Impact of Harvest Month and Drying Process on the Nutritional and Bioactive Properties of Wild Palmaria palmata from Atlantic Canada

Abstract

The macroalga Palmaria palmata could be a sustainable and nutritional food resource. However, its composition may vary according to its environment and to processing methods used. To investigate these variations, wild P. palmata from Quebec were harvested in October 2019 and June 2020, and dried (40 °C, ≃5 h) or stored as frozen controls (-80 °C). The chemical (lipids, proteins, ash, carbohydrates, fibers), mineral (I, K, Na, Ca, Mg, Fe), potential bioactive compound (carotenoids, polyphenols, β-carotene, α-tocopherol) compositions, and the in vitro antioxidant activity and angiotensin-converting enzyme (ACE) inhibition potential of water-soluble extracts were determined. The results suggested a more favorable macroalgae composition in June with a higher content of most nutrients, minerals, and bioactive compounds. October specimens were richer only in carbohydrates and carotenoids. No significant differences in antioxidant or anti-ACE inhibitory activities were found between the two harvest months. The drying process did not significantly impact the chemical and mineral compositions, resulting in only small variations. However, drying had negative impacts on polyphenols and anti-ACE activities in June, and on carotenoids in October. In addition, a concentration effect was observed for carotenoids, β-carotene and α-tocopherol in June. To provide macroalgae of the highest nutritional quality, the drying process for June specimens should be selected.

Full text

Citation: Lafeuille, B.; Tamigneaux,
É.; Berger, K.; Provencher, V.;
Beaulieu, L. Impact of Harvest Month
and Drying Process on the
Nutritional and Bioactive Properties
of Wild Palmaria palmata from
Atlantic Canada. Mar. Drugs 2023,21,
392. https://doi.org/10.3390/
md21070392
Academic Editors:
Ana Marta Gonçalves
and Leonel Pereira
Received: 2 May 2023
Revised: 26 June 2023
Accepted: 28 June 2023
Published: 3 July 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
Impact of Harvest Month and Drying Process on the
Nutritional and Bioactive Properties of Wild Palmaria palmata
from Atlantic Canada
Bétina Lafeuille 1,2,3 ,Éric Tamigneaux 2,4,5, Karine Berger 5, Véronique Provencher 2,3,6
and Lucie Beaulieu 1,2,7,*
1Département de Science des Aliments, Facultédes Sciences de l’Agriculture et de l’alimentation (FSAA),
UniversitéLaval, Québec, QC G1V 0A6, Canada; betina.lafeuille.1@ulaval.ca
2Institut sur la Nutrition et les Aliments Fonctionnels (INAF), Québec, QC G1V 0A6, Canada;
etamigneaux@cegepgim.ca (É.T.); veronique.provencher@fsaa.ulaval.ca (V.P.)
3Centre Nutrition, Santéet Société(NUTRISS), UniversitéLaval, Québec, QC G1V 0A6, Canada
4École des Pêches et de L’aquaculture du Québec, Cégep de la Gaspésie et des ˆ
Iles,
Québec, QC G0C 1V0, Canada
5Merinov, Grande-Rivière, QC G0C 1V0, Canada; karine.berger@merinov.ca
6École de Nutrition, Facultédes Sciences de l’Agriculture et de l’Alimentation (FSAA), UniversitéLaval,
Québec, QC G1V 0A6, Canada
7Québec-Océan, UniversitéLaval, Québec QC G1V 0A6, Canada
*Correspondence: lucie.beaulieu@fsaa.ulaval.ca
Abstract:
The macroalga Palmaria palmata could be a sustainable and nutritional food resource.
However, its composition may vary according to its environment and to processing methods used.
To investigate these variations, wild P. palmata from Quebec were harvested in October 2019 and
June 2020, and dried (40
◦
C,
'
5 h) or stored as frozen controls (
−
80
◦
C). The chemical (lipids,
proteins, ash, carbohydrates, fibers), mineral (I, K, Na, Ca, Mg, Fe), potential bioactive compound
(carotenoids, polyphenols,
β
-carotene,
α
-tocopherol) compositions, and the
in vitro
antioxidant
activity and angiotensin-converting enzyme (ACE) inhibition potential of water-soluble extracts were
determined. The results suggested a more favorable macroalgae composition in June with a higher
content of most nutrients, minerals, and bioactive compounds. October specimens were richer only
in carbohydrates and carotenoids. No significant differences in antioxidant or anti-ACE inhibitory
activities were found between the two harvest months. The drying process did not significantly
impact the chemical and mineral compositions, resulting in only small variations. However, drying
had negative impacts on polyphenols and anti-ACE activities in June, and on carotenoids in October.
In addition, a concentration effect was observed for carotenoids,
β
-carotene and
α
-tocopherol in
June. To provide macroalgae of the highest nutritional quality, the drying process for June specimens
should be selected.
Keywords: red macroalgae; wild; dried; antioxidant activity; ACE inhibitory activity
1. Introduction
In recent years, with the exponential increase in the world population and the per-
spective of sustainability, new food sources have been studied [
1
]. Taking into account
economic, social, and environmental components [
1
,
2
], more sustainable food alternatives
are being considered, and among them, seaweeds have become increasingly popular in
Western Countries [3,4].
Seaweeds have been an integral part of Asian culinary culture for a very long time.
Asia is the leading producer, with China accounting for 59.5% of global algal farming
in 2020 [
5
]. In Europe and North America, macroalgae consumption is more limited to
certain areas, such as Ireland, Scotland, Brittany (France), Nova Scotia (Canada) or Maine
Mar. Drugs 2023,21, 392. https://doi.org/10.3390/md21070392 https://www.mdpi.com/journal/marinedrugs

Mar. Drugs 2023,21, 392 2 of 19
(USA) [
6
,
7
]. In Canada, some coastal Indigenous groups and Irish immigration in the
19th century contributed to traditional local seaweed consumption [
6
]. In the Western
world, macroalgae are mainly used in the food industry as an ingredient, in pharmaceutical
and cosmetic production, or in animal feed [
3
,
7
], but they are still an uncommon part of
most Canadians’ eating habits [
6
]. The ever-increasing commercial availability of seaweed
food, however, could favor future positive changes [8].
Macroalgae are a large family of marine plants divided into several groups. The
Ochrophyta are brown and include 1500–2000 species, the Chlorophyta are green with
8000 species, and the Rhodophyta are red with 6000 to 7000 species worldwide [
9
,
10
].
One of the most common red seaweeds in the North Atlantic Ocean is Palmaria palmata
(P. palmata)
, also called dulse [
3
]. The macroalga P. palmata is found in sheltered and moder-
ately exposed areas of the intertidal and subtidal zones (maximal depth of 20 m). It lives
on hard substrates, like rocky shores, but can also be found as an epiphyte on the stipes of
brown seaweeds such as Laminaria hyperborea,Laminaria digitata, or Saccharina latissima. It is
a pseudo-perennial small species whose frond length usually varies from 10 to 20 cm to a
maximum of 50–70 cm, and it can regrow new fronds every year [
3
,
11
–
13
]. Red algae in gen-
eral, and especially P. palmata, are known to be a valuable source of proteins, with protein
contents ranging from 7 to 19% dry weight (DW) with approximately 30% of the essential
amino acids counted in the total amino acid fraction [
12
]. In addition, they also contain
high proportions of polysaccharides (38–74%) such as carrageenan, and a large variety of
minerals (12–37%) such as I, K, Na, Ca, Mg, or Fe [
7
,
14
,
15
]. Despite the low proportion of
lipids (0.2–3.8%), P. palmata can contain liposoluble vitamins such as
β
-carotene (provitamin
A) or tocopherol (vitamin E) and omega-3 [
7
,
16
,
17
]. In addition to their nutritional benefits,
they contain bioactive compounds responsible for different bioactivities. Peptides derived
from proteins and proteins involved in photosynthesis have demonstrated antioxidant and
ACE (angiotensin-converting enzyme) inhibition activities [
18
,
19
]. Immunomodulatory,
anticoagulant, or antihyperlipidemic effects associated with the presence of carrageenan
have also been detected with other red macroalgae [
15
,
20
]. Carotenoids such as lutein
or
β
-carotene [
16
,
17
] and polyphenols have demonstrated antioxidant properties [
16
,
21
].
Macroalgae composition and bioactivities could vary according to the growing environ-
ment, the season, the light intensity, or the availability of nutrients [
3
,
16
,
18
,
22
–
25
]. Wild
P. palmata
, in the North Atlantic regions have a seasonal life cycle with a period of reproduc-
tion and growth during winter and spring, and no or negative growth during summer and
autumn [
3
,
11
,
12
,
25
,
26
]. Changes in the composition of certain chemicals and bioactives
therefore seem linked to the concentration of photosynthetic pigment, whose synthesis is
favored during winter and spring due to environmental factors such as light irradiance and
seawater nutrient concentrations [
24
,
25
]. This strong seasonality has been investigated in
many studies [
3
,
13
,
18
,
23
–
25
,
27
,
28
] and some research has been carried out in Canada. This
includes the work of Vasconcelos et al. [
29
], which highlighted variations in the chemical
composition of Quebec macroalgae over several months. Indeed, P. palmata harvested in
Île-de-la-Madeleine (QC, Canada) had higher protein, mineral, and lipid contents during
the summer (June and July), whereas carbohydrate content was high in the autumn (Octo-
ber). More studies are needed in this area, especially in the St. Lawrence estuary, which is
an important natural cradle for the wild biomass of macroalgae.
Once macroalgae are harvested, food processing is necessary to stabilize the product
over time as it contains more than 75 to 85% water [
30
,
31
]. Moreover, in the Western world,
because crude macroalgae are not really integrated into food habits, processing methods
such as drying, blanching, freezing, etc., could favor consumption [8]. However, different
food processing methods may induce variations in macroalgae composition and bioactivity.
Overall, two major principles are applied to seaweed preservation: heat and cold. The
oldest and most widespread processing method is drying, which consists of removing
water, and reducing degradation and the growth of microorganisms [
30
,
31
]. One of the
most ancient drying methods is sun drying, which is the most accessible method, but
requires a warm and sunny climate and does not allow the constant application of a precise

Mar. Drugs 2023,21, 392 3 of 19
temperature [
6
,
32
]. Other drying methods, such as oven drying, compensate for these
disadvantages and allow precise control of the applied heat [
30
,
31
]. The impact of drying
on seaweed composition seems to be mainly related to the temperature used. This could
induce changes in the nutritional value or bioactive potential with losses of heat-sensitive
molecules such as polyphenols or carotenoids [
31
,
33
,
34
]. A previous study using another
edible red seaweed, Pyropia orbicularis, found a decrease in pigment content and antioxidant
activity when using a drying method [
35
]. The second common treatment principle is the
application of low temperature (freezing), which limits or even stops microbial metabolism
and enzymatic activities [
36
]. The most efficient preservation method for long-term storage
is freezing, corresponding to a storage temperature under the food freezing point, leading
to ice crystal formation [
36
–
38
]. This phenomenon can cause changes in membrane integrity,
leading to losses of nutritional compounds such as amino acids during thawing [
39
]. Trials
performed on P. palmata frozen at
−
20
◦
C showed a loss of over 40% of the amino acid
content compared to macroalgae held in seawater at 5
◦
C [
39
]. The overall effect of freezing
on macroalgae composition is not completely clear but seems to be more efficient than
drying for preserving bioactive compounds and bioactivities, such as antioxidant potential.
A study using Phyllariopsis brevipes (formerly Phyllaria reniformis) found a higher antioxidant
activity for frozen (
−
18
◦
C) compared to dried (38
◦
C) seaweed [
40
]. In contrast, previous
studies using S. latissima have found a similar nutritional composition and antioxidant
potential (depending on the harvest month) between frozen (
−
80
◦
C) and dried (40
◦
C)
macroalgae, whereas some losses were detected for carotenoids and polyphenols in dried
(40
◦
C) specimens [
41
]. Studies on P. palmata need to be expanded in order to understand
the impact of the month of harvest and food processing method on its quality.
As mentioned above, among the many bioactive compounds in seaweeds are proteins
and peptides [
18
,
19
]. In order to test their bioactive capacities, the extraction of these
compounds is necessary and can be carried out according to the different aqueous methods
referenced in the literature [
9
,
19
,
27
,
42
–
44
]. In addition, for the bioactivity measurements,
many protocols exist, as is the case for antioxidant capacity tests. Indeed, several tests are
used to measure the antioxidant capacity based on two principles: the electron transfer
reaction (such as the ferric reducing antioxidant capacity assay [FRAP] or the 2,2-diphenyl-
1-picrylhydrazyl [DPPH] test) or the hydrogen atom transfer (such as oxygen radical
absorbance capacity assay [ORAC] or total radical trapping antioxidant parameter test
[TRAP]) [
45
,
46
]. Since the use of a single technique is not recommended [
47
], previous
studies have used at least two tests [
19
,
27
,
42
–
44
]. In general, however, this wide variety of
extraction protocols and bioactive tests makes comparisons between studies difficult and
should be considered carefully.
The objectives of the present study aimed to determine the impact of (1) the harvesting
month and (2) the drying process on chemical (lipids, proteins, ash, carbohydrates, fibers),
mineral (I, K, Na, Ca, Mg, Fe) and bioactive compound composition (carotenoids, polyphe-
nols, tocopherol,
β
-carotene), as well as
in vitro
bioactivity potential (antioxidant and ACE
inhibitory) on wild P. palmata harvested on the shores of Eastern Canada.
2. Results and Discussion
2.1. Chemical Composition
2.1.1. Crude Palmaria palmata
The chemical composition of crude P. palmata is shown on a dry matter basis in Table 1.
Overall, crude P. palmata harvested in June was richer in most compounds, except for
carbohydrates, which were more concentrated in October. Lipids, proteins, and minerals
were significantly higher in samples harvested in June (4.64
±
0.19%, 16.19
±
0.09% and
27.26
±
0.29% dry weight (DW), respectively) than in October (1.17
±
0.18%, 11.23
±
0.17%
and 12.30
±
0.04% DW, respectively). Carbohydrates were significantly higher in specimens
harvested in October, with concentrations varying from 75.10% to 75.50% DW, whereas
their concentration was significantly lower in June (51.91
±
0.57% DW). On the other hand,

Mar. Drugs 2023,21, 392 4 of 19
fiber content was significantly higher for macroalgae harvested in June (36.88
±
0.10% DW)
compared to those harvested in October (35.81 ±0.27% DW).
Table 1.
Chemical composition of crude and processed wild P. palmata harvested in October 2019 and
June 2020.
P. palmata Lipids (%) Proteins (%) Ash (%) Carbohydrates (%) Fiber (%)
Crudes—October 1.17 ±0.18 d11.23 ±0.17 c12.30 ±0.04 c75.30 ±0.20 a35.81 ±0.27 c
Dried—October 1.65 ±0.10 c11.19 ±0.17 c12.22 ±0.09 c74.94 ±0.13 ab 36.54 ±0.18 b
Crudes—June 4.64 ±0.19 a16.19 ±0.09 b27.26 ±0.29 a51.91 ±0.52 bc 36.88 ±0.10 b
Dried—June 3.65 ±0.21 b17.32 ±0.17 a26.18 ±0.05 b52.85 ±0.43 ab 37.80 ±0.22 a
Lipids, proteins, ash, carbohydrates, and fiber are expressed as a percentage of the DW of macroalgae
(mean ±SD; n= 3). Means within each column with different letters (a–d) differ significantly (p< 0.05).
The chemical composition of October and June seaweeds were fairly consistent with
the literature. The reported proportions of protein in P. palmata varied from 7 to 19% DW,
ash varied from 12 to 37% DW, lipids varied from 0.2 to 3.8% DW, and carbohydrates
varied from 38 to 74% DW [
7
,
12
]. Furthermore, the observed chemical compositions echo
the life cycle of Atlantic P. palmata, characterized by a reproductive period and nutrient
storage in winter, growth in spring, end of growth and senescence in summer, and growth
slowdown period in autumn [
3
,
11
,
25
,
26
]. Thus, the strong seasonality of seaweed due to
environmental variations (such as light or nutrient availability) has already been described
in the literature. In fact, previous studies have shown that the highest protein content was
detected in winter and early spring, when seawater is particularly rich in nutrients, and
the lowest during summer and autumn [
24
,
25
]. Even though June seaweeds in this study
appeared to be the most protein-rich specimens compared to October seaweeds, previous
observations of P. palmata in France and Norway have shown that winter specimens can
reach approximately 25% DW [
3
]. Seasonal protein synthesis could be related to the
P. palmata
pigment study of Schmid et al. [
25
], which demonstrated that photosynthetic
pigment concentrations were dependent on the availability of light and nutrients from
seawater. Thus, winter and early spring were characterized by low irradiance and a
high nutrient seawater concentration that favor pigment synthesis, resulting in highly
active photosynthesis and nutrient storage for later months. On the other hand, high
temperature, high irradiance, and a lack of nutrients in the summer and autumn led to
loss of photosynthetic efficiency and the use of nutrient reserves [
3
,
25
]. In the macroalgae
harvest area of the present study, nutrient stratification in the water column typically
occurs from late spring to late September and reduces the availability of nutrients (such
as nitrogen) [
48
]. Thus, the observed differences in protein levels could be related to the
fact that June specimens have not consumed their reserves, while October macroalgae have
used their reserves during the summer and have not had time to replenish them. Lipid
content was also investigated and correlated to that nutrient cycle because of their role
in photosystems [
25
]. In general, it was not surprising to observe a higher level of most
chemical compounds in June compared to October because of the reserves in late spring,
which were then likely consumed during the summer. For carbohydrates, it is interesting
to note that whatever the month of harvest, both macroalgae had similar proportions of
fiber, while the total carbohydrate content was usually higher in October specimens. This
observation suggests that the difference in total carbohydrate content could be due to higher
proportions of monosaccharides such as floridosides. Floridoside is the main carbon reserve
synthesized in P. palmata due to high light exposure; it is accumulated during summer
and is important for vegetative propagation during winter [
26
]. Its synthesis seems to
have taken place after the month of June and seems to constitute the detectable reserves
in October. These results demonstrated an inverse correlation between protein/ash and
carbohydrate contents, which has previously been reported in the literature [
49
]. Moreover,
the study of Vasconcelos et al. [
29
] found similar variations in the chemical composition

Mar. Drugs 2023,21, 392 5 of 19
of P. palmata from Île-de-la-Madeleine (Gulf of Saint-Lawrence, QC, Canada) harvested
in June, July, and October, 2015. Their results showed an increase in carbohydrates and a
decrease in protein and ash during summer and autumn, and similar proportions of fiber
between July and October.
2.1.2. Dried Palmaria palmata
The chemical composition of dried P. palmata is presented on a dry matter basis in
Table 1
. First, interactions between the month of harvest and drying treatment
(p-value < 0.05) were observed, meaning that the impact of processing on macronutri-
ent content was variable between the different months. Similar to crude P. palmata, dried
samples harvested in June were significantly richer in lipids, proteins, ash, and fiber
(3.65
±
0.21%, 17.32
±
0.17%, 26.18
±
0.05% and 37.80
±
0.22% DW, respectively), and
dried October specimens were richer in carbohydrates (74.94
±
0.13% DW). In comparison
to crude P. palmata harvested in June, the results presented above for lipids and ash were
slightly but significantly lower. In contrast, proteins and fiber were higher. No significant
difference in carbohydrates was found between crude and processed seaweeds.
The results obtained for chemical composition were consistent with previous studies
on dried Quebec P. palmata. Indeed, Lafeuille et al. [
50
] reported, on specimens harvested
from the same site (Forillon, QC, Canada), an average lipid content of 0.41% DW, protein
16.05% DW, ash 20.48% DW, carbohydrate 63.05% DW, and fiber 35.26% DW. Furthermore,
the Vasconcelos et al. [
29
] study on dried P. palmata harvested from six different locations in
the Gulf of Saint-Lawrence (Rivière-au-Renard, Gaspé, Grande-Rivière, Pabos, Newport,
Sept-Îles and Île-de-la-Madeleine) during 2015 found, in terms of dry weight, less than
2.1% was due to lipids, 9.0 to 16.9% to proteins, 11.1 to 40.0% to ash, 34.6 to 63.2% to
carbohydrates, and 7.4 to 12.8% to fibers, similar to our results. Overall, the month of
harvest (and by extension, the growth environment and the life cycle phase) has the largest
effect on the chemical composition of P. palmata, with the drying method having only a
small effect. The principle of the drying method is just the removal of water, and is not
known to induce large losses of macronutrients [31]. However, environmental conditions,
as discussed above (Section 2.1.1), had a tremendous impact and almost entirely determined
the chemical compositions of dried P. palmata.
2.2. Mineral Composition
2.2.1. Crude Palmaria palmata
The mineral composition of crude P. palmata is shown on a dry matter basis in Table 2.
Mineral analysis revealed that the highest amounts of sodium (Na) (2.58
±
0.06 g/100 g),
calcium (Ca) (317.00
±
21.00 mg/100 g), magnesium (Mg) (313.00
±
9.00 mg/100 g), and
iron (Fe) (436.67
±
35.00 mg/100 g) occurred in P. palmata harvested in June, and iodine (I)
and potassium (K) occurred in similar proportions in the October and June harvests.
The proportions of Na, Mg, and I for both harvest months were close to the results
reported previously in the literature. Indeed, Mabeau and Fleurence [
51
] reported a
Na content of 1.7 to 2.5 g/100 g DW and Mg content of 170 to 500 mg/100 g DW, and
Mouritsen et al. [
32
] reported an I content of 0.5 to 1.0 mg/100 g DW. Since both Na and
Mg are involved in seaweed photosynthesis [
52
], spring seawater is known to be richer
in nutrients [
24
,
25
], and the macroalgae showed growth phase signs in June, it was not
surprising to find a higher content in the June specimens. Compared to other foods, Mg
content in the seaweeds harvested in June and October was much higher than in spinach
(a great source of Mg [
53
,
54
]), which has approximately 79 mg/100 g [
55
]. The amount
of Mg in the June macroalgae was much closer to that of dark chocolate (70–85% cocoa,
known to be rich in this mineral [
56
]), which has, on average, 228 mg Mg/100 g [
57
].
Brown macroalgae such as S. latissima are known to have a high I content [
14
,
15
], and
previous studies on Quebec specimens have detected an I content 10–100 times higher in
S. latissima [
41
] than in P. palmata. This difference could be considered an advantage that
allows a higher consumption of these red macroalgae because the maximum recommended

Mar. Drugs 2023,21, 392 6 of 19
intake of I is approximately 1.1 mg per day for an adult [
14
,
15
]. The phenomenon behind
the fact that no significant difference was found for the I content between June and October
is not clear. Seawater is richer in nutrients in spring than in autumn, but since I is involved
in the production of secondary metabolites that may play a role as an antioxidant [
52
,
58
], it
should probably have been higher in June, which was not the case. The K content from
the October harvest (3.43
±
0.06 g/100 g DW) was lower than the results of Mabeau and
Fleurence [
51
], where K proportions ranged from 7.0 to 9.0 g/100 g DW. It is interesting to
note that, as a strong source of K [
59
], bananas contain on average 358 mg K/100 g [
60
],
thus, approximately 25 times less K than the macroalgae harvested in June, in comparable
portions. The amount of Ca detected in the macroalgae harvested in both months was also
lower (560–1200 mg/100 g [
51
]). However, this was largely higher than watercress (known
to be rich in this mineral [
61
]), which contains, on average, 81 mg Ca/100 g [
60
]. As for
the Na and Mg content, these were probably higher in June because of nutrient richness
in spring and the growth phase of the seaweed. The Fe content in October seaweeds
(30.00 ±1.00 mg/100 g DW)
agreed with the results of Mabeau and Fleurence [
51
] (from
15 to 140 mg/100 g), whereas in June, the Fe content was approximately three times higher
than the maximum found in this previous study. In comparison with a good vegetable
source such as raw parsley [
62
], which contains, on average, 6.20 mg Fe/100 g, the amount
of Fe in P. palmata specimens harvested in June was higher [
63
]. The accumulation of Fe
in P. palmata could be related to the synthesis of ferredoxin, which is an essential iron-
related protein involved in photosynthesis that appears to be more active in spring than
in summer and autumn [
64
]. The Na/K ratio of diets is a risk factor for the development
of hypertension and cardiovascular disease, and according to World Health Organization
recommendations, should be less than 0.6 [
17
,
65
]. In the present study, the Na/K ratios of
wild P. palmata were usually under 0.6 and the lowest ratios were observed in June. For
comparison, the Na/K ratio of raw shrimp is 5.00 [
66
]. The observed variations in the
mineral composition of dulse within the harvest months and also reported in the literature
were probably related to seasonal changes in the environmental parameters, such as the
mineral load of seawater, which is higher in spring but lower during the summer and the
autumn [
23
,
24
,
67
]. In addition, they were likely related to physiological changes during the
life cycle of P. palmata [
3
,
11
,
25
,
26
]. In addition, deterioration of fronds during summer was
observed when the fronds were frequently covered with epiphytes, leading to P. palmata
having a less favorable overall composition at the end of summer [3].
Table 2.
Mineral composition of crude and processed wild P. palmata harvested in October 2019 and
June 2020.
P. palmata I (mg/100 g) K (g/100 g) Na (g/100 g) Ca (mg/100 g) Mg (mg/100 g) Fe (mg/100 g) Na/K
Ratio
Crudes—October 3.00 ±0.44 bc 3.43 ±0.06 bc 1.30 ±0.00 c120.00 ±13.00 c169.00 ±4.00 c30.00 ±1.00 c0.38
Dried—October 4.10 ±0.10 ab 3.53 ±0.12 ab 1.27 ±0.06 c130.00 ±13.00 c168.00 ±5.00 c28.67 ±0.56 c0.36
Crudes—June 9.60 ±1.45 ab 9.23 ±0.38 ab 2.58 ±0.06 a317.00 ±21.00 a313.00 ±9.00 a436.67 ±35.00 a0.28
Dried—June 10.73 ±2.63 a12.65 ±1.73 a2.20 ±0.04 b256.00 ±2.00 b261.00 ±4.00 b352.33 ±4.16 b0.17
Iodine (I), potassium (K), and sodium (Na) are expressed as g/100 g DW of seaweed, and calcium (Ca), magne-
sium (Mg), and iron (Fe) are expressed as mg/100 g (mean
±
SD; n= 3). Means within each column with different
letters (a–c) differ significantly (p< 0.05).
2.2.2. Dried Palmaria palmata
The mineral composition of dried P. palmata is presented in Table 2. Statistical anal-
ysis revealed the same trends as mineral composition in crude seaweed harvested over
the two months. The levels of Na (2.20
±
0.04 g/100 g), Ca (256.00
±
2.00 mg/100 g),
Mg (261.00
±
4.00 mg/100 g), and Fe (352.33
±
4.16 mg/100 g) in dried P. palmata were sig-
nificantly higher in June than in October. The levels of I and K were similar in October and
June. Thus, interactions between the harvest months and drying method
(p-value < 0.05)
were detected for the minerals investigated. In comparison to crude P. palmata harvested in
June, the amounts of Na, Ca, Mg, and Fe in dried dulse were significantly lower. On the

Mar. Drugs 2023,21, 392 7 of 19
other hand, for specimens harvested in October, no significant difference in the mineral
composition was found between crude and dried seaweeds.
The range of mineral proportions in dried P. Palmata from October and June were
consistent with the results reported in previous studies. Maehre et al.’s [
68
] study on
freeze-dried Norwegian P. palmata harvested in spring reported 360 mg Ca/100 g DW,
530 mg Mg/100 g DW
, and 0.026 g I/100 g DW. Only Fe was mostly lower (10 mg/100 g
DW) compared to the results of our study, suggesting that Quebec spring seawater was
richer in Fe. This phenomenon could be explained by the composition of the rocky bed
of the GaspéPeninsula (harvesting area), which is composed of red sandstone containing
hematite, an iron oxide [
69
]. Another study performed with freeze-dried Danish P. palmata
harvested in September detected approximately 4.11 g K/100 g, 160.00 mg Mg/100 g, 30.70
mg Fe/100 g, 0.32 g Na/100 g, and 933.00 mg Ca/100 g, with a Na/K ratio of 0.08 [
17
].
Compared to those results, Quebec P. palmata harvested in October contained similar
amounts of K, Fe, and Mg, but approximately seven times less Ca and four times more
Na. October and June dried specimens had a higher Na/K ratio compared to the results
of Parjikolaei et al.’s [
17
] study, but still remained below 0.6 [
17
,
65
]. The detected loss of
minerals in June macroalgae might be linked to the Maillard reaction or mineral binding
protein denaturation [70].
2.3. Bioactive Compound Content
2.3.1. Crude Palmaria palmata
The bioactive compound content of crude P. palmata is shown on a dry matter basis
in Table 3.P. palmata harvested in October showed a significantly higher proportion of
carotenoids (371.00
±
11.00
µ
g/g), while polyphenols (2.06
±
0.05 mg GA/g),
α
-tocopherol
(22.67 ±2.08 µg/g) and β-carotene (33.33 ±1.53 µg/g) contents were higher in June.
Table 3.
Bioactive compound contents of crude and processed wild P. palmata harvested in Octo-
ber 2019 and June 2020.
P. palmata Carotenoids (µg/g) Polyphenols (mg GAE/g) α-Tocopherol (µg/g) β-Carotene (µg/g)
Crudes—October 371.00 ±11.00 b1.10 ±0.00 c9.95 ±1.11 c12.87 ±3.09 c
Dried—October 308.00 ±3.00 c0.80 ±0.14 c9.05 ±0.63 c7.73 ±0.77 c
Crudes—June 315.00 ±22.00 c2.06 ±0.05 b22.67 ±2.08 b33.33 ±1.53 b
Dried—June 535.00 ±24.00 a1.89 ±0.17 a50.00 ±5.29 a47.67 ±2.08 a
Carotenoids,
α
-tocopherol, and
β
-carotene are expressed as
µ
g/g and polyphenols as mg GA/g (mean
±
SD;
n= 3). Means within each column with different letters (a–c) differ significantly (p< 0.05).
In the literature, several carotenoids have been detected in red seaweeds, such as
lutein, zeaxanthin, or asthaxanthin [
16
,
71
]. But since it is known to be one of the major
carotenoids [
72
], only asthaxanthin concentrations were determined and presented as
carotenoid concentrations in this study. To the authors’ knowledge, no literature is available
on the astaxanthin content of P. palmata. However, studies on other pigments have shown
that the synthesis of carotenoids in this species are favorably linked to nitrogen and
light [
17
,
28
]. In addition, a study of the red macroalga Porphyra umbilicalis from the North
Atlantic reported higher carotenoid concentrations in September and November than in
July. This was explained by the fact that the low irradiance of autumn favored pigment
synthesis [
73
] and this was probably the case for the October specimens in the present
study. On the other hand,
β
-carotene contents were higher in June, probably due to the
high nutrient content of the water at this time of the year [
28
]. However, the concentrations
detected were lower in both months than in previous studies, which reported 420
µ
g
β
-carotene /g in summer and 37
µ
g
β
-carotene /g in winter in P. palmata [
16
]. Compared
to the
β
-carotene contents in vegetables such as carrots and mango (79.7 and 31.0
µ
g/g,
respectively, which usually contain a high amount of this vitamin [
17
]), P. palmata had a
lower amount than carrot in both months, but an equal amount to mango in June and a

Mar. Drugs 2023,21, 392 8 of 19
lower amount in October [
17
]. The total phenolic content (TPC) for both months was lower
than that previously reported in the literature, probably due to variation in environmental
factors. Indeed, P. palmata harvested in Iceland had a TPC of about 5 mg GAE/g [21], and
in New Brunswick, Canada, this was 10.3 mg GAE/g [
74
]. Another study conducted on
P. palmata harvested in spring on the west and east coasts of Grand Manan Island (NB,
Canada) with variations in UV exposure, found no difference in TPC. In fact, the TPC of
the western-side seaweeds was detected at 12.8 mg GAE/g, and for eastern-side specimens
it was 12.7 mg GAE/g, suggesting a low impact of UV exposure [
13
]. Thus, the difference
detected between June and October TPC in that study should not be related to the greater
light exposure during the summer but probably to other environmental conditions, such as
nutrient availability or water temperature. To the authors’ knowledge, no studies have been
conducted on the
α
-tocopherol (vitamin E) content of P. palmata, but since
α
-tocopherol is a
phenolic compound [
13
], it was not surprising to observe a higher level in June. Previous
studies performed on red seaweeds have related varying concentrations from 0.01
µ
g/g DW
in Peruvian Chondracanthus chamissoi [
75
] to 0.18
µ
g/g DW in Danish Chondrus chrispus [
76
].
Thus, detected
α
-tocopherol amounts ranging from 9.05 to 22.67
µ
g/g DW qualify wild
P. palmata as a true vitamin-E-rich red species. Compared to common
α
-tocopherol-rich
foods such as spinach or salmon [
77
,
78
], which contain 20.3
µ
g/g DW and 35.5
µ
g/g DW
of
α
-tocopherol, respectively [
55
,
79
], P. palmata could be considered an interesting marine
plant source of vitamin E in comparable serving sizes. It is therefore important to note that
the actual consumed servings of seaweeds (80 g wet or approximately 16 g dried [
80
]) often
remain lower than for spinach or salmon. The higher vitamin content in June could be due
to the more active metabolism of the growth phase.
2.3.2. Dried Palmaria palmata
The content of bioactive compounds for dried P. palmata is presented in Table 3. As
with chemical and mineral compositions, interactions between the months of harvest and
drying method (p-value < 0.05) were observed for bioactive compounds, thus compounds
in dried seaweeds varied depending on the month of harvest. Compared to October,
the drying process of June P. palmata produced the highest concentrations of carotenoids,
polyphenols,
α
-tocopherol, and
β
-carotene (535.00
µ
g/g, 1.89 mg GA/g, 50.00
µ
g/g, and
47.67
µ
g/g, respectively). Dried October seaweeds were not significantly different from
crude samples for polyphenols,
α
-tocopherol, and
β
-carotene contents. However, they
differed in carotenoids, which were affected by the treatment. On another hand, dried
June specimens had the highest levels of carotenoids,
α
-tocopherol, and
β
-carotene, and
the lowest polyphenol content. Limited literature is available regarding the impact of
drying treatment on bioactive compounds present in P. palmata. However, it is known that
carotenoids and polyphenols are highly sensitive to heat and could be negatively affected
by the drying treatment [
33
,
34
]. This seems to have been the case for carotenoids in dried
October seaweeds and polyphenols in dried June macroalgae, but this phenomenon was
not generalized. The higher levels of carotenoids and vitamins in the dried June samples
could be due to a concentration effect related to drying. In a previous study, Parjikolaei
et al. [
17
] detected approximately 2
µ
g
β
-carotene /g, in freeze-dried P. palmata, which is
approximately 3.5 to 23.5 times less than in the macroalgae from this study. However, the
work of Machu et al. [
81
] on dried P. palmata flakes reported a TPC of 31.8 mg GAE/g,
which was generally higher than in our study.
2.4. In Vitro Bioactive Potential
2.4.1. Protein Contents and Extraction Yields of Soluble Palmaria palmata Extracts
•Crude Palmaria palmata
Extraction of water-soluble extracts >1 kDa from crude P. palmata, presented in
Table 4
,
showed no significant difference in extraction yields and protein extraction yields (PEY)
between October and June specimens. However, the protein contents of the water-soluble
extracts were significantly higher in June macroalgae (29.03
±
4.30% DW). This was

Mar. Drugs 2023,21, 392 9 of 19
not surprising because of the higher protein contents found in crude P. palmata in June
(16.19
±
0.09% DW, presented in Table 1). Whereas the objective of the extraction was
to recover macroalgae proteins and peptides, the low PEY (maximum about 12% DW)
showed limited extraction, probably due to interactions with polysaccharides [
24
,
82
,
83
].
A previous study performed protein water extraction on P. palmata harvested in July and
October, and reported significantly lower protein contents in July (1.47
±
0.04% DW) than
in October (2.97
±
0.04% DW) [
27
]. These observations regarding the amount of protein
and the seasonality differed from our results. Indeed, the protein contents in this study
were higher in June than in October, which would seem consistent with seawater nutrient
availability in the spring, allowing protein synthesis that could produce phycobiliproteins
(water-soluble pigment proteins) [
84
]. Different protein extraction results could be due
to environmental factors (Ireland versus Quebec), seaweed conditions (freeze-dried ver-
sus defrosted), extraction method (deionized water versus phosphate buffer) or protein
quantification method (Lowry method versus Dumas method).
Table 4.
Water-soluble extracts from wild and dried P. palmata: proportion of protein, extraction yield,
and protein extraction yield.
P. palmata Crudes—October Dried—October Crudes—June Dried—June
Protein (%) 15.70 ±2.31 a23.00 ±0.89 ab 29.03 ±4.30 bc 22.32 ±2.34 ab
Extraction yield (%) 8.40 ±0.24 a3.44 ±1.25 bc 5.60 ±0.47 ab 5.38 ±0.20 ab
Protein extraction yield (%)
12.64 ±1.57 a10.37 ±1.28 a7.45 ±1.16 a6.92 ±0.62 a
In the table, “Protein” represents the amount of protein that was obtained in each extract, “Extraction yield”
represents the difference between the initial mass and the mass of the extract and “Protein Extraction Yield”
represents the amount of protein in the extract as a function of the mass of the extract. Protein, extraction yield,
and protein extraction yield are expressed as a percentage of the DW of seaweed (mean
±
SD; n= 3). Means
within each row with different letters (a–c) differ significantly (p< 0.05).
•Dried Palmaria palmata
Extraction performance on dried P. palmata (Table 4) showed no significant difference
in protein content (approximately 23% DW), extraction yield (from 2.19 to 5.58% DW), and
PEY (about 8.5% DW) between the October and June samples. Thus, the drying treatment
appears to favor the extraction of a similar protein content, although the protein content
of the dried June samples was higher than in October. In comparison with the crude June
seaweeds, the drying process did not improve or limit extraction performance. This could
be related to the fact that the crude samples were frozen and that both treatments (freezing
and drying) can affect the integrity of the cell wall [
30
,
31
] and thus resulting in the same
extraction performance. However, in water-soluble extracts from dried October specimens,
the extraction yield (3.44
±
1.25% DW) was significantly lower than that of crude samples
(8.40
±
0.24% DW) but did not affect the protein extraction. Interactions between the
months of harvest and applied treatment were thus once again detected. In the literature,
the PEY on dried P. palmata has been reported as being quite variable. Hell et al. [
43
]
reported a PEY of 3.30% DW, while Bondu et al. [
19
] found 11.40
±
3.12% DW, and Wang
et al. [
21
] reported over 35% DW. The results of this study were close to those obtained by
Bondu et al. [
19
], and differences could be due to polysaccharide content, environmental
factors, drying techniques, or extraction protocols.
2.4.2. Antioxidant Capacity of Water-Soluble Palmaria palmata Extracts
•Crude Palmaria palmata extracts
Extraction ORAC and FRAP assays were performed on water-soluble crude P. palmata
extracts in order to evaluate the antioxidant potential. ORAC values detected at 1250
µ
g of
extract/mL were, on average, 30.36
±
14.69
µ
mol TE/g in October and 43.26
±
14.31
µ
mol
TE/g in June, with no significant differences. FRAP values for the lowest concentration
extracts (500
µ
g/mL) were, on average, slightly higher in both months (77.07
±
32.12
µ
mol
TE/g in October and 81.74
±
28.68
µ
mol TE/g in June), with no significant differences. The

Mar. Drugs 2023,21, 392 10 of 19
ORAC and FRAP tests produced different results but this might be related to the different
measuring mechanisms. The ORAC test measures the potential peroxyl radical scavenging
capacity while the FRAP assay detects the potential reducing capacity of the extracts [
18
].
Overall, the antioxidant potential for these extracts did not change with the seasons. This
result differed from previously reported ORAC and FRAP values for P. palmata harvested
in Ireland, where July water extracts had a significantly higher antioxidant potential
than October extracts. Harnedy et al. [
27
] reported ORAC values of 1.41
µ
mol TE/g in
October and 2.18
µ
mol TE/g in July, and FRAP values of 81.35
µ
mol TE/g in October and
296.45
µ
mol TE/g in July. Differences compared to previous studies could be due to the
extraction process, which was not identical, or due to environmental factors. In general, the
ORAC values obtained by these authors were more than ten times higher than our results,
whereas the FRAP values were close in October but lower in June [
27
]. Water-soluble
extracts from P. palmata contained peptides and proteins that had already been identified
in the literature. Beaulieu et al. [
18
] have studied the antioxidant potential of protein
hydrolysis from P. palmata and have determined that major peptides with bioactivity
were related to RuBisCo, a plant metabolic enzyme involved in photosynthesis, or to
photosynthetic pigments, such as allophycocyanins or phycocyanins. These proteins
and pigments are intrinsically linked to the rate of photosynthesis and should be more
abundant in seasons with nutrient-rich seawater and high irradiance, such as the spring.
Our results did not corroborate these published results, but the observed differences could
be due to the sizes of proteins and peptides, since enzymatic hydrolysis allowed for better
antioxidant activity [
18
] than for inherent seaweed peptide and protein synthesis. Another
study performed on wild P. palmata grown with different light exposures highlighted the
fact that reducing activity, detected with Yen and Chen’s 1995 method, was higher, with
greater luminosity [
13
]. Explanations for this phenomenon were that the increase in UV
radiation favored the synthesis of antioxidants with reducing capacity to help protect
against photooxidative stress. In addition to proteins and peptides, water-soluble extracts
also contained polyphenols and polysaccharides [
43
]. As described and discussed in
Section 2.3.1, the TPCs of crude P. palmata were only slightly higher in June, but remained
below the previously reported concentrations and did not substantially influence the
measured antioxidant potential. This finding was the same for fiber (Section 2.1.1).
•Dried Palmaria palmata Extracts
As was observed for water-soluble extracts from crude samples, water-soluble extracts
from dried P. palmata did not show significant differences in ORAC and FRAP values
between October and June, and were similar to crude/control samples. The ORAC val-
ues obtained from dried October specimens were, on average, 56.36
±
16.46
µ
mol TE/g,
and for dried June macroalgae, were 50.12
±
19.92
µ
mol TE/g at the tested concentra-
tion of
1250 µg/mL
. FRAP tests performed on dried macroalgae at 500
µ
g/mL produced
higher values of 146.52
±
65.64
µ
mol TE/g in October and 82.88
±
15.31
µ
mol TE/g
in June. Thus, the drying process did not impact the antioxidant capacity of P. palmata.
Compared to previous studies on freeze-dried P. palmata water extracts, the values we
obtained were close to the results of Wang et al. [
21
] who reported an ORAC value of
approximately
35.8 µmol TE/g
, and Harnedy and FitzGerald [
42
] who obtained an average
of
45.17 ±1.95 µmol TE/g
. Using dried P. palmata (35 to 50
◦
C), Bondu et al. [
19
] reported
ORAC values for the protein extract at the same concentration of less than 70
µ
mol TE/g,
and Beaulieu et al. [
44
] found an antioxidant potential for the protein extract, <10 kDa, of
less than 100
µ
mol TE/g. On the other hand, values were largely lower than Hell et al.’s [
43
]
results for Quebec P. palmata, which was dried at 30
◦
C for 24 h, resulting in an ORAC value
of 56.60
±
5.06 mmol TE/g. The results of the FRAP tests seem highly dependent on the frac-
tion size. Bondu et al. [
19
] reported FRAP values for chymotrypsin- and trypsin-hydrolyzed
dried P. palmata extracts of 24.82
±
1.91
µ
mol TE/g and
42.27 ±3.42 µmol TE/g
, respec-
tively. Beaulieu et al. [
44
], however, detected a higher FRAP value on the unhydrolyzed
protein extracts of dried P. palmata for the <10 kDa fraction
(170.80 ±15.21 µmol TE/g)
than for the >10 kDa fraction (26.85
±
0.23) at a concentration of 750
µ
g/mL. To the au-

Mar. Drugs 2023,21, 392 11 of 19
thors’ knowledge, no study has been conducted to investigate the effect of drying on the
antioxidant activity of P. palmata. However, a study performed on another red seaweed,
Pyropia orbicularis, found a reduction in antioxidant activity through sun drying (35 to
52.5
◦
C) and convective drying (70
◦
C, 120 min) compared to fresh specimens [
35
]. Thus,
the ORAC value of methanolic extracts from fresh seaweeds was 22.5
µ
mol TE/g, while
that of sun-dried and convective-dried samples was 9.38 µmol TE/g and 8.72 µmol TE/g,
respectively. Drying techniques appeared to negatively affect photosynthetic pigments
(such as phycocyanin) [
35
], which have previously been detected in P. palmata bioactive
extracts [
18
], resulting in a loss of antioxidant activity. However, in the present study, the
lack of a significant difference between dried and crude samples suggests a low impact of
heat (40
◦
C) or the formation of new antioxidants such as melanoidins derived from the
Maillard reaction, which may have taken place during drying [
35
]. As for the crude extracts,
the PEY of the dried extracts were similar between October and June and were similar
to the control samples, which could explain the lack of impact of the drying treatment.
Polyphenols were significantly less present in dried June samples (Section 2.3.2), which
did not impact the antioxidant potential of the dried macroalgae. Since water extracts
may contain compounds other than peptides and proteins [
43
], further tests could help to
evaluate their extraction as well as understand their actual impact on antioxidant potential.
2.4.3. ACE-Inhibition Capacity of Water-Soluble Palmaria palmata Extracts
•Crude Palmaria palmata Extracts
ACE-inhibitory percentages of crude P. palmata harvested in October and June are
presented in Figure 1. Inhibition rates averaged 42.91% in the October extracts and 17.91%
in June for both concentrations (8 and 10 mg of protein/mL corresponding to
'
38 mg of
extract/mL and
'
47 mg of extract/mL, respectively). No significant difference was found
between the two months (p-value > 0.05). A previous study performed on P. palmata har-
vested in July and in October in Ireland reported ACE IC
50
values of >1.50 mg of extract/mL
in July and >2.00 mg of extract/mL in October [
27
]. Therefore, 50% of ACE inhibition was
reached by using very small amounts of extracts compared to our results. Another study
performed on a different Irish red seaweed, Porphyra dioica, harvested in July and Novem-
ber, found ACE IC
50
values relatively close to those obtained by the authors in [
27
], with
0.90 mg of extract/mL in July and 1.62 mg of extract/mL in November [
85
]. In addition, a
significant difference was detected between these two months, with the November extracts
showing higher ACE IC
50
values. Overall, higher amounts of ACE-inhibitory compounds
appear to be more available during the autumn. However, a previous study performed
on frozen (
−
20
◦
C) Japanese P. palmata identified phycobiliproteins (phycoerythrin, phyco-
cyanin, and allophycocyanin) as the original proteins of ACE-inhibitory peptides contained
in water extracts [
86
]. Those proteins are pigment proteins that play an important role in
macroalgae photosynthesis, and appear to be present at higher levels in winter and early
spring, compared to summer and autumn [
24
,
25
], which is inconsistent with the previously
published results. A higher anti-ACE effect was also obtained following the hydrolysis of
proteins in water extracts [
18
,
27
]. Furuta et al. [
86
] showed ACE-inhibition rates went from
approximately 30% for protein extracts to over 80% for hydrolyzed extracts [
86
]. Thus, the
higher ACE-inhibition potential of crude P. palmata in autumn could be related to protein
degradation, releasing bioactive anti-ACE peptides.

Mar. Drugs 2023,21, 392 12 of 19
Mar. Drugs 2023, 21, x 12 of 19
Figure 1. Angiotensin-converting enzyme (ACE) inhibition activity in water-soluble extracts from
Palmaria palmata (crude and dried). Results are expressed as ACE-inhibition rate in percentages (%)
of water-soluble extracts (mean ± SD; n = 3). Means with different lowercase letters (a,b) differ sig-
nificantly (p < 0.05). Means with different capital letters (A,B) differ significantly (p < 0.05). No com-
parison was made between the different concentrations. Pp: Palmaria palmata.
• Dried Palmaria palmata Extracts
ACE-inhibitory percentages of dried P. palmata harvested in October and June are
presented in Figure 1. Compared to crude specimens, ACE inhibition of dried P. palmata
extracts was not significantly different. However, anti-ACE potential was significantly
different between October and July. Therefore, the impact of the drying process differed
across the seasons. Significant losses of inhibition rates were detected in June (46% loss
for 8 mg prot/mL and 14% loss for 10 mg prot/mL), while October’s inhibition rates in-
creased slightly (on average, 65.02% for 8 mg prot/mL and 62.58% for 10 mg prot/mL). A
previous study on dried P. palmata determined an IC50 of water-soluble extracts of > 1 kDa
of 27.69 mg extract/mL, corresponding to 5.31 mg proteins/mL at 50% ACE inhibition [43].
Thus, the amount of protein and peptide required to inhibit ACE in the extract was
slightly less than that required for the October extracts and considerably less than for the
June extracts [43]. Finally, in this study, the effect of the drying process appeared to be
strongly related to the harvest season and in favor of October. The loss of ACE inhibitory
effect in June indicated an absence of protein degradation by drying, and as shown by the
chemical composition of dried P. palmata (in Section 2.1.2) and with the PEY, it was prob-
ably not due to protein losses.
3. Materials and Methods
3.1. Macroalgal Biomass and Processing
Wild P. palmata (Linnaeus) Weber and Mohr were harvested in the Forillon area (QC,
Canada) on 29 October 2019 and 2 June 2020. Collected specimens were cleaned with fresh
water, and stipes and biofouling were removed. All samples were stored at 4 °C overnight.
The next day, fresh P. palmata were divided into several batches to be either air dried for
5 h at 40 °C in a dryer (Hamilton beach, Glen Allen, VA, USA) then ground into 1 to 0.5
cm2 flakes or ground without pre-drying, as control batches. All samples were stored at
−80 °C after grinding.
3.2. Chemical Composition (Lipids, Ash, Proteins, Carbohydrates, Fibers) and Mineral
Composition
The same methods were applied as described in a previous study [41]. Briefly, sea-
weeds were freeze-dried and powdered (BFP660, Breville, Sydney, Australia). Moisture
and ash contents were measured by a thermal gravimetric analysis using method no.
Figure 1.
Angiotensin-converting enzyme (ACE) inhibition activity in water-soluble extracts from
Palmaria palmata (crude and dried). Results are expressed as ACE-inhibition rate in percentages (%)
of water-soluble extracts (mean
±
SD; n= 3). Means with different lowercase letters (a,b) differ
significantly (p< 0.05). Means with different capital letters (A,B) differ significantly (p< 0.05). No
comparison was made between the different concentrations. Pp: Palmaria palmata.
•Dried Palmaria palmata Extracts
ACE-inhibitory percentages of dried P. palmata harvested in October and June are
presented in Figure 1. Compared to crude specimens, ACE inhibition of dried P. palmata
extracts was not significantly different. However, anti-ACE potential was significantly
different between October and July. Therefore, the impact of the drying process differed
across the seasons. Significant losses of inhibition rates were detected in June (46% loss
for 8 mg prot/mL and 14% loss for 10 mg prot/mL), while October’s inhibition rates
increased slightly (on average, 65.02% for 8 mg prot/mL and 62.58% for 10 mg prot/mL). A
previous study on dried P. palmata determined an IC
50
of water-soluble extracts of >1 kDa
of
27.69 mg extract/mL
, corresponding to 5.31 mg proteins/mL at 50% ACE inhibition [
43
].
Thus, the amount of protein and peptide required to inhibit ACE in the extract was slightly
less than that required for the October extracts and considerably less than for the June
extracts [
43
]. Finally, in this study, the effect of the drying process appeared to be strongly
related to the harvest season and in favor of October. The loss of ACE inhibitory effect in
June indicated an absence of protein degradation by drying, and as shown by the chemical
composition of dried P. palmata (in Section 2.1.2) and with the PEY, it was probably not due
to protein losses.
3. Materials and Methods
3.1. Macroalgal Biomass and Processing
Wild P. palmata (Linnaeus) Weber and Mohr were harvested in the Forillon area (QC,
Canada) on 29 October 2019 and 2 June 2020. Collected specimens were cleaned with fresh
water, and stipes and biofouling were removed. All samples were stored at 4
◦
C overnight.
The next day, fresh P. palmata were divided into several batches to be either air dried for 5 h
at 40
◦
C in a dryer (Hamilton beach, Glen Allen, VA, USA) then ground into 1 to 0.5 cm
2
flakes or ground without pre-drying, as control batches. All samples were stored at
−
80
◦
C
after grinding.
3.2. Chemical Composition (Lipids, Ash, Proteins, Carbohydrates, Fibers) and Mineral Composition
The same methods were applied as described in a previous study [
41
]. Briefly, sea-
weeds were freeze-dried and powdered (BFP660, Breville, Sydney, Australia). Moisture and
ash contents were measured by a thermal gravimetric analysis using method no. 950.46
AOAC (Association of Official Analytical Chemists) 2008 and method no. 938.08 AOAC
2008, respectively. Protein content was obtained by the Kjeldahl method (AOAC 2008, no.

Mar. Drugs 2023,21, 392 13 of 19
988.05) using a protein factor of 5 [
87
], and lipid content was obtained by the Bligh and
Dyer method [
88
]. Total carbohydrate content was determined by difference [
21
], and fiber
content was determined with a total dietary fiber assay kit (Megazyme, Bray, Ireland) using
method no. 985.29 AOAC 2008. Calcium, iron, magnesium, potassium, and sodium con-
tents were quantified using a flame atomic absorption spectrophotometer (220FS, Varian,
CA, USA) with AOAC 2008 method no. 968.08, and iodine content was assessed with an
iodine ion-selective electrode (Thermo Scientific 258508, Waltham, MA, USA).
3.3. Potential Bioactive Compound Determination
3.3.1. Total Phenolic Content (TPC) and Carotenoids
The same methods were used as described in a previous study [
41
]. The TPC was
determined using an adapted Folin–Ciocalteu method [
89
,
90
]. Briefly, aqueous extracts
made with 0.5 g of freeze-dried/ground macroalgae were mixed with Folin–Ciocalteu
reagent and the absorbance was read at 750 nm (Spectrafluor Plus, Tecan, Thermo Scientific,
Ottawa, ON, Canada). The results were expressed as milligram Gallic acid equivalent
(GAE)/g dry extract [
91
]. Carotenoid content was determined using Quan and Turner’s
method [
92
] where 0.5 g of macroalgae sample was used for ethanol extraction and the
absorbance of the extract was read at 468 nm (Genesys 20, Thermofisher, Waltham, MA,
USA). The final concentration was calculated using the absorbance coefficient of astaxanthin
and expressed as µg/g.
3.3.2. Vitamins (β-Carotene and α-Tocopherol)
Vitamin quantification was performed following the protocolof Sanchez-Machado [
93
].
Dried macroalgae (0.25 g) was mixed with a solution of pyrocatechol (0.2 g/mL), and
samples were saponified with 5 mL of KOH (0.5 M diluted in methanol) and heated at
80
◦
C in the dark. Vitamins were then extracted with 5 mL of hexane and transferred into a
solution of chloroform and methanol (1:49, v/v). The separation was conducted by HPLC
using a C18 Lichrospher (RP-18e 100 A, 125
×
4 mm, 5
µ
m) column coupled with a guard
column, Security GuardTM C18 4
×
3.0 mm (Phenomenex, Torrance, CA, USA). The mobile
phase was a solution of acetonitrile and methanol (70:30, v/v) heated to 25
◦
C, at a flow rate
of 1 mL/min. The detection and quantification of
β
-carotene (
450 nm
) and
α
-tocopherol
(298 nm) were carried out with a photodiode array detector (PAD) (
ProStar 330
, Varian, Palo
Alto, CA, USA) and standards (
α
-tocopherol 258024,
β
-carotene 217538, Sigma-Aldrich,
Saint-Louis, MO, USA).
3.4. In Vitro Bioactive Potential
3.4.1. Water-Soluble P. palmata Extracts
The same methods were used as described in a previous study [
41
]. Briefly, samples
were extracted twice with phosphate buffer (20 mM, pH 7) at a ratio of 1:1.665 (w/v)
for non-dehydrated specimens and 1:16.667 (w/v) for dehydrated specimens. Samples
were centrifuged at 4000
×
g, 4
◦
C for 45 min (Avanti
®
J-E high-speed centrifuge, Beckman
Coulter, Brea, CA, USA). Supernatants were collected, brought to 80% saturated ammonium
sulfate, stirred, then centrifuged at 10,000
×
g, 4
◦
C for 60 min. Pellets were solubilized in
Milli-Q water and dialyzed (1 kDa cut-off membrane unit, MWCO 1 kDa, Pur-A-Lyzer,
Sigma-Aldrich, Saint-Louis, MO, USA) at 9
◦
C for 48 h. The resulting water-soluble seaweed
extracts were lyophilized and stored in the dark in vacuum bags at
−
20
◦
C until further
use. Total protein content was quantified according to the Dumas method [
94
], with a
TruSpec N nitrogen analyzer (Leco Corporation, St. Joseph, MI, USA). All extractions were
performed in triplicate.
3.4.2. Oxygen Radical Absorbance Capacity (Orac) Assay
The methods used were the same as those described in a previous study [
41
]. Water-
soluble seaweed extracts were dissolved in phosphate buffer (75 mM, pH 7.4) at eight
concentrations (serial dilution from 5000
µ
g/mL to 39
µ
g/mL with a dilution factor of 2).

Mar. Drugs 2023,21, 392 14 of 19
Samples, or Trolox standards, or blanks (phosphate buffer) were put in black microplate
wells (96-Well, Black U-Shape, Greiner Bio-One GmbH, Frickenhausen, Germany) with
150
µ
L of fluorescein (0.1
µ
M). The microplate was incubated and a volume of 50
µ
L of
2,2
0
-azobis (2-methylpropionamidine) dihydrochloride (AAPH) solution (150 nM) was
added to each well. The fluorescence was read at a wavelength of 485 nm for excitation and
583 nm for emission and using Synergy H1 (Biotek, Winooski, VT, USA). The calculated
ORAC values were expressed in
µ
mol equivalent Trolox per gram of freeze-dried extracts
(µmol TE·g−1). Assays were performed in triplicate.
3.4.3. Ferric Ion Reducing Antioxidant Power (FRAP) Assay
The same methods were used as described in a previous study [
41
]. Macroalgae
extracts (<1 kDa) were solubilized in Milli-Q water at five different concentrations (500,
250, 100, 50, and 10
µ
g/mL). A volume of 180
µ
L of FRAP reagent was added to the
wells of a 96-well microplate (Greiner Bio-One, Frickenhausen, Germany) and incubated.
Each tested concentration, Trolox standard, or blank (Milli-Q water) was added to the
wells and incubated. The absorbance was read at 593 nm using a Microplate Absorbance
Spectrophotometer (xMark, Biorad, Hercules, CA, USA). The calculated FRAP values were
expressed as
µ
mol equivalent Trolox per gram of freeze-dried extracts (
µ
mol TE
·
g
−1
). Tests
were performed in triplicate.
3.4.4. Angiotensin-Converting Enzyme (Ace) Inhibition
The ACE-inhibitory activity was performed as described by Hayakari [
95
] and used
by Hell et al. [
43
]. Briefly, 20
µ
L of ACE (250 mU per mL of borate buffer, 1 M NaCl, pH 8.3)
and 80
µ
L of phosphate buffer (pH 8.3) were added to 20
µ
L of water-soluble macroalgae
extract at concentrations of 8 and 10 mg prot/mL (A
sample
), or to enalapril which was
used as the positive control (A
control
), then incubated at 37
◦
C for 10 min. Simultaneously,
a replicate of each sample was incubated at 95
◦
C for 10 min to inactivate ACE and
used as negative control (A
blank
). Then, 40
µ
L of Hippuryl-L-histidyl-L-leucine hydrate
6.25 mM (ACE substrate) was added to all samples and incubated for 60 min at 37
◦
C. The
enzymatic reaction was stopped by heating to 95
◦
C for 10 min. Volumes of 360
µ
L of
2,4,6-trichloro-s-triazine (TT, 3% v/v in 1,4-dioxane) and 480
µ
L of phosphate buffer (pH 8.3)
were added to each sample, vortexed, and centrifuged at 2000
×
gfor 30 s (MiniStar, VWR,
St. Catharines, ON, Canada). A transparent 96-well microplate (Clear F-Bottom, Greiner
Bio-One GmbH, Frickenhausen, Germany) was filled with 200
µ
L of each sample per
well and the absorbance was read at 382 nm (xMark, Biorad, Hercules, CA, USA). The
ACE-inhibition rate (%) was calculated using the following formula:
ACE inhibition (%)=Acontrol −Asample
Acontrol −Ablank
×100
3.5. Statistical Analysis
All laboratory analyses were performed in triplicate. All values are expressed as the
mean
±
standard deviation (SD). Statistical analyses were mostly performed using SAS
University
®
(Cary, NC, USA) using orthogonal contrasts. After checking for the normality
and homoscedasticity of samples (using the Fisher and Shapiro–Wilk tests, respectively),
ANOVA tests were performed with a threshold of
α
= 0.05, and the Tukey test was used
as a post hoc test. For some cases where the ANOVA conditions were not reached, Prism
GraphPad
®
(San Diego, CA, USA) software was used and the Kruskal–Wallis test followed
by a multiple comparison (Dunn’s test) was performed, also with a threshold of α= 0.05.
4. Conclusions
In conclusion, wild crude P. palmata showed an important seasonal variation in chemi-
cal, mineral, and bioactive compound composition, with markers of growth in June and
decreased growth during October. Indeed, June specimens were richer in lipids, proteins,

Mar. Drugs 2023,21, 392 15 of 19
ash, fiber, sodium, calcium, magnesium, iron, polyphenols,
α
-tocopherol, and
β
-carotene,
signaling an active metabolism. Conversely, macroalgae harvested in October were signif-
icantly richer in carbohydrates, which probably reflects the increased synthesis of small
sugars called floridosides. October specimens were also richer in carotenoids, probably
caused by a decrease in photoperiod and light intensity combined with an increase in
nitrate concentrations in the water. ORAC, FRAP, and ACE inhibition activities measured
for water-soluble extracts at >1 kDa, containing proteins and peptides, did not significantly
differ through the harvesting months despite significant differences in the protein content
of crude macroalgae. This indicates a weak impact of the season on the antioxidant and
ACE-inhibitory potential of P. palmata from Eastern Quebec. For better nutritional value and
health benefit potential, P. palmata might be harvested in June. However, P. palmata growth
yield and shoreline abundance should be measured to better reflect the algal industry
reality. In fact, some companies in Quebec harvest P. palmata mostly during the autumn
because, in June, the available biomass in their area is too small. Since the fiber content
was similar between the two months, harvesting P. palmata in October could be more
profitable for the valorization of polysaccharides. Moreover, crude P. palmata harvested in
both months presented higher amounts of magnesium than chocolate (70–85% of cocoa),
more calcium than watercress, more iron than parsley, and a better Na/K ratio than shrimp,
another marine product. The drying process induced only small changes for most analyzed
compounds of P. palmata. By far, the factor determining the best nutrient potential was
the harvest season. The drying process did not promote a large loss or gain of bioactive
potential, thus indicating no potential negative impact during processing at a temperature
of 40
◦
C, which limited the deleterious impact of the heat. Drying would therefore seem a
good strategy for macroalgae quality and preservation.
Author Contributions:
Conceptualization, B.L, É.T., K.B. and L.B.; methodology, B.L., É.T., K.B. and
L.B.; software, B.L.; validation, B.L., É.T., K.B. and L.B.; formal analysis, B.L.; investigation, B.L.;
resources, B.L., É.T., K.B., V.P. and L.B.; data curation, L.B.; writing—original draft preparation, B.L.;
writing—review and editing, É.T., K.B., V.P. and L.B.; visualization, B.L. and L.B.; supervision, L.B.;
project administration, L.B.; funding acquisition, É.T., K.B., V.P. and L.B. All authors have read and
agreed to the published version of the manuscript.
Funding:
This research was funded by the Odyssée Saint-Laurent program of Réseau Québec
Maritime (RQM), ministère de l’Économie et de l’Innovation (MEI) (OSL-2019-PS-06), and the Fonds
de recherche du Québec—Nature et technologies (FRQNT).
Institutional Review Board Statement: Not applicable.
Data Availability Statement:
The datasets analyzed in this study are available in a publicly accessible
repository that can be found here: Merinov and Universitédu Québec àRimouski (2022). Banque des
données sur les macroalgues marines des sites de la Gaspésie (2019) [Data set]. https://catalogue.
ogsl.ca/dataset/ca-cioos_cbf7730a-7074-4a37-92c8-0e549d6c6194?local=fr, accessed on 21 April 2023.
Acknowledgments:
The authors thank Océan de Saveurs
®
for supplying the crude seaweeds. They
also thank Ariane Tremblay, Diane Gagnon, Martha Paola Riviera Rodriguez, Julie Galarneau,
Gabriela Vollet Marson (Département des sciences des aliments, UniversitéLaval, QC, Canada),
and Marie-Élise Carbonneau (Merinov, Gaspé) for their technical expertise. This project was carried
out in partnership with the Institute of Nutrition and Functional Foods (INAF, QC, Canada) and
the industrial research center Merinov (QC, Canada). The authors would also like to thank the
Odyssée Saint-Laurent program (Réseau Québec Maritime, QC, Canada), ministére de l’Économie et
de l’Innovation (MEI), and the Fonds de recherche du Québec—Nature et technologies (FRQNT) for
their financial support.
Conflicts of Interest: The authors declare no conflict of interest.

Mar. Drugs 2023,21, 392 16 of 19
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