Content uploaded by Cécile Le Guillard
Author content
All content in this area was uploaded by Cécile Le Guillard on Apr 04, 2023
Content may be subject to copyright.
Available via license: CC BY 4.0
Content may be subject to copyright.
Citation: Le Guillard, C.; Bergé, J.-P.;
Donnay-Moreno, C.; Cornet, J.;
Ragon, J.-Y.; Fleurence, J.; Dumay, J.
Optimization of R-Phycoerythrin
Extraction by Ultrasound-Assisted
Enzymatic Hydrolysis: A
Comprehensive Study on the Wet
Seaweed Grateloupia turuturu.Mar.
Drugs 2023,21, 213. https://doi.org/
10.3390/md21040213
Academic Editor: Giuseppina
Chianese
Received: 27 February 2023
Revised: 17 March 2023
Accepted: 23 March 2023
Published: 28 March 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/).
marine drugs
Article
Optimization of R-Phycoerythrin Extraction by
Ultrasound-Assisted Enzymatic Hydrolysis: A Comprehensive
Study on the Wet Seaweed Grateloupia turuturu
Cécile Le Guillard 1, 2, *,† , Jean-Pascal Bergé3, Claire Donnay-Moreno 1, Josiane Cornet 1, Jean-Yves Ragon 1,
Joël Fleurence 2and Justine Dumay 2, *
1IFREMER Centre Ifremer Atlantique, EM3B, BP 21105, CEDEX 03, 44311 Nantes, France
2
Institut des Substances et Organismes de la Mer, ISOMER, Nantes Université, UR 2160, 44000 Nantes, France
3UPCYCLINK, 22 Rue de Mangorvenec, 56890 Saint-Avé, France
*Correspondence: cecile.leguillard@roullier.com or cecile.le.guillard@outlook.fr (C.L.G.);
justine.dumay@univ-nantes.fr (J.D.)
† Current address: Agro Innovation International, Timac Agro, 18 Avenue Franklin Roosevelt,
35400 Saint-Malo, France.
Abstract:
Enzyme-assisted extraction (EAE) and ultrasound-assisted extraction (UAE) are both recog-
nized as sustainable processes, but little has been done on the combined process known as ultrasound-
assisted enzymatic hydrolysis (UAEH), and even less on seaweed. The present study aimed to
optimize the UAEH of the red seaweed Grateloupia turuturu for the extraction of R-phycoerythrin
(R-PE) directly from the wet biomass by applying a response surface methodology based on a central
composite design. Three parameters were studied: the power of ultrasound, the temperature and
the flow rate in the experimental system. Data analysis demonstrated that only the temperature had
a significant and negative effect on the R-PE extraction yield. Under the optimized conditions, the
R-PE kinetic yield reached a plateau between 90 and 210 min, with a yield of 4.28
±
0.09 mg
·
g
−1
dry weight (dw) at 180 min, corresponding to a yield 2.3 times higher than with the conventional
phosphate buffer extraction on freeze-dried G. turuturu. Furthermore, the increased release of R-PE,
carbohydrates, carbon and nitrogen can be associated with the degradation of G. turuturu constitutive
polysaccharides, as their average molecular weights had been divided by 2.2 in 210 min. Our results
thus demonstrated that an optimized UAEH is an efficient method to extract R-PE from wet G.
turuturu without the need for expensive pre-treatment steps found in the conventional extraction.
UAEH represents a promising and sustainable approach that should be investigated on biomasses
where the recovery of added-value compounds needs to be improved.
Keywords:
seaweed; Grateloupia turuturu; R-phycoerythrin; ultrasound-assisted enzymatic hydrolysis
(UAEH); extraction process; response surface methodology
1. Introduction
Since the 1970s, seaweed production has been largely dominated by aquaculture,
representing 97% of the global production in 2019 (34.7 million tonnes of wet weight).
Red seaweeds are the main cultivated phylum, representing approximately 52.6% of the
global aquaculture production of seaweed in 2019 (18.3 million tonnes of wet weight) [
1
].
Some of them have been used for hundreds of years for various applications, such as for
human consumption (sea vegetable), animal feed and fertilizer, and since the 1940s they
have been used in industry for the production of hydrocolloids [
2
,
3
]. Currently, most of
the red seaweeds remain underexploited, especially in Europe where the consumption of
seaweed as food is regulated. Only a few species are therefore approved as sea vegetables or
ingredients; they are listed in the European Novel Food Catalogue (Novel Food Regulation
(EU) 2015/2283) [4].
Mar. Drugs 2023,21, 213. https://doi.org/10.3390/md21040213 https://www.mdpi.com/journal/marinedrugs
Mar. Drugs 2023,21, 213 2 of 16
Grateloupia turuturu is a polymorphic red seaweed belonging to the order of Haly-
meniales. It is a native species from Japan, and while commonly consumed in Asia, it is not
yet part of the authorized species in Europe. This non-indigenous species was introduced
in Brittany, the first record of which dates from 1989, and is considered at the moment as
potentially invasive on the French Atlantic coasts (Brittany) [
5
] as well as in Portugal where
its distribution has some typical features of an invasive organism [6]. However, this avail-
able biomass remains underexploited in spite of its content in compounds of interest. Some
studies have already demonstrated that G. turuturu could have a great potential of exploita-
tion in aquaculture, nutraceutical, pharmaceutical and cosmeceutical industries [
5
,
7
–
10
].
Indeed, it could be a source of nutritional compounds for humans and animals, with up
to approximately 60% of total dietary fibres [
11
], 27% dw of total proteins [
12
], including
all the essential amino acids for 38% of total amino acids [
13
], and around 5% of total
lipids [
14
], among which the presence of polyunsaturated fatty acids (PUFA) which are
mainly represented by eicosapentaenoic acid (
ω
3 PUFA) [
15
]. G. turuturu is also a source
of potential bioactive molecules, such as anti-microfouling and anti-bacterial, as well as
high added-value compounds such as the R-phycoerythrin pigment (R-PE) [
5
,
6
,
10
,
16
].
Additionally, previous studies reported that its biochemical composition is affected by
environmental factors, notably seasonal and geographical parameters [10–12,14].
R-phycoerythrin belongs to a family of light-harvesting pigment proteins, named phy-
cobiliproteins. It is responsible for the red-pink colour of red seaweeds by masking the green
pigment chlorophyll [
17
,
18
]. This accessory pigment is the most abundant phycobiliprotein
in Rhodophyta and consists of one proteinic part covalently linked to a chromophore to
form three subunit types
α
,
β
and
γ
. Theses subunits are associated to give the hexameric
structure of R-PE, with a molecular weight ranging from 240 to 260 kDa. The R-PE pos-
sesses specific spectral properties: it notably absorbs the light in the yellow-green spectral
range (between 498 and 565 nm) with absorbance maxima at 565 nm, 540 nm and 498 nm,
and it has a fluorescence emission maximum at around 575 nm [
17
,
19
,
20
]. This pigment is
already used as a fluorescent probe in advanced biotechnologies, as a natural food colorant
notably in Asian countries, and it exhibits several bioactivities which could be exploited
for the pharmaceutical field [
3
,
19
,
21
,
22
]. Due to these various potential markets, some
previous studies have more particularly investigated the extraction and the pre-purification
of R-PE from G. turuturu as a way to exploit this available biomass
[23–26]
. Thus, it has
been demonstrated that using a conventional extraction with sodium phosphate buffer on
freeze-dried G. turuturu, the R-PE extraction yield can reach up to 5.28 mg·g−1dw [23].
The conventional extraction method used on G. turuturu is performed on freeze-dried
thalli ground in liquid nitrogen and suspended in a phosphate buffer solution [
23
]. On some
conventional extracts it has been demonstrated that the use of precipitation, ultrafiltration
and anion exchange chromatography techniques was efficient for the partial purification or
purification of the R-PE, notably from G. turuturu [23,25–27].
However, alternative and innovative methods would be valuable, notably to avoid
expensive pre-treatments, and to save time and increase the extraction of other compounds.
In this context, enzyme-assisted extraction (EAE) has demonstrated its interest for R-PE
extraction from various red seaweed species [
28
–
30
], but without success on G. turuturu [
31
].
Over the past two decades, different innovative techniques such as microwave-assisted
extraction, supercritical fluid extraction and pressurized liquid extraction, have emerged to
retrieve biomolecules from seaweeds [
32
–
34
]. Our previous studies applied, for the first
time, to the wet G. turuturu one of those alternative techniques, namely ultrasound-assisted
extraction (UAE) and ultrasound-assisted enzymatic hydrolysis (UAEH), to extract water
soluble compounds such as R-PE [
13
,
35
]. Based on the literature reviews of the last ten years,
it appears that UAE is a promising and efficient process to extract plant pigments
[36–38]
and seaweed compounds [
39
–
45
], including phycobiliproteins [
45
,
46
]. Regarding the
UAEH, despite an increase in the use of this combined process on various biomasses of plant
origin (i.e., pumpkin, cassava waste, goji fruit and sugarcane bagasse) [
47
–
50
], only a few
studies have applied it to seaweeds, and they demonstrated promising results [
13
,
35
,
51
,
52
].
Mar. Drugs 2023,21, 213 3 of 16
However, UAEH requires further study because it is still difficult to understand the positive
effect and underlying mechanisms of ultrasound on enzymatic hydrolysis. It appears to be
generally accepted that during UAEH the positive effects of ultrasound can be attributed
to the turbulence and the mass transfer caused by the implosion of cavitation bubbles,
thereby increasing the access of the substrate to the enzyme [
50
,
53
–
55
]. In addition, the
efficiency of this combined process would be influenced by numerous operating parameters
related to enzymes, free or immobilized, and ultrasound devices [
38
,
53
,
55
,
56
] that need to
be systemically investigated and optimized. Previous works have already demonstrated
that experimental designs are an efficient tool to improve the R-PE extraction yields from
other red seaweeds by enzymatic hydrolysis [
28
–
30
], to optimize the UAE of compounds
from Ascophyllum nodosum [
44
,
57
], and the UAEH of compounds of plant and animal origin
such as bioactive polysaccharides from a plant leaves and molluscs [58,59].
The aim of the present work was to evaluate the potential of UAEH to extract R-PE
from wet G. turuturu. To this end, we used a central composite design to study the effects
of three UAEH parameters on the R-PE extraction yields: power, temperature and flow rate.
We demonstrated that after optimization of these parameters the extraction yield of R-PE
was significantly increased in comparison with the conventional extraction on freeze-dried
G. turuturu. This research article highlights the potential of this combined process to extract
high added-value compounds from biomasses recalcitrant to enzymatic hydrolysis, as well
as carbohydrates, carbon and nitrogen.
2. Results and Discussion
2.1. Experimental Design
The R-PE extraction yields obtained for each experiment are reported in Table 1. The
experiment N
◦
2, corresponding to the lowest level of temperature (20
◦
C) and medium
levels of power (300 W) and flow rate (145 L
·
h
−1
) led to the highest R-PE extraction yield.
Under these conditions, a marked increase of the R-PE in the first hour can be observed,
reaching up to 4.19 mg
·
g
−1
at 180 min, followed by a very slow decrease (4.06 mg
·
g
−1
at
360 min). Conversely, for experiment N
◦
7, in the same conditions of power and flow rate
(300 W and 145 L
·
h
−1
) but with the highest temperature (40
◦
C), the lowest R-PE extraction
yields were obtained over time, with 2.42 mg
·
g
−1
at 180 min, which represents a difference
of 1.7 times between experiment N
◦
2 and N
◦
7. This result is consistent with our previous
study demonstrating that a high temperature (40
◦
C) negatively affects the extraction of
R-PE by the UAEH process [
35
], and with previous studies demonstrating the thermal
sensitivity of this pigment [
24
,
35
,
60
]. Furthermore, all the experiments carried out at
36 ◦C
(experiment N
◦
s 10, 12, 14, 18) led to low R-PE extraction yields, relatively stable over time:
between 2.39 (experiment N
◦
14 at 180 min) and 2.92 mg
·
g
−1
(Experiment N
◦
18 at 60 min).
According to these results, it seems that it will be suitable to carry out the UAEH of G.
turuturu at the lowest level of temperature (20
◦
C), and the levels of power and flow rate
seem not to have a strong effect on the R-PE contents. These trends will then be checked by
the data analysis of the experiment design.
2.2. Predicted R-PE Extraction Yields
The R-PE extraction yields obtained for the 19 experiments were analysed indepen-
dently for each point of the kinetics (0, 30, 60, 120, 180, 240, 300, 360 min) (Table 1). The data
showed that the highest R-PE extraction yields were obtained for experiments between
120 and 240 min, for which a canonical analysis has been performed. The influence of
each variable on the R-PE extraction yield was determined. As illustrated by Pareto charts
(Figure 1a–c), at 120, 180 and 240 min the temperature increase has a significant negative
effect on the R-PE extraction yield (p< 0.001). At 120 min and 240 min, the temperature
is the only variable significantly affecting the extraction of R-PE, especially temperature
increase, which results in a negative effect. This confirms the trend noticed in our previ-
ous study conducted at 22
◦
C and 40
◦
C [
35
]. At 180 min (Figure 1b), in addition to this
significant negative effect of the temperature (B) (p< 0.001), a negative quadratic effect of
Mar. Drugs 2023,21, 213 4 of 16
the ultrasound power (AA) is observed (p< 0.05). Despite this quadratic effect, it appears
that the ultrasonic power does not have a significant effect on the R-PE extraction yield
in the tested conditions. The influence of the ultrasonic power is complex. Some studies
conducted on different biomass and in other experimental conditions of UAE or UAEH
have previously demonstrated that a power increase improved the extraction of pigments
up to a critical power level, from which the inverse effect was observed with a decrease
of pigment extraction yields [
36
,
37
,
61
]. The same observations have been depicted for
the UAEH of polysaccharides from plant materials [
47
,
49
,
58
] and molluscs [
59
]. Three
factors are believed to explain this phenomenon: too many cavitation bubbles, which
would reduce the energy transmission into the medium [
36
]; coalescence of cavitation
bubbles, which would implode less strongly [
37
]; and the inhibition of the enzymatic
activities [
61
]. Regarding the flow rate (Figure 1), it never has a significant effect on our
UAEH process. With the exception of a few publications, such as Zhao et al. working on
microalgae sonication [
62
], this parameter has been very under studied in the literature
because processes involving ultrasonic technologies used mainly batch reactors without
a recirculating loop. It has to be kept in mind that comparison between studies is quite
difficult due to the ultrasound parameters which present numerous differences, such as the
kind of ultrasonic reactor (more often ultrasonic baths or probes), the ultrasonic frequency
and intensity applied, the temperature of the medium, the solvent and matrix properties,
and how the process is carried out (open or close/batch reactor) [
38
,
63
]. This is even more
difficult for the UAEH process due to additional parameters induced by the presence of
different enzymes (type of enzymes, pH, temperature, enzyme/substrate ratio, etc) and the
interactions between ultrasound and enzymes [53].
Table 1.
UAEH conditions and response for R-PE extraction yields obtained for the central composite
design. Grey lines represent the central points.
Experiment
N◦
UAEH Conditions R-PE Extraction Yields (mg·g−1dw)
Power of
Ultrasound
(W)
Temperature
(◦C)
Flow
Rate
(L·h−1)
0
min
30
min
60
min
120
min
180
min
240
min
300
min
360
min
1 359 24 97 2.63 3.31 3.51 4.11 4.05 4.04 3.92 3.80
2 300 20 145 2.39 2.93 3.92 3.93 4.19 4.14 4.14 4.06
3 300 30 145 2.70 3.16 3.42 3.34 3.27 3.22 3.13 3.10
4 300 30 145 2.43 3.17 3.41 3.39 3.30 3.17 3.17 3.11
5 300 30 145 2.62 3.53 3.56 3.47 3.40 3.25 3.17 3.00
6 241 24 193 3.11 3.65 3.97 4.05 3.96 3.90 3.77 3.65
7 300 40 145 2.15 2.56 2.48 2.43 2.42 2.45 2.49 2.51
8 359 24 193 2.53 3.28 3.85 4.02 4.01 4.09 3.75 3.89
9 300 30 65 2.77 3.31 3.54 3.45 3.46 3.44 3.47 3.43
10 241 36 193 2.32 2.62 2.71 2.71 2.69 2.62 2.67 2.64
11 300 30 145 2.77 3.52 3.70 3.59 3.47 3.43 3.41 3.22
12 241 36 97 2.38 2.74 2.54 2.66 2.59 2.52 2.56 2.58
13 300 30 145 2.77 3.13 3.26 3.35 3.30 3.21 3.22 3.12
14 359 36 193 2.50 NA 12.61 2.53 2.39 2.43 2.48 2.46
15 200 30 145 2.49 3.10 2.92 3.01 2.93 2.87 2.82 2.82
16 300 30 225 2.67 3.02 3.16 3.20 3.16 3.14 3.14 2.96
17 400 30 145 2.19 3.12 3.25 3.31 3.18 3.29 3.18 3.22
18 359 36 97 2.56 2.81 2.92 2.76 2.69 2.60 2.59 2.71
19 241 24 97 2.42 3.00 3.50 3.60 3.68 3.59 3.66 3.63
1Not analyzed.
Mar. Drugs 2023,21, 213 5 of 16
Mar. Drugs 2023, 21, x FOR PEER REVIEW 5 of 17
because processes involving ultrasonic technologies used mainly batch reactors without a
recirculating loop. It has to be kept in mind that comparison between studies is quite dif-
ficult due to the ultrasound parameters which present numerous differences, such as the
kind of ultrasonic reactor (more often ultrasonic baths or probes), the ultrasonic frequency
and intensity applied, the temperature of the medium, the solvent and matrix properties,
and how the process is carried out (open or close/batch reactor) [38,63]. This is even more
difficult for the UAEH process due to additional parameters induced by the presence of
different enzymes (type of enzymes, pH, temperature, enzyme/substrate ratio, etc) and
the interactions between ultrasound and enzymes [53].
Figure 1. Pareto charts obtained at 120 min (a), 180 min (b) and 240 min (c) for the analysis of R-PE
extraction using the UAEH process according to temperature (B), ultrasonic power (A) and flow rate
(C) conditions. The significance level, with p value = 0.05, is represented by the vertical line. The
light and dark grey bars represent positive and negative effects, respectively.
After removing the insignificant effects, the equations of the model at 120, 180 and
240 min were obtained (Table 2). The adjusted R² values comprised between 85 and 92%
indicate a high robustness of the extraction paern of the R-PE using the UAEH process.
The UAEH process for the extraction of R-PE was thus optimized for these three kinetic
points. Remarkably, our model predicted identical optimized conditions at 120, 180 and
240 min (Table 2): 300 W, 20 °C, 145 L.h
−1
. These conditions are consistent with those that
allowed us to reach the highest R-PE extraction yield during the experimental design
Figure 1.
Pareto charts obtained at 120 min (
a
), 180 min (
b
) and 240 min (
c
) for the analysis of R-PE
extraction using the UAEH process according to temperature (B), ultrasonic power (A) and flow rate
(C) conditions. The significance level, with pvalue = 0.05, is represented by the vertical line. The light
grey and blue bars represent positive and negative effects, respectively.
After removing the insignificant effects, the equations of the model at 120, 180 and
240 min were obtained (Table 2). The adjusted R
2
values comprised between 85 and 92%
indicate a high robustness of the extraction pattern of the R-PE using the UAEH process.
The UAEH process for the extraction of R-PE was thus optimized for these three kinetic
points. Remarkably, our model predicted identical optimized conditions at 120, 180 and
240 min (Table 2): 300 W, 20
◦
C, 145 L
·
h
−1
. These conditions are consistent with those
that allowed us to reach the highest R-PE extraction yield during the experimental design
(Experiment N
◦
2, 4.19 mg
·
g
−1
dw at 180 min) (Table 1). Although the optimal operating
temperature is lower than the recommendations of the enzyme’s suppliers, previous studies
have shown that 24
◦
C is the optimized temperature for the enzymatic extraction of R-PE
from Palmaria palmata against the 40
◦
C recommended by the supplier for the working
activity of its enzyme [28]. It also demonstrates that a moderate temperature is preferable
as the R-PE is well known to be a heat-sensitive pigment [24,35,60].
Mar. Drugs 2023,21, 213 6 of 16
Table 2.
Equations of the models, the optimized conditions (Power (P), Temperature (T) and Flow
rate (Q)) and values statistically predicted at 120, 180 and 240 min for the extraction of R-PE from
Grateloupia turuturu using UAEH.
Time
(min) Equation of the Model Adjusted
R2(%)
Optimized Conditions Predicted Value
(R-PE mg·g−1dw)
P (W) T (◦C) Q (L·h−1)
120 3.31235 −0.535479 ×T 85.46 300 20 145 4.21
180 3.33989 −0.585277 ×T−0.0958245 ×P292.07 300 20 145 4.32
240 3.23246 −0.58251 ×T 88.17 300 20 145 4.21
The highest extraction yield was predicted at 180 min with 4.32 mg
·
g
−1
dw; a slightly
lower value was predicted at 120 and 240 min with 4.21 mg
·
g
−1
dw. Once again, a similar
trend was observed in experiment N
◦
2 (Table 1). Thus, it can be assumed that the highest
R-PE content was reached at approximately 180 min. It was therefore decided to increase
the number of kinetics points, every 30 min after 90 min, in order to refine the kinetic.
The UAEH were carried out over 210 min to avoid a decrease of the R-PE content in the
soluble fraction. Indeed, the duration of exposure to ultrasonic waves is an important
parameter to consider, particularly when sensitive compounds are targeted [
38
], such as
natural pigments [
36
,
64
]. Hence, it appears relevant to refine the kinetic to improve its
accuracy. The relevance of the optimized conditions was further tested by conducting
UAEH experiments in three independent replicates (n = 3), at 300 W, 20 ◦C and 145 L·h−1.
2.3. Validation of the Predicted R-PE Extraction Yield: UAEH Conducted under
Optimized Conditions
2.3.1. R-Phycoerythrin Content
The R-PE extraction yields obtained over time are represented in Figure 2. A significant
marked increase was observed in the first 45 min (from 2.05
±
0.22 to
3.55 ±0.22 mg·g−1dw
),
followed by a slower but significant increase up to 90 min (3.98
±
0.15 mg
·
g
−1
dw), lead-
ing to a plateau. The statistically predicted extraction yields at 120, 180 and
240 min
were 4.21, 4.32 and 4.21 mg
·
g
−1
dw, respectively, which is perfectly in line with the ex-
perimental results. Indeed, the highest extraction yield was observed at 180 min with
4.28
±
0.09 mg
·
g
−1
dw, whereas the values were slightly lower at 120 min and 210 min with
4.11
±
0.12 mg
·
g
−1
dw and 4.24
±
0.05 mg
·
g
−1
dw, respectively. In addition, the achieve-
ment of a refined kinetic was relevant as it allows one to see the break in the extraction yield
between 45 and 90 min, and also that R-PE extraction yields were similar (no significant
differences) from 90 to 210 min (included). The R-PE yield reached at
90 m
in was stable
for at least 2 h (corresponding to the end of the extraction) and that could be true for even
longer according to the results of experiment N
◦
2 (Table 1). Thus, in these conditions, the
soluble R-PE seems to be relatively stable under ultrasonic waves over time. Comparison
with classical maceration extraction has been performed to ensure the efficiency of this
UAEH process. Thus, the optimized UAEH allowed us to reach a higher R-PE extraction
yield (4.28
±
0.09 mg
·
g
−1
dw after 180 min (Figure 2)) than the conventional method
in sodium phosphate buffer (20 mM; pH 7.1) (1.90
±
0.03 mg
·
g
−1
dw) while avoiding
freeze-drying and grinding in liquid nitrogen, which enables savings in time and money.
This part of our study demonstrates once again that UAEH is an efficient and promising
alternative to the conventional technique to extract R-PE from G. turuturu. Finally, for
future works, thorough analyses would have to be carried out during UAEH to verify the
integrity and properties of R-PE through fluorescence measurements, electrophoresis and
chromatographic methods; after that, a pre-purification could be considered [12,23,27,60].
Mar. Drugs 2023,21, 213 7 of 16
Mar. Drugs 2023, 21, x FOR PEER REVIEW 7 of 17
integrity and properties of R-PE through fluorescence measurements, electrophoresis and
chromatographic methods; after that, a pre-purification could be considered [12,23,27,60].
Figure 2. Evolution of the R-PE extraction yield (210 min) in optimized conditions of ultrasound-
assisted enzymatic hydrolysis (UAEH) (300 W, 20 °C, 145 L.h−1). Values are means ± SD from three
independent experiments (n = 3). Significant differences (p < 0.05) are indicated by different leers.
2.3.2. Seaweed Liquefaction
Red seaweeds are a promising biomass for an integrated biorefinery approach due to
their worldwide distribution and their content in a wide range of chemical components,
such as minerals, vitamins, lipids, carbohydrates, proteins and high value-added pig-
ments (R-phycoerythrin and R-phycocyanin) [65]. Thus, R-PE is most likely not the only
compound of interest contained in the soluble fractions, and the seaweed liquefaction over
time has therefore been evaluated (Figure 3). Looking at the results, it is apparent that the
start of the kinetic shape for the liquefaction is close to the one obtained for the R-PE yield
(Figure 2), with a significant and marked increase during the first 90 min, from 45.50 ±
1.74% to 58.96 ± 1.72%. A smaller but significant increase between 90 min (58.96 ± 1.72%)
and 180 min (63.04 ± 1.08%) was then measured, indicating that the seaweed liquefaction
slowed down. At 210 min, 65.34 ± 1.46% of the seaweed compounds were released in the
soluble phase, corresponding to an increase of 1.4 times in comparison with the initial
level. Kadam et al. demonstrated that ultrasound improved the liquefaction of Ascophyl-
lum nodosum with a strong correlation between the protein extraction yield and the per-
centage of solubilized material, as seen here for our protein pigment [41]. Since the R-PE
yield appeared quite stable over time (Figure 2), it would therefore be interesting and ad-
visable to pursue the UAEH up to 90 min for the recovery of other valuable molecules in
a biorefinery approach. Further biochemical analyses at the end of UAEH (210 min) were
thus carried out, and the results are described in the next section (2.3.3). However, because
the liquefaction had also been investigated in a previous study [35], we first compared the
results obtained here and in our previous work. As expected, after 210 min in the opti-
mized conditions for the R-PE extraction, the percentage of solubilized material appeared
sensitively lower than the one obtained after only 180 min in harsher conditions of power
and temperature (400 W and 40 °C) (86.37 ± 1.43%); this is also the case for a temperature
closer to the one used in the present study (400 W and 22 °C) (76.75 ± 2.44%). By showing
that a reduction of power from 400 W to 300 W clearly decreased the rate and level of
liquefaction, this study provides novel clues regarding the effect of the ultrasonic power
on seaweed liquefaction by UAEH. This is in line with some studies investigating the UAE
Figure 2.
Evolution of the R-PE extraction yield (210 min) in optimized conditions of ultrasound-
assisted enzymatic hydrolysis (UAEH) (300 W, 20
◦
C, 145 L
·
h
−1
). Values are means
±
SD from
th
ree inde
pendent experiments (n = 3). Significant differences (p< 0.05) are indicated by different
letters.
2.3.2. Seaweed Liquefaction
Red seaweeds are a promising biomass for an integrated biorefinery approach due
to their worldwide distribution and their content in a wide range of chemical compo-
nents, such as minerals, vitamins, lipids, carbohydrates, proteins and high value-added
pigments (R-phycoerythrin and R-phycocyanin) [
65
]. Thus, R-PE is most likely not the
only compound of interest contained in the soluble fractions, and the seaweed liquefaction
over time has therefore been evaluated (Figure 3). Looking at the results, it is apparent
that the start of the kinetic shape for the liquefaction is close to the one obtained for
the R-PE yield (
Figure 2
), with a significant and marked increase during the first 90 min,
from
45.50 ±1.74%
to
58.96 ±1.72%
. A smaller but significant increase between 90 min
(
58.96 ±1.72%
) and 180 min (63.04
±
1.08%) was then measured, indicating that the sea-
weed liquefaction slowed down. At 210 min, 65.34
±
1.46% of the seaweed compounds
were released in the soluble phase, corresponding to an increase of 1.4 times in comparison
with the initial level. Kadam et al. demonstrated that ultrasound improved the liquefaction
of Ascophyllum nodosum with a strong correlation between the protein extraction yield and
the percentage of solubilized material, as seen here for our protein pigment [
41
]. Since the
R-PE yield appeared quite stable over time (Figure 2), it would therefore be interesting and
advisable to pursue the UAEH up to 90 min for the recovery of other valuable molecules
in a biorefinery approach. Further biochemical analyses at the end of UAEH (210 min)
were thus carried out, and the results are described in the next section (2.3.3). However,
because the liquefaction had also been investigated in a previous study [
35
], we first com-
pared the results obtained here and in our previous work. As expected, after 210 min in
the optimized conditions for the R-PE extraction, the percentage of solubilized material
appeared sensitively lower than the one obtained after only 180 min in harsher conditions
of power and temperature (400 W and 40
◦
C) (86.37
±
1.43%); this is also the case for a
temperature closer to the one used in the present study (400 W and 22
◦
C) (76.75
±
2.44%).
By showing that a reduction of power from 400 W to 300 W clearly decreased the rate and
level of liquefaction, this study provides novel clues regarding the effect of the ultrasonic
power on seaweed liquefaction by UAEH. This is in line with some studies investigating
the UAE process to recover bioactive compounds from brown and red seaweed species,
where the highest extraction yields were achieved by operating at the highest ultrasonic
powers tested [
39
,
44
,
66
]. Our results here with an UAEH process applied on wet G. tu-
Mar. Drugs 2023,21, 213 8 of 16
ruturu show a similar trend, where higher ultrasonic power increases the percentage of
solubilized material.
Mar. Drugs 2023, 21, x FOR PEER REVIEW 8 of 17
process to recover bioactive compounds from brown and red seaweed species, where the
highest extraction yields were achieved by operating at the highest ultrasonic powers
tested [39,44,66]. Our results here with an UAEH process applied on wet G. turuturu show
a similar trend, where higher ultrasonic power increases the percentage of solubilized ma-
terial.
Figure 3. Evolution of the seaweed liquefaction in optimized conditions for the extraction of R-PE
by ultrasound-assisted enzymatic hydrolysis (UAEH) (300 W, 20 °C, 145 L.h−1); results are the per-
centages of solubilized material over time (210 min). Values are means ± SD from three independent
experiments (n = 3). Significant differences (p < 0.05) are indicated by different leers.
2.3.3. Further Biochemical Analyses of Optimized Soluble Fractions
Biochemical analyses of soluble fractions have been carried out at the beginning (T0
min) and at the end of the extraction period (T210 min) in order to characterize the solu-
bilisation of the different compounds of G. turuturu as well as their recovery levels. The
results reported in Table 3 have been corrected by removing from soluble fractions the
amount of carbon, nitrogen and carbohydrates provided by the enzymatic cocktail. Thus,
in these conditions and at the end of optimized UAEH, nitrogen and carbon extraction
yields reached 55.51 ± 1.36% and 48.76 ± 1.53%, respectively. As with the percentage of
solubilized material, which was multiplied by 1.4 after 210 min, nitrogen and carbon ex-
traction yields also increased significantly over time, because they were multiplied by fac-
tors of 1.7 and 2.1, respectively. Regarding nitrogen, the use and the efficiency of enzymes
(proteases and/or carbohydrases) for the extraction of nitrogen compounds from sea-
weeds has been well documented [28,67–71], whereas the use of ultrasound with or with-
out enzymes is more recent [13,39–41,52]. However, our results indicate that the UAEH
process applied on wet G. turuturu is also very efficient for the carbon release, which has
not been well documented yet. Indeed, the carbohydrate content in the soluble phase has
significantly increased by 3.2-fold (141.86 ± 20.86 mg.g−1 dw after 210 min). This result is
not surprising, knowing that the enzymes used here are dedicated to the hydrolysis of
polysaccharides (glycosidases) and ultrasound have already been shown to intensify the
enzymatic hydrolysis of various biomass for the release of carbohydrates and polysaccha-
rides [40,47,48,50,51,54,59]. It is therefore logical that, when combined, these two pro-
cesses lead to a higher release of carbon in the soluble phase. Despite the limitations pre-
venting the direct comparison of two studies, it is important to remember that, in our
previous research work on G. turuturu [13], after 360 min of UAEH (400 W, 22 °C, 50 L.h−1),
the content of soluble carbohydrates was higher, reaching 210 ± 14 mg.g-1 dw. Based on
Figure 3.
Evolution of the seaweed liquefaction in optimized conditions for the extraction of R-
PE by ultrasound-assisted enzymatic hydrolysis (UAEH) (300 W, 20
◦
C, 145 L
·
h
−1
); results are
the percentages of solubilized material over time (210 min). Values are means
±
SD from three
independent experiments (n = 3). Significant differences (p< 0.05) are indicated by different letters.
2.3.3. Further Biochemical Analyses of Optimized Soluble Fractions
Biochemical analyses of soluble fractions have been carried out at the beginning
(T0 min) and at the end of the extraction period (T210 min) in order to characterize the
solubilisation of the different compounds of G. turuturu as well as their recovery levels.
The results reported in Table 3have been corrected by removing from soluble fractions the
amount of carbon, nitrogen and carbohydrates provided by the enzymatic cocktail. Thus,
in these conditions and at the end of optimized UAEH, nitrogen and carbon extraction
yields reached 55.51
±
1.36% and 48.76
±
1.53%, respectively. As with the percentage
of solubilized material, which was multiplied by 1.4 after 210 min, nitrogen and carbon
extraction yields also increased significantly over time, because they were multiplied
by factors of 1.7 and 2.1, respectively. Regarding nitrogen, the use and the efficiency
of enzymes (proteases and/or carbohydrases) for the extraction of nitrogen compounds
from seaweeds has been well documented [
28
,
67
–
71
], whereas the use of ultrasound with
or without enzymes is more recent [
13
,
39
–
41
,
52
]. However, our results indicate that the
UAEH process applied on wet G. turuturu is also very efficient for the carbon release,
which has not been well documented yet. Indeed, the carbohydrate content in the soluble
phase has significantly increased by 3.2-fold (141.86
±
20.86 mg
·
g
−1
dw after 210 min).
This result is not surprising, knowing that the enzymes used here are dedicated to the
hydrolysis of polysaccharides (glycosidases) and ultrasound have already been shown to
intensify the enzymatic hydrolysis of various biomass for the release of carbohydrates and
polysaccharides [
40
,
47
,
48
,
50
,
51
,
54
,
59
]. It is therefore logical that, when combined, these
two processes lead to a higher release of carbon in the soluble phase. Despite the limitations
preventing the direct comparison of two studies, it is important to remember that, in our
previous research work on G. turuturu [
13
], after 360 min of UAEH (400 W, 22
◦
C, 50 L
·
h
−1
),
the content of soluble carbohydrates was higher, reaching 210
±
14 mg
·
g
−1
dw. Based on
these two studies, it appears clear that UAEH parameters need to be optimized according to
the nature of the targeted compounds, and they have to be carefully considered beforehand.
Mar. Drugs 2023,21, 213 9 of 16
Table 3.
Biochemical analyses conducted on soluble fractions at T0 min and T210 min of ultrasound-
assisted enzymatic hydrolysis (UAEH) in optimized conditions (300 W, 20
◦
C, 145 L
·
h
−1
). These
values are corrected by the amounts provided by the enzymatic cocktail. Values are means
±
SD
from three independent experiments (n = 3). Significant differences between T0 and T210 min are
indicated by ** (p< 0.01), *** (p< 0.001).
Biochemical Analyses Time
T0 min T210 min
Nitrogen extraction yield (%) 32.88 ±1.81 55.51 ±1.36 ***
Carbon extraction yield (%) 22.78 ±0.66 48.76 ±1.53 ***
Carbohydrates (mg·g−1dw) 44.31 ±8.64 141.86 ±20.86 **
Weight-average molecular weight (Mw) (kDa) 1080 ±51 493 ±24 ***
The carbohydrate content in the extracts was further characterized by determining
the weight-average molecular weight (Mw) of the polysaccharides at T0 and T210 min
(Table 3). Of note, the average Mw value of the carbohydrates contained in the enzymatic
preparations has also been investigated, and the Mw appears to be much lower and clearly
distinctive of the Mw obtained at T0 and T210 for extracted polysaccharides. At the
beginning of the extraction, a high Mw was measured with a value of 1080
±
51 kDa.
Although the polysaccharides composing G. turuturu are not well known, some studies
reported the presence of cellulose, a small fraction of agar, and more recently hybrid
kappa/iota/theta carrageenans in the cell wall of this species [
6
,
31
]. This value can thus
be compared to the average Mw values of carrageenans ranging from 260 kDa for kappa
carrageenans to 1400 kDa for iota carrageenans [
72
], and the results obtained here seem to
be in accordance with the presence of hybrid kappa/iota carrageenans.
Regarding the Mw evolution over time (Table 3), a significant decrease of Mw oc-
curred during our UAEH process from 1080
±
51 kDa at T0 to 493
±
24 kDa at T210 min, a
2.2-fold reduction of the Mw. To explain this result, we hypothesize that high Mw polysac-
charides were released in the soluble phase at the beginning of UAEH, and those were
subsequently degraded by glycosidases as well as ultrasound. Indeed, it has been demon-
strated that 1
5 mi
n of UAE enabled the extraction of high molecular weight laminarins [
42
],
and other studies have demonstrated that sonication enabled polysaccharide depolymer-
ization
[59,73–75]
, including brown and red seaweed polysaccharides [
43
,
76
,
77
]. Contrary
to the enzymatic hydrolysis of cell wall polysaccharides, in which many parameters have
to be optimized (i.e., type of enzyme, enzyme/substrate ratio) for each seaweed specie [
69
],
sonication does not need a thorough knowledge of the polysaccharide structure to induce
its degradation [
73
]. Furthermore, this degradation of G. turuturu polysaccharides over
time could explain in part the increase of carbohydrates, carbon and nitrogen compounds
in the soluble fractions (Table 3), as well as the R-PE, because cell wall polysaccharides are
one of the main barriers to the solubilisation of protein components from seaweeds [69].
Regarding the pigment content, we assumed in a previous study [
35
] that co-extracted
compounds, notably carbohydrates, could be involved in the stability of the R-PE yield,
and the results obtained here are interesting in that regard.. Indeed, a previous study
conducted in our laboratory demonstrated that among the different food preservatives
tested, sucrose and ascorbic acid were the most efficient to preserve R-PE [
12
]. Ascorbic
is well known for its antioxidative properties, a patent has demonstrated that it allowed
photo-stabilization of phycoerythrin retrieved from Porphyridium cruentum [
78
]. Thus, it
can be assumed that natural bioactive compounds from seaweed, such as antioxidative
polysaccharides [
79
], could contribute to R-PE preservation. Moreover, the antioxidant
activity of seaweed polysaccharides can be improved by enzymatic hydrolysis [
80
] and
sonication [
76
,
81
], through their Mw decrease [
76
,
80
,
81
], and this is even more true with
the UAEH process [
59
]. The assumed link between R-PE yield and co-extracted bioactive
carbohydrates could be evaluated in the future by assessing the antioxidant properties of
our soluble fractions.
Mar. Drugs 2023,21, 213 10 of 16
3. Materials and methods
3.1. Materials
Seaweeds, G. turuturu, were harvested on 30 April 2014, in the intertidal zone of
Batz-sur-Mer on the Atlantic coast, France. Epiphytes were removed by hand and algae
were partially dewatered with a spin-dryer, then vacuum-packed (Boulanger INV 40)
and immediately frozen in plastic bags stored at
−
20
◦
C in darkness. Algae were used
for experiments within the year after the date of harvest. A part of G. turuturu was
also freeze-dried after harvesting to perform conventional extraction of R-PE. For UAEH,
four industrial carbohydrase preparations were used and combined according to their
similar pH and temperature optima and their complementarity [
13
,
35
]. The enzymatic
cocktail was thus composed of Sumizyme TG and Sumizyme MC, produced by SHIN
NIHON CHEMICAL and kindly provided by Takabio (Beaucouzé, France); Multifect
®
CX 15 L, kindly provided by DuPont
™
(Wilmington, DE, USA); and Ultraflo
®
XL, kindly
provided by Novozymes
®
(Bagsværd, Denmark). The ultrasonic flow-through reactor
(SONITUBE
®
35 kHz, 200 to 400 W) was manufactured and kindly provided by SYNETUDE
(Chambéry, France).
3.2. Ultrasound-Assisted Enzymatic Hydrolysis (UAEH)
3.2.1. Experimental System
The extraction of R-PE requires one to take into consideration some parameters af-
fecting its stability. The R-PE is stable in a wide range of pH [
24
,
60
,
82
] from pH 3 to 10
for the R-PE extracted from G. turuturu [
24
]. However, it is very sensitive to light [
24
] and
heat, thus it is recommended to not expose it to a temperature higher than 40–60
◦
C or less
(30 ◦C) depending on the duration of exposure and pH [24,35,60,83].
Prior to UAEH experiments, seaweeds were cut into small pieces (approximately
5–7 mm2
) using a cutting mill (Microcut Stephan MC 15, Germany) and subsequently stored
at
−
20
◦
C. According to the process developed in a previous study [
35
], all the experiments
were performed in a jacketed glass reactor vessel (5 L) containing approximately 3 kg
of reaction mixture, composed of 20% wet and cut seaweed homogenized in tap water
(corresponding to the minimal water quantity to obtain an effective circulation of the
reaction mixture) with the pH adjusted to 5.5 by the addition of 6 M HCl (Radiometer
analytical TitraLab
®
854, HACH
®
, Loveland, CO, USA). Homogenization was conducted
continuously at 100 rpm (Stuart
®
Overhead Stirrer SS20, Bibby Scientific Ltd., Stone, UK),
and the reaction mixture was circulated using a peristaltic pump (Heidolph PD 5006—SP
standard). An external circulation system (Hitema
®
ESE 010, Bovolenta Padua, Italy and
Memmert, Schwabach, Germany) was used to control and adjust the temperature in the
reactor during the 6 h of the process. To ensure R-PE preservation, the whole system was
kept in darkness.
The UAEH was initiated by the application of ultrasound (SONITUBE
®
—SYNETUDE,
Chambéry, France—turned on) and the simultaneous addition of the enzymatic cocktail
in the reaction mixture. One percent w/w of each enzymatic preparation related to the
weight of wet seaweed was added. Experiments were monitored for up to 360 min,
and regular sampling (
±
30 mL) was carried out throughout the experiment. Samples
were immediately centrifuged (15,500
×
g, 30 min, 20
◦
C, Beckman Coulter Avanti
®
J-
E Centrifuge) providing supernatant and sludge fractions that were weighed and then
freeze-dried. The temperature was regulated, and pH was monitored inside the reactor
continuously during the whole experiment.
3.2.2. Experimental Design
Response surface methodology (RSM) was used to investigate the influence of three
variables of ultrasound-assisted enzymatic hydrolysis on the R-PE extraction. The temper-
ature (T) was chosen because R-PE is known to be heat-sensitive and it is also a variable
affecting enzyme activities, the power of ultrasound (P) was studied as its effect has never
been, to the best of our knowledge, studied for the R-PE extraction, and finally the flow
Mar. Drugs 2023,21, 213 11 of 16
rate in the experimental system (Q), because it impacts the length of stay of seaweed and
enzymes in the SONITUBE, thus impacting how long they are subjected to ultrasonic waves.
For a second time, these three variables were optimized. A 2
3
central composite design
(CCD) was conducted, each variable taking 5 levels:
−α
,
−
1, 0, +1, +
α
. Star points (
−α
and +α) were varied systematically at high (Max) and low (Min) levels. The real values of
the factorial points (
−
1 and +1) were calculated for each variable according to
Equation (1)
and (2):
−1=Min +Max
2−1
αMax −Min +Max
2(1)
+1=Min +Max
2+1
αMax −Min +Max
2(2)
Central points (0) were the medium levels of all three factors studied (0; 0; 0); they
were used to check the linear relationship between the high and low levels of the variables
tested (Table 4).
Table 4.
Experimental design levels of independent variables used in the UAEH of Grateloupia
turuturu (α= 1.682).
Levels of Independent Variable Power of Ultrasound (W) Temperature (◦C) Flow Rate (L·h−1)
−α200 20 65
−1 241 24 97
0 300 30 145
+1 359 36 193
+α400 40 225
The measured variable response was the R-PE extraction yield in the soluble fraction.
A total of 19 experiments were carried out, corresponding to 2
3
factorial points, 2
×
3 star
points and 5 central points. Thus, the central points were performed in n = 5 (Table 1,
experiments N
◦
3, 4, 5, 11, 13) and all the other experiments were carried out in n = 1.
Kinetics (0, 30, 60, 120, 180, 240, 300, 360 min) were carried out in order to follow the
evolution of the R-PE extraction yields, but also to compare them with the kinetics of our
previous study [
35
]. The order of the experiments was fully randomized. The mathematical
analyses of data were carried out by the software Statgraphics Plus v.5 Experimental Design
(Statgraphics Technologies Inc., The Plains, VA, USA). The accuracy of the model was
further tested by conducting additional UAEH experiments.
All the optimized extractions and biochemical analyses were carried out in three
independent replicates (n = 3). Means and standard deviations (SD) are given for three
independent experiments. Pairwise comparisons were carried out using t-test (p< 0.05).
Multiple comparison tests were carried out using the Holm–Sidak test following the
ANOVA procedure (p< 0.05). Analyses were performed using the software SigmaStat 3.5
(Systat Software Inc., CA, San Jose, USA).
3.3. Conventional Extraction of R-PE
Freeze-dried G. turuturu was ground in liquid nitrogen to obtain a fine powder, and
subsequently suspended in sodium phosphate buffer (20 mM; pH 7.1). The extraction was
performed with a 1/20 ratio (w/v) for 20 min at 4
◦
C; the suspension was then centrifuged
(25,000×g, 20 min, 4 ◦C) [24].
3.4. Analyses
3.4.1. R-Phycoerythrin (R-PE)
Absorption spectra were monitored from 200 to 800 nm using a UV-VIS spectropho-
tometer (Shimadzu UV-1800). The R-PE concentrations were determined spectrometrically
Mar. Drugs 2023,21, 213 12 of 16
using the Beer and Eshel equation [
84
] (Equation (3)), where A
565
,A
592
,A
455
are the ab-
sorbances at 565 nm, 592 nm, 455 nm:
[R-PE] = [(A565 −A592)−(A455 −A592)×0.20] ×0.12 (3)
The R-PE extraction yield was expressed as mg·g−1seaweed dry weight (dw).
3.4.2. Determination of Seaweed Liquefaction
For the optimized conditions, the liquefaction of the material was determined over
time. The proportion of soluble material was obtained, for each time, by calculating the
ratio between the weight of the freeze-dried supernatant (m
1
) and the weight of the freeze-
dried supernatant (m
1
) added to the weight of the freeze-dried sludge (m
2
) [
13
], expressed
in percentage, according to Equation (4):
Solubilized material =m1
(m1+m2)×100 (4)
3.4.3. Elemental Composition: Carbon and Nitrogen
The elemental C and N composition was determined on dehydrated samples: freeze-
dried seaweed ground in liquid nitrogen (algal powder) and freeze-dried supernatants [
13
].
These dehydrated samples were weighed (1.5–5 mg) and placed in small tin capsules that
were carbonized by flash combustion at 1800
◦
C. The C and N contents were oxidized
and converted into a gaseous form, at 950
◦
C in a combustion column and at 750
◦
C in a
reduction column. The gases formed were transferred by carrier gas (helium) and analysed
by gas chromatography (FLASH 2000 NC Organic Elemental Analyzer—ThermoScientific,
Thermo Fisher Scientific Inc., Waltham, MA, USA). The results were integrated using the
Eager Xperience for Flash software Ver. 1.1 (Thermo Fisher Scientific, Inc., Waltham, MA,
USA). Carbon and nitrogen extraction yields were expressed as a percentage of the initial
carbon and nitrogen seaweed content (%).
3.4.4. Soluble Carbohydrates
The water-soluble carbohydrates were analysed using a phenol-sulfuric acid method [
85
].
Glucose was used as a standard (range from to 15 to 150 mg
·
L
−1
) and the absorbance was
measured at 490 nm (Shimadzu UV-1800, UV-VIS Spectrophotometer, Kyoto, Japan). The
extraction yield of soluble carbohydrates was expressed as mg
·
g
−1
seaweed dry weight
(dw) [13].
3.4.5. Weight-Average Molecular Weight (Mw) Determination
The weight-average molecular weight (Mw) was determined by high-performance
size exclusion chromatography (HPSEC) (HPLC system Prominence, Shimadzu, Kyoto,
Japan) coupled with a multiangle light scattering detector (MALS, Dawn Heleos-II, Wyatt
Technology, Santa Barbara, CA, USA) and a differential refractive index (RI) detector
(Optilab, Wyatt Technology, Santa Barbara, CA, USA), according to the method of Chopin
et al. [
86
]. The samples, freeze-dried supernatants at T0 and 210 min, were dissolved in
distilled water at 2 mg
·
mL
−1
and filtered through a 0.45
µ
m cellulose acetate syringe filter
before being injected.
4. Conclusions
This study confirmed the influence of temperature on the R-PE extracted by UAEH of
G. turuturu. The determined optimized conditions (300 W, 20
◦
C, 145 L
·
h
−1
) for the R-PE
extraction made it possible to obtain a 2.3 times higher yield (4.28
±
0.09 mg
·
g
−1
dw at
180 min) than with the conventional sodium phosphate buffer method, while avoiding
expensive pre-treatments of freeze-drying and cryo-grinding. The R-PE yields reached
a plateau between 90 and 210 min, and the pigment appears to be stable over time in
those optimized conditions. Furthermore, after 210 min, UAEH has not only led to an
Mar. Drugs 2023,21, 213 13 of 16
increase in R-PE extraction but also in carbohydrates as well as nitrogen and carbon with
extraction yields multiplied by 3.2, 1.7 and 2.1, respectively. The improved extraction of
R-PE and other solubilized compounds could be attributed to the enzymatic and ultrasonic
degradation of polysaccharides (Mw divided by 2.2 in 210 min), and in particular cell-
wall polysaccharides, resulting in higher material transfer to the soluble phase and a
potential increase in compounds preserving the R-PE. Based on these results, it would now
be pertinent to thoroughly characterize and purify the R-PE, and to make an economic
evaluation of this UAEH process thereafter. Finally, UAEH could thus be an efficient
alternative to the conventional extraction processes currently applied on various biomasses
(i.e., seaweed, microalgae, plants) that still present low extraction yields.
Author Contributions:
Conceptualization, C.L.G., J.-P.B. and J.D.; formal analysis, C.L.G.; funding
acquisition, J.-P.B. and J.D.; investigation, C.L.G., C.D.-M., J.-Y.R. and J.C.; methodology, C.L.G., J.-P.B.
and J.D.; resources, J.-P.B. and J.D.; supervision, J.-P.B. and J.D.; validation, C.L.G., J.-P.B. and J.D.;
visualization, C.L.G.; writing—original draft, C.L.G.; writing—review and editing, C.L.G., J.-P.B., J.F.
and J.D. All authors have read and agreed to the published version of the manuscript.
Funding:
This research was funded by the Région Pays de La Loire and the MSH Ange Guépin,
France (COSELMAR project).
Data Availability Statement:
No new data were created or analyzed in this study. Data sharing is
not applicable to this article.
Acknowledgments:
The authors thank DuPont
™
, Novozymes
®
and Takabio for kindly providing
the enzymes, SYNETUDE for providing the SONITUBE
®
. The authors thank Ewa Lukomska for
elemental analyses, Corinne Sinquin for HPSEC analyses, Régis Baron for statistical advices and
Xavier Guillory for his advice and proofreading.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
Cai, J.; Lovatelli, A.; Aguilar-Manjarrez, J.; Cornish, L.; Dabbadie, L.; Desrochers, A.; Diffey, S.; Garrido Gamarro, E.; Geehan,
J.; Hurtado, A.; et al. Seaweeds and Microalgae: An Overview for Unlocking Their Potential in Global Aquaculture Development; FAO
Fisheries and Aquaculture Circular; FAO: Rome, Italy, 2021. [CrossRef]
2.
Delaney, A.; Frangoudes, K.; Ii, S.-A. Chapter 2—Society and Seaweed: Understanding the Past and Present. In Seaweed in Health
and Disease Prevention; Academic Press: San Diego, CA, USA, 2016; pp. 7–40. ISBN 978-0-12802-772-1.
3.
Fleurence, J. Chapter 5—Seaweeds as Food. In Seaweed in Health and Disease Prevention; Academic Press: San Diego, CA, USA,
2016; pp. 149–167. ISBN 978-0-12802-772-1.
4.
Mendes, M.C.; Navalho, S.; Ferreira, A.; Paulino, C.; Figueiredo, D.; Silva, D.; Gao, F.; Gama, F.; Bombo, G.; Jacinto, R.; et al. Algae
as Food in Europe: An Overview of Species Diversity and Their Application. Foods 2022,11, 1871. [CrossRef]
5.
Stiger-Pouvreau, V.; Thouzeau, G. Marine Species Introduced on the French Channel-Atlantic Coasts: A Review of Main Biological
Invasions and Impacts. Open J. Ecol. 2015,5, 227–257. [CrossRef]
6.
Cardoso, I.; Cotas, J.; Rodrigues, A.; Ferreira, D.; Osório, N.; Pereira, L. Extraction and Analysis of Compounds with Antibacterial
Potential from the Red Alga Grateloupia turuturu.J. Mar. Sci. Eng. 2019,7, 220. [CrossRef]
7.
Almeida, J.; Ferreira, T.; Santos, S.; Pires, M.J.; da Costa, R.M.G.; Medeiros, R.; Bastos, M.M.S.M.; Neuparth, M.J.; Faustino-Rocha,
A.I.; Abreu, H.; et al. The Red Seaweed Grateloupia turuturu Prevents Epidermal Dysplasia in HPV16-Transgenic Mice. Nutrients
2021,13, 4529. [CrossRef] [PubMed]
8.
Félix, C.; Félix, R.; Carmona, A.M.; Januário, A.P.; Dias, P.D.M.; Vicente, T.F.L.; Silva, J.; Alves, C.; Pedrosa, R.; Novais, S.C.; et al.
Cosmeceutical Potential of Grateloupia turuturu: Using Low-Cost Extraction Methodologies to Obtain Added-Value Extracts. Appl.
Sci. 2021,11, 1650. [CrossRef]
9.
da Costa, E.; Melo, T.; Reis, M.; Domingues, P.; Calado, R.; Abreu, M.H.; Domingues, M.R. Polar Lipids Composition, Antioxidant
and Anti-Inflammatory Activities of the Atlantic Red Seaweed Grateloupia turuturu.Mar. Drugs 2021,19, 414. [CrossRef]
10.
Garcia-Bueno, N.; Decottignies, P.; Turpin, V.; Dumay, J.; Paillard, C.; Stiger-Pouvreau, V.; Kervarec, N.; Pouchus, Y.-F.; Aitor
Marin-Atucha, A.; Fleurence, J. Seasonal Antibacterial Activity of Two Red Seaweeds, Palmaria palmata and Grateloupia turuturu,
on European Abalone Pathogen Vibrio harveyi.Aquat. Living Resour. 2014,27, 83–89. [CrossRef]
11.
Denis, C.; Morançais, M.; Li, M.; Deniaud, E.; Gaudin, P.; Wielgosz-Collin, G.; Barnathan, G.; Jaouen, P.; Fleurence, J. Study of the
Chemical Composition of Edible Red Macroalgae Grateloupia turuturu from Brittany (France). Food Chem.
2010
,119, 913–917.
[CrossRef]
Mar. Drugs 2023,21, 213 14 of 16
12.
Munier, M. Étude du Polymorphisme Pigmentaire de l’Algue Rouge Grateloupia turuturu au Travers de la Purification et de
la Caractérisation de la R-Phycoérythrine en vue d’une Valorisation Industrielle. Ph.D. Thesis, Universitéde Nantes, Nantes,
France, 2013.
13.
Le Guillard, C.; Bergé, J.-P.; Donnay-Moreno, C.; Bruzac, S.; Ragon, J.-Y.; Baron, R.; Fleurence, J.; Dumay, J. Soft Liquefaction of
the Red Seaweed Grateloupia turuturu Yamada by Ultrasound-Assisted Enzymatic Hydrolysis Process. J. Appl. Phycol.
2016
,28,
2575–2585. [CrossRef]
14.
Munier, M.; Dumay, J.; Morançais, M.; Jaouen, P.; Fleurence, J. Variation in the Biochemical Composition of the Edible Seaweed
Grateloupia turuturu Yamada Harvested from Two Sampling Sites on the Brittany Coast (France): The Influence of Storage Method
on the Extraction of the Seaweed Pigment R-Phycoerythrin. J. Chem. 2013,2013, 568548. [CrossRef]
15.
Kendel, M.; Couzinet-Mossion, A.; Viau, M.; Fleurence, J.; Barnathan, G.; Wielgosz-Collin, G. Seasonal Composition of Lipids,
Fatty Acids, and Sterols in the Edible Red Alga Grateloupia turuturu.J. Appl. Phycol. 2013,25, 425–432. [CrossRef]
16.
Plouguerné, E.; Hellio, C.; Deslandes, E.; Véron, B.; Stiger-Pouvreau, V. Anti-Microfouling Activities in Extracts of Two Invasive
Algae: Grateloupia turuturu and Sargassum muticum: Botanica Marina. Bot. Mar. 2008,51, 202–208. [CrossRef]
17.
Dumay, J.; Morançais, M. Chapter 9—Proteins and Pigments. In Seaweed in Health and Disease Prevention; Academic Press: San
Diego, CA, USA, 2016; pp. 275–318. ISBN 978-0-12802-772-1.
18.
Dawes, C. Chapter 4—Macroalgae Systematics. In Seaweed in Health and Disease Prevention; Academic Press: San Diego, CA, USA,
2016; pp. 107–148. ISBN 978-0-12802-772-1.
19.
Dumay, J.; Morançais, M.; Munier, M.; Le Guillard, C.; Fleurence, J. Chapter Eleven—Phycoerythrins: Valuable Proteinic Pigments
in Red Seaweeds. In Advances in Botanical Research; Sea Plants; Bourgougnon, N., Ed.; Academic Press: Cambridge, MA, USA,
2014; Volume 71, pp. 321–343. ISBN 0065-2296.
20.
Morançais, M.; Mouget, J.-L.; Dumay, J. Chapter 7—Proteins and Pigments. In Microalgae in Health and Disease Prevention; Levine,
I.A., Fleurence, J., Eds.; Academic Press: Cambridge, MA, USA, 2018; pp. 145–175. ISBN 978-0-12811-405-6.
21.
Sudhakar, M.P.; Jagatheesan, A.; Perumal, K.; Arunkumar, K. Methods of Phycobiliprotein Extraction from Gracilaria crassa and
Its Applications in Food Colourants. Algal Res. 2015,8, 115–120. [CrossRef]
22.
Sekar, S.; Chandramohan, M. Phycobiliproteins as a Commodity: Trends in Applied Research, Patents and Commercialization. J.
Appl. Phycol. 2008,20, 113–136. [CrossRef]
23.
Munier, M.; Morançais, M.; Dumay, J.; Jaouen, P.; Fleurence, J. One-Step Purification of R-Phycoerythrin from the Red Edible
Seaweed Grateloupia turuturu.J. Chromatogr. B 2015,992, 23–29. [CrossRef]
24.
Munier, M.; Jubeau, S.; Wijaya, A.; Morançais, M.; Dumay, J.; Marchal, L.; Jaouen, P.; Fleurence, J. Physicochemical Factors
Affecting the Stability of Two Pigments: R-Phycoerythrin of Grateloupia turuturu and B-Phycoerythrin of Porphyridium cruentum.
Food Chem. 2014,150, 400–407. [CrossRef]
25.
Denis, C.; Ledorze, C.; Jaouen, P.; Fleurence, J. Comparison of Different Procedures for the Extraction and Partial Purification of
R-Phycoerythrin from the Red Macroalga Grateloupia turuturu.Bot. Mar. 2009,52, 278–281. [CrossRef]
26.
Denis, C.; Massé, A.; Fleurence, J.; Jaouen, P. Concentration and Pre-Purification with Ultrafiltration of a R-Phycoerythrin
Solution Extracted from Macro-Algae Grateloupia turuturu: Process Definition and up-Scaling. Sep. Purif. Technol.
2009
,69, 37–42.
[CrossRef]
27.
Dumay, J.; Morançais, M.; Nguyen, H.P.T.; Fleurence, J. Extraction and Purification of R-Phycoerythrin from Marine Red Algae.
Methods Mol. Biol. 2015,1308, 109–117. [CrossRef]
28.
Dumay, J.; Clément, N.; Morançais, M.; Fleurence, J. Optimization of Hydrolysis Conditions of Palmaria palmata to Enhance
R-Phycoerythrin Extraction. Bioresour. Technol. 2013,131, 21–27. [CrossRef]
29.
Nguyen, H.P.T.; Morançais, M.; Fleurence, J.; Dumay, J. Mastocarpus stellatus as a Source of R-Phycoerythrin: Optimization of
Enzyme Assisted Extraction Using Response Surface Methodology. J. Appl. Phycol. 2017,29, 1563–1570. [CrossRef]
30.
Mensi, F.; Ksouri, J.; Seale, E.; Romdhane, M.; Fleurence, J. A Statistical Approach for Optimization of R-Phycoerythrin Extraction
from the Red Algae Gracilaria verrucosa by Enzymatic Hydrolysis Using Central Composite Design and Desirability Function. J.
Appl. Phycol. 2012,24, 915–926. [CrossRef]
31.
Denis, C.; Morançais, M.; Gaudin, P.; Fleurence, J. Effect of Enzymatic Digestion on Thallus Degradation and Extraction of
Hydrosoluble Compounds from Grateloupia turuturu.Bot. Mar. 2009,52, 262–267. [CrossRef]
32.
Kadam, S.U.; Álvarez, C.; Tiwari, B.K.; O’Donnell, C.P. Chapter 9—Extraction of Biomolecules from Seaweeds. In Seaweed
Sustainability; Troy, B.K.T.J., Ed.; Academic Press: San Diego, CA, USA, 2015; pp. 243–269. ISBN 978-0-12418-697-2.
33. Michalak, I.; Chojnacka, K. Algal Extracts: Technology and Advances. Eng. Life Sci. 2014,14, 581–591. [CrossRef]
34.
Cikoš, A.-M.; Jokic, S.; Šubaric, D.; Jerkovic, I. Overview on the Application of Modern Methods for the Extraction of Bioactive
Compounds from Marine Macroalgae. Mar. Drugs 2018,16, 20. [CrossRef] [PubMed]
35.
Le Guillard, C.; Dumay, J.; Donnay-Moreno, C.; Bruzac, S.; Ragon, J.-Y.; Fleurence, J.; Bergé, J.-P. Ultrasound-Assisted Extraction
of R-Phycoerythrin from Grateloupia turuturu with and without Enzyme Addition. Algal Res. 2015,12, 522–528. [CrossRef]
36.
Maran, J.P.; Priya, B.; Nivetha, C.V. Optimization of Ultrasound-Assisted Extraction of Natural Pigments from Bougainvillea glabra
Flowers. Ind. Crops Prod. 2015,63, 182–189. [CrossRef]
37.
Dey, S.; Rathod, V.K. Ultrasound Assisted Extraction of
β
-Carotene from Spirulina platensis.Ultrason. Sonochem.
2013
,20, 271–276.
[CrossRef]
Mar. Drugs 2023,21, 213 15 of 16
38.
Shirsath, S.R.; Sonawane, S.H.; Gogate, P.R. Intensification of Extraction of Natural Products Using Ultrasonic Irradiations—A
Review of Current Status. Chem. Eng. Process. Process Intensif. 2012,53, 10–23. [CrossRef]
39.
Wang, F.; Guo, X.-Y.; Zhang, D.-N.; Wu, Y.; Wu, T.; Chen, Z.-G. Ultrasound-Assisted Extraction and Purification of Taurine from
the Red Algae Porphyra yezoensis.Ultrason. Sonochem. 2015,24, 36–42. [CrossRef]
40.
Fidelis, G.P.; Gomes Camara, R.B.; Queiroz, M.F.; Santos Pereira Costa, M.S.; Santos, P.C.; Oliveira Rocha, H.A.; Costa, L.S.
Proteolysis, NaOH and Ultrasound-Enhanced Extraction of Anticoagulant and Antioxidant Sulfated Polysaccharides from the
Edible Seaweed, Gracilaria birdiae.Molecules 2014,19, 18511–18526. [CrossRef]
41.
Kadam, S.U.; Álvarez, C.; Tiwari, B.K.; O’Donnell, C.P. Extraction and Characterization of Protein from Irish Brown Seaweed
Ascophyllum nodosum.Food Res. Int. 2016,99, 1021–1027. [CrossRef]
42.
Kadam, S.U.; O’Donnell, C.P.; Rai, D.K.; Hossain, M.B.; Burgess, C.M.; Walsh, D.; Tiwari, B.K. Laminarin from Irish Brown
Seaweeds Ascophyllum nodosum and Laminaria hyperborea: Ultrasound Assisted Extraction, Characterization and Bioactivity. Mar.
Drugs 2015,13, 4270–4280. [CrossRef]
43.
Flórez-Fernández, N.; López-García, M.; González-Muñoz, M.J.; Vilariño, J.M.L.; Domínguez, H. Ultrasound-Assisted Extraction
of Fucoidan from Sargassum muticum.J. Appl. Phycol. 2017,29, 1553–1561. [CrossRef]
44.
Kadam, S.U.; Tiwari, B.K.; Smyth, T.J.; O’Donnell, C.P. Optimization of Ultrasound Assisted Extraction of Bioactive Components
from Brown Seaweed Ascophyllum nodosum Using Response Surface Methodology. Ultrason. Sonochem.
2015
,23, 308–316.
[CrossRef] [PubMed]
45.
Carreira-Casais, A.; Otero, P.; Garcia-Perez, P.; Garcia-Oliveira, P.; Pereira, A.G.; Carpena, M.; Soria-Lopez, A.; Simal-Gandara, J.;
Prieto, M.A. Benefits and Drawbacks of Ultrasound-Assisted Extraction for the Recovery of Bioactive Compounds from Marine
Algae. Int. J. Environ. Res. Public Health 2021,18, 9153. [CrossRef]
46.
Mittal, R.; Tavanandi, H.A.; Mantri, V.A.; Raghavarao, K.S.M.S. Ultrasound Assisted Methods for Enhanced Extraction of
Phycobiliproteins from Marine Macro-Algae, Gelidium pusillum (Rhodophyta). Ultrason. Sonochem.
2017
,38, 92–103. [CrossRef]
[PubMed]
47.
Wu, H.; Zhu, J.; Diao, W.; Wang, C. Ultrasound-Assisted Enzymatic Extraction and Antioxidant Activity of Polysaccharides from
Pumpkin (Cucurbita moschata). Carbohydr. Polym. 2014,113, 314–324. [CrossRef]
48.
Leaes, E.X.; Zimmermann, E.; Souza, M.; Ramon, A.P.; Mezadri, E.T.; Dal Prá, V.; Terra, L.M.; Mazutti, M.A. Ultrasound-Assisted
Enzymatic Hydrolysis of Cassava Waste to Obtain Fermentable Sugars. Biosyst. Eng. 2013,115, 1–6. [CrossRef]
49.
Liu, Y.; Gong, G.; Zhang, J.; Jia, S.; Li, F.; Wang, Y.; Wu, S. Response Surface Optimization of Ultrasound-Assisted Enzymatic
Extraction Polysaccharides from Lycium barbarum.Carbohydr. Polym. 2014,110, 278–284. [CrossRef]
50.
Lunelli, F.C.; Sfalcin, P.; Souza, M.; Zimmermann, E.; Dal Prá, V.; Foletto, E.L.; Jahn, S.L.; Kuhn, R.C.; Mazutti, M.A. Ultrasound-
Assisted Enzymatic Hydrolysis of Sugarcane Bagasse for the Production of Fermentable Sugars. Biosyst. Eng.
2014
,124, 24–28.
[CrossRef]
51.
Korzen, L.; Pulidindi, I.N.; Israel, A.; Abelson, A.; Gedanken, A. Single Step Production of Bioethanol from the Seaweed Ulva
rigida Using Sonication. RSC Adv. 2015,5, 16223–16229. [CrossRef]
52.
Romarís-Hortas, V.; Bermejo-Barrera, P.; Moreda-Piñeiro, A. Ultrasound-Assisted Enzymatic Hydrolysis for Iodinated Amino
Acid Extraction from Edible Seaweed before Reversed-Phase High Performance Liquid Chromatography–Inductively Coupled
Plasma-Mass Spectrometry. J. Chromatogr. A 2013,1309, 33–40. [CrossRef] [PubMed]
53.
Delgado-Povedano, M.M.; Luque de Castro, M.D. A Review on Enzyme and Ultrasound: A Controversial but Fruitful Relationship.
Anal. Chim. Acta 2015,889, 1–21. [CrossRef] [PubMed]
54.
Yachmenev, V.; Condon, B.; Klasson, T.; Lambert, A. Acceleration of the Enzymatic Hydrolysis of Corn Stover and Sugar Cane
Bagasse Celluloses by Low Intensity Uniform Ultrasound. J. Biobased Mater. Bioenergy 2009,3, 25–31. [CrossRef]
55.
De Souza Soares, A.; Augusto, P.E.D.; de Castro Leite Júnior, B.R.; Nogueira, C.A.; Vieira, É.N.R.; de Barros, F.A.R.; Stringheta,
P.C.; Ramos, A.M. Ultrasound Assisted Enzymatic Hydrolysis of Sucrose Catalyzed by Invertase: Investigation on Substrate,
Enzyme and Kinetics Parameters. LWT 2019,107, 164–170. [CrossRef]
56.
Priya; Gogate, P.R. Ultrasound-Assisted Intensification of Activity of Free and Immobilized Enzymes: A Review. Ind. Eng. Chem.
Res. 2021,60, 9650–9668. [CrossRef]
57.
Gisbert, M.; Barcala, M.; Rosell, C.M.; Sineiro, J.; Moreira, R. Aqueous Extracts Characteristics Obtained by Ultrasound-Assisted
Extraction from Ascophyllum nodosum Seaweeds: Effect of Operation Conditions. J. Appl. Phycol.
2021
,33, 3297–3308. [CrossRef]
58.
Chen, R.; Li, S.; Liu, C.; Yang, S.; Li, X. Ultrasound Complex Enzymes Assisted Extraction and Biochemical Activities of
Polysaccharides from Epimedium Leaves. Process Biochem. 2012,47, 2040–2050. [CrossRef]
59.
Liao, N.; Zhong, J.; Ye, X.; Lu, S.; Wang, W.; Zhang, R.; Xu, J.; Chen, S.; Liu, D. Ultrasonic-Assisted Enzymatic Extraction of
Polysaccharide from Corbicula fluminea: Characterization and Antioxidant Activity. LWT-Food Sci. Technol.
2015
,60, 1113–1121.
[CrossRef]
60.
Galland-Irmouli, A.V.; Pons, L.; Luçon, M.; Villaume, C.; Mrabet, N.T.; Guéant, J.L.; Fleurence, J. One-Step Purification of
R-Phycoerythrin from the Red Macroalga Palmaria palmata Using Preparative Polyacrylamide Gel Electrophoresis. J. Chromatogr.
B Biomed. Sci. Appl. 2000,739, 117–123. [CrossRef] [PubMed]
61.
Zhang, Q.; Zhou, M.-M.; Chen, P.-L.; Cao, Y.-Y.; Tan, X.-L. Optimization of Ultrasonic-Assisted Enzymatic Hydrolysis for the
Extraction of Luteolin and Apigenin from Celery. J. Food Sci. 2011,76, C680–C685. [CrossRef]
Mar. Drugs 2023,21, 213 16 of 16
62.
Zhao, G.; Chen, X.; Wang, L.; Zhou, S.; Feng, H.; Chen, W.N.; Lau, R. Ultrasound Assisted Extraction of Carbohydrates from
Microalgae as Feedstock for Yeast Fermentation. Bioresour. Technol. 2013,128, 337–344. [CrossRef]
63. Tiwari, B.K. Ultrasound: A Clean, Green Extraction Technology. TrAC Trends Anal. Chem. 2015,71, 100–109. [CrossRef]
64.
Deenu, A.; Naruenartwongsakul, S.; Kim, S.M. Optimization and Economic Evaluation of Ultrasound Extraction of Lutein from
Chlorella vulgaris.Biotechnol. Bioprocess Eng. 2013,18, 1151–1162. [CrossRef]
65.
Baghel, R.S. Developments in Seaweed Biorefinery Research: A Comprehensive Review. Chem. Eng. J.
2022
,454, 140177.
[CrossRef]
66.
Kadam, S.U.; Tiwari, B.K.; O’Connell, S.; O’Donnell, C.P. Effect of Ultrasound Pretreatment on the Extraction Kinetics of Bioactives
from Brown Seaweed (Ascophyllum nodosum). Sep. Sci. Technol. 2015,50, 670–675. [CrossRef]
67.
Fleurence, J.; Massiani, L.; Guyader, O.; Mabeau, S. Use of Enzymatic Cell Wall Degradation for Improvement of Protein Extraction
from Chondrus crispus,Gracilaria verrucosa and Palmaria palmata.J. Appl. Phycol. 1995,7, 393–397. [CrossRef]
68.
Joubert, Y.; Fleurence, J. Simultaneous Extraction of Proteins and DNA by an Enzymatic Treatment of the Cell Wall of Palmaria
palmata (Rhodophyta). J. Appl. Phycol. 2008,20, 55–61. [CrossRef]
69.
Fleurence, J. The Enzymatic Degradation of Algal Cell Walls: A Useful Approach for Improving Protein Accessibility? J. Appl.
Phycol. 1999,11, 313–314. [CrossRef]
70.
Hardouin, K.; Bedoux, G.; Burlot, A.-S.; Nyvall-Collén, P.; Bourgougnon, N. Chapter Ten—Enzymatic Recovery of Metabolites
from Seaweeds: Potential Applications. In Advances in Botanical Research; Sea Plants; Bourgougnon, N., Ed.; Academic Press:
Cambridge, MA, USA, 2014; Volume 71, pp. 279–320.
71.
Amano, H.; Noda, H. Proteins of Protoplasts from Red Alga Porphyra yezoensis.Nippon Suisan Gakkaishi
1990
,56, 1859–1864.
[CrossRef]
72.
Kornprobst, J.-M. Substances Naturelles d’Origine Marine: Chimiodiversité, Pharmacodiversité, Biotechnologies; Tec & Doc Lavoisier:
Paris, France, 2005; Volume 1.
73.
Petit, A.-C.; Noiret, N.; Guezennec, J.; Gondrexon, N.; Colliec-Jouault, S. Ultrasonic Depolymerization of an Exopolysaccharide
Produced by a Bacterium Isolated from a Deep-Sea Hydrothermal Vent Polychaete Annelid. Ultrason. Sonochem.
2007
,14, 107–112.
[CrossRef] [PubMed]
74. Pétrier, C.; Gondrexon, N.; Boldo, P. Ultrasons et sonochimie. Tech. L’ingénieur Sci. Fondam. 2008,AFP4, 15. [CrossRef]
75.
Cheung, Y.-C.; Siu, K.-C.; Liu, Y.-S.; Wu, J.-Y. Molecular Properties and Antioxidant Activities of Polysaccharide–Protein
Complexes from Selected Mushrooms by Ultrasound-Assisted Extraction. Process Biochem. 2012,47, 892–895. [CrossRef]
76.
Zhou, C.; Wang, Y.; Ma, H.; He, R. Effect of Ultrasonic Degradation on In Vitro Antioxidant Activity of Polysaccharides from
Porphyra yezoensis (Rhodophyta). Food Sci. Technol. Int. 2008,14, 479–486. [CrossRef]
77.
Tecson, M.G.; Abad, L.V.; Ebajo, V.D.; Camacho, D.H. Ultrasound-Assisted Depolymerization of Kappa-Carrageenan and
Characterization of Degradation Product. Ultrason. Sonochem. 2021,73, 105540. [CrossRef]
78.
Minard, F. Procédéde Photo-Stabilisation de Phycobiliprotéines dans un Extrait Aqueux, Compositions Contenant des Phyco-
biliprotéines Stabilisées et Utilisation de Phycobiliprotéines Stabilisées. France Patent WO2005065697A1, 21 July 2005.
79. Michalak, I.; Chojnacka, K. Algae as Production Systems of Bioactive Compounds. Eng. Life Sci. 2015,15, 160–176. [CrossRef]
80.
Fleita, D.; El-Sayed, M.; Rifaat, D. Evaluation of the Antioxidant Activity of Enzymatically-Hydrolyzed Sulfated Polysaccharides
Extracted from Red Algae; Pterocladia capillacea.LWT-Food Sci. Technol. 2015,63, 1236–1244. [CrossRef]
81.
Zhou, C.; Yu, X.; Zhang, Y.; He, R.; Ma, H. Ultrasonic Degradation, Purification and Analysis of Structure and Antioxidant
Activity of Polysaccharide from Porphyra yezoensis Udea. Carbohydr. Polym. 2012,87, 2046–2051. [CrossRef]
82.
Liu, L.-N.; Su, H.-N.; Yan, S.-G.; Shao, S.-M.; Xie, B.-B.; Chen, X.-L.; Zhang, X.-Y.; Zhou, B.-C.; Zhang, Y.-Z. Probing the PH
Sensitivity of R-Phycoerythrin: Investigations of Active Conformational and Functional Variation. Biochim. Biophys. Acta
(BBA)-Bioenerg. 2009,1787, 939–946. [CrossRef]
83.
Orta-Ramirez, A.; Merrill, J.E.; Smith, D.M. PH Affects the Thermal Inactivation Parameters of R-Phycoerythrin from Porphyra
yezoensis.J. Food Sci. 2000,65, 1046–1050. [CrossRef]
84.
Beer, S.; Eshel, A. Determining Phycoerythrin and Phycocyanin Concentration in Aqueous Crude Extracts of Red Algae. Aust. J.
Mar. Freshw. Res. 1985,36, 785–792. [CrossRef]
85.
Chaplin, M.F. Monosaccharides. In Carbohydrate Analysis: A Practical Approach; Chaplin, M.F., Kennedy, J.F., Eds.; IRL Press:
Oxford, UK, 1986; pp. 1–36; ISBN 978-0-94794-644-9.
86.
Chopin, N.; Sinquin, C.; Ratiskol, J.; Zykwinska, A.; Weiss, P.; Cérantola, S.; Le Bideau, J.; Colliec-Jouault, S. A Direct Sulfation
Process of a Marine Polysaccharide in Ionic Liquid. BioMed Res. Int. 2015,2015, e508656. [CrossRef] [PubMed]
Disclaimer/Publisher’s Note:
The statements, opinions and data contained in all publications are solely those of the individual
author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to
people or property resulting from any ideas, methods, instructions or products referred to in the content.
