Aquaculture Production of the Brown Seaweeds Laminaria digitata and Macrocystis pyrifera: Applications in Food and Pharmaceuticals

Abstract

Seaweeds have a long history of use as food, as flavouring agents, and find use in traditional folk medicine. Seaweed products range from food, feed, and dietary supplements to pharmaceuticals, and from bioenergy intermediates to materials. At present, 98% of the seaweed required by the seaweed industry is provided by five genera and only ten species. The two brown kelp seaweeds Laminaria digitata, a native Irish species, and Macrocystis pyrifera, a native New Zealand species, are not included in these eleven species, although they have been used as dietary supplements and as animal and fish feed. The properties associated with the polysaccharides and proteins from these two species have resulted in increased interest in them, enabling their use as functional foods. Improvements and optimisations in aquaculture methods and bioproduct extractions are essential to realise the commercial potential of these seaweeds. Recent advances in optimising these processes are outlined in this review, as well as potential future applications of L. digitata and, to a greater extent, M. pyrifera which, to date, has been predominately only wild-harvested. These include bio-refinery processing to produce ingredients for nutricosmetics, functional foods, cosmeceuticals, and bioplastics. Areas that currently limit the commercial potential of these two species are highlighted.

Full text

molecules
Review
Aquaculture Production of the Brown Seaweeds
Laminaria digitata and Macrocystis pyrifera: Applications in
Food and Pharmaceuticals
Diane Purcell-Meyerink 1, * , Michael A. Packer 1, Thomas T. Wheeler 1and Maria Hayes 2


Citation: Purcell-Meyerink, D.;
Packer, M.A.; Wheeler, T.T.; Hayes, M.
Aquaculture Production of the Brown
Seaweeds Laminaria digitata and
Macrocystis pyrifera: Applications in
Food and Pharmaceuticals. Molecules
2021,26, 1306. https://doi.org/
10.3390/molecules26051306
Academic Editors: Daniel Franco
Ruiz, María López-Pedrouso and Jose
Manuel Lorenzo
Received: 15 January 2021
Accepted: 23 February 2021
Published: 28 February 2021
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
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iations.
Copyright: © 2021 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/).
1Cawthron Institute, 98 Halifax Street, Nelson 7010, New Zealand; Mike.Packer@cawthron.org.nz (M.A.P.);
Tom.Wheeler@cawthron.org.nz (T.T.W.)
2Food BioSciences, Teagasc, Ashtown, Dublin 15, Ireland; Maria.Hayes@teagasc.ie
*Correspondence: diane.purcell-meyerink@cawthron.org.nz
Abstract:
Seaweeds have a long history of use as food, as flavouring agents, and find use in traditional
folk medicine. Seaweed products range from food, feed, and dietary supplements to pharmaceuticals,
and from bioenergy intermediates to materials. At present, 98% of the seaweed required by the
seaweed industry is provided by five genera and only ten species. The two brown kelp seaweeds
Laminaria digitata, a native Irish species, and Macrocystis pyrifera, a native New Zealand species, are not
included in these eleven species, although they have been used as dietary supplements and as animal
and fish feed. The properties associated with the polysaccharides and proteins from these two species
have resulted in increased interest in them, enabling their use as functional foods. Improvements
and optimisations in aquaculture methods and bioproduct extractions are essential to realise the
commercial potential of these seaweeds. Recent advances in optimising these processes are outlined
in this review, as well as potential future applications of L. digitata and, to a greater extent, M. pyrifera
which, to date, has been predominately only wild-harvested. These include bio-refinery processing
to produce ingredients for nutricosmetics, functional foods, cosmeceuticals, and bioplastics. Areas
that currently limit the commercial potential of these two species are highlighted.
Keywords:
aquaculture; seaweed; Laminaria digitata;Macrocystis pyrifera; extraction; food;
pharmaceuticals; feed
1. Introduction
1.1. Laminaria digitata and Macrocystis pyrifera in the Context of Global Seaweed Aquaculture
Nearly three hundred seaweed species of interest have been identified for their poten-
tial commercial value [
1
], yet only ten are cultivated extensively with a handful of other
species grown for niche applications. These include three brown seaweeds Saccharina
japonica,Undaria pinnatifida, and Sargassum fusiforme (Ochrophyta, Phaeophyceae); four
red seaweeds Neopyropia/Pyropia/Porphyra spp., Eucheuma spp., Kappaphycus alvarezii, and
Gracilaria spp. (Rhodophyta); and five green seaweeds Ulva clathrata (formerly Entero-
morpha clathrata), Monostroma nitidum and Caulerpa spp., Ulva spp., Oedogonium termedium
(Chlorophyta) [
2
]. The brown seaweed commonly called Japanese kelp, Saccharina japonica,
formerly known as Laminaria japonica, was the most cultivated seaweed in the world until
2010. It still retains a considerable market share, commanding 29% of global production in
2014 and over 33% in 2018 [
3
,
4
]. However, in 2010, production of Eucheuma/Kappaphycus
surpassed 9.07 million tonnes with a value of over EUR 1,079 million [
5
], and by 2014,
Eucheuma (35%) and Kappaphycus (6%), collectively at 41% global production, were the most
cultivated species [
3
]. In the context of the cultivation advantage gained by aquaculture,
seaweed cultivation is unequalled in mariculture, as 94% of the annual seaweed biomass
used globally is from cultivated sources [6].
At present 98% of seaweed cultivated across the globe comes from five genera: Saccha-
rina, Undaria, Neopyropia/Pyropia/Porphyra, Eucheuma/Kappaphycus, and Gracilaria [
4
,
5
,
7
,
8
].
Molecules 2021,26, 1306. https://doi.org/10.3390/molecules26051306 https://www.mdpi.com/journal/molecules

Molecules 2021,26, 1306 2 of 41
These species are predominantly cultivated at sea, with a few groups including kelps and
nori requiring an extra step, often onshore, to facilitate their microscopic life cycle stage.
This step, known as the aquaculture hatchery phase, enables growth and seeding of ropes
prior to deployment at sea [9].
Seaweed products range from food to pharmaceuticals, and bioenergy intermediates
to materials. Brown and green seaweeds are predominately used in food as a source of
fibre, protein, and minerals, especially throughout Asia [
2
]. Red seaweeds have been
used as food, and as a source of agars and carrageenan which are used in food, cosmetic
ingredients, and for biomedical applications [
10
]. The global seaweed industry is worth
more than USD 6 billion per annum, of which 85% is for human consumption, and seaweed-
based polysaccharides (carrageenan, agar, and alginates) account for nearly 40% of the
world’s hydrocolloid market [
2
]. In Europe, brown seaweeds were traditionally used to
produce additives (e.g., alginates) or animal feeds in the form of meal [
11
]. L. digitata
and M. pyrifera are two brown seaweed species that are harvested and cultivated globally.
In comparison to other kelp species such as Saccharina japonica, Saccharina latissima, and
Undaria pinnatifida, M. pyrifera is a native species to New Zealand whereas Undaria pinnatifida
is an invasive species and Saccharina japonica is not a native species [
12
,
13
]. L. digitata is
a native Irish species with a wide distribution at low water on the Irish coast, when
compared to Saccharina latissima, also a native Irish species, yet not comparable to L. digitata
in distribution and abundance on the Irish coast [
14
,
15
]. In 2016, L. digitata global wild
harvest yielded ~45,000 tonnes, and M. pyrifera yield reached 31,835 tonnes; however, only
one tonne of M. pyrifera was produced through aquaculture [
3
]. Chile was the highest
producer of brown seaweed from natural populations at 300,000 dry tonnes per year by
2012 [
16
], in a global context 7% of the brown seaweed from natural populations was
provided by Macrocystis sourced in Chile and Mexico [
17
]). Seaweed is sourced mainly
from wild harvesting, with only 2.4% from cultures, which are dominated by Agarophyton
chilense [
18
]. It can be observed from these data that M. pyrifera when compared with
L. digitata has significant future cultivation potential through aquaculture. Additionally,
this aquaculture potential may assist in a reduction in the impact of wild harvesting on
M. pyrifera’s distribution off the Chilean coast.
Specifically, L. digitata has been used in Europe as a food supply for algivores in the
mariculture of abalone and sea urchins; it has also been harvested and supplied to Asia as
a dried product and used as stock for soup making [
14
]. Within the fuel and renewable
energy sector, it has been investigated for methane gas production through bioconversion
trials in France and the US [
14
]. In Australia, Chile, and the US, M. pyrifera was also
used to feed abalone [
14
,
19
,
20
]. In Mexico, M. pyrifera was used as a meal for goats. The
digestibility of this seaweed was 77% when fed to this ruminant animal [
21
]. Digestibility
increased to 85% when fed to male bovine zebu bulls [
22
]. Due to its low digestibility in
salmonids, M. pyrifera in a derived flour form was added as a food supplement at 1.5%, 3%,
and 6% dry weight (DW) of the total diet as a mineral and carbohydrate source [23].
In the US, commercial harvesting of L. digitata started in 2010, among several other
species, and now kelp aquaculture is considered one of the fastest growing industries in
the North Eastern US [
24
]. A native species of the North Atlantic coast L. digitata commonly
called oarweed was used in an offshore system cultivation trial in Ireland between 2008 and
2011. The trial found that its production using this system was commercially viable [
15
].
In Europe, L. digitata has been the main raw material used to supply the French alginate
industry, being harvested mainly from the upper sublittoral zone and around the coast
of Brittany and surrounding islands. In Norway, L. digitata grows in large masses at
the lower end of the eulittoral zone and had been previously an important industry in
Norway until seaweed market expansion required increased biomass and was replaced by
Laminaria hyperborea (L. hyperborea) forests [
25
]. In Iceland, L. digitata was grown to supply
the UK alginate industry which is based in Scotland [
25
]. In 2009, alginate production
was moved from Scotland to Norway by Pronova, a Norwegian alginate producer, but L.
hyperborea is still sourced in Scotland and Ireland and Ascophyllum nodosum is also sourced

Molecules 2021,26, 1306 3 of 41
in Scotland, Ireland, Iceland, and Norway for Pronova [
26
]. Recently, the UK’s interest
in alternatives to fossil fuels saw the inclusion of L. digitata in method development for
bioethanol production [27].
In 2012, Asia dominated the cultivation of Laminaria species. The three main producers
were China, Korea, and Japan, with China’s market share at 4.35 million tonnes, or 23% of
total global production. At this time Denmark was the only European country to cultivate
Laminaria [
28
]. Global consumption of the Laminaria genus as a food or a feed additive
accounted for over 0.9 million tonnes of the biomass produced in 2012, worth between
USD6 billion and 8 billion, or 50% of the total seaweed global revenue for 2012 [1].
Macrocystis pyrifera, commonly called the giant kelp, is presently wild harvested in
Alaska. However, aquaculture started in 1972 in California where an offshore kelp farm
was set up. It achieved limited success due to design problems, low nutrient supply,
and storms yet persevered until 1982 [
24
]. Work continued in California on M. pyrifera
between 1980 and 1986, when another group received funding and managed not only to
cultivate it effectively by modelling their system on a natural kelp bed, but also created a
planting technology using fertiliser in oligotrophic waters, and performed genetic studies
creating 800 strains [
29
]. The US Marine Biomass Program discontinued funding of projects
after 12 years and USD 20 million, as seaweed which had been grown specifically as an
alternative fuel source to fossil fuels proved to be uneconomical by 1986 [
30
]. In Southern
Argentina, M. pyrifera grows to depths of 55 m; collections of M. pyrifera were made
from these large kelp beds, primarily for alginate production, until this practise ceased
in the early 2000s [
25
,
31
]. Macrocystis pyrifera (formerly M. angustifolia) was cultivated
in South Africa on an experimental scale, as a potential resource for alginate production
and abalone feed [
25
]. Northern Chile has also harvested M. pyrifera initially in smaller
quantities than California, yet exploitation of these natural kelp beds has increased in
Chile in recent years to supply food to the abalone industry. This demand has initiated
pilot aquaculture cultivation research of M. pyrifera to a potential productivity level of 200
tonnes (fresh)/ha/year [
32
]. A further study including economic profitability calculations
noted that a cultivation system had to be of 30–50 ha in size and M. pyrifera priced at
EUR 64/tonne was essential to reach economic viability [
19
]. More recent work has found
that a 10-ha cultivation system with M. pyrifera priced at EUR 72/wet tonne would be
profitable [
33
]Macrocystis pyrifera was only cultured in Peru, with Chile the predominant
country for wild harvest collection followed by the US. Peru has one of the most productive
marine coastlines in the world, with over 34 recognised brown seaweeds found there;
4% of annual seaweed biomass landings in Peru are produced through seaweed farms,
yet this has been declining since 2012 [
34
]. The US has used M. pyrifera harvested off the
Californian coast as a source of alginate since at least 1913; the harvests have varied from
325,157 wet tonnes in 1918, to 214 tonnes in 1931, to 90,718 in 1984 [
35
]. In comparison with
Laminaria, Chile’s Macrocystis harvest was 23,587 tonnes [
28
]. Macrocystis pyrifera, similar to
Laminaria, are used as foods or food additives [1].
This review describes the state of play of brown seaweeds in the context of global
seaweed production and outlines the present seaweed studies in the literature on the two
brown kelp species M. pyrifera and L. digitata. In addition to previous reviews, it examines
the differences that occur when one species, M. pyrifera, is predominantly wild harvested,
versus L. digitata which has been grown extensively via aquaculture, and the impact that
has on the variety of products being sourced and developed from each species. It details
the best practice to be applied to aquaculture cultivation of these two species, and lists
the food, feed, and pharmaceutical products produced from M. pyrifera and L. digitata.
Additionally, it makes suggestions where potential improvements could be applied to
expand the seaweed bioproducts sector.

Molecules 2021,26, 1306 4 of 41
1.2. Aquaculture methods for Brown Kelps
1.2.1. Macrocystis pyrifera
The aim of seaweed aquaculture is to meet the global demand for seaweed which
cannot be fulfilled from wild harvest of seaweed alone. World production of farmed
seaweeds doubled between 2000 and 2012; the FAO reported a global increase of over
20 million tonnes of seaweed production since 2001 [
10
,
28
]. This growth has coincided
with the largest increase in global aquaculture, where almost 10 million tonnes of seaweed
product came from aquaculture between 2011 and 2015 [
10
]. In 2015, of the total 30.4 million
tonnes of seaweed produced, the aquaculture industry produced 29.4 million tonnes with
only 1.1 million tonnes produced from wild harvest [
10
]. The predominant cultivators
of seaweed are countries throughout Asia and a market which has shown considerable
expansion is Indonesia [28].
Other countries have been investigating and trialling the most efficient cultivation
systems to produce commercially efficient seaweed cultivation. A proposed Canadian
offshore kelp farm producing Laminaria sp. in close proximity to a salmon farm was
studied using a mathematical model [
36
]. This proposed 1060 m of ropes, on either end
of a salmon cage. The annual biomass yield from the model was 1600 kg dry weight, and
it was deemed that a return on investment would be obtained after 6 years of seaweed
introduction on the farm. Additionally, if increased kelp production occurred on several
salmon farms, this would result in improved productivity of kelp and a profitable net
return. Environmental impacts such as nitrogen and oxygen levels did not increase above
background levels. Alternative species were also suggested for cultivation including
Macrocystis and Nereocystis [
36
]. Another study noted that M. pyrifera protein content
increased from 9 to 13% when cultured in proximity to salmon farms in Chile [
37
]. This
finding is potentially economically advantageous as M. pyrifera is used as a food source for
abalone [14,19,20].
Extensive work on cultivation of M. pyrifera in Chile has been essential to protect
the natural kelp beds still present which have been harvested extensively. Best practice
for efficient production of M. pyrifera requires starting with healthy juvenile sporophytes.
This has been optimised to a growing period of only 45 days, a reduction from 60 days.
Wild-sourced sori are used as seed material and once sporulation has occurred and a
spore density of 40,000 cells ml
−1
has been reached, sterile spores are transferred to 10 L
rectangular tanks containing polyvinyl chloride (PVC) cylinders wrapped in 1.5 mm nylon
string. Once settlement has occurred after 24 h, they are transferred to new clean 10 L tanks
with seawater enriched with Provasoli’s Enriched Seawater (PES) culture media. These
cultures are maintained at an optimum photoperiod of 16:8 light day cycle, temperature of
12
◦
C, photo irradiance of 12
µ
mol
−1
s
−1
m
2
, and aeration rate of 414 L h
−1
. After 45 days,
juvenile sporophytes of 4–5 mm in size are produced and can be harvested for human
consumption at this stage. If being grown to produce biomass for fuel, these optimised
conditions enable the open ocean growing season to be extended by a month, increasing
biomass [38].
The optimal out-planting method uses long lines of 50–100 m in length. These juvenile
sporophytes are visible as a brown “fuzz” on the nylon lines at this stage and can be
transferred to long lines by wrapping the nylon tightly around the long lines, which are
a rope-based material. The long lines are then suspended in the nearshore environment,
secured at both ends with floatation buoys to maintain buoyancy in the water column and
then weights maintain position [
39
]. This out-planting method is similar to the traditional
method used in East Asia.
A second method of nearshore cultivation uses individual juvenile kelps, produced
from a free-floating cultivation of the sexual phase (gametophytes) producing unattached,
floating sporophytes, which are grown in tanks for several months, to a size of about 8
cm. These are then attached manually to long lines [
40
]. Comparisons between these
two cultivation methods in locations in Chile have found this second method produced
up to three times the biomass than the previous method [
41
]. The authors conclude

Molecules 2021,26, 1306 5 of 41
that the increased productivity is due to these gametophyte plants being in a unialgal
environment, resulting in less impact of bacteria and disease as they grow. Additionally,
the increased size to 8 cm in comparison to 4–5 mm (juvenile sporophytes) means they can
outgrow the epifauna and epiflora that they encounter on introduction to the nearshore
environment [41].
1.2.2. Laminaria digitata
Laminaria digitata is a common kelp predominantly found along the North European
and Eastern American coast [
14
]. Similar to M. pyrifera, it has a complex life cycle that
includes a microscopic gametophyte phase of growth requiring a hatchery system to sup-
port efficient reproduction for farming of the macroscopic growth phase. The sporophytes
are then out-planted in the near-shore ocean environment [
15
]. This cultivation method is
specific to L. digitata practiced off the Irish coast.
This Irish study comprehensively outlines the best practice to follow when cultivating
L. digitata throughout its life cycle. This study, “The seaweed hatchery project”, was
carried out in Ireland over a two-year period. It investigated the potential of L. digitata
as a commercial seaweed aquaculture crop using new techniques and improving existing
practices. Conditions for establishing optimum growing gametophytes requires wild-
sourced sori tissue collected on a low spring tide in the lower intertidal and subtidal
regions off the Irish coast. This sori tissue is then cleaned, all epiphytes are removed, and
then it is placed in the cold (~
−
10
◦
C) dark for 18–24 h. Then, these pieces are chopped
further into smaller pieces of 4–5 cm in size and are placed in a 1 L beaker with sterile
seawater for 35–40 min to allow zoospore release, being stirred occasionally with a sterile
glass rod. When the water appears cloudy, this indicates spores have been released. The
rehydrated sori tissue is then filtered out and transferred to zoospore solution in a suitable
culture vessel. The solution is then aerated in PES media which are changed every two
weeks. Light is provided, the plantlets are covered in red cellophane, and irradiance at the
surface of the glassware is approximately 15–20
µ
mol m
−2
s
−1
, with day length starting
from a 16:8 light–dark cycle then progressing to 24 h of complete light and a temperature
of 10
◦
C. Cultures are maintained for 3–5 months to produce optimal biomass for spraying
of the macroscopic growth phase of juvenile sporophytes onto the string [15].
Prior to out-planting, reproduction of the gametophytes must be induced. This is
achieved by supplying new media, changing the light spectrum from red to blue, maintain-
ing the incubation conditions described in the previous paragraph but reducing the day
length to a 12:12 light–dark cycle. These conditions are maintained for 12–15 days or until
reproductive structures, i.e., fertilised eggs or developing sporophytes, are observed. At
this stage, the culture is ready to be sprayed onto string. The string, in 30 cm lengths, is
wrapped around 65 mm of square plastic PVC drainpipe that has 4–5 cm holes made in it
in as many areas as possible without reducing the structural integrity of the collector. Once
the culture is sprayed, it must immediately be placed vertically into tanks in the hatchery
for 30–45 days. Conditions are maintained at 10
◦
C, day length at 12:12, but irradiance and
aeration are incrementally increased. Irradiance starts at 35–40
µ
mol m
−2
s
−1
from day 0
to 3, then increases to 60–70
µ
mol m
−2
s
−1
for the remaining 30–45 d. No aeration occurs
from day 0 to 3, then from day 3 to 14, it is turned on to a low setting and is progressively
increased to a moderate setting. After day 14, aeration is increased towards a high setting.
Deployment of the now grown juvenile sporophytes at sea is the same method as that
used from M. pyrifera, with the nylon being wrapped around large anchored and floated
longlines between October and December. These lines are maintained and checked every
two months with optimum harvest after 5–6 months, usually in April–May (Northern
hemisphere spring), just before the water temperature increases, to avoid seaweed being
infested with epiphytes [15].
Optimum growing depth was 2 m for out-planting unialgal gametophyte cultures
of L. digitata in Helgoland, North Sea, Germany, with optimum blade growth noted in
spring and summer. L. digitata also had a longer growing season, only reducing growth in

Molecules 2021,26, 1306 6 of 41
September compared to July for both L. hyperborea and S. latissima (formerly L. saccharina),
potentially indicating an adaptation to life in the sublittoral fringe [42].
Wave exposure impact on growth rates of L. digitata found a reduction in blade growth
at the lowest and highest wave velocities, which may occur as a trade-off to increase blade
strength [
43
]. A comparable wave velocity study on Undaria pinnatifida sporophytes on
seeding strings noted that total biomass increase was significantly higher than total length,
indicating seawater velocity encourages greater biomass productivity over length increase
in sporophytes [
44
]. Alginate is a component of the cell wall of brown seaweeds and is
partly responsible for the flexibility of the seaweed; therefore, species which grow in more
exposed and turbulent sites usually contain higher alginate content [
25
]. L. digitata is found
in the upper sublittoral zone in rocky wave-exposed locations, and it is considered to have
a high alginate content [14,25].
Several L. digitata studies have looked specifically at the impact of temperature and
produced guidelines regarding tolerance levels for this species. For the microscopic ga-
metophyte growth phase, its temperature range is quite restricted with reductions in
growth at 20–21
◦
C, with death observed after one week at 22–24
◦
C [
42
,
45
]. Other studies
from Helgoland, North Sea, Germany found that optimal temperatures for vegetative
gametophyte growth were 10–18
◦
C, during a 16:8 light–dark cycle to simulate summer
photoperiods, with induction of gametogenesis at between 5 and 15
◦
C, and maximum
sporophyte development during summer photoperiods with enriched nutrient regimes,
even though the fastest gametogenesis was at 10–15
◦
C and the highest sporophyte recruit-
ment was at 5
◦
C [
46
]. Previous work found sporophytes were tolerant to conditions in
North European waters, with optimum growth from 5 to 15
◦
C, but at 20–22
◦
C, a 25%
reduction in growth rate occurs [
47
]. Projected increase in sea temperatures due to climate
change will significantly impact the L. digitata populations on the European coast, with
model projections predicting a push to the verge of local extinction in the first half of the
21st century, consequently causing a decline in species whose life cycle depends on this
seaweed for survival [48].
2. Current and Future Uses
2.1. Food and Feed Uses of Brown Seaweeds
Seaweeds are traditionally cooked and used as sea vegetables in Asian countries, yet
their consumption by Western countries is minimal [
49
]. The nutritional content of fats, pro-
teins, and carbohydrates in brown seaweeds depends on the season and location of harvest.
Specifically, fat content in seaweeds is less than 5% dry weight (DW), with L. digitata and S.
latissima harvested in Ireland containing only 1% and 0.5% fat, respectively [
14
,
50
]. Protein
percentage values range from 5 to 20%. L. digitata collected off the UK coast had a protein
content of 15.9%(DW) [
51
], and Undaria pinnatifida, commonly called wakame, collected in
spring on the northwest Iberian coast, had a protein content of 16.8% of seaweed DW [
52
].
Carbohydrates account for 13–60% of the DW of brown seaweeds [
53
,
54
]. A study of four
brown species sampled off the Isle of Seil in Scotland, on eight occasions between August
2010 and October 2011, found carbohydrate content ranged from 34.6
±
3.1%, 33.2
±
3.8%,
28.5
±
3.9, and 37.4
±
4.0% DW in L. digitata, L. hyperborea, Saccharina latissima, and Alaria
esculenta, respectively. Interestingly when protein content was compared between the
species, the values were higher than previously reported in the literature at 6.9
±
1.1% in
L. digitata, 6.8
±
1.3% in L. hyperborea, 7.1
±
1.7% in Saccharina latissima, and 11.0
±
1.4% in
Alaria esculenta. Results noted the range of protein levels were highest in the first quarter
and lowest in the third quarter of the year [
55
], indicating potential seasonal impact on
protein levels detected in this study.
The contribution of brown seaweeds to the food and animal feed industry has been
primarily as whole foods or as additives to feeds. In 2009, the 78,109 tonnes of hydrocol-
loids that were traded included 58% carrageenan and 11% agar, both from red seaweed
and 31% alginate from brown seaweed [
56
]. The main commercial seaweed extracts are
hydrocolloids which include agar, alginates, and carrageenans. The main use of alginates is

Molecules 2021,26, 1306 7 of 41
as thickening or gelling agents and emulsion stabilisers [
56
]. Hydrocolloids sourced from
algae are called phycocolloids. The most useful phycocolloid derived from brown seaweed
in the food industry is alginate [
25
]. The phycocolloids found in the brown seaweeds Macro-
cystis sp.,Laminaria sp., and Ascophyllum sp. include the alginates which are a soluble source
of fibre [
57
]. L. digitata total fibre content when compared to whole foods provides 6.2
g/100 g wet weight; this value exceeds other whole foods including brown rice (3.8 g/100
g weight) with the exception of brown lentils (8.2 g/100 g weight) [
58
,
59
]. In comparison to
the dietary fibre guideline daily allowance (GDA) set by the American Association of Ana-
lytical Chemists (AOAC) fibre recommendation, using an 8 g portion size, L. digitata can
provide 12.5% of that fibre [
58
]. Fucoidan is another phycocolloid and soluble fibre source
predominantly found in the brown seaweed species Saccharina religiosa (formerly Laminaria
religiosa) and Nemacystus decipiens [
57
]. Fucoidan is an acidic heteropolysaccharide, and the
biological activities of fucoidan are multi-factorial. Its biological activity is utilised through
its low molecular weight and sulphate groups. Structural skeletal characterisation is still
needed to locate specific branching sites of the sulphate groups [60].
M. pyrifera and L. digitata were used previously to produce alginic acid powder for use
in diet biscuits to provide a feeling of fullness [
25
]. Both species were utilised for alginate
production of gels for the food industry [
25
,
61
]. Alginate extracted from M. pyrifera has also
been used as a stabiliser for food products such as ice cream, yogurt, and cream, as well as
in foods as an emulsifier and gelling agent for sauces and dressings [
62
]. A food study used
M. pyrifera, also called “huiro” in Chile, as an ingredient in Chilean recipes, specifically
for huiro fritters and breadstick recipes to test whether seaweeds improved nutritional
content, compared with the usual ingredients. Between 3 and 28% of the ingredients were
replaced using dried ocean-sourced M. pyrifera; the results found that protein was lower in
the huiro fritters at 6.9% DW, but the breadsticks with huiro showed high protein levels
at 9.5% of DW. This study did taste test all products yet products were not considered a
commercial success [
63
]. Saccharina japonica’s dried seaweed powder extract was added at
1% to breakfast sausage ingredients and an improvement in physiochemical and sensory
properties of the sausages was noted as well as improving the ash content [
64
]. Table 1lists
the current commercial feed, food, and functional food products produced from M. pyrifera
and Laminaria spp.
Seaweeds or sea vegetables are a great source of B-group vitamins (mainly B
1
, B
12
),
along with the lipophilic vitamin A (derived from the carotenoid
β
-carotene) and vitamin
E (tocopherol). M. pyrifera contains quantities of
α
-tocopherol (the most biologically
active form of vitamin E) comparable and in some cases higher than plant oils which are
considered to be abundant in this vitamin, such as sunflower seed and soybean oil [
65
,
66
].
M. pyrifera and L. digitata were used to produce crude alginate for use as binding agents for
salmon feeds [
25
]. M. pyrifera provided a carbohydrate and mineral supplement in a flour-
derived meal ingredient added as 3% DW of the salmonid fish species diet [
23
]. Juvenile
white shrimp were given a M. pyrifera-based diet supplement. The dose was calculated
based on shrimp body weight at a concentration of (33.3 g/kg) of M. pyrifera, then 1.6 g
of this concentration was fed to the shrimp over 28 days; a protein efficiency level of 1.7
was recorded [
67
]. M. pyrifera is also used to feed abalone as an in situ ocean-based food
comprising 9–13% crude protein [
14
,
19
,
20
]. L. digitata was also used as a fresh ocean-based
food source for the algivores, abalone and sea urchin, in Ireland (Table 1) [
14
]. M. pyrifera
was used in ruminant diets due to its high digestibility rates. When incorporated at 30% DW
of the goat diet, it had a 70% digestibility value; for bovine zebu bulls, a meal supplement
provided 8.5% of their dietary protein, and this achieved 85% digestibility [
21
,
22
]. Two
Laminaria species, L. digitata and L. hyperborea, provided a complete daily diet of 1.4 kg wet
weight (WW) for six North Ronaldsay sheep in Scotland resulting in 79.6% digestibility [
68
].
L. digitata powder was also used as a dietary supplement (at a concentration of 0.001 kg per
day) for rabbits [69] (Table 1).
The main uses of L. digitata have been for laminarin and fucoidan extract genera-
tion [14]. In France, it is predominately used to supply the alginate industry. The alginate

Molecules 2021,26, 1306 8 of 41
content of brown seaweeds from Irish waters is 32% DW and fucoidan accounts for up
to 15% DW, yet this is dependent on the extraction method used [
14
,
57
]. In addition to
alginates, Laminaria species have additional polysaccharides with commercial value; in
comparison to M. pyrifera, L. digitata contains 32.2% alginic acid, 5.5% fucoidan, 14.4%
laminarin, and 13.3% mannitol [57].
Earlier studies on weaning pigs and piglets used a combination of laminarin, fucoidan,
and ash to supplement diets for improved growth performance. Laminarin and fucoidan
were tested with a range of lactose concentrations within the daily diet of weaning pigs
for 21 days to see the effect on weight, average daily gain (ADG), and food conversion
ratio (FCR). Results noted that the inclusion of the laminarin and fucoidan extract may
reduce the need for high lactose diets of animals less than 60 kg in weight, and lessen other
common problems which occur in pigs post weaning [70] (Table 1).
More recently, seaweed extracts have been commercially patented by companies such
as Ocean Harvest Technology based in Ireland and Olmix based in France, to name just
two. Ocean Harvest Technology have made a seaweed extract from brown, green, and
red seaweeds to produce OceanFeed Swine
®
(OFS), which was tested in Chile on 1809
pigs. OFS was supplied as a flour supplement at 5 g/kg dose per day from day 21 to 55
of swine age. Results indicated their daily average weight gain (ADG) increased by 26
g and improved their feed efficiency (FE) by 0.07. In addition, there was an increase in
the slaughter weight to (92.38 kg
±
0.47) from the control group weight of (90.97
±
0.47
kg), showing statistical significance with a P-value of (<0.05). A reduction in the bacteria
Escherichia coli and an increase in Lactobacillus sp. were also observed in these pigs [
71
].
Olmix have made a piglet feed supplement called Ecopiglet using the green seaweed Ulva
sp. They tested 833 piglets between day 5 and day 21 of life with a 50 g dose of Ecopiglet
per animal twice a day. Results noted no change in the ADG, survivability, or microbial
gut community but did noticed a significant decrease in observed diarrhoea incidence [
72
].
Table 1.
Current commercial feed, food, and functional food products produced from Macrocystis pyrifera and Laminaria spp.
Brown Seaweed
Species
Feed or
Food Food Group
Product
Produced/Product
Quality
Function of Product Reference
M. pyrifera Feed Fresh seaweed Fresh seaweed/
9–13% Crude Protein Food supply for abalone [14,19,20]
L. digitata Feed Fresh seaweed Fresh seaweed Food supply for abalone and
sea urchins [14]
L. digitata and L.
hyperborea Feed Fresh seaweed 1.4 ±0.2 kg WW per
day/79.6% Digestibility
North Ronaldsay sheep breed
complete daily food supply;
dry matter degradation
(DMD, 71.7%, at 48 h)
[68]
M. pyrifera Feed Dried seaweed,
included in meal
30% DW of Diet/77%
Digestibility 30%DW of goat daily diet [21]
L. digitata Feed
Dried seaweed
supplement to
daily diet
Powder: 0.001 kg/day
Rabbits showed a significant
effect of lowering total
cholesterol, lipoprotein,
especially triglyceride
[69]
Saccharina japonica Food Dried seaweed
supplement Powder (1, 2, 3, 4%)
Ash content increased; 1%
seaweed added to breakfast
sausages were the most
improved for physiochemical
and sensory properties
[64]
M. pyrifera Food
Dried seaweed
3–28%
Food recipe
ingredient
Huiro fritters/6.9%DW of
protein Protein supplement

Molecules 2021,26, 1306 9 of 41
Table 1. Cont.
Brown Seaweed
Species
Feed or
Food Food Group
Product
Produced/Product
Quality
Function of Product Reference
M. pyrifera Food
Dried seaweed
3–28%
Food recipe
ingredient
Breadsticks/9.5%DW of
protein Protein supplement
M. pyrifera Feed Macrocystis meal
food supplement
Macrocystis Meal
concentration of
(33.3 g/kg) based on
shrimp weight, was fed
(1.6 g over 28
days)/Protein efficiency
ratio of 1.7 purified
Dietary supplement for
juvenile white shrimp
(Litopenaeus vannamei)
[67]
M. pyrifera Feed Complementary
meal
Meal dietary supplement
with 8.5% crude
protein/85% Digestibility,
Dietary supplement for male
bovine zebu bulls [22]
M. pyrifera Feed
Food supplement
in the form of
derived flour
3% DW of daily diet/low
digestibility for salmonids
fish
3% DW of daily diet as dietary
supplement for minerals and
carbohydrates needed by
salmonids fish species
[23]
M. pyrifera Food Carbohydrates
Phycocolloids/
medium or high viscosity
alginate
Thickening agent for food
products [25,61]
M. pyrifera and
Laminaria spp. Food Carbohydrates Phycocolloids/
Alginic acid powder
Ingredients for dietary
biscuits, to induce feeling of
fullness
[25]
Laminaria spp. Food Carbohydrates
Phycocolloids/
Alginate: soft to medium
strength gel
Thickening agent for food
products [25,61]
Macrocystis sp. Food Carbohydrates Phycocolloids/
Alginate
Emulsifying, gelling, stabiliser
yoghurts, ice creams [62]
M. pyrifera and
Laminaria spp. Feed Carbohydrates Phycocolloids/
crude alginate
Binding agent in salmon and
other fish feeds [25]
Laminaria spp. Feed
Carbohydrates.
Prebiotics:
Seaweed extract as
a dietary
supplement
Polysaccharides:
(Laminarin and Fucoidan)
1–4 g/kg/day
Supplement to daily diet
weanling pigs for 21 days:
alleviated the need to use of
high-lactose diets for
(>60 g/kg) for weanling pigs,
and alleviated common
problems occurring
post-weaning
[70]
Laminaria spp. Feed Carbohydrates.
Food Supplement
Polysaccharides:
Laminarin (0.001 kg),
Fucoidan (0.0008 kg), and
ash (0.0082 kg) = Total
supplement weight 0.01
kg/day
Enhanced piglet immune
function and colonic
microflora at weaning
[73]

Molecules 2021,26, 1306 10 of 41
Table 1. Cont.
Brown Seaweed
Species
Feed or
Food Food Group
Product
Produced/Product
Quality
Function of Product Reference
L. digitata Feed
Carbohydrates.
Seaweed extract:
dietary
supplement:
spray-dried (SD)
and wet forms
(WS)
Polysaccharides:
Laminarin: 0.5 g/kg feed;
Fucoidan: 0.42 g/kg
Supplement to basal diet
(Complete daily basal diet
was SD = 1.9 kg/day; WS
= 1.8 kg/day)
Reduction in lipid oxidation
in the muscle tissue in 75% of
pigs consequently improved
the quality of pork steaks
[76]
L. digitata Food
Carbohydrates.
Seaweed extract:
dietary
supplement:
spray-dried (SD)
and wet forms
(WS)
Laminarin (9.3%) and
fucoidan (7.8%), added to
mince pork patties
Reduced the appearance of
surface redness of fresh
patties, significantly
decreased lipid oxidation in
cooked patties
[77]
L. digitata Feed
Carbohydrates.
Dietary
supplement
seaweed extract
Polysaccharides:
Laminarin and fucoidan:
of 1.5 g/kg addition to the
basal diet
Reduced the enterobacteria,
bifidobacteria, and lactobacilli
populations in the caecum
and colon, while only
marginal effects on the
immune response was
observed in weaned pigs
[74]
L. digitata Feed
Carbohydrates.
Dietary
supplement
Structural
polysaccharides: Purified
β-glucans
of 0.25 g/kg addition to
the basal diet
Reduced the
Enterobacteriaceae population
and pro-inflammatory
markers in the colon in pigs
[75]
Other studies on pigs have supplemented sow diets with a 0.001 kg/day seaweed
extract consisting of laminarin (0.001 kg), fucoidan (0.0008 kg), and ash (0.0082 kg). This
seaweed extract was administered to ten sows for 107 days of gestation, followed by 24
days of neonatal piglet growth. The study found piglets had enhanced immune function
and colonic microflora at weaning [
73
]. Laminarin and fucoidan extract from L. digitata
was added to the basal diet of weaning pigs in a 1.5 g/kg dietary supplement. Similar
to the sow study, the bacterial microflora of the colon improved with a reduction in
enterobacteria, bifidobacteria and lactobacilli populations in the caecum and colon, yet
unlike the sow study, only marginal effects on the immune response were noted [
74
].
β
-glucans are complex polysaccharides extracted from L. digitata. They were added as a
pig dietary supplement at 250 mg/kg of body weight, which lead to a reduction in the
Enterobacteriaceae population and pro-inflammatory markers in the pig’s colon [
75
]. An
L. digitata-based extract with 500 mg/kg of laminarin and 420 mg/kg of fucoidan was used
to supplement pig diets for 21 days pre-slaughter. A 75% reduction in lipid oxidation in
muscle tissue, which improved pork steak quality, was observed [76] (Table 1).
2.2. Pharmaceutical Uses of Brown Seaweeds
Pharmaceuticals are well-defined molecules that are used for medical purposes to
cure, treat, or prevent disease [
78
]. The use of marine algae is noted in the Chinese “Materia
Medica” of Shen-nung 2700 B.C. Seaweeds were used in folk medicines for the treatment of
goitre, nephritic diseases, anthelmintic, catarrh, vermifuge, and skin diseases [
79
]. The use
of brown seaweeds to treat medical ailments were documented in the 1750s by a physician
who used ash from kelp fronds, which is rich in iodine, to treat goitre [80].

Molecules 2021,26, 1306 11 of 41
Phycocolloids are used in wound dressings as a gelling agent to assist in blood
coagulation; M. pyrifera and L. digitata provide sodium and calcium alginates with a range
of viscosities from low to high which enable wound dressings to stop bleeding by assisting
blood clotting [
25
,
81
]. Alginic acid powder has been used for relieving acid indigestion
and to treat gastroesophageal reflux (GERD) disease [
25
,
61
]; calcium alginate beads were
used to control the release of medicinal drugs and other chemicals and are sourced from
M. pyrifera and L. digitata [
25
,
82
]. Sodium alginate from L. digitata is used to produce a soft
and elastic gel, which may be used in microparticles to aid drug delivery [
83
] (Table 2). The
current commercial pharmaceutical products from M. pyrifera and Laminaria spp. are listed
in Table 2.
Fucoidans are fucose-rich sulphated polysaccharides found in the extracellular matrix
(ECM) of brown seaweeds. Purported fucoidan properties include anti-inflammatory,
antioxidant, antibacterial, lipid inhibition, and immunological activities [
84
–
87
]. L. digi-
tata was one of the brown species where extracts from it exhibited strong antithrombotic,
anti-inflammatory activities, and a decrease in tumour proliferation, with results of an 80%
(P-value, 0.01) reduction in tumour cell adhesion to human platelets under static condi-
tions [85]. Another laminaria species, Saccharina japonica, was found to cause a significant
reduction in thrombus lysis time when oral administration of fucoidan (molecular weight
300 kDa) at a dosage of 400 mg per day for 5 weeks was carried out [
88
]. Fucoidan extracted
from M. pyrifera has also been reported to be a powerful immune modulator, enabling
delays in apoptosis and promotion of pro-inflammatory cytokine production in human
neutrophils at low concentration (2
×
10
5
), as well as activation of dendritic cells (DCs)
and T-cells [89] (Table 2).
Fucoidan is also used to alleviate metabolic syndrome, and benefits angiogenesis and
bone health [
60
]. Metabolic syndrome (MetS) commonly refers to the pathological state
in which proteins, fats, carbohydrates, and other substances in the body are metabolically
disordered. These disorders are the pathological basis of cardiovascular and cerebrovas-
cular disease and diabetes. To treat MetS, a multi-drug treatment approach is required
yet individual risk factors remain unmanageable [
90
]. Natural products including marine
polysaccharides have been claimed to reduce MetS. Fucoidan can alleviate MetS-related
disorders, including obesity, hyperlipidaemia, hyperglycaemia, and hypertension. Fu-
coidan has been intensively investigated as a potential hypoglycaemic agent to assist in
type 2 diabetes treatment; specifically, fucoidan extracted from the brown seaweed Fucus
vesiculosus can be used in the treatment of type 2 diabetes [
91
]. Low molecular weight
fucoidan (LMWF) has been reported to possess bioactive compounds which can protect
vascular endothelial function and reduce the basal blood pressure in diabetic rats. There-
fore, fucoidan is a possible candidate drug for protection of the endothelium in diabetic
cardiovascular complications [
92
]. In protection of the gastrointestinal tract, fucoidan’s
pharmacological activity was discovered in the treatment of inflammatory bowel disease
(IBD). Fucoidan was able to reduce crypt destruction and mucosal damage in the colon of
dextran sodium sulphate-treated mice to treat chronic colitis [
93
]. Fucoidan from seaweed
Cladosiphon okamuranus improved chronic colitis by downregulating the expression of the
proinflammatory cytokine IL-6 in the colonic epithelial cells of IBD mice [94].
Fucoidan inhibits angiogenesis through control of the expression of vascular endothe-
lial growth factor (VEGF) and endothelial cell plasminogen activator inhibitor-1. The
brown seaweed Sargassum fusiforme produces a fucoidan that can inhibit the angiogenesis
of human microvascular endothelial cells in a dose-reliant manner [
95
]. Regarding the
improvement of bone health, fucoidan is thought to reduce blood vessel development
in bone tumours such as osteosarcomas [
96
]. Low molecular weight fucoidan (LMWF),
extracted from fresh Sargassum hemiphyllum, increased bone density and ash weight in C57
BL/6J female mice. These findings allow conclusions to be drawn regarding fucoidan as a
promising osteogenic drug [97].
A crude polysaccharide-rich seaweed extract from L. digitata was tested for its effect
on metabolic activity of human gut microbiota. Results observed improved gut microbiota

Molecules 2021,26, 1306 12 of 41
composition and increased short-chain fatty acids [
98
]. An M. pyrifera extract-derived
product range was proposed on a US patent in 2012; the products included a high purity
fucoidan 75–90% kelp oil and/or kelp concentrate, and one product had 50% krill oil added.
These products were proposed to provide total antioxidant protection as pharmaceutical
products [
99
]. Several L. digitata pharmaceutical products, also based on seaweed extract,
which act as moisturising agents for the skin with protecting, soothing, and smoothing
properties and another product, which can be used as a homeopathic remedy, are produced
by two companies: Actipone®® and Boiron [100,101] (Table 2).
Secondary metabolites are primarily excretory products made under different stress
situations, such as exposure to ultraviolet (UV) radiation, variations in temperature and
salinity, or environmental contaminants. The main secondary metabolites manufactured
in algae tissues are phenolic compounds, halogenated compounds, sterols, terpenes, and
small peptides, as well as other bioactive compounds [
53
,
102
–
104
]. At present, more
than one hundred metabolites have been identified within forty-nine species of brown
seaweed [105].
Phlorotannins are a large and varied group of naturally occurring polyphenolic com-
pounds and are also secondary metabolites found in brown seaweeds [
106
]. Phlorotannins
are tannin derivatives made of several phloroglucinol units connected to each other in
distinct ways [
103
,
107
]. Phlorotannins, display effective antioxidant activity through their
ability to scavenge reactive oxygen species [
108
]. Furthermore, due to the inhibitory effect
of hyaluronidase (HAase) activation, phlorotannins exhibit antiallergic, bleaching, anti-
wrinkle, and skin antiaging actions [
105
]. Phlorotannins are components of cell walls and
are also found in vesicles in the cytoplasm (physodes). These compounds can make up
to 20% of the dry weight of seaweeds [
109
]. Fucus spp., Sargassum spp., and Ascophyllum
nodosum are three brown seaweeds with the highest phenolic compounds which range
from 12.2 to 14% DW; when compared, L. digitata’s range was considerably lower, between
~0.2 and 5.3% DW of phenolic compounds [
53
,
106
]. Fucus spiralis was found to have higher
molecular weight phlorotannins, which usually exhibit the strongest lipid peroxidation
inhibitory activity, when compared to three other brown seaweeds: Gongolaria nodicaulis
(formerly Cystoseira nodicaulis), Ericaria selaginoides (formerly Cystoseira tamariscifolia), and
Gongolaria usneoides (formerly Cystoseira usneoides [
110
]. Recent work on L. digitata found
that its phlorotannin content was ~4.5% of dry matter [
111
]. Phlorotannins from Saccharina
japonica were found to be effective in proliferation control of human tumour cells [
112
].
Laminaria hyperborea phlorotannins exhibited efficacy at wound sealing and reconstruction
during wound healing. Additionally, the application of photosynthetically active radiation
(PAR), and PAR + UV radiation, induces a high-stress response and resulted in an increase
in physodes in the epidermal cells of the seaweed frond, and a resulting photoprotective
response [
113
]. M. pyrifera has also been found to contain two phlorotannins, phloroeckol
and tetrameric phloroglucinol, both demonstrating antidiabetic and antioxidant activity as
well as preventing skin aging [114] (Table 2).
Table 2. Current commercial pharmaceutical products from Macrocystis pyrifera and Laminaria spp.
Seaweed Types and
Species Compound of Interest Product Produced Function of Product Reference
M. pyrifera
Phycocolloids/
medium or high
viscosity alginate
Sodium and calcium
alginate Wound dressings [25,81]
M. pyrifera Phycocolloids/
Alginate Alginic acid powder
Aid in relieving acid indigestion;
treatment of gastroesophageal
reflux (GERD) disease
[25,61]

Molecules 2021,26, 1306 13 of 41
Table 2. Cont.
Seaweed Types and
Species Compound of Interest Product Produced Function of Product Reference
M. pyrifera Phycocolloids/
Alginate Calcium alginate bead Controlled release of medicinal
drugs and other chemicals [25,82]
Laminaria spp.
Phycocolloids/
Alginate: soft to
medium strength gel
Sodium and calcium
alginate fibres Wound dressings [25,81]
Laminaria spp. Phycocolloids/
Alginate Alginic acid powder
Aid in relieving acid indigestion;
treatment of gastroesophageal
reflux (GERD) disease
[25,61]
Laminaria spp. Phycocolloids/
Alginate Calcium alginate bead Controlled release of medicinal
drugs and other chemicals [25,82]
L. digitata Phycocolloids/
Alginate Sodium alginate Soft and elastic gels; potential
drug delivery via microparticles [83]
M. pyrifera Sulphated
polysaccharides Fucoidan
Immune modulator, causing
delays in apoptosis and
promoting pro-inflammatory
cytokine production
[89]
Saccharina japonica Sulphated
polysaccharides Fucoidan Significant reduction in
thrombus lysis time [88]
Brown seaweeds
(including L. digitata,
Saccharina japonica;
M. pyrifera)
Sulphated
polysaccharides Fucoidan
Anti-inflammatory, antioxidant,
antibacterial, and immunological
activity; lipid inhibition; obesity
prevention or treatment
[84–87]
Saccharina japonica Secondary metabolite Phlorotannin Anti-proliferation of human
tumour cells [112]
L. hyperborea Secondary metabolite Phlorotannin
Wound sealing and
reconstruction during wound
healing
[113]
M. pyrifera Secondary metabolite
Phlorotannin:
phloroeckol, tetrameric
phloroglucinol
Antidiabetic; antioxidant
activity; prevention of skin aging
[114]
L. digitata
crude
polysaccharide-rich
seaweed extract
Crude extract and
depolymerised extract
Improved gut microbiota
composition; increase in
short-chain fatty acids
[98]
Fucus vesiculosus,
M. pyrifera; Saccharina
japonica,
Seaweed extract:
Maritech ®® extract
Fucus vesiculosus 85%,
w/w;M. pyrifera 10%,
w/w; Saccharina
japonica, 5%, w/w; zinc
vitamin B6 and
manganese)
Dose-dependent decrease
in osteoarthritis in 5 females and
7 males
[115,116]
M. pyrifera Seaweed extract
High purity Fucoidan
75–90% purity; Kelp
Oil and/or Kelp
Concentrate: Krill oil
Total antioxidant protection [99]
L. digitata
Seaweed Extract:
L. digitata thallus
prepared in glycerine
and water.
Actipone®®
A moisturising agent and
stimulant, skin protecting,
soothing, and smoothing
properties.
[100]
L. digitata Seaweed Extract:
1 DH Boiron Homeopathic medicine [101]

Molecules 2021,26, 1306 14 of 41
2.3. Other Uses of Brown Seaweeds
M. pyrifera and several Laminaria spp. have been used for other purposes beyond the
food, feed, and pharmaceutical industries. The renewable energy and fuel industry have
investigated the ability to use the polysaccharides laminarian and mannitol to make ethanol
from L. hyperborea [
117
]. Both M. pyrifera and Saccharina latissima also known as Laminaria
saccharina Linnaeus Lamour, have been investigated for methane production mostly via
anaerobic fermentation. The mannitol and alginate content of the kelp was key to methane
production; the higher the mannitol content, the better gas yield produced [
25
]. Saccharina
latissima was also converted to methane using anaerobic fermentation, with methane yield
doubling from autumn to spring. This study found laminarian and mannitol levels were
reduced to 5% yet alginate was only reduced to 30% by the anaerobic fermentation methane
yield and was dependent on the total carbohydrate content of the raw seaweed [
118
] (Table
3). Other current uses of Macrocystis sp. and Laminaria sp. are listed in Table 3.
Table 3. Other current uses of brown seaweeds Macrocystis sp. and Laminaria spp.
Seaweed Types and
Species Compound of interest Product Produced Function of Product Reference
M. pyrifera, Mannitol and alginate Methane Fuel [25]
Saccharina latissima Laminarian and
mannitol, alginate Methane: Natural Gas Fuel [118]
L. hyperborea Laminarian and
mannitol Ethanol Fuel [117]
Macrocystis sp. and
Laminaria sp. Dried seaweed Dried seaweed Removal of copper, zinc, and
cadmium ions from solution [119]; [120]
L. digitata Alginate Beads covered in
calcium alginate
Removal of heavy metals,
cadmium, and copper from
single and binary solutions
[121]
Macrocystis sp. and
Laminaria sp. Alginates Alginates
Cosmetic uses: as gelling
colloids, emulsion stabilisers,
immunostimulating agents,
moisturising, protective
phycocolloids
[82,122]
Laminaria sp. Laminarians Laminarians
Cosmetic uses: antioxidant,
anticellulite, and
anti-inflammatory agents
[104]
M. pyrifera Seaweed extract High purity Fucoidan
75–90% purity
Antioxidant and health and
wellness benefits (for potential
use in cosmetics and
nutraceuticals)
[99]
Removal of toxic metal ions has been successfully achieved using both Macrocystis sp.
and Laminaria sp. as dried seaweed to trap metal ions in solution [
119
,
120
]. Additionally,
L. digitata contains calcium alginate that was shown to effectively remove copper and
cadmium from single and binary solutions [121] (Table 3).
In the cosmetics industry, alginates from M. pyrifera and Laminaria sp. have been used
for their gelling, emulsion stabilising properties, and more recently, their immunostimulat-
ing properties [
82
,
122
]. Specifically, laminarian from Laminaria sp. is used in cosmetics for
its ability to act as an antioxidant, anticellulite, and anti-inflammatory agent [
104
]. Nutri-
cosmetics are defined as products and/or ingredients which are nutritional supplements
for the care of the skin, nails, and hair. They function by working within the body to
promote beauty from within [
123
]. A high purity fucoidan rich extract from M. pyrifera was
proposed in a patent in 2012 by a company called KNOCEAN Sciences, Inc. It is claimed

Molecules 2021,26, 1306 15 of 41
to be an antioxidant booster and health and wellness additive to be added to cosmetics,
functional foods, and pharmaceuticals [99] (Table 3).
2.4. Functional Foods Applications
2.4.1. Protein Content and Applications
A nutritional study in Chile catalogued the amino acid content of three edible Chilean
seaweeds: Codium fragile (Chlorophyta), Agarophyton chilense (formerly Gracilaria chilensis)
(Rhodophyta), and M. pyrifera, which contained proteins at 13.7–10.8% and amino acid
contents at 1879.6–1417.7 mg/100 g dry algae. Specifically, M. pyrifera had 13.2
±
0.30%DW
protein [
66
]. Protein sourced from seaweed contains all essential amino acids, and specifi-
cally glycine, alanine, arginine, proline, glutamic, and aspartic acids. In seaweeds, their
essential amino acids account for almost half of the total amino acids and their protein
profile is similar to that of egg protein [
124
,
125
]. Laminaria sp. contains 13 g of aspartic
acid/16 g N and 24 g glutamic acid/16 g N, and when compared to other brown seaweed
species Undaria pinnatifida and Sargassum fusiforme (formerly Hizikia fusiforme), Laminaria
sp. had significantly higher aspartic acid content [
125
]. Seaweed as an alternative protein
source to animal protein has been postulated [
126
]. In the context of functional foods, the
addition of seaweed supplements to improve the nutritional value of a food to deliver
essential amino acids appears to have potential. Regarding regulations, in the US, the Food
and Drug Administration (FDA) uses the designation GRAS (generally recognised as safe),
and both M. pyrifera and L. digitata are listed as GRAS for human consumption as flavour
enhancers and flavour adjuvants, with concentrations in food not exceeding current good
manufacturing practice (GMP) [
127
]. Lectins and phycobiliproteins are two protein families
which have notable bioactive properties and have been detected in seaweeds [
128
,
129
];
however, brown seaweeds do not contain either lectins or phycobiliproteins.
2.4.2. Carbohydrate Content and Applications
The carbohydrate content of M. pyrifera was reported as 75.3
±
0.2% DW previ-
ously [
66
]. When compared to the daily consumption of fruit and vegetables, this resembles
the carbohydrate contribution of dried fruit [130].
2.4.3. Tocols Applications
Tocols are liposoluble metabolites which include
α
-,
β
-,
γ
-, and
δ
-tocopherol and
their isomers
α
-,
β
-,
γ
-, and
δ
-tocotrienols. They are made by plant cells with antioxidant
action, and in the human body,
α
-tocopherol acts as vitamin E [
66
]. Alpha tocopherol
was tested at a dose of (50 mg/day) as a dietary supplement and showed potential to
prevent prostate and colorectum cancer. A 34% reduction in prostate cancer and 16%
reduction in colorectum cancer was observed, yet was not effective at treating stomach
cancer where a 25% increase in cancer occurred [
131
]. Recent work comparing three edible
seaweeds in Chile found the total tocols content for Codium fragile, Agarophyton chilense,
and M. pyrifera ranged between 391.9 and 1617.6
µ
g/g lipids, with the highest value for
Codium fragile, the lowest for Agarophyton chilense, and M. pyrifera reaching 1457
µ
g/g
lipids. The most abundant fatty acid found in M. pyrifera was
α
-tocopherol at 1327.7
±
4.4
µ
g/g lipid [
66
]. The lipid fraction of M. pyrifera can be considered to have a high tocol
content when compared to arachis oil, grapeseed oil, palm oil, and sunflower seed oil.
Interestingly, M. pyrifera contains 1327.7
±
4.4
µ
g/g of
α
-tocopherol, compared to seed
oils such as arachis oil which contains between 49 and 373
µ
g/g
α
-tocopherol, grapeseed
oil 16–38
µ
g/g
α
-tocopherol, palm oil 4–193
µ
g/g
α
-tocopherol, soyabean 9–352
µ
g/g
α
-tocopherol, and sunflower seed oil 403–935
µ
g/g
α
-tocopherol, which is the closest
α
-tocopherol content to M. pyrifera [
65
,
66
]. M. pyrifera had only 0.7
±
0.3% DW lipid
content, yet the value of vitamin E is important, as it provides stability to the PUFA present
in this seaweed, preventing the development of free radicals and therefore, converting
this seaweed into a potential complimentary food in light of its important contribution of
vitamin E and PUFA [66].

Molecules 2021,26, 1306 16 of 41
2.4.4. Pigment Applications
Pigments including
β
-carotene have been recommended as dietary supplements to
prevent cardiovascular disease (CVD) and cancer. One study recommended
≥
0.4
µ
mol/l
β
-carotene and
≥
0.5
µ
mol/l
α
+
β
-carotene for primary prevention of CVD and cancer [
132
].
Conversely, another study on
β
-carotene tested the effectiveness of supplementation at 20
mg/d on cancer prevention in 29,133 male smokers between the ages of 50 and 69 for 5–8
years. Results found an increase in lung (18%), prostate (23%), and stomach (25%) cancers.
This study concluded that
β
-carotene supplements at high doses should be avoided by
smokers, who also had high alcohol consumption, as it may increase the incidence of lung
cancer, yet additional advice for smokers was the most effective mode of action which was
to stop smoking to avoid lung cancer [
131
,
133
]. The potential causal mechanism between
β
-
carotene supplements and alcohol consumption to increase lung cancer risk have observed
ethanol-related changes in carotenoid metabolism, and hepatocellular toxicity in response
to
β
-carotene supplements when consumed with extremely high alcohol intake (i.e., 50% of
calories as ethanol), yet further work is needed as to how these responses would enhance
the risk of lung cancer [
134
,
135
]. These studies highlight the use of
β
-carotene as a dietary
supplement primarily for prevention of disease; the contributary factors such as smoking
20 cigarettes a day and drinking 11 g of alcohol a day increase the incidence of developing
lung disease, potentially outweighing the preventative impact of a dietary supplement
such as β-carotene [131,133].
The principal carotenoid in M. pyrifera is
β
-carotene. When compared to other edible
seaweed species in the same study,
β
-carotene was lowest in M. pyrifera at 17.4 mg/g dry
algae, highest in Codium fragile at 197.9 mg/g dry weight, with Agarophyton chilense in the
middle at 113.7 mg/g beta carotene in dry algae [
66
]. The
α
-tocopherol and
β
-carotenoid
contents in M. pyrifera are beneficial because they can perform as both vitamins and
antioxidants [
66
]. The literature recommends a 15 mg/day dose of
β
-carotene to contribute
to a healthy diet, and so 75 g of Codium fragile would complement the daily recommended
requirements of
β
-carotene [
136
]. Regarding optimal times to harvest seaweed species,
β
-carotene and tocopherol isolated from Saccharina japonica were highest from July through
to September and less during the winter [137].
2.4.5. Phlorotannins Applications
M. pyrifera contains the secondary metabolites, phlorotannins, and specifically phloroeckol
and tetrameric phloroglucinol, which display both antidiabetic effect and antioxidant ac-
tivity and can contribute to the prevention of skin aging [
114
]. Phloroeckol has also been
reported to prevent Alzheimer’s disease, yet this has only been tested using phlorotannins
from the brown seaweed Ishige okamurae [138].
2.4.6. Polysaccharides—Soluble Fibre Phycocolloids
Dietary fibres are complex carbohydrates. They are mainly sourced in vegetables,
fruits, grains, nuts, and root crops and are a vital part of a healthy diet. Since dietary fibre
is not digested by digestive enzymes, it does not provide direct nutrition in the human
body. Yet, dietary fibre indirectly helps human nutrition by involving it in some important
functions to promote digestive health during its passage through the gastrointestinal track.
These functions consist of reduction in incidences of colorectal cancers, suppression of
bowel inflammations and related abdominal disorders, facilitation of bowel movement,
and growth promotion of health-promoting gut microflora. Recommended average daily
intake of dietary fibre is between 25 and 30 g in the US and >18 g in the UK [
139
]. The
health benefits of seaweed-derived dietary fibre have been focussed mainly on components
in humans, and specifically on potential anti-obesity effects, such as enhanced repletion, de-
layed nutrient absorption, and delayed gastric emptying, yet the effects of whole seaweeds
containing alginate appear to be restricted [
140
–
142
]. In the US, dietary fibre is considered
a nutrient under the Nutrition and Education Act of 1990 [143].

Molecules 2021,26, 1306 17 of 41
Fucoidans are L-fucose-containing sulphated polysaccharides found in the cell walls
of brown algae. Structures of fucoidans vary as does their complexity amongst different
species. Generally, brown seaweed fucoidans consist of one of two types of homofucose
backbone chains, either the repeating
α
(1->3)-linked L-fucopyranose residues or an al-
ternation of
α
(1->3) and
α
(1->4)-linked L-fucopyranosyls, which, in either case, may be
substituted with sulphate or acetate and/or have side branches containing fucopyranoses
or other glycosyl units, e.g., glucuronic acid [
85
,
144
]. The first type of fucoidan backbone
was isolated from seaweeds Saccharina latissima, Chorda filum, Cladosiphon okamuranus, and
L. digitata [
145
]. Other fucoidans reported in the literature include small amounts of various
other monosaccharides, e.g., glucose, galactose, xylose, and/or mannose [144].
At present, fucoidan’s profile as a diet supplement from disease assistance is improv-
ing due to the extensive preclinical testing being undertaken. Its typical activities include
antitumour, antioxidant, anticoagulant, anti-inflammatory, antiviral, and immunoregu-
latory [
60
]. Fucoidan is also used to alleviate metabolic syndrome, for protection of the
gastrointestinal tract, and for benefiting angiogenesis and bone health [
60
]. In protec-
tion of the gastrointestinal tract, fucoidan’s pharmacological activity was discovered in
the treatment of inflammatory bowel disease (IBD). Fucoidan was able to reduce crypt
destruction and mucosal damage in the colon of dextran sodium sulphate-treated mice
to treat chronic colitis [
93
]. Fucoidan from seaweed Cladosiphon okamuranus improved
chronic colitis by downregulating the expression of the proinflammatory cytokine IL-6 in
the colonic epithelial cells of IBD mice [94].
Laminarin is another polysaccharide which contains soluble fibre in the form of
phycocolloids; brown seaweeds noted for their laminarin content are Saccharina japonica
and Saccharina latissima [
57
]. When comparing the fibre content of foods from terrestrial
plants, seaweed has similar or even higher levels of dietary fibre. The average total dietary
fibre content in seaweed can vary from 36 to 60% based on its dry matter [
146
]. Almost
55–70% of its total dietary fibre is represented by the soluble fibre fraction which primarily
contains agar, alginates, and carrageenan at varying amounts depending on the type of
seaweed and the seasonal growing conditions [
6
,
53
,
147
–
149
]. The brown seaweed genera,
Fucus and Laminaria, have the highest content of insoluble dietary fibre among the other
commercially harvested seaweed used in the food industry [
150
]. The typical daily portion
size of the seaweeds consumed in Asian cuisines on a dry matter basis is about 8 g [
57
].
Seaweed can provide 12–15% of daily dietary requirements of fibre in the human diet, with
brown seaweeds contributing the highest amount at 14.28%, reds providing the lowest at
10.64%, and greens slightly higher at 12.10% [
148
]. This is a large amount compared to that
of other food sources when compared on a weight-for-weight basis [57].
2.4.7. Polysaccharides—Prebiotic Potential
Brown seaweeds have been investigated for their bioactive properties. A bioactive
compound is a substance that has a biological activity [
151
] linked to its ability to regulate
one or more metabolic processes, which results in the promotion of improved health
conditions [152].
L. digitata is rich in polysaccharides that function well as prebiotics to improve human
gut biota populations [
98
]. Prebiotics are compounds in food that induce or support the
growth or activity of microorganisms such as bacteria and fungi deemed beneficial to a
host [
153
]. This is most often by consumption of the prebiotic by the microorganism as
carbon sources. Laminarins sourced from L. digitata have been used in several studies
to access their effectiveness as prebiotics. One study on rats used a treatment of 1 g of
laminarins from L. digitata. The results noted no changes to gut biota; however, an increase
occurred in the colon luminal mucin content, and also a decrease in luminal mucin in
the jejunum, ileum, and the caecum [
154
]. Another study which used laminarin isolated
from L. digitata found significant changes in gut biota with an increase in parabacteroides,
fibrobacter, and lachnospiracease and a decrease in streptococcus, ruminococcus, and

Molecules 2021,26, 1306 18 of 41
peptostreptococcaceae [
98
]. M. pyrifera does not contain Laminarin, which is only found in
the brown seaweed genera Laminaria and Fucus [155].
Fucoidans from brown seaweed have shown bioactivity including, e.g., anti-inflammatory,
antioxidant, antibacterial, and immunological activity; lipid inhibition; obesity prevention
or treatment [
84
–
86
]. Unlike laminarins, fucoidans from brown seaweeds have not shown
any prebiotic characteristics useful for improving gut biota communities.
2.4.8. Polyunsaturated Fatty Acids
The carbon-18 (C
18
) polyunsaturated fatty acids (PUFA) have been found in seaweeds
and plants and are important in human and fish nutrition as neither can synthesize them.
They are exceptional sources of n-3 fatty acids with 18 to 20 or more carbons, such as
eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) [
156
,
157
]. Yet, fish can
lengthen and desaturate dietary fatty acids (18:2n-6 and 18:3n-3) [
158
]. The health benefits
of n-3 fatty acids, also known as omega-3 fatty acids EPA and DHA, are numerous and
include proper foetal development and neuronal, retinal, and immune function [
159
,
160
].
Other potential uses of EPA and DHA are prevention of mild Alzheimer’s disease and
obesity [
159
,
161
–
165
]. Seasonal impacts on the PUFA content in Saccharina japonica have
found the PUFA, (n-6) family, was highest during warm months, while (n-3) PUFAs were
highly abundant during the colder months when seaweed thalli were very young; a de-
crease occurs progressively toward October when sori development was noted (Hafting,
2015) [
137
]. Fatty acid methyl esters (FAMES) analysis on M. pyrifera found that the highest
PUFA present was linoleic acid (18:2n-6) at 43.41%; it also detected the monounsaturated
fatty acids (MUFA) and found 18:1n-9c (oleic acid) was highest at 19.64% [
66
]. The advan-
tage of this seaweed is that it contains a suitable PUFA n-6/n-3 relation of high impact in
human and animal nutrition [66].
3. Current Extraction Strategies
3.1. Seaweed Cell Wall Structure
The cell wall structure of seaweeds requires considered approaches to break and remove
the complex polysaccharides without losing the vital proteins compounds of interest. Protein
content in brown seaweeds varies from 5 to 20% dry weight [
53
,
54
,
167
]. Brown algae have
evolved a cell wall (Figure 1) which shares elements with both plants and animals due to
its multicellular eukaryotic nature. The evolution of an extracellular matrix (ECM) enabled
development, established defence systems conferring innate immunity, and provided a
boundary for nonself recognition [
168
]. In eukaryotes such as seaweed, this ECM was
organised usually as a three-dimensional network of fibres embedded in fluid components.
The cell wall structure of brown seaweeds contains plant cellulose, yet these crystalline
fibres only account for 1–8% of the thallus (DW) [
169
]. The cell wall main components are
anionic polysaccharides, i.e., alginates and fucoidans [168,170–172]. Alginates consist of two
uronic acids,
β
-1,4-D-mannuronate and
α
-1,4-L-guluronate, arranged in blocks along the
polysaccharide chain (Figure 1a) [166].

Molecules 2021,26, 1306 19 of 41
Molecules 2021, 26, x FOR PEER REVIEW 18 of 41
Other potential uses of EPA and DHA are prevention of mild Alzheimer’s disease and
obesity [159,161–165]. Seasonal impacts on the PUFA content in Saccharina japonica have
found the PUFA, (n-6) family, was highest during warm months, while (n-3) PUFAs were
highly abundant during the colder months when seaweed thalli were very young; a de-
crease occurs progressively toward October when sori development was noted (Hafting,
2015) [137]. Fatty acid methyl esters (FAMES) analysis on M. pyrifera found that the high-
est PUFA present was linoleic acid (18:2n-6) at 43.41%; it also detected the monounsatu-
rated fatty acids (MUFA) and found 18:1n-9c (oleic acid) was highest at 19.64% [66]. The
advantage of this seaweed is that it contains a suitable PUFA n-6/n-3 relation of high im-
pact in human and animal nutrition [66].
3. Current Extraction Strategies
3.1. Seaweed Cell Wall Structure
Figure 1. The main polysaccharide structures of brown alga: (a) alginate; (b) sulphated fucan from
Fucales; (c) sulphated fucan from Ectocarpales; (d) proposed model of the biochemical organisa-
tion of brown alga cell wall structure. Reproduced with permission from [166].
The cell wall structure of seaweeds requires considered approaches to break and re-
move the complex polysaccharides without losing the vital proteins compounds of inter-
est. Protein content in brown seaweeds varies from 5 to 20% dry weight [53,54,167]. Brown
algae have evolved a cell wall (Figure 1) which shares elements with both plants and ani-
mals due to its multicellular eukaryotic nature. The evolution of an extracellular matrix
(ECM) enabled development, established defence systems conferring innate immunity,
and provided a boundary for nonself recognition [168]. In eukaryotes such as seaweed,
this ECM was organised usually as a three-dimensional network of fibres embedded in
fluid components. The cell wall structure of brown seaweeds contains plant cellulose, yet
these crystalline fibres only account for 1–8% of the thallus (DW) [169]. The cell wall main
components are anionic polysaccharides, i.e., alginates and fucoidans [168,170–172]. Algi-
nates consist of two uronic acids, β-1,4-D-mannuronate and α-1,4-L-guluronate, arranged
in blocks along the polysaccharide chain (Figure 1a) [166].
3.2. Functional Food Extraction
3.2.1. Protein Extraction
M. pyrifera was studied for its nutritional value in Chile with two other species—
Codium fragile and Agarophyton chilense. The study analysed M. pyrifera’s protein content
using a proximal analysis standard method for proteins (N 6.25, AOAC 954.01) [173]. Ad-
ditionally, the amino acids were analysed using a simple and fast HPLC method [174].
Figure 1.
The main polysaccharide structures of brown alga: (
a
) alginate; (
b
) sulphated fucan from
Fucales; (
c
) sulphated fucan from Ectocarpales; (
d
) proposed model of the biochemical organisation
of brown alga cell wall structure. Reproduced with permission from [166].
3.2. Functional Food Extraction
3.2.1. Protein Extraction
M. pyrifera was studied for its nutritional value in Chile with two other species—
Codium fragile and Agarophyton chilense. The study analysed M. pyrifera’s protein content
using a proximal analysis standard method for proteins (N 6.25, AOAC 954.01) [
173
].
Additionally, the amino acids were analysed using a simple and fast HPLC method [
174
].
The sample was dried and milled prior to extraction. The samples were then ground
further using a mortar and pestle. A sample equivalent to 2 mg of protein was weighed
into a hydrolysis tube, then 4 ml of 6.0 M hydrochloric acid. The internal standard used
was D,L-
α
-aminobutyric acid. The solution was gassed with nitrogen and sealed and then
incubated in an oven at 110
◦
C for 24 h. Once complete, the amino acid hydrolysate was
dried in a rota-evaporator and then, the resulting sample was dissolved in 25 mL borate
buffer (1 M, pH 9.0). Five millilitres of this sample were derivatised with 4
µ
L diethyl
ethoxymethylene malonate at 50
◦
C for 50 min and shook vigorously. Of this sample,
20
µ
L was subsampled and injected directly into the HPLC system [
174
]. Of these three
seaweeds tested, M. pyrifera had the second highest protein content at 13.2%. The amino
acid profiles of M. pyrifera recorded the lowest content of amino acids of all three species at
0.8–1827.3 mg 100
−1
g dry weight, with essential amino acids corresponding to 38.9% of
total protein content [66].
3.2.2. Phlorotannins and Polyphenol Extraction
To decipher the most effective phlorotannin extraction to perform on M. pyrifera, an
orthogonal design set of experiments was performed previously [
114
]. Parameters impor-
tant to optimal extraction conditions were investigated; these included pre-treatment use,
solvent type, drying temperature, particle size, solid/liquid ratio, temperature, and extrac-
tion time. Recommended conditions included pre-treatment with hexane, extraction using
water, 40
◦
C drying temperature, <1.4 mm particle size, extraction temperature of 55
◦
C for
4 h, and a solid–liquid ratio of 1:15 [
114
]. Using these parameters, phlorotannin content was
200.5
±
5.6 mg gallic acid equivalent (GAE)/100 g for dry seaweed and total antioxidant
activity was (TAA) 38.4
±
2.9 mg Trolox equivalent TE/100 g for dry seaweed. Using
HPLC-ESI-MS, two phlorotannins were detected: phloroeckol and tetrameric phloroglu-
cinol [
114
]. More recently, M. pyrifera was investigated for its phlorotannin properties in
another study. Extraction yields of carbohydrates and phlorotannins were 81.02
±
8.9%
and 1.62
±
0.13% w/w, respectively. The phlorotannin fraction activity was concluded to be
useful as a natural antioxidant and an antibacterial compound [175].
Four seaweed species, Fucus serratus, L. digitata (Ochrophyta, Phaeophyceae), Gracilaria
gracilis (Rhodophyta), and Codium fragile (Chlorophyta), were tested for antioxidant ac-

Molecules 2021,26, 1306 20 of 41
tivity and total phenol content (TPC) using solid–liquid extraction (SLE) and pressurised
liquid extraction (PLE). These extraction methods were evaluated using 2,2-diphenyl-
1-picrylhdrazyl (DPPH) and ferric reducing antioxidant power (FRAP) assays and the
Folin–Ciocalteu total phenol content (TPC) assay. Results indicated that Fucus serratus
had TPC and antioxidant activities thirty times higher than the other species. Only low
TPC levels were observed for L. digitata,G. gracilis, and C. fragile from both SLE and PLE
extracts, yet the SLE extracts did retain higher FRAP and DPPH activities than PLE extracts.
This study concluded that pressures and high temperatures in PLE did not improve the
antioxidant activities when compared to SLE extraction [176].
3.2.3. Carbohydrate Extraction
Commercially viable fucoidan is extracted from a range of brown seaweed species
including M. pyrifera and Laminaria sp. by a company in Tasmania, Marinova Pty Ltd.
Marinova have developed a patented process called Maritech
TM
, which uses a coldwater
extraction process that is species-specific, enabling up to 95% purity levels of fucoidan
to be attained. The Marinova product is reported to retain optimal bioactivity due to
the nature-like high molecular weight molecules, which can be used as components for
cosmetic or functional food applications [145].
One method used to extract polysaccharides from L. digitata begins with a hot acid
extraction applied to freeze-dried frozen samples. This process previously used powdered
ground seaweed and suspended it in 0.1 M HCl at a ratio of 1:10 (w/v). The sample was
incubated at 70
◦
C in an orbital shaker at 175 rpm for three successive time periods of
3, 3, and 24 h. Extracts were filtered using a muslin bag with the extract removed after
each period and fresh solvent added to the retentate. All three extract batches were pooled
and underwent further filtering with cotton and glass wool using a funnel, Buchner flask,
and vacuum pump. The final filtrates were neutralised with the addition of 20 M NaOH
and freeze-dried. An ethanol precipitation was undertaken to produce a crude protein
extract (CE). This was compared with a depolymerised extract (DE), which had been put
through a further purification using a Fenton reaction [
98
]. Dietary soluble and insoluble
fractions were also measured. Results found both extracts produced microbiota-associated
metabolic and compositional changes, indicating putative beneficial health benefits of
L. digitata
in vitro
, yet further work is needed to clarify if fibre can positively alter the gut
microbiota and cause health benefits in vivo [98].
Another study focused on extracting polysaccharides, laminarin, and fucoidan from
Saccharina japonica. Their aim was to produce a simple quick and reliable method using
high-performance size exclusion chromatography (HPSEC). The initial extraction was
similar to the aforementioned L. digitata method that used a ground sample, which was re-
suspended in 30.0 mL solvent in a flask. The suspension was heated and stirred constantly,
then after cooling to room temperature, the suspension was centrifuged. An ethanol
precipitation with a 3:1 ethanol to filtrate ratio was applied, sediments were washed
twice using acetone then ether sequentially. The depurated sediments were dried at 60
◦
C and polysaccharides were obtained for further analysis. The polysaccharides were
re-dissolved in the solvent. The extraction solution was then filtered (0.45 mm) prior to
injection into the HPLC system. Each sample was injected, in triplicate, to assess the
precision and accuracy of the analysis. Results produced 169.2 mg g
−1
of fucoidan and
383.8 mg g
−1
of laminarin [
177
]. Sodium alginate was the compound of interest when
extracted from a Moroccan strain of L. digitata. Different conditions including temperature
and sample size were used during the extractions. The alginates were purified by re-
precipitation with ethanol and characterised by
1
H-NMR, fluorescence spectroscopy, and
infrared spectroscopy. The highest pure alginate yield 51.8% was reached using a < 1mm
sample size and a temperature of 40 ◦C [83].

Molecules 2021,26, 1306 21 of 41
3.3. Pharmaceutical Extraction
3.3.1. Protein Extraction
Proteins contain amino acids, essential amino acids, and peptides. Seaweed proteins
account for between 5 and 20% [
53
]. Bioactive peptides activity has been linked to a
range of health benefits including antimicrobial activity and blood pressure-lowering
including angiotensin converting enzyme-1 (ACE-I) and renin inhibitory bioactivities,
anti-atherosclerotic, antioxidant, antithrombotic, and immune-modulatory activities [
178
].
Ultrasound-assisted solvent extraction and using ultrasound as a pre-treatment before an
acid or alkaline treatment have improved recovery of seaweed proteins [
179
]. Extracting
bioactive peptides from these proteins requires hydrolysis to release functional peptide
fragments with their specific bioactivities intact. Additionally, enzymatic hydrolysis is
favoured by the pharmaceutical and functional food industry as it avoids severe chemical
and physical treatments and retains both functional and nutritional properties [
180
,
181
]. To
extract ACE and antioxidant peptides, proteolytic enzymes are used; flavourzyme, corolase
PP, and other enzymes were reportedly used previously for hydrolysis, which resulted in
the release of encrypted peptides which range in size from 2 to 20 amino acids. Further
concentration and fractionation into their specific molecular weight cut off have achieved
through a ultrafiltration membrane and gel permeation chromatography, yet ion exchange,
affinity chromatography, and high performance liquid chromatography (HPLC) are the
most vital chromatographic approaches that are employed [180–182].
Bioproteins and peptides have been extracted and characterised using the aforemen-
tioned procedures from the seaweeds Ulva lactuca (Chlorophyta), Solieria chordalis, Pal-
maria palmata (Rhodophyta), and Saccharina longicruris (Ochrophyta, Phaeophyceae) [
183
].
Enzyme-assisted extraction using cellulase optimised protein extract from M. pyrifera,
which demonstrated antioxidant activity and potential antihypertensive activity [
184
]. To
date, studies of bioactive activity from L. digitata have focussed on its carbohydrates, with
no studies on protein or peptide bioactivity for pharmaceutical applications.
3.3.2. Phlorotannins and Polyphenol Extraction
A study conducted on phlorotannins from M. pyrifera used macroporous resins as a
purification method, with the potential application to expand the use of phlorotannins as a
bioactive substance in the food, functional food, and pharmaceutical sectors. To prepare
the M. pyrifera phlorotannins extract for purification, M. pyrifera was dried at 40
◦
C and
ground to <0.5 mm. The dried sample was extracted using a solution 0.5 M of NaOH,
solid/liquid ratio of 1/20 at 100
◦
C for 3 h. The mixer was filtered using Whatmann N
0
1
paper and the liquid phase was stored at 4
◦
C. The phlorotannin concentration present
in the extract was 1800 mg of phloroglucinol equivalent (PGE)/L [
185
]. The six resins
tested were Diaion HP-20, Sepabeads SP850, Amberlite XAD-7, XAD-16N, XAD-4, and
XAD-2 (from Sigma-Aldrich). Prior to use, all resins were washed with 70% ethanol at 25
◦
C for 12 h. To test the adsorption of phlorotannins, a static adsorption was performed,
using 2 g of each resin placed in a tube with 30 mL of phlorotannin extract. The tube was
shaken in a shaking incubator, at 300 rpm, at 25
◦
C to reach adsorption equilibrium. After
adsorption, the resins were filtered for the subsequent desorption of phlorotannins, and
the concentration of phlorotannins in the extract was measured [
186
]. The phlorotannin
concentration in the extracts was determined according to the Folin–Ciocalteu (FC) assay,
and then adapted to a 96-well plate, with phloroglucinol concentrations ranging from 20
to 100 mg/L. The plate was then loaded with samples and standards (20 mL) separately,
each well containing 100 mL of Folin–Ciocalteu’s reagent diluted with water (10 times)
and 80 mL of sodium carbonate (7.5% w/v). The plate was then mixed and incubated at
45
◦
C for 15 min. Absorbance was measured at 765 nm on a UV–visible spectrophotometer.
The phlorotannin concentration was then determined by the regression equation of the
calibration curve and expressed using mg of phloroglucinol equivalent (mg PGE/L).
Phloroglucinol quantification was performed using a reverse phase C18 column (150
×
4.6 mm, 5 mm) and an HPLC system with an ultraviolet (UV) detector according to the

Molecules 2021,26, 1306 22 of 41
method by [
187
]. Adsorption time for the resins on average was 9 h, the highest level of
purification for phlorotannins was 42% with XAD-16N resin, with an adsorption capacity
of 183
±
18 mg PGE/g resin, and a desorption ratio of 38.2
±
7.7%. The best temperature
was 25
◦
C according to the adsorption isotherm; the Freundlich model best described the
adsorption properties [186].
A study of antibacterial activity of phlorotannins from two brown seaweeds Ascophyl-
lum nodosum and Fucus serratus used two methods to detect phlorotannin concentration, the
aforementioned FC assay with detection using UV
−
vis spectroscopy, and second method
1
H-NMR and
13
C-NMR spectroscopy. The
1
H-NMR and
13
C-NMR spectroscopy method is
both a qualitative and quantitative method which detects TPC and the linkages between
phlorotannins present in the extract, which is then purified by solid phase extraction (SPE).
Phenolic content was measured by quantitative NMR (qNMR) using milligram of phenolics
per gram of seaweed (mg/g); however, the FC assay uses phloroglucinol equivalents per
gram of seaweed (PGE/g). In this study, the concentration of phenolics in Ascophyllum
nodosum was significantly higher than Fucus serratus at 37.35 and 17.00 mg/g, respectively,
based on the
1
H-NMR analysis. Conversely, the FC assay noted the opposite trend, with the
phenolic content for Ascophyllum nodosum being 30.68 (
±
0.55) PGE/g, and Fucus serratus
being higher in this analysis at 36.68 (
±
1.33) PGE/g.
13
C-NMR spectra of the phenolic
extract prepared using SPE from each species were compared to determine the difference
in linkages of the phenolics between these seaweed species [188].
L. digitata has been investigated for its phlorotannin content [
111
]. The characterisation
method used
13
C and
1
H-NMR spectroscopy for linkage characterisation and to determine
extract purity. Phlorotannin fractions were obtained using NP-flash chromatography,
followed by ESI-MS and MALDI-TOF-MS to decipher structural and molecular weight as
well as identifying the fucol-to-phlorethol linkage ratio [111].
3.3.3. Carbohydrate Extraction
M. pyrifera, although a member of the kelp family, Laminariaceae, it is typically not one
of the kelp species investigated for pharmaceutical product content. Most studies in the
literature focus on species from the Laminaria genus, such as Saccharina japonica, or L. digitata.
A study analysed laminarian from L. digitata fucoidan, sodium alginate from M. pyrifera,
and fucoidan from Fucus vesiculosus for biological activity, i.e., antitumour, cytotoxicity,
and humoral immune response. Except fucoidan from M. pyrifera, all compounds were
sourced already extracted from chemical companies, Kelco and Sigma. The fucoidan from
M. pyrifera needed to be extracted. The extraction method for fucoidan used a dried milled
sample from Argentina using a 30% ethanol extraction then split the sample into a dialysed
and non-dialysed sample, then using the un-dialysed sample, split it into precipitate and
supernatant. All extracts were tested in vivo in mice and in vitro for efficacy [189].
A patent filed in 2012 by the company Knocean Sciences, based in Texas, claimed
it could provide total antioxidant protection using fucoidan extract from M. pyrifera at
a commercial scale. Several products were proposed, and the extraction method used
fresh kelp which was chopped and then milled before being pumped to a separation tank
for 12–24 h, which enables gravity separation to liberate the kelp oil from the chopped
milled kelp. The kelp oil is then drained and pumped to a holding tank, and the solid
content at this stage is 4–7%. This kelp oil is then pumped to a thin film evaporator, then a
wiped film evaporator, to pre-concentrate the solids in the kelp oil to 25–35% increasing
viscosity. This was followed by a spray drying step, with an inlet temperature of 180
◦
C and
an outlet temperature of 90
◦
C; this was optimised to the inlet/outlet feed temperatures,
which can be increased to 225/105
◦
C with only a minimal loss of functionality and ORAC
value (approximate 10% decrease) of the final product—a kelp concentrate powder. For
the other products to be produced, the chopped and milled fresh kelp that is left over
from the kelp oil separation process is then dried using a range of procedures including
geothermal drying, wind drying, solar drying, and mechanical drying. This drying step
continues until the product is 85–90% solids, usually 2–6 days, dependent on weather

Molecules 2021,26, 1306 23 of 41
conditions. The dried product is placed in a hopper and fed into a milling system to
produce a finely milled product which is tested for fucoidan content at this stage. This is
the final product which is milled to the appropriate mesh size needed for the specific use;
high purity fucoidan requires a mesh “size of 40”. The uses of this product range include
as a food, dietary supplement, skin cosmetic, and pharmaceutical treatments for viruses
or oxidation control [
99
]. Several L. digitata extract-based pharmaceutical products have
been manufactured by two companies, Actipone
®
and Boiron, yet extraction details were
unavailable [100,101].
The goal of the exploration of seaweed polysaccharides with potential bioactive
compounds is to prepare a crude seaweed extract for precise detection, identification, and
quantification of target analytes applying chromatographic and spectrometric methods.
This process includes efficient extraction, elimination of seaweed matrices, concentration,
or dilution of the extracts. Quantitative chemical analysis of key groups of compounds in
seaweeds are performed using chromatographic techniques, such as LC and GC coupled
to MS and NMR, with TLC used for sample screening. When sample preparation and
chromatography are appropriately selective, it is advantageous to rely on cheaper and
more accurate spectroscopic detection procedures [
190
]. For successful seaweed product
development, it is a necessity to become commercially viable; therefore, extraction methods
must be both economical and environmentally friendly.
3.4. Extraction Improvements—Including Less Environmentally Impactful Strategies
Protein has usually been extracted from seaweed using aqueous acid, or alkaline
methods, or by enzymatic hydrolysis of dried powdered biomass. These proteins are
then recovered through ultrafiltration and precipitation using ammonium sulphate or
chromatography techniques [
191
]. To reduce the need for solvents’ enzymatic extractions,
use enzymes such as proteases, cellulases, amylases, glucanases or endoproteases [
192
,
193
]
to degrade the seaweed matrix structure and release the proteins. The presence of phenolic
compounds, specifically phlorotannins in brown seaweeds, has a considerable influence on
protein digestibility. Oven-drying and freeze-drying treatments were used on three brown
seaweeds Sargassum hemiphyllum, Sargassum henslowianum, and Sargassum patens. Results
found no significant differences in the amount of essential or individual amino acids from
the two treatments; however, there was a significant difference in the total amino acids
between treatments with the oven-dried samples significantly lower than the freeze-dried
samples. In addition, properties such as swelling, water holding, and oil holding capacity
of freeze-dried samples were significantly higher than the oven-dried samples, indicating
the potential for freeze-dried seaweeds use as food ingredients in food products [
194
].
To improve the bioavailability of amino acids from seaweeds, heat treatments have been
employed. One brown and red seaweed, Alaria esculenta and Palmaria palmata, were boiled
for 15, 30, and 60 min. Results found an increase in accessible amino acids by 86–109% post
treatment for the red seaweed Palmaria palmata; however, no equivalent results were noted
for the brown seaweed Alaria esculenta, possibly due to its resilient cell wall physiology [
195
].
Other extraction methods also being investigated include chemical hydrolysis or subcritical
water hydrolysis [192,196,197].
To reduce cost and extraction time, new methods such as ultrasound-assisted extrac-
tion (UAE), pulsed electric field (PEF), and microwave-assisted extraction (MAE) are being
used. Ultrasound-assisted extraction (UAE) is a low cost and low solvent use method
which is targeted towards high-value compounds, and works across a range of frequencies,
from 10 to 20 MHz [
198
]; ultrasound creates bubbles which change in size depending on
frequency, and creates increases in temperature of (5000
◦
C) and pressure of 2000 atmo-
spheres. When the cavitation bubbles implode, the temperature and pressure released
cause violent reactions which, if happening in proximity to cells, causes significant cell
wall damage, if not complete cell rupture. If cavitation bubbles rupture in proximity to
compounds, similar damage levels occur causing compound degradation and particle
breakdown [
198
]. Ascophyllum nodosum protein extraction noted increased recovery when

Molecules 2021,26, 1306 24 of 41
assisted by the use of ultrasound at an amplitude level of 68.4
µ
m as in a pre-treatment step;
when compared with acid and alkaline treatment alone, recovery improved by 540% and
27%, respectively, and processing time was also reduced from 60 to 10 min [
179
]. Regarding
commercial utilisation, UAE is used extensively in food processing at commercial scale,
potentially enabling it to be used for seaweed protein extraction [199].
Pulsed electric field (PEF) is considered an emerging technology regarding intracellular
extraction from seaweeds; it has been used effectively as a cell disruptor method for
microalgae, yet its main use has been in lipid extraction to produce biofuels. Pulsed electric
field (PEF) functions by the application of high electric currents to create holes in a cell wall
or cell membrane, causing reversible or irreversible electroporation [
200
]. Initial studies
had focussed on lipid extraction from green microalga, with one study which used the
green seaweed Ulva for quantitative protein extraction, concluding that PEF was selective
in its ability to extract and damage specific proteins [
201
,
202
]. It does show future potential
for protein extraction from seaweeds due to the absence of both heat and solvents required
by the method [203].
The use of MAE for protein extraction has been limited in seaweeds. The elevated
temperatures up to 100
◦
C for several minutes coupled with high frequencies of 2450 MHz,
creating bubbles under high pressure which can then rupture and disrupt the cell contents,
may be effective at lipid extraction, but would not work for protein extraction [204,205].
Other methods which provide more promising solutions incorporate membrane
filtration systems, which include the use of membrane technologies such as microfiltration,
ultrafiltration, nanofiltration, and reverse osmosis. Filtration systems are controlled by the
size of a particle, specifically their molecular weight, enabling exclusion of, for example
larger to smaller particles, which differ in molecular weight. This system could be quite
useful for the enrichment of algal proteins. These membrane filtration technologies do
not create any thermal impact on the proteins and are environmentally friendly due
to the lack of solvents used [
206
]. Additionally, regarding other potential applications,
ultrafiltration has been utilised for polysaccharide purification for the brown seaweed
Sargassum pallidum [207].
Polysaccharide extraction requires the liberation of these complex polysaccharides
from the cell wall structure (Figure 1). The cell wall of seaweeds represents at a minimum
50% DW of the seaweed [
62
]. The cell wall structure is well known in seaweeds; these
differences encompass numerous factors including differences in specific polysaccharide
components based on the species, the physiological component of the algae being consid-
ered, the developmental and life cycle stage, as well as the season and habitat. Within
seaweeds lineages, the classes of polysaccharides are extremely varied based on their
degree of sulfation, esterification, molecular weight, and sugar residue configuration [
168
].
To date, the conventional extraction methods for polysaccharides used have included
dilute aqueous acids, alkaline solutions, and other solvent-based extractions [
144
,
208
].
Newer methods include microwave, ultrasonic, hydrothermal, and enzyme-assisted meth-
ods, which have become established due to the increased yield, bioactivity, and the indus-
trial and therapeutic applicability of seaweed polysaccharides. These technology-driven
rather than chemical methods also assist in maintaining the chemical composition, their
interior structure, and other vital properties. Aqueous-based extractions fulfil both the cost
requirements and reduction in environmental impact for products using polysaccharides,
but the efficiency of the yield is much lower than traditional chemical methods [
209
,
210
].
Other uses of aqueous-based extractions are present in the functional food sector, where
conventional solvent extraction methods use chemicals such as chloroform, butanol, and
hexane, which are not acceptable for these kinds of products.
A study on brown seaweed Ecklonia radiata to improve antioxidant activities used
microwave-assisted enzymatic extraction. The study found that in comparison to conven-
tional acidic extraction, they had significantly higher yields in total phlorotannin content
(TPC) and antioxidant activities, and an extraction yield of 52%. Utilising these two

Molecules 2021,26, 1306 25 of 41
techniques in concert has created the opportunity for this brown seaweed to be used in
producing value-adding nutritional products [211].
Microwave-assisted extraction (MAE) was used as an efficient and rapid method for
the separation and purification of fucoxanthin from three seaweeds. The MAE method
was used in conjunction with a high-speed counter current chromatography (HSCCC)
system with a two-phase solvent system consisting of hexane-ethyl acetate-ethanol-water.
Extraction from for Saccharina japonica, Undaria pinnatifida, and Sargassum fusiforme occurred
in <75 mins for producing weights of 0.83, 1.09, and 0.20 mg of fucoxanthin, respectively.
The fucoxanthin purity was 90% and detected using HPLC, with the structure further
identified by liquid chromatography electrospray ionisation-mass spectrometry (LC-ESI-
MS) and hydrogen 1-nuclear magnetic resonance (
1
H-NMR) [
212
]. Presently, the techniques
being applied to extract polysaccharides from seaweeds are CSE, MAE, UAE, HAE, and
EAE [209,213].
Liquid chromatography (LC) is used extensively in the detection of carbohydrates,
yet for low quantities to separate carbohydrates and peptides, ion exchange chromatog-
raphy (IEC) is used. To separate and then purify alginates and sulphated polysaccha-
rides, anion-exchange chromatography (AEC) is used. Once purified, detection is per-
formed using a range of methods including electrospray ionisation-tandem mass spec-
trometry (ESI-MS/MS) and matrix-assisted laser desorption/ionisation (MALDI) [
214
].
Ultra-performance liquid chromatography (UPLC) works similarly to high performance
liquid chromatography (HPLC) but analyses particles of >2
µ
m in diameter to acquire
better resolution, speed, and sensitivity than HPLC. It also reduces time and solvent use,
making it a more attractive greener method when compared to HPLC [
215
]. Recently,
UPLC was used to purify bioactive compounds and characterise amino acids from 21
seaweed species in Norway; it was also used to determine the monosaccharide compo-
sition of six seaweeds including L. digitata for their anticoagulant properties [
216
,
217
].
Using multiple chromatography methods is typical; for example, in the characterisation of
fucoidan carbohydrates with anticancer activity from the brown seaweed Padina boryana,
ion exchange chromatography (IEC) was followed by ESI-MS/MS [
218
]. To characterise
carbohydrates, specifically to identify the number of monosaccharide units present, the
resonances of
1
H-NMR in the anomeric region (4.4–5.5 ppm) and the
13
C-NMR spectra
(95–110 ppm) provide crucial information [219].
Refinement and improvement in purification and detection methods have enabled
more novel chromatographic and spectroscopy techniques to be used for characterisation
and have been refined to tackle the complex structures of polysaccharides and protein
from seaweeds. For future progression of seaweed product research and development,
the utilisation of these novel techniques including UPLC, NMR spectroscopy (
1
H and
13C-NMR), ESI, and MALDI will be crucial [214].
Biorefinery Application—Optimised Biomass Utilisation
The manufacturing units of bio-economies are biorefineries. A biorefinery works
with one or several feedstocks by providing biomass from which a range of products are
produced. These products may include foods, food ingredients, agrochemicals, biomate-
rials, and biofuels [
9
]. When applying biorefineries to the marine environment, seaweed
would be used as the main feedstock, and could provide alternative sources of biofuels,
creating more sustainable alternatives to fossil-based resources [
220
]. Both M. pyrifera
and Laminaria spp. have been previously investigated for their sustainable fuel-making
capabilities, using anaerobic fermentation to produce methane or natural gas; additionally,
L. hyperborea has also been studied for ethanol production [25,117,118].
As outlined previously, both M. pyrifera and L. digitata contain commercially viable
polysaccharides, the alginates, which are used globally in the food, animal feed, and
pharmaceutical industries [
25
], providing viable commercial products worth extracting as
part of a biorefinery model. Macrocystis sp. and Laminaria sp. have been used to remove
copper, zinc, and cadmium ions from solutions [
119
,
120
], potentially including these species

Molecules 2021,26, 1306 26 of 41
as useful species in bioremediation. Based on the range of present commercial products
produced from both, these brown seaweeds have potential to produce a biorefinery system
for these species. For a future biorefinery for these brown seaweeds, the most crucial
considerations to address would be that the biorefinery system being used to extract all
products functions without significant loss in biomass, and a reduction in chemical solvent
use.
Using L. digitata as a food and energy source within a biorefinery was investigated [
221
].
The study used enzymatic hydrolysis to solubilise the carbohydrates and convert them to
simple sugars providing a 78.23% recovery of sugar in the seaweed hydrolysate. To aid
production of a carbon source in the form of succinic acid, this seaweed hydrolysate was
fermented using Actinobacillus succinogenes producing a yield conversion 86.49% (g/g
−1
of
total sugars) and an overall productivity of 0.50 g L
−1
h
−1
; these results provide potential
uses of L. digitata as a bio-based product and energy producer in biorefineries [221].
A recent biorefinery model study on the green seaweed Ulva lactuca used a cascading
biorefinery process focussed on the protein extraction process and included extraction
of five chemical products in this order starting with minerals, lipids, ulvan, protein, and
cellulose [
222
]. The study found protein digestibility was 85% using an
in vitro
digestibility
assay reaction of o-phthalaldehyde (OPA) and
β
-mercaptoethanol with primary amines,
thus enabling the basis of a bioeconomy to be formed [
222
,
223
]. Another study on the
green seaweed Ulva lactuca (formerly Ulva fasciata) (Chlorophyta) compared the direct and
sequential extractions for mineral-rich liquid extract (MRLE), lipid, ulvan, and cellulose.
They found that their sequential extraction system used 66% of the available biomass, while
the direct extraction approach was less efficient, using between 3 and 30% of the biomass
available [224].
An M. pyrifera study investigated the use of enhanced hydrolysis on M. pyrifera by
integrated hydroxyl radicals and hot water pre-treatment (IHRHW) to produce monosac-
charides or polysaccharides for use as potential fuel feedstocks. This pre-treatment method
IHRHW uses a central composite design and is reported to be able to disrupt both cellu-
lose and hemicellulose enzymatic hydrolysis barriers, preserve the pentose fractions, and
minimize chemical demand and costs. The predicted optimum pre-treatment conditions
were 113.95
◦
C for 29.1 min with the addition of 12.25 mM of ferrous sulphate. Enzymatic
hydrolysis was performed on both pre-treated and untreated M. pyrifera using the desired
15FPU cellulase/g biomass loading. Hydrolysis was conducted in 50 mL Erlenmeyer
flasks, with a 20 mL working volume. A 5% substrate concentration (w/v) was maintained.
Sodium azide (0.5%) was added to the reaction mixtures to prevent microbial contami-
nation. Samples were removed at regular intervals and the supernatants were boiled to
denature enzymatic activity. Samples were then filtered using a 0.22
µ
m filter for glucose
content analysis. Samples were incubated in triplicate at 50
◦
C and rotated at 150 rpm. A
comparison was undertaken with untreated samples. Residues were separated from the
liquid by centrifugation, decantation, and filtration after hydrolysis. The sugar in the liquid
was then analysed. This enabled all glucan and xylan to be recovered as monosaccharides
or polysaccharides and the overall yield was three times higher than the untreated samples
of M. pyrifera. In addition, the digestibility achieved was 92.1% [
225
]. A recent investigation
has claimed that phlorotannins, carbohydrate, and fertiliser fractions can be provided by
using the biorefinery process on M. pyrifera [
175
]. This emerging method effectively works
on disrupting the cellulose structure in the seaweed cell wall using hydroxyl radicals and
without producing fermentation inhibitors, such as hydroxymethyl furfural or furfural.
This high yield product is then ready for saccharification without requiring neutralisation.
IHRHW provides an alternative to acid or alkaline pre-treatment, by reducing the util-
ity cost, improving the yield, and being environmentally friendly. The attributes of this
method make it a very attractive candidate for inclusion in a potential commercially viable
seaweed biorefinery [225].

Molecules 2021,26, 1306 27 of 41
4. Regulation and Legislation
To ensure public safety when purchasing products, regulatory bodies such as the Eu-
ropean Food Safety Authority (EFSA) in Europe and the US (FDA) publish lists of products
or ingredients that are safe for consumption as food or functional food or pharmaceuticals
and for use as novel foods or health ingredients.
In accordance with the online European Novel Food Catalogue, a list of twelve
seaweed species (scientific and common names) are categorised as accepted for use as
food (non-novel) and are not subject to Novel Food Regulation (EC) No. 258/97, or the
updated Regulation (EU) 2015/2283; Novel Food Catalogue, 2018. This occurs due to
specific seaweed species having a history of significant consumption as a food or food
ingredient before 15 May 1997 in the EU. L. digitata is listed in this food catalogue as both
a food and a non-food as of the 15 May 1997. However, M. pyrifera is not present in this
European Novel Food Catalogue. To have a species added to this food catalogue list,
the individual or company must undergo the authorisation process through Regulation
2015/2283, and normally, national authorities may assist with this process [5].
The FDA uses the designation GRAS (generally recognised as safe) to classify a
substance to be used in food for humans or animals under Section 201 [
226
]. M. pyrifera
and L. digitata have GRAS recognition for human consumption as flavour enhancers and
flavour adjuvants [127], enabling their addition to functional foods being sold in the US.
Food regulations in France, have authorised 21 seaweed species including L. digitata
and 3 species of the microalgae genus Arthrospira, formerly known as Spirulina, to be
classified as vegetables and condiments. In relation to metal intake, defined maximum
allowed values are as follows: lead (5 mg/kg), cadmium (0.5 mg/kg DW), tin (5 mg/kg
DW), mercury (0.1 mg/kg DW), inorganic arsenic (5 mg/kg DW), and iodine (2000 mg/kg
DW) [
227
]. An arsenic range (4–106 mg/kg) was found when ten seaweeds were surveyed
at two locations in New England, USA. L. digitata was one of the ten seaweeds sampled and
when extracted using a weak acid extraction with microwave heating, was found to contain
high levels (2.8–20 mg/kg) of inorganic arsenic [
228
]. Another study of L. digitata and
Laminaria hyperborea sampled on North Ronaldsay, one of the Orkney Islands off Scotland,
detected arsenic of 74
±
4 mg/kg DW, which formed the main diet for a sheep population
on the island [
229
]. In the US, L. digitata is used in agriculture and livestock feed; therefore,
monitoring of inorganic arsenic is recommended [228].
Fucoidan has been classified by the European Food Safety Authority (EFSA) as a novel
food, making it a potential contender as a developing functional food ingredient [
230
]. The
present extensive use of seaweed-derived fibres in the food industry confirms they are
safe for human consumption, according to the European Food Safety Authority (EFSA)
and the US Food and Drug Administration (FDA) [
231
–
233
]. Conversely, other seaweed
fibres, including xylan, laminarin, and ulvan, have not received official EFSA approval to
date [148]; consequently, their inclusion in food or functional food will be delayed.
To safeguard human health, regulators have put guidelines in place for specific prod-
ucts. Prebiotic is not yet a term recognised by the US Food and Drug Administration (FDA).
In the US, prebiotics are regulated based on the category of product their intent and design
dictates [234].
In humans, to elicit an effect from the use of most prebiotics, an oral dose of >3 g per
day is required [
235
]. Any dose lower than this should not be termed a prebiotic unless the
low dose has been proven to produce specific effects on the microbiota and related health
aspects [234].
5. Delivery Methods and Applications of Seaweeds
5.1. Food and Feed Delivery and Applications
L. digitata and M. pyrifera have been approved as food flavour additives by the FDA
as dehydrated or ground products, for direct addition to food for human consumption
as a source of iodine or dietary supplement [
127
]. Delivery of these two seaweeds in
food is in an unrefined form as dried seaweed and is added to food ingredients such as

Molecules 2021,26, 1306 28 of 41
breadsticks and huiro fritters [
63
]. Delivery of seaweed extracts such as the phycocolloid,
alginate, in food has been in the form of powder or gel, to be applied to biscuits, yoghurts,
and ice creams, and as a food thickening and emulsifying agent [
25
,
61
,
62
]. The extracted
polysaccharides of laminarin and fucoidan from L. digitata were delivered as wet and
dry spray and applied to mince pork patties to improve appearance and reduce lipid
oxidation [77].
The use of L. digitata,L. hyperborea, and M. pyrifera for animal feed was delivered
as fresh seaweed and applied as the main food source for abalone and sea urchin and
North Ronaldsay sheep; unrefined dried seaweed or flour seaweed was added as a dietary
supplement for goats, bulls, rabbit, fish, and shrimp diets [
14
,
19
–
23
,
67
–
69
]. Extracted
phycocolloid, called crude alginate, from M. pyrifera and Laminaria spp., was used in fish
feeds as a binding agent [25].
Dietary supplements for sows and piglets were delivered using a seaweed extract
from Laminaria sp. containing polysaccharides laminarin and fucoidan [
70
,
73
]. L. digitata
also provided feed supplements for pigs through seaweed extracts containing laminarin
and fucoidan, and purified
β
-glucans were added to their basal diets [
74
–
76
]. These
polysaccharides were used as a prebiotic dietary supplement to improve the microbial gut
populations of pigs, and specifically, weaning piglets [74–76].
5.2. Pharmaceutical Delivery and Applications
Macrocystis pyrifera is used to treat thyroid conditions, anaemia in pregnancy in the
US, and hypertension in Japan; an oral dose of 300 mg of iodine is recommended for
hypertension treatment, with M. pyrifera reported to contain iodine at 0.1 to 0.5%. Medicinal
preparations of iodine from M. pyrifera should be taken from the thallus [
236
]. Conversely,
over consumption of seaweed was found to coincide frequently with medical ailments
including goitre, hypothyroidism, and Hashimoto’s thyroiditis in countries where seaweed
is used traditionally as food [237].
Alginates sourced from both L. digitata and M. pyrifera have been delivered in gels,
powders, beads, and fibres and applied in wound dressings, indigestion control, and drug
delivery [
25
,
61
,
81
–
83
]. With a growing global population, these alginate uses will continue
to increase in demand and will undoubtedly require improved technology in their mode of
delivery of these products. For example, the drug delivery speed or mode of action may
need to be increased or consumed by a different method; in the case of wound dressings,
alginates and sodium alginate are used in hydrogel which has the potential to contain
bioactive compounds to improve the healing process [238].
Fucoidan has been extracted from M. pyrifera, L. digitata, and Saccharina japonica, and
has shown bioactive properties, potentially making these fucoidans suitable to act as
therapeutic agents for cancer and infectious diseases by assisting the immune system’s
response, as an antitumour remedy, anti-inflammatory, antioxidant, and antibacterial, and
to assist in lipid inhibition [
89
,
239
]. Fucoidan is a highly polar polysaccharide which limits
its transport through the intestinal epithelial cells. Administration orally is then considered
the easiest method; however, due to its molecular weight, oral bioavailability is considered
low. To harness the clinical potential of fucoidan, a better understanding of preparation,
quality standards, and administration must be acquired [60].
Secondary metabolites, phlorotannins extracted from several laminaria species in-
cluding S. japonica and L. hyperborea, have been used in the effective control of human
tumour cell proliferation, wound sealing, and reconstruction [
112
,
113
]. M. pyrifera, has
also had two phlorotannins, phloroeckol and tetrameric phloroglucinol, identified as
demonstrating antidiabetic and antioxidant activity as well as preventing skin aging [
114
].
Regarding application of phlorotannins, a study on the seaweed Ishige okamurae, which
contains phlorotannins, suggested inclusion in potential functional food ingredients or
nutraceuticals [138].
Seaweed extracts from L. digitata have been applied as a treatment to improve gut
microbiota, and in commercial products as a skin moisturiser and as a homeopathic

Molecules 2021,26, 1306 29 of 41
medicine [
98
,
100
,
101
]. A combination seaweed extract using M. pyrifera, Fucus vesiculosus,
Saccharina japonica, zinc, and vitamin B6 was used to treat osteoarthritis, with positive
results [
115
,
116
]. M. pyrifera seaweed extract in combination with krill oil was proposed to
be useful as a pharmaceutical product with total antioxidant protection [99].
5.3. Other Applications
5.3.1. Fuel
Three seaweed species were listed as potential fuel producers in Table 3:M. pyrifera,
Saccharina latissima, and Laminaria hyperborea [
25
,
117
,
118
]. Saccharina japonica, another com-
mercially grown brown seaweed species, has been investigated for its fuel potential using
a novel engineered microbial platform. S. japonica was used as a model brown seaweed
species, due to the high alginate content found in brown seaweeds and was fermented
to produce ethanol. The novelty in this platform was the use of genetically modified
Escherichia coli to produce an alginate-degrading enzyme called “Aly”, followed by con-
solidated bioprocessing (CBP), which incorporates enzyme production, with feedstock
degradation (in this case, alginate) and metabolism, (through fermentation at a temperature
range of 25–30
◦
C), resulting in an ethanol yield of ~4.7% v/v [
240
]. S. japonica has also been
used as a feedstock for the production of bio-oil, gas, and char using fast pyrolysis, with
the highest yield of 40.91 wt% at a temperature of 350
◦
C and sweeping-gas velocity of 300
mL/min [
241
]. Whether either of these processes will be implemented as a commercial fuel
production system remains to be seen. Interestingly, M. pyrifera, like S. japonica, has been
trialled for its fuel producing capacity using a similar process to CBP. A pilot study used 75
L fermentation of genetically modified E. coli on M. pyrifera biomass. The higher alginate
to mannitol content in M. pyrifera required a four-stage process to exploit more of the car-
bohydrates present; this included acid leaching, de-polymerisation, saccharification, and
fermentation steps, which yielded 0.213 kg ethanol kg
−1
dry macroalgae, reaching 64% of
the maximum theoretical ethanol yield [
242
]. Whether these systems will gain commercial
success remains to be seen. More recently, the biorefinery process has been proposed and
trialled in an attempt to utilise seaweeds, completely reducing any waste products during
extraction [
9
]. This extraction system, which is still in the emerging technology stage, may
enable a significant increase in seaweed biomass usage during extraction than a single
product production allows [
224
]. The caveat is that the seaweed species being used in the
biorefinery must produce a fuel feedstock. As M. pyrifera has already proven its potential
as a fuel, this opens the utility of it as a biorefinery species.
5.3.2. Nutricosmetics and Cosmeceuticals
Fucoidan’s well documented anti-inflammatory properties have been investigated
as potential additional ingredients in nutricosmetics, with a focus towards anti-aging
or sunscreen products. Marinova, an Australian-based seaweed bioproduct-producing
company, utilises a cold water-based extraction for the polysaccharide fucoidan, which is
supplied to the functional food and cosmetics industry [
145
]. Marinova also uses clinical
trials to investigate the utility of fucoidan as anti-inflammatory and anti-aging ingredients
in nutricosmetic products [
243
]. For potential nutricosmetics products, seaweeds and their
extracts are well placed to be utilised for their inhibition of glycation, elastin calcification,
and matric enzymes, as well as anti-inflammatory activity, all properties which assist in
providing cosmetic anti-aging benefits [243].
Alginates extracted from Macrocystis sp. and Laminaria sp., and laminarins extracted
from Laminaria sp., have been used in the cosmetics industry for their range of uses as
gelling colloids and emulsion stabilisers for inclusion in lotions and moisturisers as well as
immunostimulating, antioxidant, anticellulite, and anti-inflammatory agents [
82
,
104
,
122
].
There is further potential product development in this sector, with new applications of
alginates and laminarins being explored and polysaccharides-rich extracts being included
in skin care products to reduce the effects of aging and blemishes [244].

Molecules 2021,26, 1306 30 of 41
L. digitata (sea kelp) extract with glycerine added has been used to create a certified
organic product by the USDA, which can be used in a range of products: anti-aging
creams, serums, and face masks, hair treatments, root treatment, and shampoos [
245
]. Juice
extracted from the whole M. pyrifera plant has been classified as a cosmetic ingredient for
skin conditioning, now sold in several products including those for skin aging [246].
Interestingly, both M. pyrifera and L. digitata are listed in the FDA Product Category—
Brown Algae-Derived Ingredients. M. pyrifera is listed in the ingredients of bath products,
oils, salts, skin and eye products including masks, make-up, and self-tanning lotions,
shampoo, foot products, nail lotions, nail polish, and aftershave, and its content ranges
from 0.009 to 5% and has a higher content for eye lotions with 36.4%. L. digitata is also
listed in the same products as M. pyrifera but has some additional products including
hair bleaches, hair sprays, and for face and neck products, a concentration of 40%. Other
product concentrations range from 0.0004 to 5% [127].
5.3.3. Bioplastics
Sourcing bioplastics from L. digitata and M. pyrifera has not been documented to
date; however, emerging uses for glycans extracted from the green seaweed Ulva include
bioplastic. The Australian company Phycohealth already produces seaweed products
including cosmetics, food, and food supplements and are behind this bioplastic product.
The bioplastic they are working on is made using a glycan-based extract from Ulva, which
is used to make thread which is then woven to produce a plastic film; other applications
include potential 3D fabrication of materials [247].
5.3.4. Bioremediation
Bioremediation of ions from metals is another use of both M. pyrifera and L. digi-
tata [
119
–
121
]. In the context of aquaculture, the potential dual purpose of these species
is to be cultivated as an aquaculture crop and be used to remove metals that may be a
potential environmental hazard is promising.
5.3.5. Aquaculture
The addition of these species to an Integrated Multi-Trophic Aquaculture system
(IMTA) could potentially improve the productivity of the seaweeds, while also producing
multiple aquaculture products from one environmental footprint. The IMTA initial concep-
tual design is a system that allows several species to be grown within the same encloser
system, utilising nutrients from each trophic level to the level below it. The system includes
a fed aquaculture tank, for example, fin fish, beside a shellfish growing station (called or-
ganic extractive aquaculture). This takes advantage of the enrichment in particulate organic
matter (POM) from the fish tank; next to the shellfish is the seaweed growing station, called
the inorganic extractive aquaculture, which gains nutritional advantage from the dissolved
inorganic nutrients (DIN) [
248
]. An integrated approach enables efficient nutrient cycling
to take place, attempting to partially close the nutrient loop, reducing external nutrient
supply to the system and improving efficiency, and minimising environmental impact to
the local ecosystem by utilising the fish waste within the other trophic levels. Chopin de-
scribed this system as extremely flexible, and it could be applied to land-based, freshwater,
or marine, and could be termed “aquaponics”. Implementation of the IMTA system was
trialled in Chile using the red seaweed Agarophyton chilense as a biofilter for nitrogen on an
open-water salmon farm. The study found a significant reduction in nitrogen at 9.3 g M
−1
per m of line. These long lines were positioned within 800 m of the salmon pens within the
effluent flow. Seaweed tissue analysis for nitrogen noted up to 2% of the daily weight (DW)
in the Agarophyton chilense that were growing within 800 m of the salmon pens. This site
had the highest level of nitrogen in the seaweed tissue and the highest growth rates of up
to 4% DW in summer and 2% DW in winter. This Chilean study concluded that IMTA was
a successful biofiltration platform using red seaweed Agarophyton chilense and proposed

Molecules 2021,26, 1306 31 of 41
that a 100 hectare (ha) Agarophyton chilense farm would effectively reduce the inorganic N
inputs of a 1500-tonnes salmon farm [249].
6. Conclusions
Suggestions for the Future Potential of Commercial Brown Seaweed Cultivation
In the context of the future commercial viability of new seaweed products, it is impor-
tant to factor in the commercially important phycocolloids sourced from seaweed which
are alginates, agar, and carrageenan. The seaweeds from which these phycocolloids are
extracted are Ascophyllum, Durvillaea, Ecklonia, Lessonia, Laminaria, Macrocystis, Sargassum,
and Turbinaria. The brown seaweeds of interest in this review, Laminaria and Macrocys-
tis, produce alginate. Alginate production is a global industry and was worth USD 213
million annually in 2009, yet none of the alginate-yielding seaweeds were produced by
aquaculture at that time, as they were not grown by vegetative propagation. Cultivation
of alginophytes, especially their reproductive cycles involving alternation of generation,
requires more research. In China, S. japonica was the only alginate-producing seaweed to
be cultivated primarily for food, while some excess was used for alginate extraction [
250
].
The seaweed industry requires large quantities and high-quality seaweed raw material
that exerts pressure on the existing natural seaweed resources. Aquaculture cultivation
of seaweed has grown considerably since 2009 to meet product demand and to protect
wild kelp beds that are still under threat from over-exploitation and climate change, which
has caused increases in ocean water temperatures [
251
]. To produce high value products
(HVP) from seaweed requires considerable cultivation control to retain quality biomass, not
possible in offshore systems; therefore, onshore aquacultures have been proposed to fulfil
this need [
252
]. FAO reported that of the 29.5 million tonnes of seaweed harvested in 2016,
only 0.5 million tonnes were wild-harvested, with 29 million tonnes from aquaculture [
3
].
Yet Chile, Peru, and Mexico still depend primarily on harvesting natural kelp beds [
253
].
Asia still dominates global seaweed production, extending productivity through cultivation
of Kappaphycus and Eucheuma in Southeast Asia [
3
]. Further global development of seaweed
aquaculture cultivation may benefit the preservation of wild kelp beds.
Additional suggestions have been made to further enable consistent successful aqua-
culture biomass production of seaweeds:
Extending knowledge of nutrient uptake and assimilation in the species of interest,
which could be effectively employed when considering the utilisation of seaweed polycul-
ture and Integrated Multi-Trophic Aquaculture (IMTA) [
254
]. Environmental conditions
vary significantly in locations on the same coastline as has been demonstrated in Chile
where biomass of M. pyrifera varied considerably based on local fluctuations in environmen-
tal variables and the occurrence of epiphytes [
18
]. Species-specific knowledge of nutrient
uptake within the context of local environmental conditions could be vital in maintaining
IMTA systems of cultivation and nutrient cycling within coastal systems.
To address concerns regarding the lack of nearshore space to accommodate future
aquaculture sites as demand increases and due to the labour-intensive nature of seaweed
harvesting, experts advise the use of offshore locations, where technology-based moni-
toring and harvesting can be carried out, reducing the necessity of a nearshore seaweed
aquaculture location and labour-intensive harvesting. Saccharina latissima, a European kelp
species, has been successfully cultivated offshore in Portugal in challenging conditions,
producing growth rates of 3.3–4.5% day
−1
from January to May [
255
]. To implement
and maintain successful ongoing offshore seaweed cultivation systems, incorporation of
technology is necessary.
Understanding of seaweed life cycles and where to control them is crucial to efficient
aquaculture biomass productivity. Nori is the common name for the red seaweed Porphyra
purpurea and nori production from Pyropia/Porphyra spp. is used as a successful example of
this. The use of algal phytohormones to control the switch from vegetative-to-reproductive
transition and to increase the speed of reproduction was performed on the red seaweed
Grateloupia imbricata using methyl jasmonate. Maturation was reduced from three weeks

Molecules 2021,26, 1306 32 of 41
to forty eight hours, and a 7.5-fold increase in cystocarp number was observed [
256
].
Further research into the understanding and manipulation of lifecycle stages and triggers
to enhance reproductive success and speed of biomass accumulation should be considered.
A major challenge for the established commercially successful euchematoid (two gen-
era of red seaweeds, Kappaphycus and Eucheuma, are known collectively as eucheumatoids)
cultivation is the delay in the establishment of cultivars or strains with higher produc-
tivity and/or resistance to disease [
257
], which remains a challenge also for the brown
and red seaweeds. Knowledge can be gained from progress made by other seaweed
species. Using key commercial species, S. japonica, Pyropia spp.,Undaria spp.,Cladosiphon
okamurarus, and Nemacystus decipiens, it took 21 years for Japan, Korea, and China to
produce 47 certified seaweed cultivars to be commercially cultivated. These cultivars
were produced to improve productivity; however, work is now continuing to produce
higher quality seaweeds through aquaculture [
258
]. Enabling the expansion required for
successful extensive bioproduct production from all three seaweeds groups will require
the cultivation of domesticated seaweed cultivars, as well as the domestication of new
species, and further streamlining of cultivation techniques, to include more comprehensive
quality control methods, to enable higher quality seaweed bioproducts to be produced [
6
].
A temperature-tolerant mutant strain for Neopyropia tenera (formerly Pyropia tenera) was
produced using gamma irradiation [259]. The potential to produce heat-tolerant seaweed
strains for commercial seaweed species could become extremely important, with climate
change affecting increases in ocean temperatures across the globe.
As an industry, the potential efficiencies that could be gained from these suggestions
cannot be underestimated. The potential loss of natural kelp beds to over-exploitation
and temperature increases due to climate change are serious concerns which need to be
addressed in the immediate future. Independent cultivar cultivation and the development
of species resistant to disease and epiphytes that are thermo-tolerant have the potential to
significantly improve commercial opportunities for the seaweed industry.
Author Contributions:
Writing—original draft preparation, D.P.-M.; writing—review and editing,
D.P.-M., M.H., M.A.P. and T.T.W.; funding acquisition, D.P.-M. All authors have read and agreed to
the published version of the manuscript.
Funding:
Dr. Diane Purcell-Meyerink has received funding from the Research Leaders 2025 pro-
gramme co-funded by Teagasc and the European Union’s Horizon 2020 research and innovation
programme under the Marie Skłodowska-Curie grant agreement number 754380.
Acknowledgments:
Author acknowledges support from the Cawthron Institute Staff, who assisted
in reading the manuscript prior to submission.
Conflicts of Interest: The authors declare no conflict of interest.
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