marine drugs
Article
Microwave-Assisted Hydrothermal Processing of
Rugulopteryx okamurae
Tania Ferreira-Anta, Noelia Flórez-Fernández
, Maria Dolores Torres * , José Mazón and Herminia Dominguez
CINBIO, Universidade de Vigo, Department of Chemical Engineering, Facultade de Ciencias, Campus Ourense,
As Lagoas, 32004 Ourense, Spain
* Correspondence: matorres@uvigo.gal; Tel.: +34-988-387-047
Abstract: One possible scheme of Rugulopteryx okamurae biomass valorization based on a green,
rapid and efficient fractionation technique was proposed. Microwave-assisted pressurized hot
water extraction was the technology selected as the initial stage for the solubilization of different
seaweed components. Operation at 180 ◦ C for 10 min with a 30 liquid-to-solid ratio solubilized
more than 40% of the initial material. Both the alginate recovery yield (3.2%) and the phenolic
content of the water-soluble extracts (2.3%) were slightly higher when distilled water was used
as solvent. However, the carbohydrate content in the extract (60%) was similar for both solvents,
but the sulfate content was higher for samples processed with salt water collected from the same
coast as the seaweeds. The antiradical capacity of the extracts was related to the phenolic content
in the extracts, but the cytotoxicity towards HeLa229 cancer cells was highest (EC50 = 48 µg/mL)
for the extract obtained with distilled water at the lowest temperature evaluated. Operation time
showed a relevant enhancement of the extraction performance and bioactive properties of the soluble
extracts. The further fractionation and study of this extract would be recommended to extend its
potential applications. However, due to the low extraction yield, emphasis was given to the solid
residue, which showed a heating value in the range 16,102–18,413 kJ/kg and could be useful for the
preparation of biomaterials according to its rheological properties.
Keywords: Rugulopteryx okamurae; microwave; sea water; bioactive compounds; biobased materials
Citation: Ferreira-Anta, T.;
Flórez-Fernández, N.; Torres, M.D.;
Mazón, J.; Dominguez, H.
Microwave-Assisted Hydrothermal
Processing of Rugulopteryx okamurae.
Mar. Drugs 2023, 21, 319. https://
doi.org/10.3390/md21060319
Academic Editors: Faiez Hentati and
Laurent Vandanjon
Received: 14 April 2023
Revised: 19 May 2023
Accepted: 23 May 2023
Published: 25 May 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/).
1. Introduction
Rugulopteryx okamurae (Dictyota marginata, Dilophus marginatus, Dictyota okamurae,
Dilophus okamurae, Dictyotales, Phaeophyceae) is a brown seaweed native from the warm
temperate western Pacific Ocean [1], which in recent years colonized the southwestern
coasts of Europe [2] and became an aggressive invader [3–5]. The Strait of Gibraltar is
an area exposed to multiple environmental changes including other invasive seaweed
species, but in this area R. okamurae has displaced the local biota and substantial amounts
of beach coast material, generating a great ecological impact and also affecting human and
economic activities [3]. The urgent need to implement management measures has been
highlighted [6], and solutions integrated into a circular economy model are preferred [7].
This high invasive potential has been ascribed to the chemical defenses, especially
to diterpenoids [8]. Dilkamural, a compound with deterrent properties and harmful
effects on generalist herbivores, can be found at high concentrations in this seaweed
and was not described previously in the invaded area [9]. The diterpenoid dilkamural,
obtained by chromatographic purification of an ethyl acetate extract of this alga, showed
antimicrobial activity [10]. Other terpenoids, rugukadiol A, rugukamurals A–C, and
ruguloptones A–F, have been identified [11]. Among secondary metabolites, the terpene
and polyphenolic compounds stand out, presenting photoprotective, antioxidant, and
immune-stimulant properties [12]. Different solvent extracts from Rugulopteryx okamurae
have shown selective cytotoxicity to human leukemic cells [13], with inhibitory effects on
Mar. Drugs 2023, 21, 319. https://doi.org/10.3390/md21060319
https://www.mdpi.com/journal/marinedrugs
Mar. Drugs 2023, 21, 319
2 of 18
α-glucosidase, but were non-cytotoxic on mouse pre-adipocytes cells [14]. However, data
on the biomedical potential are scarce, and only antibacterial activity and anti-inflammatory
have been reported [11].
The extremely rapid expansion of this brown seaweed in coasts of Southwest Europe
has been compared to that of Sargassum sp. [15] and the future impact of such tides could be
quite different if they become regarded as potential crops rather than harmful weeds [16].
The biomass valorization has been proposed to lower the expansion of this invasive species;
De la Lama-Calvente et al. (2021) proposed an anaerobic co-digestion of R. okamurae
biomass with olive mill solid waste to enhance both the methane yield and biodegradability
of both substrates [15] and De la Lama Calvente et al. (2023) proposed a new mechanical
pretreatment with zeolite and a thermal pretreatment at 120 ◦ C for 45 min to improve the
process [7]. A microwave irradiation pretreatment before enzyme hydrolysis for obtaining
reducing sugars was successful to produce volatile fatty acids in dark fermentation [17].
Santana et al. (2022) developed bio-based plastic materials, processed by injection molding
blends of the raw seaweed with glycerol to obtain environmentally friendly materials [18].
Patón et al. (2023) proposed traditional composting and black soldier fly larvae composting
to eliminate algae, providing fertilizers and animal proteins, because toxins in the algae
do not affect the long-term survival, growth, or reproduction of these invertebrates [19].
Alternatives based on the cascade fractionation of this resource have not been addressed,
although this biorefinery approach to obtain fertilizers, bioactive compounds, and biofuels
proved technically and economically feasible for Sargassum biomass [20,21]. Later authors
indicated a wide range of potential applications of Rugulopteryx okamurae key components
such as fucoidans and phenolic compounds for the development of pharmaceutical, cosmetic, and nutraceutical products. The corresponding alginates could be employed from
food packaging to wound dressing.
The main aim of this work is to propose one of the multiple possibilities for an integral cascade valorization sequence of the invasive brown seaweed Rugulopteryx okamurae
biomass following a biorefinery approach. The major novelty is the preliminary exploration
of the extraction of the alginate, sulfated oligosaccharides and phenolic compounds as well
as the formulation of printable alginate-based biomaterials using the residual solids after
hydrothermal treatment.
2. Results and Discussion
2.1. Raw Material
The dried algae contained 8.06 ± 0.16% moisture and the proximal composition in dry
basis is shown in Table 1. The major fractions were protein (16.43%) and carbohydrates, the
most abundant constituent being glucose (11.69%), followed by galactose (2.76%), fucose
(6.38%), mannose (1.91%), uronic acids (1.65%), and xylose (0.68%).
Mercado et al. (2022) reported a highly variable N content in R. okamurae ranging from
1.4% to 4.5%, suggesting that this species has high N storage capacity that is potentially
usable when the external N concentration decreases [22]. This storage capacity of N is
a common feature of bloom-forming algae, which allows them an opportunistic growth
when the external conditions are favorable.
A relatively high lipid content (6.2%) was observed, in relation to the values found
for other brown seaweeds. The fatty acid profile was composed of palmitic acid (50.1%),
myristic acid (22%), 9-hexadecenoic acid (11%), 9-octadecenoic (12%), stearic acid (2.6%),
and eicosanoic acid (2.3%).
The solvent used for the extractives’ quantification had a notable impact on the results:
those recovered after ethanol and the mixture of methanol, acetone, and water exhibited
higher values (above 11%) than those treated with hexane (1.7%).
Brown seaweeds contain polysaccharides (alginates, laminarin, and fucoidan), phlorotannins, terpenoids, minerals, vitamins, and fatty acids. No information on the sulfated polysaccharides of this seaweed has been found in the literature.
Brown seaweeds contain polysaccharides (alginates, laminarin, and fucoidan), phlorotannins, terpenoids, minerals, vitamins, and fatty acids. No information on the sulfated
polysaccharides of this seaweed has been found in the literature.
Mar. Drugs 2023, 21, 319
3 of 18
Table 1. Proximal composition of Rugulopterix okamurae in dry basis (w/w), except moisture content.
Fraction
Content
Metal
Content
Moisture (%)
8.06 ± 0.16
Ca (mg/g)
15.90 ± 1.33
Table 1. Proximal composition of Rugulopterix okamurae in dry basis (w/w), except moisture content.
Ash (%)
11.56 ± 0.68
Mg (mg/g)
3.20 ± 0.12
Protein
16.43
± 0.70
Fe (mg/g)
0.55
± 0.06
Fraction(%)
Content
Metal
Content
Lipid
(%)
6.17
±
0.15
Na
(mg/g)
0.53
±±
0.02
Moisture (%)
8.06 ± 0.16
Ca (mg/g)
15.90
1.33
Acid
insoluble residue, AIR (%)
30.81
0.40
0.02
Ash (%)
11.56±±1.00
0.68 K (mg/g)
Mg (mg/g)
3.20±±
0.12
Protein (%) (%)
16.43 ± 0.70 Cu (µg/g)
Fe (mg/g)
0.55±±0.98
0.06
Extractives
23.98
Lipid (%)
6.17 ± 0.15
Na (mg/g)
0.53 ± 0.02
Ethanol (96%)
11.24 ± 0.14
As (µg/g)
5.82 ± 0.40
Acid insoluble residue, AIR (%)
30.81 ± 1.00
K (mg/g)
0.40 ± 0.02
Hexane
1.73
±
0.66
Pb
(µg/g)
0.97
±±
0.02
Extractives (%)
Cu (µg/g)
23.98
0.98
MeOH
Acetone + H2O (3:1:1)
12.73
0.15
0.05
Ethanol+(96%)
11.24±±0.27
0.14 Cd (µg/g)
As (µg/g)
5.82±±
0.40
Hexane
1.73 ± 0.66
Pb (µg/g)
0.97±±
0.02
Carbohydrates
(%)
B (mg/g)
0.041
0.001
MeOH
+
Acetone
+
H
O
(3:1:1)
12.73
±
0.27
Cd
(µg/g)
0.15
±
0.05
2
Glucose
11.69 ± 0.12
Cu (mg/g)
0.034 ± 0.001
Carbohydrates (%)
B (mg/g)
0.041 ± 0.001
Galactose
2.76
± 0.07
Hg (µg/g)
0.016
± 0.001
Glucose
11.69 ± 0.12
Cu (mg/g)
0.034 ± 0.001
Fucose
6.38
±
0.02
Galactose
2.76 ± 0.07
Hg (µg/g)
0.016 ± 0.001
Fucose
6.38±±0.05
0.02
Mannose
1.91
Mannose
1.91±±0.02
0.05
Xylose
0.68
Xylose
0.68 ± 0.02
Acetyl groups
2.36 ± 0.01
Acetyl groups
2.36 ± 0.01
Uronic
(glucuronicacid
acid
eq.)
1.65
Uronic acids
acids (glucuronic
eq.)
1.65±±0.01
0.01
Sulfate
2.79
Sulfate (%)
(%)
2.79±±0.07
0.07
2.2. Microwave-Assisted
Microwave-Assisted Extraction
Extraction
2.2.
The aqueous
aqueousextraction
extractionoperating
operating
under
pressurized
conditions
microwave
The
under
pressurized
conditions
with with
microwave
heatheating
canproposed
be proposed
a first
stage
for the
fractionation
ofokamurae
R. okamurae
biomass
(Figure
ing
can be
as a as
first
stage
for the
fractionation
of R.
biomass
(Figure
1).
1). This
approach
previously
selected
forextraction
the extraction
of bioactive
compounds
This
approach
has has
previously
beenbeen
selected
for the
of bioactive
compounds
from
from other
seaweeds
[23–25].
other
brownbrown
seaweeds
[23–25].
Figure 1.1.Flow
Flowdiagram
diagram
of proposed
the proposed
sequence
the microwave-assisted
hydrothermal
Figure
of the
sequence
for thefor
microwave-assisted
hydrothermal
fractionfractionation of Rugulopteryx okamurae biomass.
ation of Rugulopteryx okamurae biomass.
Microwave heating provides a more rapid and uniform heating than conventional
heating, allowing shorter treatment times and more accurate control of the process. Because
dielectric heating results from both dipole orientation and ionic conductivity, the addition
of salts can increase the heating rate [26]. The rate and yield of carbohydrates and protein
solubilization is highly dependent on the conditions, and the presence of salts could also
influence the protein extraction [27].
cause dielectric heating results from both dipole orientation and ionic conductivity, the
addition of salts can increase the heating rate [26]. The rate and yield of carbohydrates
and protein solubilization is highly dependent on the conditions, and the presence of salts
could also influence the protein extraction [27].
Mar. Drugs 2023, 21, 319
2.2.1. Extraction Yield and Characteristics of the Liquid Fraction
4 of 18
The influence of the operation temperature on the extraction yield and composition
of2.2.1.
the liquid
fraction
the performance
of the Fraction
hydrothermal process was assessed.
Extraction
Yieldand
and on
Characteristics
of the Liquid
The solubilization
with temperature
at 180 °Cyield
(severity
3.4), more markThe influence yield
of the increased
operation temperature
on the extraction
and composition
edly
when
distilled
water
was
used
as
a
solvent
(Figure
2).
Note
here
that
the severities
of the liquid fraction and on the performance of the hydrothermal process was assessed.
◦
The solubilization
with
temperature
at 180 study
C (severity
3.4),
more markedly
increased
from 2.5yield
(160increased
°C) to 3.8
(180
°C). In a recent
using
a microwave
pretreatwhen
distilled
water
was
used
as
a
solvent
(Figure
2).
Note
here
that
the
severities
in-sugars
ment, Fernández-Medina et al. (2022) found maximum solubilization of reducing
◦ C) to 3.8 (180 ◦ C). In a recent study using a microwave pretreatment,
creased
from
2.5
(160
at higher severity, 220 °C at 20 min, but under those conditions, the monomeric sugars
Fernández-Medina et al. (2022) found maximum solubilization of reducing sugars at
were
degraded [17]. In our work, the aim was to explore the possibility of obtaining olihigher severity, 220 ◦ C at 20 min, but under those conditions, the monomeric sugars
gosaccharides, and such elevated temperatures were not considered based on preliminary
were degraded [17]. In our work, the aim was to explore the possibility of obtaining
experiments
and previous
behaviortemperatures
of other brown
where
hydrothermal
oligosaccharides,
and such elevated
wereseaweeds
not considered
based
on prelimi- treatments
at
comparable
severities
were
used
(2.2
at
160
°C–4.0
at
220
°C)
[28].
However, the
nary experiments and previous behavior of other brown seaweeds where hydrothermal
◦
◦
influence
ofat
time
was analyzed
forwere
the highest
°C),However,
achieving the
treatments
comparable
severities
used (2.2tested
at 160 temperature
C–4.0 at 220 (180
C) [28].
the influence
of time was
analyzed
for the highest
testeddifferences
temperatureafter
(180 ◦30
C),min
achieving
the
highest
performance
at 20
min without
significant
independently
highest
performance
at
20
min
without
significant
differences
after
30
min
independently
of the water used as the solvent agent.
of the water used as the solvent agent.
Figure2.2.Effect
Effect of
of the
the processing
onon
thethe
extraction
yieldyield
and pH
theof
liquid
fractionfraction
Figure
processingconditions
conditions
extraction
andofpH
the liquid
obtainedafter
aftermicrowave-assisted
microwave-assisted hydrothermal
treatment.
obtained
hydrothermal
treatment.
As expected, the total phenolic content of the extracts increased with increasing
As
expected, the total phenolic content of the extracts increased with increasing temtemperature regardless of the solvent used (Figure 3a,b). Additionally, Fernández-Medina
perature regardless of the solvent used (Figure 3a,b). Additionally, Fernández-Medina et
et al. (2022) found this trend and obtained a higher phenolic concentration after microwaveal.assisted
(2022) treatment
found thisattrend
obtained
a higher
phenolic
concentration
microwave200 ◦ Cand
for 20
min [17].
The phenolic
extraction
yields areafter
equivalent
assisted
treatment
at
200
°C
for
20
min
[17].
The
phenolic
extraction
yields
are
to those reported by those authors. The values attained were lower in the presence ofequivalent
salt
towater.
thoseOther
reported
by those
The values
were lower
in the impact
presence
authors
found authors.
that an increase
in saltattained
content involved
a negative
on of salt
the recovery
of the phenolic
compound
for different
as chickpea
and beans
water.
Other authors
found that
an increase
in saltpulses,
contentsuch
involved
a negative
impact on
using
microwave-assisted
extraction
in a saline
mediumpulses,
[29]. The
lossas
ofchickpea
total phenolic
the
recovery
of the phenolic
compound
for different
such
and beans
suggesting
content
from
beans
during
soaking
and
cooking
was
also
found
[30],
using microwave-assisted extraction in a saline medium [29]. The loss of totalthat
phenolic
the
presence
of
salts
led
to
the
alkylation
of
the
phenolic
compounds
to
other
hydroxy
content from beans during soaking and cooking was also found [30], suggesting that the
propyl by-products. Studies on the stability of phenolic compounds of different families
presence of salts led to the alkylation of the phenolic compounds to other hydroxy propyl
during microwave-assisted extraction indicated an acceleration of the degradation of these
by-products.
Studies
on the[31,32].
stability
of phenolic
compounds
differentthat
families
fractions during
processing
Later
authors found
that thoseofphenolics
have aduring
microwave-assisted
extraction
indicated
an
acceleration
of
the
degradation
of
these fraclarger number of hydroxyl-type substituents are more easily degraded under microwave
tions
duringThe
processing
Later authors
found
that thoseofphenolics
that haveasa larger
treatment.
influence[31,32].
of the inorganic
salts on
the hydrolysis
other biopolymers
chitosan in a microwave field implied a promotion of their degradation [33].
Mar. Drugs 2023, 21, 319
(a)
temperatures, and time studied. The selected treatment time is low to solubilize the proteins. Note here that waters used as solvent agents presented the following composition:
DW (dry content: 47 mg/L; CaCO3: n.d.; HCO3: <10 mg/L; fluorides: <1 ppm; chlorides: 1
number of hydroxyl-type substituents are more easily degraded under microwave treatppm; nitrates: <1 ppm; nitrites: <0.1 ppm; phosphates: <1 ppm; sulfates: <1 ppm) and SW
ment. The influence of the inorganic salts on the hydrolysis of other biopolymers as chi(dry content: 39 g/L; CaCO3: n.d.; HCO3: <150 mg/L; fluorides: <1 ppm; chlorides: 20,794
tosan in a microwave field implied a promotion of their degradation [33].
18
ppm; nitrates: <1 ppm; nitrites: <0.1 ppm; phosphates: <1 ppm; sulfates: <2812 ppm).5 of
The
The protein content remained under 0.4 g/100 g extract in distilled water and under
phenolic compounds seem to be responsible for the antiradical properties (Figure 3c).
0.2 g/100 g extract obtained in sea water, with a slight trend to increase with medium, high
temperatures, and time studied. The selected treatment time is low to solubilize the pro(b)
(c)
teins. Note here that waters used as solvent agents presented the following composition:
DW (dry content: 47 mg/L; CaCO3: n.d.; HCO3: <10 mg/L; fluorides: <1 ppm; chlorides: 1
ppm; nitrates: <1 ppm; nitrites: <0.1 ppm; phosphates: <1 ppm; sulfates: <1 ppm) and SW
(dry content: 39 g/L; CaCO3: n.d.; HCO3: <150 mg/L; fluorides: <1 ppm; chlorides: 20,794
ppm; nitrates: <1 ppm; nitrites: <0.1 ppm; phosphates: <1 ppm; sulfates: <2812 ppm). The
phenolic compounds seem to be responsible for the antiradical properties (Figure 3c).
Figure 3. Effect of the processing conditions on the phloroglucinol content (PC), antioxidant activity
Figure
3. Effect
of theand
processing
conditions
the phloroglucinol
content
antioxidant
(AC,
such
as TEAC
DPPH values),
andon
protein
content of the
liquid(PC),
fraction
obtainedactivafter
ity (AC, such as
TEAC and
DPPH
values),
and
proteinsolvents:
content of
liquidwater
fraction
after
hydrothermal
treatment
of R.
okamurae
using
different
(a)the
distilled
andobtained
(b) sea water.
hydrothermal
of R.PC
okamurae
different
solvents:
(a) distilled
water
(b) sea(filled
wa(c)
Correlationtreatment
between the
and theusing
AC of
the soluble
extracts
obtained
withand
distilled
ter.
(c)
Correlation
between
the
PC
and
the
AC
of
the
soluble
extracts
obtained
with
distilled
(filled
symbols) and with sea (open symbols) water. Data represent mean ± standard deviation (n ≥ 3).
symbols) and with sea (open symbols) water. Data represent mean ± standard deviation (n ≥ 3).
The protein content remained under 0.4 g/100 g extract in distilled water and under
The liquid
extracts
separated
hydrothermal
showed no
monomeric
0.2 g/100
g extract
obtained
in seaafter
water,
with a slighttreatment
trend to increase
with
medium,
units.temperatures,
The oligosaccharide
increased
with treatment
temperature
studied the
for
high
and timecontent
studied.
The selected
timeinisthe
lowrange
to solubilize
hydrothermal
systems
treated
for
5
min:
from
48%
to
60%
when
distilled
water
was
used
Figure
3.
Effect
of
the
processing
conditions
on
the
phloroglucinol
content
(PC),
antioxidant
activproteins. Note here that waters used as solvent agents presented the following composition:
ity
(AC,
such
as TEAC
and
DPPH
values),
and
content
of (Figure
the liquid
as solvent
and
from
to 45%
when
sea protein
water was
used
4).fraction obtained after
DW
(dry
content:
47 38%
mg/L;
CaCO
3 : n.d.; HCO3 : <10 mg/L; fluorides: <1 ppm; chlorides:
hydrothermal
treatment
of
R.
okamurae
using
different
solvents:
(a)
distilled
water and
sea wa1 ppm; nitrates: <1 ppm; nitrites: <0.1 ppm; phosphates: <1 ppm; sulfates:
<1(b)
ppm)
and
ter. (c) Correlation between the PC and the AC of the soluble extracts obtained with distilled (filled
SW (dry content: 39 g/L; CaCO3 : n.d.; HCO3 : <150 mg/L; fluorides: <1 ppm; chlorides:
symbols) and with sea (open symbols) water. Data represent mean ± standard deviation (n ≥ 3).
20,794 ppm; nitrates: <1 ppm; nitrites: <0.1 ppm; phosphates: <1 ppm; sulfates: <2812 ppm).
The phenolic compounds seem to be responsible for the antiradical properties (Figure 3c).
The liquid extracts separated after hydrothermal treatment showed no monomeric
The liquid extracts separated after hydrothermal treatment showed no monomeric
units. The oligosaccharide content increased with temperature in the range studied for
units. The oligosaccharide content increased with temperature in the range studied for
hydrothermal systems treated for 5 min: from 48% to 60% when distilled water was used
hydrothermal systems treated for 5 min: from 48% to 60% when distilled water was used
as solvent and from 38% to 45% when sea water was used (Figure 4).
as solvent and from 38% to 45% when sea water was used (Figure 4).
(a)
(b)
Figure 4. Effect of temperatures and solvents ((a) distilled water and (b) sea water) during microwaveassisted hydrothermal extraction on the oligosaccharide composition of the liquid fractions obtained
from R. okamurae and further dialyzed in 0.5 kDa membranes.
The highest oligosaccharide values were identified for liquid extracts treated at 180 ◦ C
for 10 min using DW (75%) and SW (62%). The soluble sulfate content was notably lower
in distilled water. The low glucose content in the extract could suggest that the cellulose
obtained from R. okamurae and further dialyzed in 0.5 kDa membranes.
Mar. Drugs 2023, 21, 319
The highest oligosaccharide values were identified for liquid extracts treated at 180
°C for 10 min using DW (75%) and SW (62%). The soluble sulfate content was notably
lower in distilled water. The low glucose content in the extract could suggest that the cel6 of 18
lulose remains in the solid phase. Galactose and fucose were the major monosaccharides.
The crude extracts with the highest performance exhibited a Gal:Fuc:Man:Xyl:Glu ratio
varying between (1.0:0.5:0.5:0.4:0.3) and (1.0:0.6:0.5:0.5:0.4) for the systems extracted using
remains
in the
solid
phase.
Galactose
and fucose
werethat
the major
monosaccharides.
The
DW and
SW.
This
behavior
is consistent
with
reported
for crude fucoidans
crude
extracts
with
the
highest
performance
exhibited
a
Gal:Fuc:Man:Xyl:Glu
ratio
varying
(1.0:0.8:0.1:0.1:0.1) of other Fucales recovered after a pre-treatment with organic solvents
between
(1.0:0.5:0.5:0.4:0.3)
and (1.0:0.6:0.5:0.5:0.4)
followed
by a hot water extraction
stage [34]. for the systems extracted using DW and
SW. This behavior is consistent with that reported for crude fucoidans (1.0:0.8:0.1:0.1:0.1)−1of
Data in Figure 5 confirm the presence of sulfate ester bonds at 1249 and 873 cm for
other Fucales recovered after a pre-treatment with organic solvents followed by a hot water
samples extracted in the presence of distillate water independently of the microwave proextraction stage [34].
cessing temperature, describing an asymmetrical S=O stretching vibration [35]. All −
proData in Figure 5 confirm the presence of sulfate ester bonds at 1249 and 873 cm 1
files exhibited a major band around 1600 cm−1 typically present in the alginate fractions
for samples extracted in the presence of distillate water independently of the microwave
related to the uronic acids. Those recovered using distillate water also presented a band
processing temperature,
describing an asymmetrical S=O stretching vibration [35]. All
at 1029 cm−1 related to the C-O and C-C stretching vibrations of pyranoses, whereas this
profiles exhibited a major band−1around 1600 cm−1 typically present in the alginate fractions
band was shifted to 1150 cm for crude extracts separated using sea water as an extractive
related to the uronic acids. Those recovered using distillate water also presented a band at
agent.−The
molecular weight of the solubilized polysaccharides is higher than 300 kDa,
1029 cm 1 related to the C-O and C-C stretching vibrations of pyranoses, whereas this band
with the signal from−1the peaks corresponding to treatments with salt water being more
was shifted to 1150 cm for crude extracts separated using sea water as an extractive agent.
intense with higher molecular weights than those from distilled water treatments (Figure
The molecular weight of the solubilized polysaccharides is higher than 300 kDa, with the
S1). from the peaks corresponding to treatments with salt water being more intense with
signal
higher molecular weights than those from distilled water treatments (Figure S1).
(a)
(b)
Figure 5. Impact of the MAE extraction treatment in R. okamurae brown seaweed using two solvents,
Figure 5. Impact of the MAE extraction treatment in R. okamurae brown seaweed using two solvents,
(a)(a)
distilled
water
(DW)
andand
(b) (b)
seasea
water
(SW),
for for
the the
FTIR-ATR
spectra
of the
processed
at
distilled
water
(DW)
water
(SW),
FTIR-ATR
spectra
of extracts
the extracts
processed
different
temperatures
and
times.
at different temperatures and times.
2.2.2. Cytotoxicity
2.2.2. Cytotoxicity
The extracts obtained with distilled water at 170 ◦ C and at 180 ◦ C showed 49 ± 2%
The extracts obtained with distilled water at 170 °C and at 180 °C showed 49 ± 2%
and 43 ± 2% growth inhibition of cell line HeLa 229 at 0.1 mg/mL. The IC50 could only
and 43 ± 2% growth inhibition of cell line HeLa 229 at 0.1 mg/mL. The IC50 could
only be
be calculated for samples obtained with sea water or with distilled water at 160 ◦ C, which
calculated for samples obtained with sea water or with distilled water at 160 °C, which
showed a maximum activity with EC50 = 0.048 mg/mL (Table 2). Note here that extracts
showed a maximum activity with EC50= 0.048 mg/mL (Table 2). Note here that extracts
obtained after longer hydrothermal treatments (10–30 min) exhibited growth inhibition of
obtained
after
longer
treatments
(10–30
min)
growth
inhibition
cell
line HeLa
229
at 0.1hydrothermal
mg/mL below
41% (10 min),
45%
(20exhibited
min), and
35% (30
min). Aof
cell
line
HeLa
229
at
0.1
mg/mL
below
41%
(10
min),
45%
(20
min),
and
35%
(30
min).
comparable activity has been reported for S. muticum fucoidans, showing 0.074 mg/mL
[36],A
comparable
activity
has
been
reported
for
S.
muticum
fucoidans,
showing
0.074
mg/mL
and activity was lower for those from U. pinnatifida, with an IC50 of 0.76 mg/mL [37]. These
values are in the range of those found for other natural polysaccharides. Additionally,
phenolic compounds might contribute to the cytotoxic activity against this cell line [38],
but the lyophilized methanolic crude extracts from Dilophus okamurae showed weak selective cytotoxic activity to murine L1210 cells and to human leukemic cells, HL60 and
MOLT-4 [13].
Mar. Drugs 2023, 21, 319
These values are in the range of those found for other natural polysaccharides. Additionally, phenolic compounds might contribute to the cytotoxic activity against this cell line
[38], but the lyophilized methanolic crude extracts from Dilophus okamurae showed weak
selective cytotoxic activity to murine L1210 cells and to human leukemic cells, HL60 and
MOLT-4 [13].
7 of 18
Table 2. Inhibitory efficacy of cell growth (Emax) and IC50 for extracts obtained by MAE in the tumoral
cell line HeLa 229.
Table 2. Inhibitory efficacy of cell growth (Emax ) and IC50 for extracts obtained by MAE in the tumoral
cell line HeLa 229.
1
Samples 1
Emax (% Inhibition)
IC50 (mg/mL)
160 DW
160 SW
170 SW
180 SW
Cisplatin
65 ± 2
60 ± 1
53 ± 1
60 ± 3
94 ± 1
0.048 ± 0.01
0.689 ± 0.13
0.317 ± 0.02
0.715 ± 0.14
0.80 ± 0.03 µM
Extracts recovered after 5 min of hydrothermal treatment.
2.2.3. Alginate Recovery and Characterization
Alginate
yield of
Rugulopteryx
okamurae brown alga obtained from the liquid phases
2.2.3. Alginate
Recovery
and Characterization
after microwave treatment varied between 3.2% and 2.3% for those using distilled and sea
Alginate yield of Rugulopteryx okamurae brown alga obtained from the liquid phases
water as an extractive agent for 5 min, respectively. The values attained with distilled waafter microwave treatment varied between 3.2% and 2.3% for those using distilled and sea
ter were
3.0as
± an
0.1%,
3.2 ± 0.1%,
and
± 0.2%
at 160 °C,The
170values
°C, and
180 °C,with
respectively.
water
extractive
agent
for2.9
5 min,
respectively.
attained
distilled water
Whenwere
salt water
as 0.1%,
the solvent,
recovery
yields
2.3 ◦±C,
0.1%,
2.5
◦ C, 170
◦ C, were
3.0 ± was
0.1%,used
3.2 ±
and 2.9the
± alginate
0.2% at 160
and 180
respectively.
± 0.1%,
and
2.4
±
0.1%
at
160
°C,
170
°C,
and
180
°C,
respectively.
Therefore,
no
notable
When salt water was used as the solvent, the alginate recovery yields were 2.3 ± 0.1%,
microwave
temperature
alginate
was◦ C,
observed.
NoteTherefore,
here that there
◦ C,
2.5 ± 0.1%,
and 2.4 influence
± 0.1% at in
160the
170 ◦ C, yield
and 180
respectively.
no notable
were microwave
also no notable
differences
with
increasing
extraction
time.
These
low
values
of that
cal- there
temperature influence in the alginate yield was observed. Note
here
cium were
alginate
are
consistent
with
those
previously
reported
for
other
invasive
brown
algae
also no notable differences with increasing extraction time. These low values of
such as
Sargasum
muticum
under conventional
processing
calcium alginate
are consistent
with thosehydrothermal
previously reported
for [39].
other invasive brown
FTIR-ATR
spectra
of
the
corresponding
extracted
alginates
as
representative
of realgae such as Sargasum muticum under conventional hydrothermal processing [39].
covered biopolymers
are
presented
in
Figure
6,
exhibiting
similar
structural
features
inof
allrecovFTIR-ATR spectra of the corresponding extracted alginates as representative
−1 attributed to the C=O and
cases.ered
Typical
signals
of
the
biopolymer
are
found
at
1600
cm
biopolymers are presented in Figure 6, exhibiting similar structural features in all
−1 attributed
carboxylate
O–C–Osignals
asymmetric
vibration
ofat
uronic
acids
[40,41]. The
cases. Typical
of the stretching
biopolymer
are found
1600 cm
to absorpthe C=O and
−1
−1 (C–C–H and O–C–H
tion bands
at 1436O–C–O
cm (C–OH
deformation
vibration),
cmacids
carboxylate
asymmetric
stretching
vibrationatof1300
uronic
[40,41]. The absorption
−1 −1(C–OH
−1 −1(C–C–H
deformation),
1147
(C-O stretching
vibrations),
and
1033
(C-O and
bands at at
1436
cmcm
deformation
vibration),
at at
1300
cmcm
andC-C
O–C–H
stretching
vibrationsatof1147
pyranoses)
werestretching
also identified.
The characteristic
at C-C
deformation),
cm−1 (C-O
vibrations),
and at 1033 weak
cm−1signals
(C-O and
−1 (α-L-guluronic asymmetric ring vibration) and at 813 cm−1 (β-mannuronic acid)
890 cm
stretching vibrations of pyranoses) were also identified. The characteristic weak signals at
−1 (α-L-guluronic
were 890
alsocm
observed
[42].
asymmetric ring vibration) and at 813 cm−1 (β-mannuronic acid)
were also observed [42].
Figure 6. FTIR-ATR spectra for the alginates recovered by calcium chloride precipitation.
Figure 6. FTIR-ATR spectra for the alginates recovered by calcium chloride precipitation.
Data from 1 H NMR spectra confirmed the characteristic signals of the extracted
alginates allowing the determination of the biopolymer block structure (Table 3) in terms
of the individual mannuronic (FM) and guluronic (FG) acids, the M/G ratio, and the four
diad frequencies (FMM, FGG, FMG, FGM) [43].
Mar. Drugs 2023, 21, 319
8 of 18
Table 3. Data from 1 H NMR spectra of the recovered alginates from microwave-assisted hydrothermal
treatment of R. okamurae.
Alginate
Alg-160 DW
Alg-170 DW
Alg-180 DW
Alg-160 SW
Alg-170 SW
Alg-180 SW
M/G
c
0.56
0.69 b
0.82 a
0.47 d
0.58 c
0.69 b
FM
FG
c
0.36
0.41 b
0.45 a
0.32 d
0.37 c
0.41 b
FMM
b
0.64
0.59 c
0.55 d
0.68 a
0.63 b
0.59 c
c
0.25
0.29 b
0.69 a
0.24 c
0.28 b
0.30 b
FGG
FMG
b
0.53
0.47 c
0.42 d
0.60 a
0.54 b
0.48 c
0.11 a
0.12 a
0.13 a
0.08 b
0.09 b
0.11 a
In all cases, standard deviations of the estimated data were <0.01. FMG = FGM. Data values in a column with
different superscript letters were significantly different at the p ≤ 0.05 level.
The magnitude of FM and M/G rose significantly with rising hydrothermal processing temperature, exhibiting lower values for alginates where sea water was used as the
extractive agent. However, M/G ratios varying between 0.47 and 0.82 are consistent with
those found in the literature for other brown algae from 0.2 to 1.6 [43–45]. The magnitude
of this parameter is relevant to have an idea of the rheological features of the extracted
biopolymers because those alginates with higher M values tend to present lower viscosities
and provide more flexible matrices, whereas those with higher G values involve higher
viscosities and more resistant gels [40]. The low values of alternating blocks together with
intermediate FGG and low FMM homopolymeric fractions were reported to promote the
development of gelled matrices [46].
Figure 7 shows the corresponding viscous profiles at 25 ◦ C of the above alginates,
extracted after 5 min of hydrothermal treatment, with a commonly used biopolymer
content (2%), confirming the hypothesis made with the structural properties. It should be
noteworthy that no differences were found with those extracted at higher hydrothermal
extraction times. At a fixed shear rate, the apparent viscosity was higher for alginates
extracted using sea water than for those processed at the same temperature with distilled
water. The highest viscosity values were identified for Alg-160 SW followed by Alg-170
SW (Alg-160 DW) > Alg-180 SW (Alg-170 DW) > Alg-180 DW. At low shear rates, alginate
recovered after hydrothermal treatment with both extractive agents showed a Newtonian
plateau. In all cases, a shear-thinning behavior was observed above 5 s−1 . It should be noted
that no hysteresis loops were identified in tested alginates. The measured viscosity values
were almost half those reported for commercial alginate solutions at similar biopolymer
content, but in the range of those found for other crude alginates [39,47].
Figure 7.
Flow curves
curves for
for extracted
extracted alginates
alginates after
after hydrothermal
hydrothermal treatment.
Figure
7. Flow
treatment.
2.3. Characteristics of the Solid Fraction
2.3. Characteristics of the Solid Fraction
Figure 8 shows representative images of the surface morphology of R. okamurae brown
Figure 8 shows representative images of the surface morphology of R. okamurae
alga used as a raw material as well as the corresponding residual solids after hydrothermal
brown alga used as a raw material as well as the corresponding residual solids after hydrothermal treatments at different temperatures in the presence of both extractive agents.
The untreated alga presents a regular mosaic-like morphology (Figure 8a–c), which was
notably modified during thermal processing. More irregular patterns with larger roughness were identified for the residual solid phases. The presence of small crystalline pre-
Figure 8 shows representative images of the surface morphology of R. okamurae
brown alga used as a raw material as well as the corresponding residual solids after hydrothermal treatments at different temperatures in the presence of both extractive agents.
The untreated alga presents a regular mosaic-like morphology (Figure 8a–c), which was
notably modified during thermal processing. More irregular patterns with larger rough9 of 18
ness were identified for the residual solid phases. The presence of small crystalline precipitates from the extraction treatment in those processed in the presence of sea water was
also observed. The creation of surface micro-pores and small pieces of debris on the surface
suggesting
the structural
damageinhas
been reported
for milder
temperatures
(120
treatments
at different
temperatures
thealso
presence
of both extractive
agents.
The untreated
°C),
enough
to
enhance
the
methane
production
[7].
alga presents a regular mosaic-like morphology (Figure 8a–c), which was notably modified
during thermal processing. More irregular patterns with larger roughness were identified
for the residual solid phases. The presence of small crystalline precipitates from the
extraction treatment in those processed in the presence of sea water was also observed.
The creation of surface micro-pores and small pieces of debris on the surface suggesting
the structural damage has also been reported for milder temperatures (120 ◦ C), enough to
enhance the methane production [7].
Mar. Drugs 2023, 21, 319
(a) Raw Material (magnifications from ×100 (left) to ×5000 (right))
(b) Solid MAE 160 °C DW
(c) Solid MAE 170 °C DW
(e) Solid MAE 160 °C SW
(f) Solid MAE 170 °C SW
(d) Solid MAE 180 °C DW
(g) Solid MAE 180 °C SW
Figure
8. SEM images of R. okamurae raw material with different magnifications (a) and the correFigure 8. SEM images of R. okamurae raw material with different magnifications (a) and the corresponding residual solids after microwave-assisted hydrothermal treatment at different processing
sponding residual solids after microwave-assisted hydrothermal treatment at different processing
conditions with distilled water (b–d) and with salt water (e–g).
conditions with distilled water (b–d) and with salt water (e–g).
Nitrogen
Nitrogenand
andcarbon
carboncontents
contentswere
werehigher
higherfor
forsolids
solidsremaining
remainingafter
afterextraction
extractionwith
with
sea
water
(Table
4).
A
slight
increase
was
observed
with
increasing
temperature
for
both
sea water (Table 4). A slight increase was observed with increasing temperature for both
extracting
solid
residue
waswas
proposed
for energetic
valorization
and for
extractingsolvents.
solvents.The
The
solid
residue
proposed
for energetic
valorization
andthe
for
formulation
of
bioplastics.
Data
of
the
estimated
HHV
(higher
heating
value)
were
slightly
the formulation of bioplastics. Data of the estimated HHV (higher heating value) were
higher
than
for the
residues
remaining
after this
treatment
on a red
varslightly
higher
thansolid
for the
solid residues
remaining
after
this treatment
onagarophyte
a red agarophyte
ying
between
14,615
and
15,343
kJ/kg
[48].
varying between 14,615 and 15,343 kJ/kg [48].
Table 4. Solid residue after microwave-assisted hydrothermal treatment of Rugulopteryx okamurae.
Mar. Drugs 2023, 21, 319
10 of 18
Table 4. Solid residue after microwave-assisted hydrothermal treatment of Rugulopteryx okamurae.
Distilled
water
Sea water
T (◦ C)
Nitrogen
(%, w/w)
Carbon
(%, w/w)
Hydrogen
(%, w/w)
HHV (kJ/kg)
160 1
3.28 ± 0.16 a
43.16 ± 0.21 c
5.89 ± 0.30 a
17,508 ± 62 f
170 1
3.31 ± 0.09 a
44.07 ± 0.08 b
6.10 ± 0.18 a
16,102 ± 188 h
180 1
3.39 ± 0.01 a
45.44 ± 0.46 a
6.03 ± 0.08 a
17,867 ± 48 e
180 *
4.10 ± 0.13
46.84 ± 0.50
6.32 ± 0.18
19,123 ± 56 c
180 **
4.10 ± 0.07
49.58 ± 0.14
6.17 ± 0.15
20,266 ± 98 a
180 ***
4.11 ± 0.01
49.34 ± 0.13
6.11 ± 0.06
20,141 ± 102 a
160 1
2.83 ± 0.04 c
38.94 ± 0.59 e
5.16 ± 0.01 b
16,045 ± 150 h
170 1
2.86 ± 0.05 c
38.82 ± 0.42 e
5.26 ± 0.05 b
18,413 ± 177 d
180 1
3.13 ± 0.06 b
41.42 ± 0.28 d
5.43 ± 0.03 a, b
16,900 ± 108 g
180 *
4.36 ± 0.08
46.80 ± 0.50
6.00 ± 0.11
19,085 ± 121 c
180 **
4.35 ± 0.17
48.57 ± 0.46
6.11 ± 0.03
19,843 ± 103 b
180 ***
4.20 ± 0.02
49.19 ± 0.23
6.25 ± 0.08
20,133 ± 99 a
Data are provided as mean ± standard deviation. Values in a column with different superscript letters are
statically different (p ≤ 0.05). 1 Extracts recovered after 5 min of hydrothermal treatment. Microwave extraction
time: * 10 min, ** 20 min, *** 30 min.
The potential of solid residue for the development of biomaterials was also studied
based on the results previously found for wild R. okamurae alga blended with glycerol [18].
Figure 9a shows the viscoelastic profiles obtained for the blends before printing treatment.
In all cases, the elastic behavior prevailed over the viscous one, confirming the marked
elastic character. A slight frequency dependence was observed in both moduli with a slope
around 0.15 ± 0.03 for G′ and about 0.10 ± 0.02 for G”. These dependences are like those
previously reported for the blends with the wild seaweed, with an elastic modulus frequency slope of 0.25 before and 0.16 after the heat treatment [18]. Although, the magnitude
of both moduli was lower for the blends with the solid residue (about two decades) when
compared with the data previously reported for later authors. At a fixed frequency, systems
made with solid residues from sea water treatment exhibited higher values than those
recovered after distilled water processing (Figure 9b). The strongest viscoelastic features
were observed for those prepared with 160 SW followed by 170 SW (160 DW) > 180 SW
(170 DW) > 180SW
DW.
These
results
evidence
the suitability
ofsuitability
proposedofprocess(170
DW)preliminary
> 180 DW. These
preliminary
results
evidence the
proposed
ing methods for
these blends.
Further
necessary
theoptimize
mechanical
processing
methods
for thesestudies
blends. are
Further
studies to
areoptimize
necessary to
the mechanical
properties
of the developed
R. okamurae
properties of the
developed
bio-based
materialsbio-based
from R. materials
okamuraefrom
depending
ondepending
their finalon
their
final applicationtofrom
control–release
application from
control–release
food
packaging. to food packaging.
(a)
(b)
Figure 9. Representative
(a)profiles
viscoelastic
profiles
and (b) variation
of Hz)
G′0 (1of
Hz)
of solid
phases
Figure 9. Representative
(a) viscoelastic
and
(b) variation
of G′ 0 (1
solid
phases
of of
R.R.
okamurae
blended
with
glycerol
(60/40)
after
different
hydrothermal
processing
conditions.
Symbols:
okamurae blended with glycerol (60/40) after different hydrothermal processing
conditions.
Symbols:
closed (elastic modulus), open (viscous modulus), blue (distilled water), green (sea water).
closed (elastic modulus), open (viscous modulus), blue (distilled water), green (sea water).
Operating under optimal microwave-assisted hydrothermal conditions (180 °C, 10
min), the major seaweed fraction remains insoluble, up to 3 g alginate/100 g seaweed
could be obtained by CaCl2 precipitation, and a product with up to 75 and 62 g galactofucans/100 g extract could be obtained when distilled and sea water were used, respectively
(Figure 10).
Mar. Drugs 2023, 21, 319
Figure 9. Representative (a) viscoelastic profiles and (b) variation of G′0 (1 Hz) of solid phases of R.
okamurae blended with glycerol (60/40) after different hydrothermal processing conditions. Symbols:
closed (elastic modulus), open (viscous modulus), blue (distilled water), green (sea water). 11 of 18
Operating under optimal microwave-assisted hydrothermal conditions (180 °C, 10
min), the
major under
seaweed
fraction
remains insoluble,
up to 3 gconditions
alginate/100
seaweed
Operating
optimal
microwave-assisted
hydrothermal
(180g◦ C,
10 min),
could
be
obtained
by
CaCl
2
precipitation,
and
a
product
with
up
to
75
and
62
g
galactofuthe major seaweed fraction remains insoluble, up to 3 g alginate/100 g seaweed could be
cans/100
extract
be obtained
distilled
and
water
used, respectively g
obtainedg by
CaClcould
andwhen
a product
with
upsea
to 75
and were
62 g galactofucans/100
2 precipitation,
(Figure
extract10).
could be obtained when distilled and sea water were used, respectively (Figure 10).
Figure
Figure10.
10.Flow
Flowdiagram
diagramofofthe
theproposed
proposedprocess
processaimed
aimedatatseparating
separatingalginate
alginateand
andother
otherbioactive
bioactive
compounds
compoundsfrom
fromR.R.okamurae
okamuraeand
andtotoformulate
formulatenovel
novelbiopolymers
biopolymerswith
withthe
theresidual
residualsolids
solidsblended
blended
with glycerol (60/40) after hydrothermal processing under selected conditions.
with glycerol (60/40) after hydrothermal processing under selected conditions.
3.3.Materials
Materialsand
andMethods
Methods
3.1.Raw
RawMaterial
Material
3.1.
Rugulopteryxokamurae
okamuraewas
wasmanually
manuallycollected
collected in
in July
July 2021
Rugulopteryx
2021 (Valdevaqueros
(Valdevaquerosand
andBolonia
BoloBeaches,
Tarifa,
Cádiz,
Spain).
Algae
were
separated
from
extraneous
material,
washed
with
nia Beaches, Tarifa, Cádiz, Spain). Algae were separated from extraneous material,
tap
water,
oven
dried,
ground
(
≤
100
µm),
and
stored
in
plastic
bags
in
the
darkness
at
room
washed with tap water, oven dried, ground (≤100 µm), and stored in plastic bags in the
temperature until use. The sea water was collected at the La Caleta Beach (Cádiz, Spain).
3.2. Microwave Assisted Extraction
The extraction was performed using a microwave reactor (Anton Paar Monowave
450, Austria). The ground seaweed was introduced with distilled or sea water in the vials
using a solid/liquid ratio of 1:30 (w/w) following a previous work [37]. The samples were
heated until the selected temperature, which was kept for 5 min at 800 rpm and 850 W prior
to cooling down to 50 ◦ C. The extraction temperatures were 160 ◦ C, 170 ◦ C, and 180 ◦ C.
Longer extraction times (up to 30 min) were assessed at the temperature conditions that
led to the best extraction results. Liquid and solid phases were split by vacuum filtration.
The solid phases or solid residue were dried and stored at room temperature.
Severity factor, R0 , was estimated according to the following equation,
(Z
! #)
"Z t
tmax
T (t) − Tre f
T ′ (t) − Tre f
f inal
log R0 = log
dt
(1)
exp
dt +
exp
ω
ω
0
tmax
where tmax (min) is the required time to achieve the target temperature, tfinal (min) is the
total time for the hydrothermal processing, T(t) and T ′ (t) are the thermal profiles in the
heating and cooling steps, respectively, Tref (100 ◦ C), and ω (14.75 ◦ C).
3.3. Alginate Precipitation
The alginate fraction was precipitated from the liquid fractions obtained after extraction
by MAE using CaCl2 (Sigma-Aldrich, St. Louis, MO, USA) at 1% (w/w) overnight, after
Mar. Drugs 2023, 21, 319
12 of 18
the fraction was recovered by centrifugation at 4500 rpm for 40 min (Rotixa 50RS, Hettich
Zentrifugen, Tuttlingen, Germany) and dried at 70 ◦ C in the laboratory oven for 48 h.
3.4. Analytical Methods for Raw Material and Solid Residue
Moisture and ash content were gravimetrically determined. In the case of moisture,
the raw material was introduced in a laboratory oven for 48 h at 105 ◦ C, and ash content
was calculated after calcination of the samples at 575 ◦ C for 6 h in a muffle.
Total nitrogen was analyzed using a Flash EA 1112 Elemental Analyser (Thermo
Electron, absorbed for Thermo Fisher Scientific, Waltham, MA, USA) equipped with a
multiple analysis column (6 × 5 mm, 2.0 m; Cromlab, Barcelona, Spain). The result was
converted to protein using the factor 5.38, specific for brown seaweeds [49].
The content of carbon and hydrogen was determined using an elemental analyzer
(Thermo Flash EA 1112, Thermo Fisher Scientific, Waltham, Massachusetts, USA). The
operation conditions used for determination were 130 mL/min for helium gas, 100 mL/min
for reference, and 250 mL/min for oxygen. The oxidation and reduction temperatures were
900 and 680 ◦ C using a laboratory oven. The analyses were conducted using a column
(6 × 5 mm, 2.0 mm) from Cromlab (Spain). The temperature selected was 50 ◦ C and 420 s
was selected as the chromatogram time, using aspartic acid as a pattern. Nitrogen (N),
hydrogen (H), and carbon (C) content were acquired from the solid phases to determine
the higher heating values (HHV) following the equation described elsewhere [48].
The total lipids content was gravimetrically determined after extraction with chloroform:methanol following the Folch method [50]. Briefly, samples were contacted with a
chloroform:methanol solution (2:1) in a solid–liquid ratio of 1:20. The mixture was centrifuged for 10 min at 300 rpm and 15 ◦ C. After filtering, 5 mL of distilled water was added,
forming a biphasic system that was separated by centrifugation using the same conditions
as in the previous stage. The lipid content was calculated after the chloroform (lower phase)
was evaporated by rotary evaporation (320 mbar at 40 ◦ C) and dried in an oven (80 ◦ C)
for 1 h.
Fatty acid methyl esters (FAMEs) were analyzed by means of transmethylation of
lipid samples (10 mg) using a solution (1 mL) of sodium hydroxide (1%) in methanol with
heating (55 ◦ C, 15 min). Then, a solution (2 mL) of hydrogen chloride (5%) in methanol
(2 mL) (55 ◦ C, 15 min) and water (1 mL) as previously detailed (Carreau and Dubacq, 1978)
were added. FAMEs were extracted using hexane prior to evaporation of the organic phase
under reduced pressure and measured using a GC-MS Trace GC Ultra (Thermo Fisher,
Waltham, MA, USA) with a ZB-WAX column (60 m × 0.25 mm internal diameter × 0.25 µm,
Zebron by Phenomenex).
The minerals of the raw material were analyzed by different methodologies. At first,
acidic digestion was performed: 0.3 g ash mixed with 10 mL HNO3 and 1 mL H2 O2 were
introduced in a Marsxpress (CEM), and the conditions of the protocol used were 1600 W
for 15 min, keeping at 200 ◦ C for 10 min. In the case of Ca, Fe, Cu, and Mg, the atomic
absorption spectroscopy technique was used, and Na and K were analyzed by atomic
emission spectroscopy (spectrometer SpectrAA-220 Fast Sequential from Varian, Palo Alto,
CA, USA). Inductively coupled plasma mass spectrometry (ICP-MS, X Series, Thermo
Scientific, Waltham, Massachusetts, USA) was used to determine Cd content.
Extractives were determined using conventional Soxhlet extraction. One part of the
ground seaweed was extracted in Soxhlet using organic solvents, ethanol (96%), hexane,
and MeOH + Acetone + H2 O (3:1:1) (Sigma-Aldrich, USA) until the solvent turned colorless.
The oligosaccharide composition of the raw material was performed by HPLC in a
1260 series Hewlett Packard chromatograph (with an IR detector); the column used was an
Aminex HPX-87H column (300 × 7.8 mm, BioRad, Hercules, CA, USA) with a pre-guard,
operating at 60 ◦ C with 0.003 M H2 SO4 at 0.6 mL/min. The content of oligosaccharides
in the raw material was determined after quantitative hydrolysis (72% sulfuric acid at
30 ◦ C, 1 h), after the solution was diluted until a concentration of 2% and introduced in
an autoclave at 121 ◦ C for 1 h. Samples were filtered, the liquid phase was analyzed in
Mar. Drugs 2023, 21, 319
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the HPLC to quantify the oligosaccharide content, the solid phase was introduced in a
laboratory oven at 105 ◦ C for 24–48 h, and the residue was gravimetrically quantified as
acid-insoluble residue (AIR).
3.5. Analytical Methods for Liquid Samples
The pH of the samples was determined in triplicate with a GLP 21 pH meter (Crison
instruments, Barcelona, Spain).
3.6. Phloroglucinol Content
This analysis was performed by spectrophotometry following the protocol described
previously [51]. In brief, the liquid sample (1 mL) was introduced in a test tube, and 1 mL
of Folin Ciocalteu reagent and 2 mL of Na2 CO3 at 20% were added. The test tube with
all the reagents was stirred in a vortex and incubated in darkness at room temperature
for 45 min. The standard used was phloroglucinol (Sigma-Aldrich, St. Louis, MO, Spain),
performing a standard curve to determine the phloroglucinol content in the samples. The
absorbance was measured at 730 nm (Evolution 201 UV–vis, Thermo Scientific, Waltham,
MA, USA).
3.7. Sulfate Content
The protocol known as gelatin-barium chloride method [52] was performed to spectrophotometrically determine the content of sulfate. Briefly, gelatin-BaCl2 reagent was
made in two steps: (1) 0.5 g gelatin powder (Scharlau, Barcelona, Spain) was dissolved in
100 mL of distilled water (previously heated until 70 ◦ C), and this solution was kept at 4 ◦ C
for at least 6 h (or overnight). After this time, 0.5 g BaCl2 (Sigma-Aldrich, Spain) was added,
obtaining a cloudy solution, and after 2–3 h, the solution was ready to use. A mixture of
0.2 mL of liquid samples or distilled water (blank), 3.8 mL of trichloroacetic acid solution at
10%, and 1 mL of gelatine-BaCl2 reagent was prepared in a test tube and incubated at room
temperature for 15 min. The standard curve was performed with potassium sulfate and the
absorbance was measured at 500 nm (Evolution 201 UV–vis, Thermo Scientific, USA).
3.8. Antioxidant Activity
The trolox equivalent antioxidant capacity (TEAC) value was determined following
the protocol described previously to measure the ABTS radical scavenging capacity [53]. At
first, the TEAC reagent was prepared: 34.8 mg of ABTS and 6.62 mg of potassium persulfate
were dissolved in 10 mL of PBS. The solution was stirred in darkness for 16 h (or overnight).
This reagent was diluted with PBS (also used as blank) until the value of 0.7 of absorbance
(at 734 nm). The samples or blank (20 µL) and diluted TEAC reagent (2 mL) were mixed in a
test tube and incubated at 30 ◦ C for 6 min. The standard curve was performed using trolox
(6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) as standard. The absorbance was
read at 734 nm (Evolution 201 UV–vis, Thermo Scientific, USA). The inhibition percentage
was studied following the protocol developed by von Gadow, Joubert, and Hansmann
(1997) to scavenge the DPPH radical (α, α-Diphenyl-picrylhydrazyl) [54]. In brief, in a test
tube, 2 mL methanolic solution of DPPH at 6 × 10−5 M was added above a 50 µL sample
solution. The absorbance was read at 10 and 16 min, at 515 nm.
3.9. Soluble Protein
The content of soluble protein was analyzed following the protocol known as the
Bradford method. The standard curve was performed using Bovine Serum Albumin (BSA,
Sigma Aldrich, USA) as standard. Bradford Reagent was used according to the protocol
supplied by Sigma-Aldrich. The test tubes were incubated at room temperature for 35 min.
The absorbance was read at 595 nm (Evolution 201 UV–vis, Thermo Scientific, USA).
Mar. Drugs 2023, 21, 319
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3.10. Oligosaccharide Determination
The oligosaccharide content in the hydrolysate samples (previously diafiltered (Spectra/Por Float-A-Lyzer Dialysis Device MWCO: 100–500 Da, SpectrumLabs)) was determined by performing a posthydrolysis with sulfuric acid at 4% at 121 ◦ C in an autoclave
(P-Selecta, Spain) for 20 min. The liquid samples were filtrated (0.45 µm, Sartorius, Madrid,
Spain) and the determination was performed by High-Pressure Liquid Chromatography
(HPLC) using a 1100 series Agilent chromatograph (St. Clara, CA, USA) described above.
In this case, the column used was an Aminex HPX87H column (300 × 7.8 mm, BioRad,
USA), operating at 60 ◦ C, the mobile phase was 0.003 M H2 SO4 (Sigma-Aldrich, USA), and
the flow rate was 0.6 mL/min.
3.11. Molar Mass Distribution
High-Performance Size Exclusion Chromatography (HPSEC) was performed to analyze the molar mass distribution of the liquid samples. The column used was a SuperMultipore PW-H column (6 mm × 15 cm) with a guard column SuperMP (PW)-H
(4.6 mm × 3.5 cm) from TSKgel by Tosoh Corporation (Japan) operating at 40 ◦ C, fitted in
the HPLC described above. The mobile phase was Milli-Q water at 0.4 mL/min. Polyethylene oxide was used as standard, from 23.6 to 786 kDa (Tosoh Corporation, Tokio, Japan).
3.12. Fourier-Transform Infrared Spectroscopy
The freeze-dried samples and alginate fractions obtained from MAE were studied by
FTIR (Nicolet 6700, source: IR, detector: DTGS KBr). The software used was OMNIC. The
extracts were blended with potassium bromide. The spectra were obtained from 400 to
2000 nm, with the resolution of 4 cm−1 and 32 scans/min.
3.13. Proton Nuclear Magnetic Resonance
1H
NMR was performed using a spectrometer (ARX400, Bruker BioSpin GmbH, Germany) for the alginate fractions’ recovery, the concentration of the solutions was 10 mg/mL
using deuterated water as solvent and 3-(trimethylsilyl)-L-propane sulfonic acid (SigmaAldrich, USA), and the assays were conducted at 75 ◦ C, 400 MHz. For the anomeric protons
of the mannuronic (M) and guluronic (G) acids, the signals of 4.70 ppm and 5.08 ppm were
found, respectively, and the M/G ratio was estimated.
3.14. Cell Inhibition Assay
The cell viability was evaluated using the cervix carcinoma (HeLa 229) cell line. These
cells were cultured in DMEM (Dulbeco Modified Eagle’s Medium) and supplemented with
FBS (10%) and L-Glutamin (2 mM) and incubated at 37 ◦ C (95% air:5% CO2 atmosphere).
The assay performed was the MTT (3-[4,5-dimethylthiazol-2-yl]-2,-5 diphenyltetrazolium bromide) test. HeLa 229 cells were seeded in a 96-well plates with 4000 cells/well
and incubated (4–6 h). The extracts were dissolved in Milli-Q water and incubated at 37 ◦ C
for 48 h (95% air:5% CO2 atmosphere).
After incubation, MTT solution (10 µL) prepared at 5 mg/mL in PBS was added to
the well plate and the mixture was incubated (4 h). Finally, SDS dissolved in 0.01 M HCl
(100 µL; 10%) was added. Then, an incubation of the well plate for 12–14 h was necessary.
The absorbance was read at 595 nm (Tecan Infinite M1000 Pro).
3.15. Rheology
The solid phase after hydrothermal treatment was proposed for the development
of bioplastics adapted from the procedure previously reported for Rugulopteryx okamurae
brown seaweed [18]. Following their achievements, the solid phases were blended with
glycerol (60/40) at 50 rpm for 15 min before printing on a Foodini 3D printer (Natural
Machines, Barcelona, Spain) at 90 ◦ C with a syringe extruder (0.8 mm nozzle) using a
rectangular model (60 × 20 × 10 mm).
Mar. Drugs 2023, 21, 319
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Rheological measurements in terms of frequency sweeps (from 0.1 to 10 Hz) were
conducted on the blends before and after processing using a controlled stress rheometer
(MCR302, Anton Paar Physica, Austria). Note here that the apparent viscosity of extracted
alginate solutions at a commonly used biopolymer content (1%) was also measured [55].
Monitoring of the viscoelastic profiles was made by means of a sand-blasted parallel-plate
geometry (25 mm diameter, 1 mm gap). Both elastic (G′ ) and viscous (G”) moduli were
recorded at 25 ◦ C within the linear viscoelastic region (15 Pa).
3.16. Statistical Analysis
Data were studied using one-factor analysis of variance using the PASW Statistics v.22
software (IBM SPSS Statistics, New York, NY, USA). If means showed differences with 95%
confidence (p < 0.05), a Scheffé post hoc test was performed.
4. Conclusions
To conclude, this study confirms the potential of a chemical-free hydrothermal treatment for the initial fractionation of Rugulopteryx okamurae biomass, a stage that could
be directly applied to the wet material using sea water. Despite the low yields, alginate
with interesting rheological properties can be obtained. The possibility of valorizing the
phenolic and the sulfated polysaccharides fraction should be studied. Further purification
of bioactive compounds in the water-soluble fraction and exploration of potential uses
would be of interest to obtain high value-added products. The residual solids accounted for
the major fraction after the microwave-assisted hydrothermal treatment, and exploration
of the uses of the biomaterials formulated are encouraged.
Supplementary Materials: The following supporting information can be downloaded at https://www.
mdpi.com/article/10.3390/md21060319/s1, Figure S1: Effect of the extraction process in R. okamurae
brown seaweed by MAE using two solvents, (a) distilled water (DW) and (b) sea water (SW), for the
profiles of molecular weight distribution (in Da) of the extracts processed at different temperatures
and times.
Author Contributions: Conceptualization, M.D.T., J.M. and H.D.; methodology, N.F.-F., M.D.T.
and H.D.; formal analysis, T.F.-A., N.F.-F., M.D.T. and H.D.; investigation, T.F.-A. and M.D.T.;
writing—original draft preparation, N.F.-F., M.D.T. and H.D.; writing—review and editing, M.D.T.,
J.M. and H.D.; supervision, N.F.-F., M.D.T. and H.D.; funding acquisition, M.D.T. and H.D. All authors
have read and agreed to the published version of the manuscript.
Funding: Authors acknowledge FTIR, NMR, and macro/micro-elements measurements to the
services of analysis of Universidade de Vigo (CACTI). Authors acknowledge Xunta de Galicia GRCED431C 2022/08. M.D.T. acknowledges the Ministry of Science, Innovation and Universities of
Spain for her postdoctoral grants (RYC2018-024454-I) and the Consellería de Cultura, Educación
e Universidade da Xunta de Galicia (ED431F 2020/01). N.F.F. thanks the Xunta de Galicia for her
postdoctoral grant (ED481D-2022/018).
Data Availability Statement: The data presented in this study are available on request from the
corresponding author.
Acknowledgments: The authors acknowledge Nieves Iglesias, who collected and sent the sea water
from Cádiz (Spain).
Conflicts of Interest: The authors declare no conflict of interest.
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