Official method 923.03/2005. The fat content was de-
termined by the AOAC Official method 945.16/2000. The protein
content was determined by the Kjeldahl method. The starch content
was determined by the Ewers polarimetric method and sugar con-
tent by the Luff-Schoorl method.
2.3 | Extraction techniques
The extraction was performed using two different types of extrac-
tion: Cold pressing (CP) and supercritical CO2 extraction (SFE). All
extraction methods were performed in triplicate.
2.3.1 | Cold pressing
Raspberry seed oil was obtained by pressing the 5 kg of raspberry
seeds using screw type expeller (Oriental Motor, Gearhead DY9
97,575, Tokyo, Japan). It was necessary to use the whole seeds on
MARIĆ et al.
this semi-industrial press, because grounded seeds would clog the
press. The temperature inside the expeller was 70°C, while the oil
has 40–45°C. This operation was carried out three times and the
extracted oil was quantified. Afterward, all the extracted oil was
combined and prepared for analysis. Fine particles in the expressed
oil were separated by vacuum filtration (Vacuumbrand, ME 2C,
Wertheim, Germany) with a double layer of Whatman filter paper
No. 1.
2.3.2 | Supercritical CO2 extraction
The SFE of the 70 g of milled (200–400 µm) Willamette raspberry
seeds was performed as Krulj et  al.  (2016) described, on a lab-
oratory-scale unit (NOVA-Swiss, high-pressure extraction plant
565.0156; Nova Werke Ltd, Effretikon, Switzerland). The water
content in seeds was 5.71%. The extraction process was conducted
under the following operating conditions: Pressure 300 bar, tem-
perature 40°C, and CO2 mass flow 0.194 kg/h. Extract samples
were collected in the glass collector in the extractor after 3h of
process. Then, samples were transferred into adequate glass vials
using a Pasteur pipette and stored at −20°C for further use in gas
chromatography analysis.
2.4 | GC-FID determination of the fatty acid profile
FA methyl esters were prepared from the extracted lipids using a
method based on 14% boron trifluoride–methanol solution. Nitrogen
gas was used for drying and removing solvents from FA methyl es-
ters. The obtained samples were analyzed using a GC Agilent 7890A
system with a flame ionization detector (FID) (Agilent Technologies,
Santa Clara, CA, USA), an auto-injection module for liquid, equipped
with a fused-silica capillary column (100 m × 0.25  mm; 0.20  μm).
Helium was used as a carrier gas (purity >  99.99%, flow rate of
1.26  ml/min). The FA peaks were identified by the comparison of
retention times with those of standards from the Supelco GRAIN
fame mix. Results were expressed as the mass of FA or FA group (g)
in 100 g of FA (% (wt/wt)).
2.5 | Functional quality of raspberry seeds oil
The functional quality of raspberry seeds oil was determined by three
indices obtained and calculated from FA profile. The ratio between
hypocholesterolemic and hypercholesterolemic FAs (H/H) was cal-
culated according to the following equation (Cunha et al., 2019):
H∕H =
C18: 1ω9 + C18: 2ω6 + C20: 4ω6 + C18: 3ω3 + C20: 5ω3 + C22: 5ω3 + C22: 6ω3
C14: 0 + C16: 0
(1)
Furthermore, atherogenicity index (AI) and thrombogenic-
ity index (TI) were calculated according to the following formulas
(Cunha et al., 2019):
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C12: 0 + 4 (C14: 0) + C16: 0
AI = ∑ (2)
MUFA + 𝜔 − 3 + 𝜔 − 6
∑ ∑
C14: 0 + C16: 0 + C18: 0
TI =
(3)
�∑ �
𝜔−3
0.5( MUFA) + 3 𝜔 − 3 + 0.5 𝜔 − 6 + ∑ 𝜔−6
∑ ∑ ∑
where C16:0 is palmitic acid, C18:0 is stearic acid, C18:1 is oleic acid,
C18:2 is linoleic acid, C18:3 is α-linolenic acid, ∑MUFA is the sum of
monounsaturated FAs, Σω-3 is the sum of the polyunsaturated ω-3
FAs, and Σω-6 is the sum of the polyunsaturated ω-6 FA.
2.6 | Tocopherol analysis
Tocopherol analysis was performed by standard SRPS EN 12,822:2014
method with some modifications. About 0.025 mg of oil was weighed
in an Eppendorf tube and then 1 ml of n-hexane was added to the tube.
Afterward, the samples were filtrated through a 0.45 µm membrane
filter in vials and were analyzed by HPLC/FLD (Agilent Technologies,
Santa Clara, CA, USA). The HPLC measurements were performed
using a normal phase column (Phenomenex Luna Silica, 5 μm, 250 mm
× 4.6 mm), eluent 4% tetrahydrofuran in n-hexane, flow rate 1 ml/min,
excitation at λex = 290 nm, and emission at λem = 330 nm.
2.7 | HPLC-DAD analysis of ellagic acid
Sample preparation for the determination of free ellagic acid was
performed according to Bobinaite, Viškelis, and Venskutonis (2012)
method with modifications: Residues after CP and SFE were ho-
mogenized and aliquots of 1  g were weighed and transferred into
a 50 ml laboratory flask and diluted in 5 ml of 90% methanol. Then
the samples were mixed on laboratory mixer (Orbital Shaker PSU-
10i, Biosan, Riga, Latvia) for 30 min. After mixing the supernatants
were filtered to 100 ml Erlenmeyer flasks. Then, another 5 ml of 90%
methanol was added to the residue and mixed for another 30 min.
After the second mixing, the samples were filtered into 100  ml
Erlenmeyer flasks. Filtrates were combined and filled to 10 ml with
90% methanol. Afterward, the samples were dried by nitrogen flow
and 2.5 ml of 90% methanol was added to a dry residue. Then the
samples were mixed on a vortex mixer for 3 min and sonicated for
5  min. Before identification and quantification by HPLC-DAD, the
samples were filtered through a 0.45 µm membrane filter.
The samples for the determination of total ellagic acid were pre-
pared by the method of Määttä-Riihinen, Kamal-Eldin, and Törrönen
(2004) with some modifications: The residues after CP and SFE were
homogenized and aliquots of 0.1  g were weighed and transferred
into 5 ml laboratory flasks and diluted in 1 ml of 2 M HCl in metha-
nol. Afterward, acid-hydrolysis was performed for 2 hr at 85°C with
reflux. Then the samples were quantitatively transferred into lab-
oratory flasks and filled to 20 ml with 2 M HCl in methanol. The
samples were sonicated for 30 min and filtered through a 0.45 µm
membrane filter.
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4 of 8       MARIĆ et al.
Samples were analyzed using an Agilent 1260 series HPLC-DAD
system (Agilent Technologies, Santa Clara, CA, USA), using a C18 col-
umn (Agilent, 4.6 mm × 50 mm, 1.8 μm particles). The injection volume
was 5 μl and the temperature was at 30°C. Solvent A was 1% aqueous
solution of formic acid and solvent B was acetonitrile. The used gra-
dient was as follows: 0–6 min, 15% of B; 6–28 min, 15%–50% of B.
The post-run was set to 5 min. The good purity and separation were
achieved in raspberry samples using this gradient (flow rate 0.5  ml/
min). Ultraviolet-visible spectra (ranging from 190 to 540  nm) were
recorded for all peaks. Triplicate analyses were performed for each
sample. Ellagic acid was detected at 254  nm, and identified accord-
ing to peak retention time and UV/Vis spectra, which were compared
with those of the standard. The quantities of ellagic acid were calcu-
lated using the calibration curve and expressed as mg/100 g defatted
weight. For easier comparison free and total ellagic acid content in
whole raspberry seeds and in residue after oil extraction were ex-
pressed on defatted weight. Theoretical oil content determined by
Soxhlet apparatus was 17.52%,
2.8 | Statistical analysis
Statistica 13 Software (StatSoft Inc., Tulsa, Oklahoma) was used for
statistical data processing using one-way ANOVA. The comparison
of mean values was performed by the Tukey’s test. Differences were
considered significant if p < .05.
3 |  R E S U LT S A N D D I S CU S S I O N
3.1 | Chemical composition of raspberry seeds
The chemical composition of raspberry seeds is shown in Table 1.
Seeds contain the largest amount of dietary fibers (65.79%), and
significant amounts of fat (14.90%) and proteins (6.95%). Dry seeds
from raspberry may contain up to 25% of fat and 20% of proteins, and
therefore it can be an interesting material to receive oily extracts rich
in unsaturated and polyunsaturated FA (Mazurek et al., 2017).
3.2 | Extraction yield
The higher extraction yield was obtained using CP (10.25%), while the
extraction yield for SFE was 8.82%. It might be explained by the ex-
traction parameters of SFE. Previous researches show that the pres-
sure has a significant influence on extraction yield and this yield might
TA B L E 1   Chemical composition of red raspberry seeds
Raw proteins
Water (%) (%) Ash (%) Starch (%)
5.71 ± 0.19 6.95 ± 0.91 1.38 ± 0.10 1.04 ± 0.43
Note: The results are presented as mean ± SD, n = 3.
be the result of the lower pressure in the extraction system (Zahedi &
Azarpour, 2011). According to other research, using Soxhlet extraction
method with hexane as a solvent may give the extraction yield up to
17.52% for this matrix, which clearly indicated that the material was
not completely exhausted by SFE nor by CP (Unpublished results), and
further experiments regarding SFE optimization would be required.
The main advantage for using examined extraction techniques is that
there is no need for using harmful organic solvents which may persist
in the product in traces (Table 2).
3.3 | Influence of different extraction methods
on the fatty acid content
The FA profiles of oils obtained by both extraction methods were
similar (Table  3). The most abundant FA from raspberry seed oil
extracted with both extraction methods were linoleic – C18:2n6c
(47.36%–48.06%), α-linolenic – C18:3n3 (28.48%–29.84%), and
oleic – C18:1n9c (14.47%–16.32%), acids (Table  3). Two other
FA that occurred in significant proportions were palmitic acid –
C16:0 (4.67%–4.91%) and stearic acid – C18:0 (1.53%–1.91%),
while other FA were detected in traces. The oleic, palmitic, and
stearic acid were more persistent in oils obtained by CP, while the
content of the linoleic and α-linolenic acid was higher in oil ob-
tained by SFE. The FA composition of the oils recovered by CP
and SFE was similar to previous studies of Johansson et al. (1997),
as they have found that the composition of oleic acid was 10.6%,
α-linolenic acid was 33.5%, and linoleic acid was 49.5%. The com-
position of palmitic acid is slightly higher in this study, considering
that Johansson et  al.  (1997) found that the composition of pal-
mitic acid was 2.5%. Mazurek et al. (2017) founded that raspberry
seeds contain 47.14% linoleic, 35.82% α-linolenic, 8.87% oleic, and
3.38% palmitic acid. Parry et  al.  (2005) founded that raspberry
seeds contain 53% linoleic, 32.4% α-linolenic, 12.4% oleic, and
1.3% palmitic acid. Higher content of linoleic acid indicates good
nutritional properties of red raspberry seeds. Comparing to grape
seed oil, apple seed oil, and watermelon seed oil, the Willamette
raspberry seed oil expressed significantly higher values of ω-3 FA,
as well as significantly lower ω-6 FA content (Górnaś & Rudzińska,
2016). Furthermore, in comparison to caraway, and hemp seed
oils, raspberry seed oil expressed significantly higher content of
ω-3 FA, and significantly lower concentration of ω-6 FA (Parker,
Adams, Zhou, Harris, & Yu, 2003). Comparing to Lycium ruthenicum
seed oil, RSO has significantly lower ω-6 FA content, while, moreo-
ver, significantly higher ω-3 FA content, which suggests that the
RSO, from the point of human health aspects, has a significantly
Fat (%) Sugars (%) Dietary fibers (%)
14.90 ± 0.90 3.27 ± 1.18 65.79 ± 2.16
MARIĆ et al. |
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better ω-6/ω-3 ratio (Liu et  al.,  2019). Moreover, cucurbita pepo
seed oil had significantly lower ω-3 FA comparing to raspberry
seed oil (Younis, Ghirmay, & Al-Shihry,  2000). The ω-6/ω-3 ratio
was 1.61 and 1.66 in oils recovered by SFE and CP, respectively.
These results show a much better ratio of ω-6/ω-3 FA in compari-
son with sunflower oil (71), corn oil (57), safflower oil (14), olive
oil (9), and soybean oil (6.75) (Bhardwaj, Verma, Trivedi, Bhardwaj,
& Shukla, 2016). Therefore, raspberry seed oil has proven to be a
significant source of ω-3 FA, with ω-6/ω-3 ratio which can be con-
sidered as optimal for human consumption, according to Valfré,
Caprino, and Turchini (2003).
3.4 | Functional quality of fatty acids composition

Table  4 shows that the PUFA/SFA ratio was 11.12 and 12.56 for
TA B L E 2   Yield of oil extracted from raspberry seeds with CP
and SFE oils recovered by CP and SFE, respectively. According to Cunha
et al. (2019), the oils that has PUFA/SFA ratio lower than 0.45 should
Extraction Yield (%)
be not used for human food due to potential risk for increase of cho-
CP 10.25 ± 0.13b lesterol in blood. Therefore, oils obtained by performed extraction
a
SFE 8.82 ± 0.17 methods could be considered as suitable for human consumption.
Note: The results are presented as mean ± SD, n = 3. Different letters Furthermore, hypocholesterolemic and hypercholesterolemic FAs
within the same column indicate significant differences (p < .05), (H/H) were 18.77 and 19.78 for oils recovered by CP and SFE, re-
according to the Tukey's HSD test. spectively. The high values of H/H are directly related to the benefit
offered to the metabolism of cholesterol. Moreover, the lower AI
(0.05–0.0525) and TI (0.05–0.06) values are more desirable for oil
TA B L E 3   Fatty acid content in oil after extraction from
raspberry seed by CP and SFE quality (Pinto et al., 2018). According to Valfré et al. (2003), AI and
TI values below 1 in edible oils could be considered as healthy for
FA CP SFE consumption in that regard.
C16:0 4.91 ± 0.005b 4.67 ± 0.001a In comparison to different oils, raspberry seed oil exhibited
C18:0 1.91 ± 0.012b 1.53 ± 0.010a better functional quality than pacaxi, palm, cupuassu, and passion
C18:1n9c 16.32 ± 0.053b 14.47 ± 0.041a fruit oil (Bezerra, da Cruz Rodrigues, de Oliveira, da Silva, & da Silva,
C18:2n6c 47.36 ± 0.055a 48.06 ± 0.055b 2017). Also, the results of AI and TI show a slightly better functional

C18:3n3 28.48 ± 0.066 a
29.84 ± 0.031 b quality of raspberry seed oil in comparison with oil recovered from
different Ribes species. (Piskernik et al., 2018). Therefore, CP and
ω6/ω3 1.66 ± 0.002b 1.61 ± 0.001a
SFE could be successfully performed methods for the extraction of
Note: The results are presented as mean ± SD, n = 3. Different letters
raspberry seed oil with appropriate functional quality.
within the same row indicate significant differences (p < .05), according
to the Tukey’s HSD test.

3.5 | Influence of different extraction methods

TA B L E 4   Functional quality indices of raspberry seed oil
obtained by CP and SFE
on the tocopherols content

CP SFE Tocopherols are the main reason for the antioxidant properties of
a b
PUFA/SFA 11.12 ± 0.007 12.56 ± 0.008 plant oil (Baldioli, Servili, Perretti, & Montedoro, 1996). Obtained
H/H 18.77 ± 0.012 c
19.78 ± 0.018 d results show that the major tocopherol content has γ-isomer in
AI 0.0525 ± 0.0001 a
0.05 ± 0.0002 a oil obtained from both extraction method, and its concentrations

TI 0.06 ± 5.37e−5 a
0.05 ± 2.64e−5 b were 75.22 and 90.38 mg/100 g for oils recovered by SFE and CP,
respectively (Table  5). It shows that raspberry seed oil is very rich
Note: Different letters within the same row indicate significant
with γ-tocopherol, since its level is higher than those reported for
differences (p < .05), according to the Tukey's HSD test.
Abbreviations: AI, atherogenicity index; H/H, other vegetable oils, such as soybean oil, canola oil, and sunflower
hypocholesterolemic and hypercholesterolemic fatty acids ratio; oil, and foods such as broccoli, corn, lettuce, tomatoes, etc. (Chun,
PUFA/SFA, polyunsaturated and saturated fatty acids ratio; TI, Lee, Ye, Exler, & Eitenmiller, 2006; Grilo, Costa, Gurgel, Beserra,
thrombogenicity index.

TA B L E 5   The content of tocopherols

Extraction α [mg/100 g] γ [mg/100 g] δ [mg/100 g] TTC [mg/100 g]
in oil extracted from raspberry
a b a
seeds gained by CP and SFE CP 64.12 ± 4.38 90.38 ± 3.81 25.79 ± 1.12 180.29 ± 9.31
SFE 61.81 ± 0.61a 75.22 ± 1.72a 25.72 ± 0.84a 162.75 ± 3.17

Note: The results are presented as mean ± SD, n = 3. Different letters within the same column
indicate significant differences (p < .05), according to the Tukey's HSD test.
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TA B L E 6   Concentration of
Concentration [mg/100 g dw] Concentration [mg/100 g dw]
free ellagic acid in raspberry seeds residue
Extraction (free ellagic acid) (total ellagic acid)
after CP and SFE
CP 10.66 ± 0.89a 836.84 ± 67.10a
SFE 14.88 ± 0.07b 841.18 ± 18.59a
c
Raspberry seeds 34.75 ± 0.90 863.71 ± 14.18a

Note: The results are presented as mean ± SD, n = 3. Different letters within the same column
indicate significant differences (p < .05), according to the Tukey's HSD test.
Abbreviation: dw, defatted weight.

Almeida, & Dimenstein, 2014). The biologically active α-tocopherol
were 61.81 and 64.12 mg/100 g for oils recovered by SFE and CP,
respectively (Table 5). In comparison to other oils obtained by fruit
processing by-products, it can be seen that the Japanese quince
seed and sea buckthorn seed oils has significantly higher content
of α-tocopherol than raspberry seed oil, while pomegranate seed,
watermelon seed, and gooseberry seed oils have significantly lower
content of this tocopherol isomer (Górnaś, Soliven, & Segliņa, 2015).
Furthermore, grape seed oil has higher amount of α-tocopherol
(Sabir, Unver, & Kara, 2012). Concentrations of δ isomer were 25.72
and 25.79 mg/100 g for oils recovered by SFE and CP, respectively,
which is higher than its content in gooseberry seed, grape seed,
pomegranate seed, and watermelon seed oils (Górnaś et al., 2015).
In comparison to other plant seed oils, previous researches reported
significantly higher concentration of the δ-isomer from different va-
rieties of the pumpkin seed oil, while dates (deglet nour and allig
cultivars) seed oils had a significantly lower concentration of this
tocopherol isomer than raspberry seed oil (Besbes et al., 2004;
Stevenson et al., 2007). The high concentration of all tocopherol
isomers can be explained by the fact that CO2 used in SFE, pre-
vents contact with O2 and oxidation of tocopherols. The amount
of γ-tocopherol is slightly lower than Oomah et al. (2000) reported
(137–272  mg/100  g of γ-tocopherol), concentrations of α-isomer
were in accordance with these authors which reported the amounts
of 46–71 mg/100 g of α, while concentrations of and δ-isomer were
higher than these authors reported (7–17.5 mg/100 g of δ tocoph-
erol). β-tocopherol was not determined in oil obtained by any extrac-
tion method and it is in consistency with previous research (Oomah
et al., 2000). The amounts of α-tocopherol and γ-tocopherol obtained
by CP and SFE were higher than Bushman et al. (2004) research,
who reported 12.6 mg/100 g of α-tocopherol and 19.4 mg/100 g
of γ-tocopherol. Moreover, comparing to Lycium ruthenicum
seeds oil, which has 10.84–21.35  mg/100  g of α-tocopherol,
2.22–9.22 mg/100 g of γ-tocopherol, and 0.03–1.18  mg/100  g of
δ-isomer depending on plant origin, RSO has higher content of all
three tocopherol isomers (Liu et al., 2019).
However, amount of free ellagic acid is negligible comparing to
relatively high concentrations of total ellagic acid which were de-
termined in residues after both extraction methods, 836.84 and
841.18 mg/100 g for CP and SFE, respectively. Large differences
between results for free and total ellagic acid in each sample shows
that Willamette raspberry seeds have a significant amount of ellagic
acid in the form of ellagitannins and ellagic acid glycosides (Zafrilla,
Ferreres, & Tomás-Barberán, 2001). The concentration of free el-
lagic acid and total ellagic acid in whole raspberry seeds was 34.75
and 863.71 mg/100 g defatted weight (dw), respectively. Suggesting
that the small amount of free ellagic acid was extracted with the oil,
but the concentration of total ellagic acid has not undergone signifi-
cant changes. It might be explained by SFE suitability for the extrac-
tion of non-polar compounds, because CO2 used in SFE is non-polar
and it can be used for the extraction of oil, but not the extraction
of ellagic acid which can be extracted by other extraction methods
(e.g., ultrasound-assisted extraction) afterward (Ramić et al., 2015).
Previous research show the content of free ellagic acid in
raspberry seeds was 27.5  mg/100. Furthermore, the total el-
lagic acid content in hydrolyzed fruit extracts for raspberries was
150  mg/100  g (Daniel et  al.  1989). Koponen, Happonen, Mattila,
and Törrönen (2007) research show the 3.7 mg/100 g of free ellagic
acid and 263.7  mg of total ellagic acid per 100  g of fresh raspber-
ries. Compared to the ellagic acid content in other fruits, that is, in
strawberries, blackberries, and cranberries which has 63 mg/100 g,
150  mg/100  g, and 120  mg/100  g, of ellagic acid, respectively
(Daniel et al. 1989), longan seeds and mango kernel which has
156 mg/100 g and 118 mg/100 g of ellagic acid, respectively (Soong
& Barlow, 2006), and pomegranate peel which is one of the best natu-
ral sources of ellagic acid and has 117 mg/100 g – 1,810.0 mg/100 g,
depending on the cultivar (Serenjeh & Hashemi, 2017), it can be con-
cluded that “Willamette” raspberry seeds are a significant source of
ellagic acid, which, according to Nguyen et al. (2019), has strong an-
tioxidant activity.
4 | CO N C LU S I O N S

3.6 | Influence of different extraction methods
on the ellagic acid content
The results indicate that higher amount of free ellagic acid is left
after oil recovery SFE compared to residue left after CP (Table 6).
Raspberry seeds are food processing by-product which can be
widely used through the valorization of oil and the residue after oil
extraction. The results indicated that it is very important to choose
the right method for oil extraction to gain the highest yield and opti-
mal content of oil. The CP proved to be the most suitable extraction
MARIĆ et al.
method due the efficiency and “green chemistry” aspect. This ex-
traction method is the most effective to gain the optimal content of
FA, tocopherols in oil, and ellagic acid content in the residue. SFE also
has “green chemistry” properties and shows highest functional qual-
ity indices, but the lack of SFE in comparison to CP is lower yield. The
obtained results show that raspberry seeds are a significant source
of oil, which shows very good functional quality indices, and is a good
source of valuable ω-3 FA and tocopherols. Furthermore, research
shows that the extraction residue contains significant amounts of
ellagic acid, which is known for its health beneficial characteristics.
These properties make raspberry seeds a promising functional food
and nutraceutical ingredient that is valuable for human health.
AC K N OW L E D G M E N T
This work is financed by the Ministry of Education, Science
and Technological Development, Republic of Serbia, Grant No.
451-03-68/2020-14/200222.
C O N FL I C T O F I N T E R E S T
The authors have declared no conflicts of interest for this article.
ORCID
Marija Bodroža-Solarov  https://orcid.org/0000-0001-8151-6276
Nemanja Teslić  https://orcid.org/0000-0002-8219-1909
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How to cite this article: Marić B, Abramović B, Ilić N, et al.
Valorization of red raspberry (Rubus idaeus L.) seeds as a
source of health beneficial compounds: Extraction by
different methods. J Food Process Preserv. 2020;00:e14744.
https://doi.org/10.1111/jfpp.14744