Sequential Biorefining of Bioactive Compounds of High Functional Value from Calafate Pomace (Berberis microphylla) Using Supercritical CO2 and Pressurized Liquids

Abstract

A biorefinery process was developed for a freeze-dried pomace of calafate berries (Berberis microphylla). The process consisted of extraction of lipophilic components with supercritical CO2 (scCO2) and subsequent extraction of the residue with a pressurized mixture of ethanol/water (1:1 v/v). scCO2 extracted oil from the pomace, while pressurized liquid extraction generated a crude extract rich in phenols and a residue rich in fiber, proteins and minerals. Response surface analysis of scCO2 extraction suggested optimal conditions of 60 °C, 358.5 bar and 144.6 min to obtain a lipid extract yield of 11.15% (d.w.). The dark yellow oil extract contained a good ratio of ω6/ω3 fatty acids (1:1.2), provitamin E tocopherols (406.6 mg/kg), and a peroxide index of 8.6 meq O2/kg. Pressurized liquid extraction generated a polar extract with good phenolic content (33 mg gallic acid equivalents /g d.w.), anthocyanins (8 mg/g) and antioxidant capacity (2,2-diphenyl-1-picrylhydrazyl test = 25 µg/mL and antioxidant activity = 63 µM Te/g). The extraction kinetics of oil by scCO2 and phenolic compounds were optimally adjusted to the spline model (R2 = 0.989 and R2 = 0.999, respectively). The solid extracted residue presented a fiber content close to cereals (56.4% d.w.) and acceptable values of proteins (29.6% d.w.) and minerals (14.1% d.w.). These eco-friendly processes valorize calafate pomace as a source of ingredients for formulation of healthy foods, nutraceuticals and nutritional supplements.

Full text

Citation: Ortiz-Viedma, J.;
Bastias-Montes, J.M.; Char, C.; Vega,
C.; Quintriqueo, A.; Gallón-Bedoya,
M.; Flores, M.; Aguilera, J.M.;
Miranda, J.M.; Barros-Velázquez, J.
Sequential Biorefining of Bioactive
Compounds of High Functional
Value from Calafate Pomace (Berberis
microphylla) Using Supercritical CO2
and Pressurized Liquids. Antioxidants
2023,12, 323. https://doi.org/
10.3390/antiox12020323
Academic Editor: Alessandra
Napolitano
Received: 22 December 2022
Revised: 12 January 2023
Accepted: 24 January 2023
Published: 30 January 2023
Copyright: © 2023 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/licenses/by/
4.0/).
antioxidants
Article
Sequential Biorefining of Bioactive Compounds of High
Functional Value from Calafate Pomace (Berberis microphylla)
Using Supercritical CO2and Pressurized Liquids
Jaime Ortiz-Viedma 1,* , JoséM. Bastias-Montes 2, † , Cielo Char 1, Camila Vega 1, Alejandra Quintriqueo 1,
Manuela Gallón-Bedoya 3, Marcos Flores 4, * , JoséM. Aguilera 5, JoséM. Miranda 6,†
and Jorge Barros-Velázquez 6, †
1Departamento de Ciencia de los Alimentos y Tecnología Química, Facultad de Ciencias Químicas y
Farmacéuticas, Universidad de Chile, Dr. Carlos Lorca 964, Santiago 8320000, Chile
2Departamento de Ingeniería en Alimentos, Universidad del Bio-Bio, Avda Andrés Bello 720,
Chillan 3800708, Chile
3
Facultad de Ciencias Agrarias, Universidad Nacional de Colombia, Sede Medellín, Medellín 050034, Colombia
4Departamento de Ciencias Básicas, Facultad de Ciencias, Universidad Santo Tomás, Talca 3460000, Chile
5Departamento de Ingeniería Química y Bioprocesos, Universidad Católica de Chile, V. Mackenna 3860,
Santiago 8940000, Chile
6Departamento de Química Analítica, Nutrición y Bromatología, Facultad de Veterinaria,
Universidad de Santiago de Compostela, 27002 Lugo, Spain
*Correspondence: jaortiz@uchile.cl (J.O.-V.); marcosflores@santotomas.cl (M.F.)
† These authors contributed equally to this work.
Abstract:
A biorefinery process was developed for a freeze-dried pomace of calafate berries (Berberis
microphylla). The process consisted of extraction of lipophilic components with supercritical CO
2
(scCO
2
) and subsequent extraction of the residue with a pressurized mixture of ethanol/water
(1:1 v/v). scCO
2
extracted oil from the pomace, while pressurized liquid extraction generated a
crude extract rich in phenols and a residue rich in fiber, proteins and minerals. Response sur-
face analysis of scCO
2
extraction suggested optimal conditions of 60
◦
C, 358.5 bar and 144.6 min
to obtain a lipid extract yield of 11.15% (d.w.). The dark yellow oil extract contained a good ra-
tio of
ω
6/
ω
3 fatty acids (1:1.2), provitamin E tocopherols (406.6 mg/kg), and a peroxide index
of 8.6 meq O
2
/kg. Pressurized liquid extraction generated a polar extract with good phenolic
content (
33 mg gallic acid equivalents /g d.w.
), anthocyanins (8 mg/g) and antioxidant capacity
(2,2-diphenyl-1-picrylhydrazyl test = 25 µg/mL and antioxidant activity = 63 µM Te/g). The extrac-
tion kinetics of oil by scCO
2
and phenolic compounds were optimally adjusted to the spline model
(
R2= 0.989
and R
2
= 0.999, respectively). The solid extracted residue presented a fiber content close to
cereals (56.4% d.w.) and acceptable values of proteins (29.6% d.w.) and minerals (14.1% d.w.). These
eco-friendly processes valorize calafate pomace as a source of ingredients for formulation of healthy
foods, nutraceuticals and nutritional supplements.
Keywords:
antioxidant capacity; bioactive components; biorefinery; calafate residual pomace;
supercritical extraction; pressurized solvents; functional ingredients
1. Introduction
Berries are an important source of bioactive secondary metabolites such as dietary
antioxidants and nutrients such as fiber and polyunsaturated fatty acids, associated with
health benefits [
1
,
2
]. In addition, berries, due to their high-water content (>80%), are
low in calories [
3
]. Bioactive compounds found in berries, classified as phenolic acids,
stilbenes, flavonoids, tannins and lignans, vary according to genetic factors, environmental
conditions, stages of maturity, harvest time, postharvest handling and storage conditions
of the fruit [
3
]. In recent years, the term “superfruit” has gained popularity and has
Antioxidants 2023,12, 323. https://doi.org/10.3390/antiox12020323 https://www.mdpi.com/journal/antioxidants

Antioxidants 2023,12, 323 2 of 19
been used to promote the health benefits of “exotic fruits” that grow wild under certain
climatic conditions or are cultivated on a small scale by local people [
4
]. This is the case
for acai (Euterpe oleracea), acerola (Malpighia emarginata), camu-camu (Myrciaria dubia),
goji (Lycium barbarum) and blueberries (Vaccinium sect. Cyanococcus), among others [
4
].
Patagonian berries maqui (Aristotelia chilensis), murta (Ugni molinae Tuscz) and calafate
(Berberis microphylla) are considered “super fruits” due to their high content of phenolic
compounds, including several anthocyanins [
5
,
6
]. Calafate is a shrub that produces a
dark-skinned berry and grows extensively in southern Chilean and Argentine Patagonia [
7
].
Several studies have shown the strong antioxidant potential of the calafate fruit due to the
content of anthocyanins, polyphenols and hydroxycinnamic acids [
5
,
7
–
16
]. Brito et al. [
8
]
reported that calafate berries had a higher content of anthocyanins than six other berries.
Ramirez et al. [
5
] compared the antioxidant activity of six Chilean berries and determined
that calafate inhibited lipid peroxidation in human erythrocytes, mitigating the spread
of oxidative stress [
5
]. Other work [
6
] found 18 types of anthocyanins in an extract of
calafate, exceeding the levels described for maqui and murtilla [
6
]. Additionally, Speisky
et al. [
16
] determined the antioxidant activity (oxygen radical absorbance capacity (ORAC))
of more than 120 species/varieties of fruits and found that calafate was the fruit with the
greatest antioxidant potential. Associated with its high antioxidant capacity and content
of phenolic compounds, mainly anthocyanins, calafate has been reported to exhibit anti-
inflammatory [
9
,
12
,
13
,
15
], antiproliferative [
9
], vasodilatory [
17
] and anti-atherosclerotic
effects on tumor cells [18].
In addition, calafate extracts have been shown to restore insulin-induced protein ki-
nase B (AKT) phosphorylation and glucose tolerance in a diet-induced obesity model using
mice [
15
] and to inhibit the enzyme
α
-glucosidase, affecting carbohydrate digestion and
thus controlling postprandial hyperglycemia. The authors determined that the administra-
tion of calafate extract increased the concentration of 16 antioxidant phenolic acids in mice
plasma [7,10].
Calafate berries can be consumed fresh or processed in products such as jellies, juices,
jams and alcoholic beverages [
9
,
11
]. When processing berries, byproducts are generated
that can be composted, used as components in the formulation of animal feed or discarded
in landfills [
19
], losing a considerable amount of nutrients and phytochemicals [
20
]. To
recover valuable compounds and valorize byproducts, zero-waste green technologies have
been applied based on biorefining. Supercritical fluid extraction (SFE) is a well-known
technology, with several applications in foods. scCO
2
is considered a green technology
since it has minor impacts on the environment and CO
2
is a solvent generally recognized
as safe. scCO
2
has been proposed for the extraction of many bioactive compounds from
plant material such as phenols, coumarins and alkaloids, among others [
21
,
22
]. CO
2
above
its critical temperature and pressure makes compound recovery very easy and provides
a solvent-free analysis [
23
]. scCO
2
extraction is efficient for the complete recovery of
neutral lipids from various plant raw materials depending on their particle size [
24
] as
well as constituents of microalgae. Extracted bioactive compounds find application in the
nutraceutical, food and energy industries, among others [25].
Pressurized solvents or enzymatic methods have also shown promising results in
extraction from rowanberry byproducts [
19
], blackcurrant [
1
], blackberry, lingonberry [
20
],
raspberry [
25
] and cranberry [
26
]. Supercritical extraction with carbon dioxide (scCO
2
) and
pressurized solvents utilizes nontoxic, relatively inexpensive, readily available, environ-
mentally friendly and food-grade safe (GRAS) solvents [
1
,
20
]. In scCO
2
, CO
2
penetrates
solid particles faster than liquid solvents, and extraction can be carried out at low tempera-
tures, maintaining the properties of heat-sensitive compounds [
1
]. In pressurized solvent
technology, high pressure keeps solvents in the liquid phase, and if temperature is applied,
contact between solvent and matrix can be maximized by increasing diffusion rates for
mass transfer to the solvent [
27
]. In berries, the seeds and the skin contain high levels of
polyphenolic compounds, fiber, lipophilic compounds and minerals [
26
]. By combining
extraction technologies, it has been possible to obtain extracts with different compositions.

Antioxidants 2023,12, 323 3 of 19
scCO
2
extraction is used for the extraction of lipophilic components [
1
]. On the other
hand, in pressurized solvent extraction, the appropriate choice of a polar solvent allows
the extraction, for example, of anthocyanins [
28
]. In the particular case of calafate, the
extraction of bioactive compounds from byproducts or residues that include the seed and
the skin using green technologies has not been extensively reported to date. Only small
studies have been published, such as Ruiz et al. who studied the profile and concentration
of flavonols in calafate skin, pulp and seed, reporting lower concentrations of flavonol
in seed compared to pulp and peel [
29
]. Additionally, Mazzuca et al. described the fatty
acid profile of seed oil from two species of Argentine calafate (Berberis buxifolia and Berberis
heterophylla), where linoleic and oleic acids predominated [
30
]. Similar results were reported
by Olivares-Caro et al.; therefore, the components of the calafate byproduct represent a po-
tential source of functional ingredients for food and other uses in nutraceuticals, cosmetics
and pharmaceuticals [18].
The objective of this study was to design a biorefining process for bioactive components
which could constitute a source of functional ingredients applicable in the development of
healthy foods, nutraceuticals and nutritional supplements from waste (pomace) from the
powdered calafate industry. The process consisted of two sequential extractions applied to
the same sample. To obtain lipophilic extracts, extraction with scCO
2
was first applied, and
later, to obtain hydrophilic extracts, accelerated hydroalcoholic extraction with pressurized
liquid extraction (PLE) was applied. The scCO
2
extraction was optimized by the response
surface method (RSM). Furthermore, both scCO
2
and PLE extraction kinetics were modeled
by the spline method described by Jesus et al. [
31
]. Fatty acid profiles, tocopherols and
physicochemical properties were determined for the lipophilic extracts. Total phenols,
anthocyanins and antioxidant capacity were determined in the hydrophilic extract. Finally,
the residual solid from the two extractions was converted into flour, and its nutritional value
was determined to define its use as a food ingredient rich in fiber, minerals and proteins.
2. Materials and Methods
2.1. Raw Material
The raw material used corresponded to dry pomace (6.65% w/wmoisture) of calafate
(Berberis microphylla) composed of seeds, skins and fruit pulp, which was harvested in
October 2021 and provided by Patagonia Superfruits S.A. (XI Region of Aysén, Chile). The
average particle size of the residual calafate pomace (CR) was 589
±
35
µ
m, obtained by
sieving in an automatic shaker (Erweka-Apparatebau GMBH 6056, Heusenstamm, Germany).
2.2. Biorefining of Calafate Pomace
The biorefining process began with extraction of the oil using scCO
2
and then pro-
ceeded to extraction of the defatted calafate product (DCP) with a pressurized ethanol:water
mixture (1:1 v/v) to obtain a bioactive extract high in polyphenolic components. The wet
residue of DCP (DCPw) was dried in an oven at 30
◦
C for 30 min to obtain a flour rich in
fiber, protein and minerals (Figure 1).
2.3. Supercritical Extraction with Carbon Dioxide
The supercritical extraction process was performed as described by Basegmez et al. [
1
],
with some modifications using a Speed SFE-2 model 7071 supercritical extractor (Applied
Separations, Allentown, PA, USA) coupled to a chiller system (F-200, Julabo USA Inc.,
Allentown, PA, USA). The 50 mL extraction cell was loaded with 16 g of calafate pomace.
Liquid CO
2
(purity 99.99%, Indura SA, Santiago, Chile) was used at a superficial speed of
1 mm/s. The temperature, pressure and extraction time were programmed as established
in the experimental design.

Antioxidants 2023,12, 323 4 of 19
Antioxidants 2023, 12, x FOR PEER REVIEW 4 of 21
Figure 1. Scheme of the sequential biorefinery extraction by scCO2 and pressurized liquid extraction
(PLE) of calafate pomace to obtain oil, bioactive extract and flour rich in fiber, protein and minerals.
DCP: defatted calafate pomace (waste), DCPw: wet defatted calafate pomace.
2.3. Supercritical Extraction with Carbon Dioxide
The supercritical extraction process was performed as described by Basegmez et al.
[1], with some modifications using a Speed SFE-2 model 7071 supercritical extractor (Ap-
plied Separations, Allentown, PA, USA) coupled to a chiller system (F-200, Julabo USA
Inc., Allentown, PA, USA). The 50 mL extraction cell was loaded with 16 g of calafate
pomace. Liquid CO₂ (purity 99.99%, Indura SA, Santiago, Chile) was used at a superficial
speed of 1 mm/s. The temperature, pressure and extraction time were programmed as
established in the experimental design.
Experimental Design for Extraction with scCO2
Optimal conditions of temperature, pressure and time for oil extraction from calafate
pomace were determined by response surface methodology (RSM) following a Box‒
Behnken design at three levels of the independent variables: extraction temperature (Te:
30, 45, 60 °C), extraction pressure (P: 300, 350, 400 bar) and extraction time (Ti: 60, 105, 150
min). A total of 15 experiments were performed. Oil yield was considered a dependent
variable based on the following polynomial equation of second order.
𝑌=𝛽
+𝛽

𝐴
+𝛽
𝐵+𝛽
𝐶+𝛽

𝐴
𝐵+𝛽

𝐴
𝐶+𝛽
𝐵𝐶 + 𝛽
𝐴
+𝛽
𝐵+𝛽
𝐶 (1)
where β0 is the intercept; βA, βB and βC are the coefficients of the factors; βAB, βAC and βBC
are the coefficients of interactions between factors; and βA2, βB2 and βC2 are the coefficients
of the double interactions. The model was determined using the lack of fit method and
the coefficient of determination R2.
2.4. Pressurized Liquid Extraction
Extraction with PLE was performed as described by Basegmez et al. [1] with some
modifications, using Dionex ASE®300 equipment (Thermo Fisher Scientific, Waltham,
MA, USA) with adjustment and control of pressure, temperature and time. A total of 4.1
+ 0.1 g of DCP from the supercritical extraction process was mixed with 1 g of celite in an
extraction cell. The PLE extraction process was carried out with the addition of 40 mL of
Figure 1.
Scheme of the sequential biorefinery extraction by scCO
2
and pressurized liquid extraction
(PLE) of calafate pomace to obtain oil, bioactive extract and flour rich in fiber, protein and minerals.
DCP: defatted calafate pomace (waste), DCPw: wet defatted calafate pomace.
Experimental Design for Extraction with scCO2
Optimal conditions of temperature, pressure and time for oil extraction from calafate
pomace were determined by response surface methodology (RSM) following a Box-Behnken
design at three levels of the independent variables: extraction temperature (Te: 30, 45, 60
◦
C),
extraction pressure (P: 300, 350, 400 bar) and extraction time (Ti: 60, 105, 150 min). A total
of 15 experiments were performed. Oil yield was considered a dependent variable based
on the following polynomial equation of second order.
Y=β0+βAA+βBB+βCC+βAB AB +βAC AC +βBC BC +βA2A2+βB2B2+βC2C2(1)
where
β0
is the intercept;
βA
,
βB
and
βC
are the coefficients of the factors;
βAB
,
βAC
and
βBC
are the coefficients of interactions between factors; and
βA
2,
βB2
and
βC2
are the
coefficients of the double interactions. The model was determined using the lack of fit
method and the coefficient of determination R2.
2.4. Pressurized Liquid Extraction
Extraction with PLE was performed as described by Basegmez et al. [
1
] with some
modifications, using Dionex ASE
®
300 equipment (Thermo Fisher Scientific, Waltham,
MA, USA) with adjustment and control of pressure, temperature and time. A total of
4.1 + 0.1 g
of DCP from the supercritical extraction process was mixed with 1 g of celite in
an extraction cell. The PLE extraction process was carried out with the addition of 40 mL of
ethanol/water mixture (1:1 v/v) per cycle. The number of extraction cycles was evaluated
by determining the total phenol content measured by the Folin–Ciocalteu methodology at
the end of each cycle. Each cycle of 5 min was carried out at a standard pressure of 1500 psi
and 25 ◦C.
2.5. Modeling of Extraction Kinetics
2.5.1. Modeling of the Extraction Kinetics with scCO2
Extraction curves of oil with scCO
2
and phenolic extracts with PLE were fitted to the
spline model described by Jesus et al. [
31
] using MATLAB R2020b software (CA, USA). The

Antioxidants 2023,12, 323 5 of 19
model describes three consecutive stages defined by the rate of extraction and associated
with the release mechanisms. The first stage corresponds to a constant extraction rate (cer)
described by convection, the second to a falling extraction rate (fer) defined by convection
and diffusion, and the third to a period controlled by diffusion (dc). Each extraction stage
is described by straight lines represented by Equations (2)–(4).
Y =b0 + b1 ∗t for t ≤tcer (2)
Y = b0 −tcer ∗b2 + (b1 + b2) ∗t for tcer < t ≤tfer (3)
Y = b0 −tcer ∗b2 −tfer ∗b3 + (b1 + b2 + b3) ∗t for tfer < t (4)
where Y corresponds to the oil extraction yield per scCO
2
bi (i = 0, 1, 2, 3) are the linear
coefficients of each stage; tcer is the time for constant extraction rate; and tfer is the time
period for falling extraction rate.
2.5.2. Modeling of the Extraction Kinetics with PLE
Likewise, the modeling of the hydrophilic extraction kinetics by PLE uses the same
Equations (2)–(4), where Y = yield expressed as total polyphenols of the hydrophilic extract.
2.6. Chemical Analysis
Nutritional Characterization
Calafate pomace and the residual product of the successive extractions by scCO
2
and PLE were subjected to proximal analysis according to the official methods (AOAC,
2005) [
32
]. Moisture and ash content were determined by gravimetric methods, proteins by
Kjeldahl, and lipids by the Soxhlet method with petroleum ether. Carbohydrates were deter-
mined by the Antrona colorimetric method after digestion with analytical grade perchloric
acid. The colored complex formed between anthrone and the soluble sugars resulting from
the hydrolysis of starch was read at 760 nm and expressed in g glucose/100 g [33].
2.7. Characterization of the Lipid Extract Obtained by scCO2
2.7.1. Fatty Acid Profile
The fatty acid profile was determined by gas chromatography according to the official
method Ce 2-66 AOCS (1998) [
34
] using an HP-5890 gas chromatograph (Hewlett-Packard,
Palo Alto, CA, USA) with a 50 m long bpx-70 fused silica column, 0.25
µ
m film thickness
and 0.25 mm internal diameter, with an Fid detector, and a split injection system, calibrated
90:10. The fatty acid methyl esters (FAMEs) obtained from Sigma-Aldrich (St. Louis, MO,
USA) were prepared as follows: 100 mg of oil was mixed with 5 mL of 0.5 N sodium
hydroxide solution in methanol and held in a thermoregulated bath for 5 min at 100
◦
C.
Then, 5 mL of 12.5% boron trifluoride in methanol was added and heated for 3 min. Finally,
1.5 mL of petroleum ether and saturated sodium chloride solution were added. After gently
shaking, the mixture was allowed to stand and promote phase separation to extract the
FAME dissolved in petroleum ether.
2.7.2. Tocols
Tocols composed of tocopherols and tocotrienols, were determined by high-performance
liquid chromatography–fluorescence detector (HPLC-FL) as described in the official method
Ce 8-89 AOCS (1998) [
34
]. A solution of 0.5% 2-propanol in hexane was used as the mobile
phase. To prepare the sample, 100 mg of extracted oil was weighed into a 10 mL amber flask
and brought to volume with hexane. Measurement runs were made for 35 min, injecting
80
µ
L of the sample. Tocols content was determined using
α
,
β
,
γ
and
δ
tocopherol and
tocotrienol standard solutions (Calbiochem Merck, Darmstadt, Germany). The results were
expressed in µg/g oil.

Antioxidants 2023,12, 323 6 of 19
2.7.3. Saponification Value
The saponification value (SV) was determined by the official method Cd 3-25 AOCS
(1993) [
35
]. Briefly, 5 g of oil and 50 mL of alcoholic KOH solution were added to an
Erlenmeyer flask connected to an air condenser to boil the mixture for 30 min. Once cool,
the mixture was titrated with 0.5 M HCl using phenolphthalein. The results were expressed
in mg KOH/g oil.
2.7.4. Iodine Value
For the iodine value (YV), the Wijs method was used, as described in the official
method Cd 1d-25 AOCS (1993) [
35
]. One hundred milligrams of completely dry and
filtered lipid extract was dissolved in 15 mL of chloroform, 25 mL of Wijs iodide solution
was added, and the samples were left to stand in the dark at 25
◦
C. Subsequently, 20 mL
of KI solution was added, and the solution was titrated under constant stirring using a
standard 0.1 M Na2S2O3 solution until the yellow color disappeared. Then, 1 to 2 mL of
starch indicator solution was added, and the titration was continued until the blue color
disappeared. The results were expressed in g I2/100 g oil.
2.7.5. Free Fatty Acids
Free fatty acids (FFAs) were determined by titration according to the official method
Ca 5a-40 AOCS (2009) [
36
] by mixing 10 g of the extracted oil with 1 mL of ethanol and
1 mL of diethyl ether neutralized with 0.1 N. Three drops of phenolphthalein were added,
and titration was carried out with 0.1 N KOH until the color of the sample changed. The
results were expressed as g oleic acid/100 g oil.
2.7.6. Peroxide Value
The peroxide value (PV) was determined as described in the official method Cd 8-53
AOCS (2009) [
36
]. Five grams of oil and 30 mL of a 3:2 mixture of acetic acid:chloroform
were added to a 250 mL Erlenmeyer flask containing 0.5 mL of saturated KI solution, and
the sample was slowly titrated with a 0.1 M Na
2
S
2
O
3
solution until the yellow color almost
disappeared. Finally, 0.5 mL of a 1% starch solution was added, and the titration continued
under vigorous stirring until all the I2 was released from the chloroform layer. The results
were expressed as milli-equivalents of O2/kg oil.
2.7.7. Oil Color Analysis
The determination of the oil color was carried out according to the official method
Cc 13e-92 AOCS (2009) [
36
] using an oil tintometer (Lovibond
®
brand PFXi-195/1, FL,
USA). First, a cell was standardized to zero with no sample, and then the yellow standard
was read, and finally, the oil sample. The equipment gives the color parameters of the
oils measured in CIEL*a*b* coordinates where L*: is the luminosity (L* = 100, perfect
white;
L* = 0
, black); a* measures redness (a* > 0, red; a* = 0, gray; a* < 0, green); and b*
green-yellow tendency (b* > 0, yellow; b* = 0, gray; b* < 0, green).
2.8. Characterization of the Bioactive Extract Obtained by Pressurized Liquid Extraction
2.8.1. Total Phenols
Determination of the total polyphenol content (TPC) was carried out by means of
the Folin–Ciocalteu method, as described by Singleton and Rossi (1965) [
37
]. A total of
0.1 mL of the hydroalcoholic extract obtained by PLE was mixed with 4.9 mL of distilled
water and 0.5 mL of Folin–Ciocalteu reagent. The sample was left to stand for 3 min, and
1.7 mL of 20% w/vanhydrous sodium carbonate solution was added. The absorbance of
the sample was measured at 765 nm. The concentration of total phenols was determined by
means of a calibration curve with gallic acid solutions between 50 and 800
µ
g/mL, and the
results were expressed as mg gallic acid equivalents per dry weight of extract (mg GAE/g
extract dw).

Antioxidants 2023,12, 323 7 of 19
2.8.2. 2,2-Diphenyl-1-Picrylhydrazyl Test
The antiradical capacity was measured by the 2,2-diphenyl-1-picrylhydrazyl (DPPH)
test, as described by Brand-Williams et al. [
38
]. Briefly, 0.1 mL of extract and 3.9 mL of
1 mg/mL DPPH solution were added to a 15 mL tube. The solution was diluted to an
absorbance range of 0.480 to 0.600. The mixture was left to stand in the dark for 30 min at
room temperature. Subsequently, the absorbance at 517 nm was measured. Results were
expressed as mg quercetin-3-rhamnoside per gram of dry extract (mgc3-o-glu/g dw).
The efficiency of the PLE extract as a free radical scavenger was determined by means
of Equation (5).
%Discoloration =Ac−Am
Ac
·100 (5)
where Acis the absorbance of the control and Amis the absorbance of the sample.
2.8.3. Oxygen Radical Absorbance Capacity Test
The antioxidant capacity was measured by the oxygen radical absorbance capacity
(ORAC) method according to the methodology described by Huang et al. [
39
]. Twenty-five
microliters of sample and 150
µ
L of fluorescein solution were incubated for 30 min at
37
◦
C. Subsequently, 25
µ
L of 4.6% AAPH solution (2,2
0
-azobis(2-methylpropionamidine)
dihydrochloride) in phosphate buffer was added to initiate the reaction. The fluorescence
intensity of the samples was recorded every 1 min using a 485 nm excitation filter with
a 20 nm bandwidth and a 528 nm emission filter with a 20 nm bandwidth. The antiox-
idant capacity by ORAC was calculated by interpolation of the net area generated by
the variation of the fluorescence intensity of the fluorescein of the samples in the linear
regression of the areas under the curve generated by the kinetic variation of the fluorescein
that was incubated with different concentrations of a Trolox standard (6-hydroxy-2,5,7,8-
tetramethylchroman-2-carboxylic acid).
2.8.4. Total Anthocyanins
The total anthocyanin content (TAC) was determined by the differential pH method
proposed by Lee et al. [
40
] with some modifications. Extracts were diluted with the buffers
KCl 0.025 M pH 1.0 and sodium acetate 0.4 M, pH 4.5, adjusting the pH of both solutions
with HCl 0.01 M. Then, the absorbances of the diluted extracts were measured at 530 and
700 nm. These values were used in Equations (6) and (7) to obtain the anthocyanin content
in equivalents of cyanidin-3-glucoside (EC-3G).
A= (A530 −A700 )pH1, 0 −(A530 −A700 )(6)
C−3Gmg
ml =A·MW·D·103
(ε·I)(7)
where Ais obtained from Equation (6); MW is the molecular weight of cyanidin-3-glucoside
(449.2 g/mol); Dis the extract dilution factor; 10
3
is the conversion factor from grams to
milligrams;
ε
is the molar extinction coefficient of cyanidin-3-glucoside; and Iis the path
length in 1 cm.
2.9. Statistical Analysis
The experiments and the characterization of the oil extracted by scCO
2
and the hy-
droalcoholic extract obtained by PLE were carried out in triplicate. Results are expressed as
means with standard deviation. For the response surface analysis, the analysis of variance
(ANOVA) was considered with a confidence level of 95%, using the Statgraphics Centurion
XVI software.

Antioxidants 2023,12, 323 8 of 19
3. Results
3.1. Response Surface Methodology Optimization of Oil Extraction by scCO2
A graph of the response surface for the extraction process of oil from calafate’s pomace
by means of scCO
2
with the variables extraction temperature (te), extraction time (ti) and
pressure (P) is shown in Figure 2. It is evident that the highest yields (11.5%) were presented
for higher values of ti and te, while the lowest yields (9.5%) were for the entire range of
te, when ti had the minimum values. According to the Pareto diagram (Figure 2), it is
clearly shown that time, its quadratic interaction, temperature and pressure influence the
extraction in a positive way, that is, these factors increase the yield.
Antioxidants 2023, 12, x FOR PEER REVIEW 8 of 21
where A is obtained from Equation (6); MW is the molecular weight of cyanidin-3-gluco-
side (449.2 g/mol); D is the extract dilution factor; 103 is the conversion factor from grams
to milligrams; ε is the molar extinction coefficient of cyanidin-3-glucoside; and I is the
path length in 1 cm.
2.9. Statistical Analysis
The experiments and the characterization of the oil extracted by scCO2 and the hy-
droalcoholic extract obtained by PLE were carried out in triplicate. Results are expressed
as means with standard deviation. For the response surface analysis, the analysis of vari-
ance (ANOVA) was considered with a confidence level of 95%, using the Statgraphics
Centurion XVI software.
3. Results
3.1. Response Surface Methodology Optimization of Oil Extraction by scCO2
A graph of the response surface for the extraction process of oil from calafate’s pom-
ace by means of scCO2 with the variables extraction temperature (te), extraction time (ti)
and pressure (P) is shown in Figure 2. It is evident that the highest yields (11.5%) were
presented for higher values of ti and te, while the lowest yields (9.5%) were for the entire
range of te, when ti had the minimum values. According to the Pareto diagram (Figure 2),
it is clearly shown that time, its quadratic interaction, temperature and pressure influence
the extraction in a positive way, that is, these factors increase the yield.
Figure 2. Response surface diagram of the experimental design of oil extraction by supercritical CO2
(indicating optimization zone in red) and Pareto diagram of interactions between variables. Te: ex-
traction temperature, ti: extraction time, and P: pressure.
The results of the ANOVA, carried out for the data obtained from the scCO2 of pom-
ace of calafate oil, showed that the model expressed by Equation (8) had a determination
coefficient (R2) of 93.9% and a nonsignificant lack of fit (p value > 0.05), which indicates
that the model adequately represents the experimental data.
Yield = 10.72 + 0.2733 ∗ Te + 0.2557 ∗ P + 0.4817 ∗ ti−0.2501 ∗ Te2−0.1873 ∗ Te ∗ P +
0.4526 ∗ Te ∗ ti − 0.196 * P2−0.534 * ti2
(8)
The experimental optimization for the theoretical lipid extract yield of 11.15% dry
weight (d.w.) yielded optimal extraction conditions with scCO2 at a temperature of 60 °C,
358.5 bar and 144.6 min.
Figure 2.
Response surface diagram of the experimental design of oil extraction by supercritical
CO
2
(indicating optimization zone in red) and Pareto diagram of interactions between variables.
Te: extraction temperature, ti: extraction time, and P: pressure.
The results of the ANOVA, carried out for the data obtained from the scCO
2
of pomace
of calafate oil, showed that the model expressed by Equation (8) had a determination
coefficient (R
2
) of 93.9% and a nonsignificant lack of fit (pvalue > 0.05), which indicates
that the model adequately represents the experimental data.
Yield = 10.72 + 0.2733 ∗Te + 0.2557 ∗P + 0.4817 ∗ti−0.2501 ∗Te2−0.1873 ∗Te ∗P +
0.4526 ∗Te ∗ti −0.196 * P2−0.534 * ti2 (8)
The experimental optimization for the theoretical lipid extract yield of 11.15% dry
weight (d.w.) yielded optimal extraction conditions with scCO2at a temperature of 60 ◦C,
358.5 bar and 144.6 min.
The oil yield found in this study is higher than the 8.7% reported in supercritical
extraction of cranberry pomace at temperatures of 53
◦
C, 158 min and 42.4 MPa [
26
]. On
the other hand, for comparison, yields of 19.1, 14.6 and 6.6% oil have been obtained in
washed, unwashed and dried berry pomaces from Viburnum opulus L., respectively, with
optimal scCO2conditions of 55–57 MPa, 120–131 min and 50 ◦C [41].
3.1.1. Kinetic Model of Oil Extraction by Supercritical CO2
The spline model suggests that the yield of oil extraction from calafate pomace tends to
increase over time (see Figure 3), although the greatest changes occurred between times 0 to
30 min, when the yield values went from 0 to 11%. These results are within the values for oil
extraction of bilberry, blackcurrant, raspberry, highbush blueberry, lingonberry, cranberry,
and American cranberry pomaces using scCO2which vary between 12 and 18% [42].

Antioxidants 2023,12, 323 9 of 19
Antioxidants 2023, 12, x FOR PEER REVIEW 9 of 21
The oil yield found in this study is higher than the 8.7% reported in supercritical
extraction of cranberry pomace at temperatures of 53 °C, 158 min and 42.4 MPa [26]. On
the other hand, for comparison, yields of 19.1, 14.6 and 6.6% oil have been obtained in
washed, unwashed and dried berry pomaces from Viburnum opulus L., respectively, with
optimal scCO
2
conditions of 55–57 MPa, 120–131 min and 50 °C [41].
3.1.1. Kinetic Model of Oil Extraction by Supercritical CO
2
The spline model suggests that the yield of oil extraction from calafate pomace tends
to increase over time (see Figure 3), although the greatest changes occurred between times
0 to 30 min, when the yield values went from 0 to 11%. These results are within the values
for oil extraction of bilberry, blackcurrant, raspberry, highbush blueberry, lingonberry,
cranberry, and American cranberry pomaces using scCO
2
which vary between 12 and 18%
[42].
Figure 3. Experimental extraction curves fitted to the spline model. (a) Oil extraction curve with
supercritical CO
2
(358 bar and 60 °C). (b) Extraction of bioactive components from defatted calafate
pomace (DCP) by pressurized liquid extraction (1500 psi and 25 °C) with an ethanol/water mixture
(1:1 v/v).
At higher times, the performance remained practically constant and with slight in-
creases that reached 11.15 after 165 min. The parameters tcer, tfer and Mcer defined by the
fit to the spline model are shown in Table 1. The calculated values for the parameters
describe very precisely the kinetics of the oil extraction curve for the two slope regions
between 0–30 min and 30–165 min. The spline model is a simple strategy to model extrac-
tion curves [43]. Despite corresponding to an empirical model [43], the experimental and
modeled curves manage to define three regions: a constant rate of extraction (cer) associ-
ated with mass transfer by convection, a decrease in the rate of extraction (fer) described
by control of mass transfer by diffusion and convection, and a period where the extraction
rate is controlled by diffusion (dc) [31]. This stage is identified by the mass transfer rate,
defined as Mcer, corresponding to parameter b1 of the spline model equations. In general,
the greatest extraction occurs at this stage, with values between 70 and 90% being ob-
served [31]. In the extraction of calafate oil during the cer period, an 88.1% extraction was
obtained. The tcer defines the minimum time that an extraction cycle must last, which in
this case corresponded to 29.8 min. This allows us to reduce the extraction time and the
consumption of solvents [44].
Figure 3.
Experimental extraction curves fitted to the spline model. (
a
) Oil extraction curve with
supercritical CO
2
(358 bar and 60
◦
C). (
b
) Extraction of bioactive components from defatted calafate
pomace (DCP) by pressurized liquid extraction (1500 psi and 25
◦
C) with an ethanol/water mixture
(1:1 v/v).
At higher times, the performance remained practically constant and with slight in-
creases that reached 11.15 after 165 min. The parameters tcer, tfer and Mcer defined by
the fit to the spline model are shown in Table 1. The calculated values for the parameters
describe very precisely the kinetics of the oil extraction curve for the two slope regions
between 0–30 min and 30–165 min. The spline model is a simple strategy to model extrac-
tion curves [
43
]. Despite corresponding to an empirical model [
43
], the experimental and
modeled curves manage to define three regions: a constant rate of extraction (cer) associated
with mass transfer by convection, a decrease in the rate of extraction (fer) described by
control of mass transfer by diffusion and convection, and a period where the extraction rate
is controlled by diffusion (dc) [
31
]. This stage is identified by the mass transfer rate, defined
as Mcer, corresponding to parameter b1 of the spline model equations. In general, the
greatest extraction occurs at this stage, with values between 70 and 90% being observed [
31
].
In the extraction of calafate oil during the cer period, an 88.1% extraction was obtained.
The tcer defines the minimum time that an extraction cycle must last, which in this case
corresponded to 29.8 min. This allows us to reduce the extraction time and the consumption
of solvents [44].
Subsequently, between 29.8 and 93.2 min, a second slope associated with the fer period
is obtained, and at 93.2 min, a third slope corresponding to the dc period is obtained.
The extraction rates in the periods fer (constant b2) and dc (constant b3) correspond to
negative values, indicating that the diffusive extraction mechanisms were irrelevant, and
the greatest contribution to the extraction yield is given by convection during the cer stage.
This behavior has been studied in a similar way in elderberry, where three phases in the
extraction kinetics were identified (Kitryte et al.) [
20
]. Similar results were reported by
Tamkute et al. [
26
] for the extraction of cranberry pomace and for the graph of the extraction
kinetics of currant pomace oil [
1
] and for several other fruits and berries in which it has
been concluded that the rate of extraction is controlled by internal diffusion through the
cell walls [45].

Antioxidants 2023,12, 323 10 of 19
Table 1.
Spline model parameters for oil extraction by scCO
2
and polyphenols by PLE from calafate.
Parameter scCO2
Oil Extraction
PLE
Polyphenol Extract
tCER (min) 29.8 5.7
tFER (min) 93.2 17.5
b06.17 ×10−4
(kg oil)
0
(g GAE)
b1—MCER 4.17 ×10−4
(kg oil/min)
0.526
(g GAE/min)
b2—MFER
−4.01 ×10−4
(kg oil/min)
−0.446
(g GAE/min)
b3—Mdc
−4.90 ×10−6
(kg oil/min)
−0.048
(g GAE/min)
error 0.220 0.0096
R20.9890 0.9999
GAE: gallic acid equivalents.
3.1.2. Kinetic Model of Extraction of Bioactive Compounds by Pressurized
Liquid Extraction
Similar to the extraction curve of calafate oil by scCO
2
, the spline model adequately
described (R
2
> 0.9999) the extraction curve of water-soluble bioactive components of the
DCP defatted residue PLE (Figure 1). Figure 3b shows the adjusted experimental curve for
extraction by PLE based on the content of total phenols, which comprised six extraction
cycles of 5 min each applied to the same sample of defatted calafate pomace (DCP). In
the three initial PLE extraction cycles applied to the same DCP residue, the quantified
total polyphenol content was 2.81, 0.56 and 0.27 g GAE/100 g d.w., respectively, giving
an accumulated total of 3.37 g GAE/100 g bw. Because the content of total phenols in the
extract after carrying out the sixth cycle only gave an accumulated
4.32 g GAE/100 g d.w.
,
it could be concluded that it was enough to carry out the third extraction to obtain almost
85% of the extract rich in polyphenols. Modeling the experimental curve using the spline
method allowed us to determine the three extraction stages defined by tcer and tfer (Table 1).
For the optimal extraction time, defined at 5.7 min (tcer), an extraction yield of 69.1% was
obtained, while at the end of the fer period (tfer = 17.5 min), the yield reached 90.9%.
Similar results were obtained when extracting polyphenols from orange peel using PLE
and fitting the three-stage spline model [44].
On the other hand, the extraction times and yield obtained with PLE were more
efficient for DCP than those obtained by applying ultrasound assisted PLE to the extraction
of phenolic compounds from passion fruit bagasse [
43
]. This would indicate that the
operating conditions applied in the DCP residue (1500 psi = 10.3 MPa and 25
◦
C) facilitated
entry of the solvent into the plant structure. In addition, the extraction rates of the period
fer (constant b2) and dc (constant b3), associated with diffusive mechanisms, were not
relevant in the total extraction since only with three cycles of 5 min of extraction, over
85% of the content of bioactive compounds expressed as total phenols of calafate pomace
was obtained.
3.2. Chemical Characterization of the Products
3.2.1. Characterization of Pomace Oil from Calafate
Table 2shows the characterization of the oil extracted from calafate pomace under
optimal operating conditions using the supercritical fluid methodology. Mainly monounsat-
urated and polyunsaturated fatty acids (MUFAS and PUFAS) were identified with a value
of approximately 88.7% of the total methyl esters. Some benefits of the consumption of
ω
3
and
ω
6 have been studied, including the regulation of blood pressure, vascular function,
control of tumor cell growth and help in neuronal development [
46
,
47
]. The content of
MUFAS given mainly by oleic acid was very similar to that of blackberry and close to that
of cranberry and goldenberry oils [
48
–
50
]. On the other hand, the total PUFAS content was

Antioxidants 2023,12, 323 11 of 19
very similar to that of goldenberry (Physalis peruviana L.) [
50
]. A good ratio of
ω
-6/
ω
-3
(1:1.2) was evidenced, with high values of
α
-linolenic acid (36.7
±
0.2%) and linoleic acid
(30.0 ±0.1%).
Table 2.
Chemical characterization of the oil extracted from calafate pomace by supercritical CO
2
and
its comparison with fatty acids from other berries.
Methyl Esters (%)
Calafate
Pomace Maqui [51] Murta [52] Cranberry [48]Blackberry
[49]
Rosa Mosqueta
[50]
Goldenberry
[53]
C16:0 8.1 ±0.8 9.1 ±0.0 2.5 ±0.3 3.7 ±0.3 4.6 ±0.4 3.1 ±0.1 12.4 ±0.1
C18:0 2.7 ±0.1 3.0 ±0.0 0.8 ±0.1 1.3 ±0.2 4.2 ±0.1 1.9 ±0.2 4.3 ±0.0
C18:1 22.2 ±0.7 38.3 ±0.1 7.7 ±0.2 15.9 ±0.3 19.9 ±0.5 14.3 ±0.1 16.5 ±0.1
C18:2ω6 30.0 ±0.1 42.7 ±0.0 88.2 ±0.9 55.9 ±0.3 58.6 ±0.8 44.2 ±0.1 63.2 ±0.2
C18:3ω3 36.7 ±0.2 0.9 ±0.0 0.8 ±0.7 22.8 ±0.5 9.1 ±0.3 31.7 ±0.8 0.4 ±0.0
FAS 10.8 12.1 3.3 5.0 8.8 5.0 16.7
MUFAS 22.2 38.3 7.7 15.9 19.9 14.3 16.5
PUFAS 66.7 43.6 89 78.7 81.4 75.9 63.6
ω6/ω3 1:1.2 1:0.0 1:0.0 1:0.4 1:0.1 1:0.7 1:0.0
Physical-chemistry parameters
Calafate
pomace Blackberry [48]Blackcurrant
[48]Blueberry [48]Strawberry
[48]Grapeseed [53]Golden berry
[53]
YV 159 ±1 148 ±1 177 ±4 167 ±1 180 ±1 127.5 ±4.5 116.3
SV 176 ±10 190 ±1 190 ±5 190 ±1 194 ±1 188 ±11.3 Nd
Calafate
Pomace Maqui [48] Blackberry [54] Cranberry [54] Nut [55] Grapeseed Goldenberry
L* 0.9 ±0.0 39.4 ±0.8 10.1 ±0.2 1.8 ±0.1 93.68 _ _
a* 1.2 ±0.0 −0.9 ±0.0 7.7 ±1.2 2.5 ±0.2 −3.6 ±0.0 _ _
b* 4.7 ±0.0 10.1 ±0.6 16.2 ±0.3 3.0 ±0.2 18.2 ±0.0 _ _
Color Dark yellow Soft yellow Dark red Dark yellow Bright yellow
Quality Parameters
Calafate Blueberry [56]Cranberry [56]Raspberry [56]Nut [55]Grapeseed Goldenberry
[53]
Pomace
PV 8.6 ±0.4 8.7 ±0.0 7.3 ±0.1 8.4 ±0.0 1.2 ±0.0 _ nd
FFA 0.6 ±0.1 2.1 ±0.0 1.7 ±0.0 4.1 ±0.0 0.4 ±0.0 _ 2.9
Data are expressed as the mean
±
standard deviation (n= 3). YV= iodine value (g I
2
/100 g oil); SV= saponification
value (mg KOH/g lipid); PV = peroxide value (milli-equivalent O
2
/Kg lipid); FFA = free fatty acid (g Oleic
acid/100 g lipid).
The linoleic acid content of calafate pomace oil was lower compared to maqui, murta,
blackberry, cape gooseberry and rosehip berries but higher in linolenic acid, which trans-
lates into an optimal
ω
-6/
ω
-3 [
48
,
51
,
52
,
55
]. On the other hand, it has been reported in
oils from other fruit seeds from the southern zone of Chile, such as blackberry (6.3:1) and
blueberry (1.5:1), a greater content of
α
-linolenic acid (18:3
ω
3) than in calafate pomace
oil [57,58].
3.2.2. Tocols Content
Table 3presents the tocopherol and tocotrienol content of the oil extracted from calafate
pomace by scCO
2
and its comparison with the tocols of other oils obtained from different
berry seeds. In calafate oil extracted by scCO
2
, a total content of 406.4 ppm tocols was
determined, composed of 18, 31 and 50%
α
-tocopherol (
α
-T),
α
-tocotrienol (
α
-T3) and
γ-tocotrienol (γ-T3), respectively.

Antioxidants 2023,12, 323 12 of 19
Table 3.
Tocopherol and tocotrienol content* of oil extracted from calafate pomace by supercritical
CO2and comparison with tocols from different berry seed oils.
Tocols (µg/g Oil)
α−Tα−T3 γ−Tγ−T3 δ−Tδ−T3 Total
Calafate
pomace 75.4 ±3.8 127.7 ±4.0 _ 203.7 ±10.5 _ _ 406.4
Maqui [59] 169.3 ±11.3 323.8 ±20.3 56.7 ±2.9 5.7 ±1.0 13.5 ±3.5 53.9 ±7.4 622.9
Blackberry [60] 25.4 ±6.5 _ 1.311 ±15.5 20.0 ±1.7 31.7 ±1.5 _ 1.388
Blueberry [60] 4.4 ±0.2 _ 34.4 ±0.1 330.4 ±11.4 _ 6.0 ±1.0 375.2
Cranberry [60] 48.3 ±4.5 152.7 ±5.8 90.7 ±2.1 1.235 ±6.1 _ _ 1.532
Rasberry [60] 407.0 ±22.9 _ 1.640 ±86.9 7.2 ±0.3 53.3 ±3.2 _ 2.112
Strawberry [60] _ _ 260.3 ±13.7 _ 20.0 ±3.8 _ 280.3
* Data are expressed as the mean
±
standard deviation (n= 3). [
60
].
α
-T,
γ
-T,
δ
-T;
α−
,
γ−
,
δ
- Tocopherols.
α
-T3,
γ-T3, δ-T3; α−,γ−,δ−Tocotrienols.
The oil obtained presents mainly
γ
-T3 tocols in its composition, which has been shown
to have a higher antioxidant capacity than
α
-tocopherol at high temperatures when added
to corn oil [
61
]. In vegetable oils, tocotrienols are scarce, particularly
γ
-T3, but cranberry
and blueberry seed oils, as well as calafate oil, contain
γ
-T3. Calafate has a lower content
of
α
-T and
α
-T3 compared to other berries, such as raspberry and maqui [
48
,
59
]. On the
other hand, calafate pomace did not present
γ
-T, unlike maqui, and most other berries,
including rosehip, where it is present at approximately 78% of the total content (1460
µ
g/g)
of tocols [
48
,
57
]. The tocols present in the calafate oil extract by scCO
2
can be observed in n
the chromatogram of Figure 4.
Antioxidants 2023, 12, x FOR PEER REVIEW 13 of 21
(1460 µg/g) of tocols [48,57]. The tocols present in the calafate oil extract by scCO
2
can be
observed in n the chromatogram of Figure 4.
Figure 4. Chromatogram of the tocols present in the calafate sample by scCO
2
. α-T: Alfa-tocopherol,
α-T3: alpha-tocotrienol, γ-T3: gamma-tocotrienol.
3.2.3. Quality Characteristics of Calafate Oil
The polyunsaturation degree of calafate pomace oil (Figure 2) with a YV = 159 was
within the range reported by Firestone (2012) for blackberry, blackcurrant, blueberry and
blackberry oils [48]. Strawberry’s YV (116 to 180) is justified by its high unsaturation pro-
vided by linoleic and α-linolenic acid [48]. The SV of 176 was representative of the average
molecular weight of the fatty acids in the oil but lower than that of blueberry, raspberry
[60] and other reported berry and fruit seed oils of similar composition [48,53,58,60]. This
may be due to the analysis of the saponification value, which also includes the free fatty
acids present in the oil. Regarding the quality characteristics of the lipid extract (Table 2),
the values of the PV and FFA in calafate seed oil were 8.6 ± 0.4 meq O
2
/kg and 0.4 ± 0.1
mg/kg, respectively, which are within the range of fresh oils according to Chilean legisla-
tion [53] and values reported by other authors [48]. On the other hand, in addition to the
oil present in berry seeds, high percentages of essential oils have been found in tissues
from other parts of dark blue berries, including α-pinene (11.1%), linalool (11.6%), α-ter-
pineol (15.7%), methyl eugenol (6.2%) and geraniol (3.7%) and in white berry oils,
mirtenyl acetate (20.3%) [56].
3.2.4. Color of Calafate Oil
The colors of the oils extracted by scCO
2
were compared with those of other cold-
pressed oils (Table 2). The L*a*b* color parameters indicated that the calafate pomace oil
presented a dark yellow tone very similar to the color of cranberry but darker than that of
maqui oil [51,54,62]. Possibly, the dark color of the oil was due to the migration of pig-
ments such as carotenoids, chlorophylls, anthocyanins or other flavonoids from residues
of skin and pulp. Components that could be present in the plant tissue that makes up
pomace of calafate and influence the color of calafate seed oil during extraction with
scCO
2
.
3.3. Characterization of the Phenolic Extracts Obtained by PLE
Figure 5 shows the content of TPC, TAC, DPPH, and yield of calafate pomace com-
pared with pomaces of other berries obtained by pressing [42,63,64]. The yield of the ex-
tracts obtained from calafate pomace obtained by PLE was close to the yield reported for
extracts obtained by cold pressing of other berries but lower in the cases of blueberry, bog
cranberry and bilberry. On the other hand, the phenol content of the calafate pomace ex-
tract obtained by PLE was similar to that of most berry pomaces, e.g., 80% of the blueberry
Figure 4.
Chromatogram of the tocols present in the calafate sample by scCO
2
.
α
-T: Alfa-tocopherol,
α-T3: alpha-tocotrienol, γ-T3: gamma-tocotrienol.
3.2.3. Quality Characteristics of Calafate Oil
The polyunsaturation degree of calafate pomace oil (Figure 2) with a YV = 159 was
within the range reported by Firestone (2012) for blackberry, blackcurrant, blueberry and
blackberry oils [
48
]. Strawberry’s YV (116 to 180) is justified by its high unsaturation
provided by linoleic and
α
-linolenic acid [
48
]. The SV of 176 was representative of the
average molecular weight of the fatty acids in the oil but lower than that of blueberry, rasp-
berry [
60
] and other reported berry and fruit seed oils of similar
composition [48,53,58,60]
.
This may be due to the analysis of the saponification value, which also includes the free
fatty acids present in the oil. Regarding the quality characteristics of the lipid extract
(Table 2), the values of the PV and FFA in calafate seed oil were 8.6
±
0.4 meq O
2
/kg and
0.4 ±0.1 mg/kg
, respectively, which are within the range of fresh oils according to Chilean
legislation [
53
] and values reported by other authors [
48
]. On the other hand, in addition
to the oil present in berry seeds, high percentages of essential oils have been found in
tissues from other parts of dark blue berries, including
α
-pinene (11.1%), linalool (11.6%),

Antioxidants 2023,12, 323 13 of 19
α
-terpineol (15.7%), methyl eugenol (6.2%) and geraniol (3.7%) and in white berry oils,
mirtenyl acetate (20.3%) [56].
3.2.4. Color of Calafate Oil
The colors of the oils extracted by scCO
2
were compared with those of other cold-
pressed oils (Table 2). The L*a*b* color parameters indicated that the calafate pomace oil
presented a dark yellow tone very similar to the color of cranberry but darker than that of
maqui oil [
51
,
54
,
62
]. Possibly, the dark color of the oil was due to the migration of pigments
such as carotenoids, chlorophylls, anthocyanins or other flavonoids from residues of skin
and pulp. Components that could be present in the plant tissue that makes up pomace of
calafate and influence the color of calafate seed oil during extraction with scCO2.
3.3. Characterization of the Phenolic Extracts Obtained by PLE
Figure 5shows the content of TPC, TAC, DPPH, and yield of calafate pomace compared
with pomaces of other berries obtained by pressing [
42
,
63
,
64
]. The yield of the extracts
obtained from calafate pomace obtained by PLE was close to the yield reported for extracts
obtained by cold pressing of other berries but lower in the cases of blueberry, bog cranberry
and bilberry. On the other hand, the phenol content of the calafate pomace extract obtained
by PLE was similar to that of most berry pomaces, e.g., 80% of the blueberry and bilberry
pomaces (Figure 5). The results indicated that the antiradical activity against DPPH
of calafate pomace was considerably higher than that reported for most pressed berry
pomaces [64].
Antioxidants 2023, 12, x FOR PEER REVIEW 14 of 21
and bilberry pomaces (Figure 5). The results indicated that the antiradical activity against
DPPH of calafate pomace was considerably higher than that reported for most pressed
berry pomaces [64].
Figure 5. Yield and composition of the pomace extract from calafate obtained by pressurized liquid
extraction and other hydroalcoholic extracts from the pomace of cold-pressed berries. * Muceniece
et al. [65]; ** Klavins et al. [42].
These differences in extraction yield, polyphenol and anthocyanin content, and anti-
oxidant capacity could be because calafate pomace is a residue obtained from sieving the
freeze-dried fruit, in which the proportion of skin and pulp would be lower than that
present in pomaces obtained by pressing berries. The lower content of pulp and skin
would be reflected in a lower content of anthocyanins in the calafate pomace extract since
these compounds are found mainly in the skin [3]. In blueberries, it has been observed
that pressing and grinding prior to extraction break the epidermal tissue where the antho-
cyanins associated with the cell wall are found, increasing extraction [65]. Several studies
have reported that the main anthocyanins in calafate are delphinidin-3-glucoside, pe-
tunidin-3-glucoside and malvidin-3-glucoside, with a smaller proportion of other poly-
phenols such as flavonols and flavan-3-noles [17,62]. On the other hand, it should be con-
sidered that some of the bioactive compounds of calafate pomace could have been
dragged during the extraction of oil with scCO
2
.
Figure 5.
Yield and composition of the pomace extract from calafate obtained by pressurized liquid
extraction and other hydroalcoholic extracts from the pomace of cold-pressed berries. * Muceniece
et al. [65]; ** Klavins et al. [42].

Antioxidants 2023,12, 323 14 of 19
These differences in extraction yield, polyphenol and anthocyanin content, and an-
tioxidant capacity could be because calafate pomace is a residue obtained from sieving
the freeze-dried fruit, in which the proportion of skin and pulp would be lower than that
present in pomaces obtained by pressing berries. The lower content of pulp and skin would
be reflected in a lower content of anthocyanins in the calafate pomace extract since these
compounds are found mainly in the skin [
3
]. In blueberries, it has been observed that press-
ing and grinding prior to extraction break the epidermal tissue where the anthocyanins
associated with the cell wall are found, increasing extraction [
65
]. Several studies have
reported that the main anthocyanins in calafate are delphinidin-3-glucoside, petunidin-3-
glucoside and malvidin-3-glucoside, with a smaller proportion of other polyphenols such
as flavonols and flavan-3-noles [
17
,
62
]. On the other hand, it should be considered that
some of the bioactive compounds of calafate pomace could have been dragged during the
extraction of oil with scCO2.
Figure 6compares the composition, antioxidant capacity and extraction yield of
calafate pomace by PLE with the yield reported for hydroalcoholic extracts of fresh berry
fruits [
6
,
7
]. The yield (2.6%) of the crude extract, obtained from calafate pomace by PLE,
was close to half the yield obtained from the whole calafate fruit and at least a third of the
yield of other fresh Chilean berries [
7
]. The anthocyanin content of the pomace was only
15% of that reported for the calafate fruit and was only higher than the content reported for
murta and chequeen [
8
]. On the other hand, the antioxidant power given by the ORAC
method for the calafate pomace extract was 85% with respect to that reported for the fresh
calafate fruit and 71% of that presented by the fresh maqui fruit [6].
Antioxidants 2023, 12, x FOR PEER REVIEW 15 of 21
Figure 6 compares the composition, antioxidant capacity and extraction yield of
calafate pomace by PLE with the yield reported for hydroalcoholic extracts of fresh berry
fruits [6,7]. The yield (2.6%) of the crude extract, obtained from calafate pomace by PLE,
was close to half the yield obtained from the whole calafate fruit and at least a third of the
yield of other fresh Chilean berries [7]. The anthocyanin content of the pomace was only
15% of that reported for the calafate fruit and was only higher than the content reported
for murta and chequeen [8]. On the other hand, the antioxidant power given by the ORAC
method for the calafate pomace extract was 85% with respect to that reported for the fresh
calafate fruit and 71% of that presented by the fresh maqui fruit [6].
Figure 6. Oxygen radical absorbance capacity (ORAC), total polyphenols (TPC), total anthocyanin
content (TAC), and yields of calafate pomace extracts obtained by pressurized liquid extraction
compared with hydroalcoholic extracts from different Patagonian berry fruits. * Brito et al. [8] and
** Ruiz et al. [6].
These results for the calafate pomace extract agree with the higher antioxidant capac-
ity shown by calafate fruit extracts compared to other fruits and berries native to Patago-
nia [16]. Similar values have been reported for calafate extracts collected between Decem-
ber (2009) and February (2010) from different localities (Temuco, Lonquimay, Mañiguales,
El Blanco) in Chilean Patagonia (Aysén, XI Region) that were in a range of 3.3 at 9.4 mg
TE/g d.w. [11].
Figure 6.
Oxygen radical absorbance capacity (ORAC), total polyphenols (TPC), total anthocyanin
content (TAC), and yields of calafate pomace extracts obtained by pressurized liquid extraction
compared with hydroalcoholic extracts from different Patagonian berry fruits. * Brito et al. [
8
] and
** Ruiz et al. [6].

Antioxidants 2023,12, 323 15 of 19
These results for the calafate pomace extract agree with the higher antioxidant capacity
shown by calafate fruit extracts compared to other fruits and berries native to Patagonia [
16
].
Similar values have been reported for calafate extracts collected between December (2009)
and February (2010) from different localities (Temuco, Lonquimay, Mañiguales, El Blanco)
in Chilean Patagonia (Aysén, XI Region) that were in a range of 3.3 at 9.4 mg TE/g d.w. [
11
].
Other factors that influence the phenolic composition and antioxidant capacity of
berries are variety, genetics, maturity, plant nutrition, harvest season and climate [
3
,
5
].
Climate is a fundamental factor considering the environmental stresses associated with
Patagonia that would increase the synthesis of phenols [11].
On the other hand, it has been reported that the concentration of bioactive components
in the seeds of berries is lower than that in the pulp and skin. One of the predominant
flavonols found in calafate seed corresponds to quercetin-3-rhamnoside [
6
]. Although
compounds derived from hydroxycinnamic acids have not been determined, the presence
of delphinidin-3-glucoside, rutin and isorhamnetin rutinoside has been highlighted [10].
3.4. Nutritional Content of the Residual Flour of Calafate Pomace
Figure 7shows the nutritional composition of calafate fruit (d.w.), the pomace and
its residual flour (68
±
1%) obtained after successive extraction with scCO
2
and PLE.
The protein content was quite high in the pomace before and after extraction by scCO
2
and PLE, with values close to those of rice flour but lower than those of wheat, oats and
corn [
66
,
67
]. However, the fiber contribution from calafate pomace and residual flour was
higher than that provided by cereal flours [
66
,
67
]. The mineral content was higher than that
of cereals (wheat, oats and corn) in the residual flour of calafate. In general, the nutritional
contribution of the calafate flour was within the range of other flours, such as hazelnut,
lentil, bean and soybean flours, used in formulations and nutritional supplements for
human and animal nutrition [67].
Antioxidants 2023, 12, x FOR PEER REVIEW 16 of 21
Other factors that influence the phenolic composition and antioxidant capacity of
berries are variety, genetics, maturity, plant nutrition, harvest season and climate [3,5].
Climate is a fundamental factor considering the environmental stresses associated with
Patagonia that would increase the synthesis of phenols [11].
On the other hand, it has been reported that the concentration of bioactive compo-
nents in the seeds of berries is lower than that in the pulp and skin. One of the predomi-
nant flavonols found in calafate seed corresponds to quercetin-3-rhamnoside [6]. Alt-
hough compounds derived from hydroxycinnamic acids have not been determined, the
presence of delphinidin-3-glucoside, rutin and isorhamnetin rutinoside has been high-
lighted [10].
3.4. Nutritional Content of the Residual Flour of Calafate Pomace
Figure 7 shows the nutritional composition of calafate fruit (d.w.), the pomace and
its residual flour (68 ± 1%) obtained after successive extraction with scCO2 and PLE. The
protein content was quite high in the pomace before and after extraction by scCO2 and
PLE, with values close to those of rice flour but lower than those of wheat, oats and corn
[66,67]. However, the fiber contribution from calafate pomace and residual flour was
higher than that provided by cereal flours [66,67]. The mineral content was higher than
that of cereals (wheat, oats and corn) in the residual flour of calafate. In general, the nutri-
tional contribution of the calafate flour was within the range of other flours, such as ha-
zelnut, lentil, bean and soybean flours, used in formulations and nutritional supplements
for human and animal nutrition [67].
Figure 7. Content (d.w.) of protein, fiber and minerals in the fruit, pomace and calafate residual
flour obtained after the scCO2-PLE extraction steps and its comparison with the nutritional compo-
sition of cereal flours [66,67]. * Corresponds to oatmeal, wheat, rye and corn [66,67].
Calafate has been praised for its large content of bioactive compounds. It is also no-
table for its high content of soluble solids, approximately 25–31° Brix, which is much
Figure 7.
Content (d.w.) of protein, fiber and minerals in the fruit, pomace and calafate residual flour
obtained after the scCO
2
-PLE extraction steps and its comparison with the nutritional composition of
cereal flours [66,67]. * Corresponds to oatmeal, wheat, rye and corn [66,67].

Antioxidants 2023,12, 323 16 of 19
Calafate has been praised for its large content of bioactive compounds. It is also
notable for its high content of soluble solids, approximately 25–31
◦
Brix, which is much
higher than most other consumed berries [
67
,
68
]. Sugars present are largely fructose and
glucose [
6
]. The beneficial high fiber content may prevent chronic noncommunicable
diseases such as diabetes, colon cancer and hypercholesterolemia [
68
]. The protein and
mineral contents of the residual calafate flour were relatively lower than those of cereal
flours and other berries, such as murta [68].
4. Conclusions
Biorefining of calafate pomace using scCO
2
and PLE produced lipidic and hydrophilic
extracts and a residual flour-type supplement for human or animal nutrition. The optimal
extraction conditions with scCO
2
were 60
◦
C, 358.5 bar and 144.6 min, and a lipid extract
yield of 11.15% (d.w.). The lipid extract presented a good content and ratio of
ω
-6/
ω
-3 fatty
acids as well as tocopherol precursors of vitamin E. The oil exhibited good physical charac-
teristics and a low oxidative state. This product could be used as a specialty ingredient in
food formulations or as a nutraceutical. The hydroalcoholic extraction by PLE generated
an extract with good phenolic content (80% of TPC) and antioxidant capacity comparable
to that obtained in pressed pomace from other berries. The extraction kinetics from oil by
scCO
2
and phenolics by PLE were optimally adjusted to the spline model (R
2
= 0.989 and
R2= 0.999
, respectively). The final residual flour from the biorefinery process had a high
fiber content and acceptable values of proteins and minerals, suitable for the development
of nutritional supplements. This study verified the feasibility of using eco-friendly pro-
cesses to recover oil, bioactive compounds and a high-fiber product from calafate pomace
that may be used as ingredients in the development of healthy foods.
Author Contributions:
Conceptualization, J.O.-V. and C.C.; methodology, C.V. and A.Q.; software,
A.Q., M.G.-B. and J.M.B.-M.; validation, M.F., J.M.A. and J.M.M.; formal analysis, J.O.-V. and J.B.-V.;
investigation, C.V., C.C. and M.G.-B.; resources, J.O.-V.; data curation, J.M.B.-M., J.M.A. and M.F.;
writing—original draft preparation, J.O.-V. and C.C.; writing—review and editing, J.M.A., M.F. and
J.M.M.; visualization, J.O.-V., M.F. and J.B.-V.; supervision, J.O.-V. and C.C.; project administration,
J.O.-V.; funding acquisition, J.O.-V. All authors have read and agreed to the published version of
the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement:
The data presented in this study are available on request from the
corresponding author.
Acknowledgments: The collaboration of Mauricio Manríquez Vera and the contribution of raw ma-
terial by the company Patagonia Superfruits SA, Aysén Region, Chile, are appreciated. Additionally,
J.M. Aguilera is grateful for the technical support of the Cape Horn International Center (CHIC-ANID
PIA/BASAL PFB210018).
Conflicts of Interest: The authors declare no conflict of interest.
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