Flórez-Fernández Et Al., 2020 Cap Book

1093
44
Extraction and Purification of Fucoidan from Marine Sources

Noelia Flórez-Fernández, Elena M. Balboa and Herminia Domínguez
Department of Chemical Engineering, University of Vigo (Campus Ourense), Edificio Politécnico, As Lagoas, 32004, Ourense, Spain
44.1 Introduction et al., 2017), antitumoral (Yang et al., 2008a;

Synytsia et al., 2010; Foley et al., 2011; Wang
Fucoidans, sulfated fucose-rich polysaccha- et al., 2015; Liu et al., 2016; Palanisamy et al.,
rides found in brown seaweeds, contain fucose 2017), antiviral (Béress et al., 1993; Ponce et al.,
as a major monomer, and several other mono- 2003; Lee et al., 2004; Hemmingson et al.,
saccharides, such as galactose, xylose, arab- 2006; Santoyo et al., 2011), anti-inflammatory
inose, mannose, glucose, or glucuronic acid. (Ananthi et al., 2010; Kang et al., 2011; Lee et al.,
These type of polysaccharides are not found in 2012; Sanjeewa et al., 2017), immunomodula-
other divisions of algae or in land plants, tory (Ha et al., 2008), wound healing properties
although related compounds occur in marine (Fujimura et al., 2000) and anti-angiogenic
invertebrates belonging to Echinodermata. effects (Cong et al., 2016).
Whereas animal polysaccharides or fucan The chemical composition, including the
sulfates, are a linear regular sequence contain- molecular weight, monosaccharides and sul-
ing only fucose and sulfate groups, seaweed fate content of fucoidans. Their structures and
fucoidans show a great structural heterogene- biological characteristics are taxonomically
ity with ramifications and different substitu- dependent (Duarte et al., 2001; Ponce et al.,
ents (Usov and Bilan, 2009; Pomin, 2012). 2003; Sakai et al., 2003; Bilan et al., 2006; Usov
Fucoidans and their lower molecular weight and Bilan, 2009) and influenced by both mac-
oligosaccharide derivatives are attracting roalgal biology (species, growth stage, part of
increasing interest for their low toxicity (Ha the alga, seasonal and geographical condi-
et al., 2008) and broad range of biological activi- tions) and different extraction/purification
ties with potential health benefits and therapeu- techniques (Morya et al., 2012; Liu et al., 2012).
tic applications (Jiao et al., 2011; Wijesekara The importance of the extraction technique is
et al., 2011; Hahn et al. 2012; Liu et al., 2012; key not only on the yields, but also on the
Fitton et al., 2015), in particular anticoagulant nature and structure of the product, signifi-
(Silva et al., 2005; Camara et al., 2011; Dore et al., cantly influenced by the extraction parameters
2013; Obluchinsksya et al., 2015; Chen et al., (Usov and Bilan, 2009; Ale et al., 2013).
2012), antioxidant (Rupérez et al., 2002; Kim Brown seaweeds contain alginic acid,
et al., 2007; Ananthi et al., 2010; Chattopadhyay fucoidans and laminarins. The gelling proper-
et al., 2010; Hou et al., 2012; Balboa et al., 2013; ties of alginate and laminarin constitute a phys-
Hifney et al., 2016; Huang et al., 2016; Palanisamy ical barrier, limiting the extraction efficiency of
Encyclopedia of Marine Biotechnology: Five Volume Set, First Edition. Edited by Se-Kwon Kim.
© 2020 John Wiley & Sons Ltd. Published 2020 by John Wiley & Sons Ltd.
1094 44 Extraction and Purification of Fucoidan from Marine Sources
bioactives, which could remain entrapped in walls. The adequate optimization of this stage
the gel matrices. The extraction and purification is determinant for attaining high yields and
stages are necessary when the objective is the purity of the target solutes. The major varia-
extraction for the detailed structural analysis of bles affecting the extraction performance are
fucoidans and also for industrial applications. summarized in Table 44.1.
Fucoidans are normally extracted from brown The fucoidan content and type varies with
algae in multistep processes using water or the raw material and is also determined by the
dilute acid, at high temperature and long environmental, seasonal and collecting char-
reaction times. Since the prolonged times and acteristics, the storage and preparation, includ-
temperatures during these processes can be del- ing the mechanical and thermal conditioning
eterious for the biological activity of sulfated to remove water and to make the solid matrix
polysaccharides, alternative extraction tech- more accessible to the solvent. A general dia-
niques are required to conserve its structure and gram of the whole extraction and purification
biological properties. The increasing conscious- process to obtain fucoidan from brown sea-
ness of health and environmental issues has led weeds is summarized in Figure 44.1.
to the development of efficient and eco-friendly When the target compounds are fucoidans,
novel and innovative techniques, which can aqueous solvents are the most suited.
improve the performance of the extraction and Therefore, the moisture content of the raw
purification process (Hahn et al., 2012; García- material is not a limitation. However, the stor-
Vaquero et al., 2017). This chapter presents and age stability of the raw material can be
overview on the extraction and purification con- enhanced if the material is dry. Since seaweeds
ventional and emerging technologies. contain more than 90% (wb) water, the drying
stage, which is highly energy consuming, has
to be considered in detail, particularly the tem-
44.2 Extraction of Seaweed perature and time influence the stability of
bioactives, although in the case of fucoidans
Fucoidans
this influence is less marked than in the phe-
nolic or carotenoids fractions. In a mass trans-
44.2.1 Fundamentals of Solid–Liquid
fer controlled process as this, particle size is a
Extraction
key factor, determining the contact area and
Solid-liquid extraction is a heterogeneous mass diffusion pathway. The influence of the pH is
transfer operation to separate components commented for the conventional solvent extrac-
and/or fractions from a solid matrix using a tion processes and the influence of specific
selective solvent. Many solutes are found operational variables for the intensification
inside cells and others are forming part of the
cell walls, usually in polymerized form. Table 44.1 Major aspects to be considered
Therefore, degradation of the cell walls, which for the optimization of the solid-liquid extraction
of fucoidans from seaweed.
act as a barrier, facilitates the mass transfer.
Among the different stages relevant for
Part of seaweed, collection area
fucoidan extraction: (a) solvent diffusion
and season, particle size, moisture
inside the solid matrix; (b) hydrolysis and solu- Solid content, drying technology
bilization of target components in the solvent;
(c) diffusion of the solute through the solid Solvent Selectivity, inertness, low toxicity,
matrix; and (d) mass transfer to the bulk solu- cost, availability, renewable
character, solvent:solid ratio
tion; stage (c) is usually the rate limiting step.
Internal mass transfer can be favored by reduc- Operational Homogeneous mixing,
conditions temperature, time, chemicals
ing the particle size and degradation of cell
44.2 Extraction of Seaweed Fucoidan 1095
separation and concentration operations.

Therefore, a solvent to seaweed of 10–35 w/w
Brown seaweed or v/w (db.) was considered the appropriate
extraction ratio.
Conditioning (washing, milling, drying)
Extraneous materials, moisture 44.2.2 Sample Conditioning
Pre-extraction
The most common initial stage usually con-
sists on washing the fresh seaweeds with
deionized, distilled or tap water in order to
Filtration or centrifugation remove salt, adhering sand, epiphytes, extrane-
Pigments, oils ous matter and necrotic parts from the surface
(Lee et al., 2004; Chattopadhyay et al., 2010;
Defatted solids
Vasquez et al., 2012; Anastyuk et al., 2014;
Imbs et al., 2015). Rinsing with seawater
Extraction (Deniaud-Bouët et al., 2014), a sequence of
Conventional Innovative washings in seawater and then rinsing in dis-
Water Pressurized hot water tilled water (Ananthi et al., 2010) or the use of
Acid Microwave assisted running tap water followed by distilled water
Alkali Enzyme assisted (Palanisamy et al., 2017) have been proposed.
Ethanol Ultrasound assisted The conditioning stages, aimed at facilitat-
ing the further extraction, include the destruc-
Filtration or centrifugation tion and degradation of the cell wall under
Exhausted solids mild conditions so that the intrinsic properties
of polysaccharides remain unchanged. The
Purification samples are usually dried (air, oven, freeze-
drying) and ground from coarse to fine powder
Precipitation (ethanol, acetone)
to cause cell wall destruction and increase the
Liquid: liquid fractionation
specific area of the solids (Hahn et al., 2012;
Ion-exchange chromatography
Imbs et al., 2015). However, for some processes
Size-exclusion chromatography
only cutting was required, i.e. Sargassum ful-
Affinity chromatography
vellum samples before autoclaving (Jo and
Membrane filtration
Choi, 2014). Even when extraction is typically
performed using pre-treated dried macroalgae
Fractions and compounds
or defatted biomass, also direct extraction of
algal pieces before water extraction was pro-
Figure 44.1 Flow diagram of the sequence of
posed (Lee et al., 2011), or homogenization of
stages required to obtain the fucoidan fractions. the on-site milled alga to facilitate storage of
the homogenate could be stored until required
techniques is further discussed. The liquid to (Foley et al., 2011). Also the fresh or deep-fro-
solid ratio is a critical factor affecting the yield zen seaweeds can be used (Zvyagintseva et al.,
and concentration of the products, the mini- 2003). Freezing can be proposed before freeze-
mum value should provide adequate mixing, drying (Heo et al., 2005; Quitain et al., 2013) or
without limitation of mass transfer or solubil- for storage purposes. More recently, a compres-
ity. However, the increased values are not sional-puffing stage (140–220°C during 10 s)
desirable since the solute can be obtained in was applied to the dried, crumbled and sieved
highly diluted solutions, thus leading to costly Sargassum glaucescens, which was further
ground and stored at 4°C before hot water some cases, previous or further washing with
extraction. This treatment decomposes the cel- acetone to remove lipids and phenols was pro-
lular structure of algae, facilitates the release posed (Bilan et al., 2002, 2004 and 2006; Ale
of fucoidan by hot water extraction and offers et al., 2012; Hahn et al., 2012; Mak et al., 2013;
advantages derived from the simplicity of the Eluvakkal et al., 2014; Lim et al., 2014).
procedure, the absence of reagents, reduced A sequence of increasing temperatures with
pollution, and feasibility for continuous pro- 80% ethanol first at room temperature then at
duction (Huang et al., 2016). Laminarin and 70°C was used to extract mannitol and some
fucoidan could be purified from the exudate of salts (Rioux et al., 2007; Foley et al., 2011;
the fresh Fucus vesiculosus (Béress et al., 1993). Hahn et al., 2012; Yuan and Macquarrie,
2015a). However, this previous defatting stage
is unpractical for some applications, since
44.2.3 Pre-Extraction
there was a higher extraction yield when it is
In order to prevent the coextraction of other not performed, although the fucose and sulfate
algal compounds during the aqueous isolation content was lower. This lack of remarkable dif-
of fucoidan, different stages have been pro- ference in cytotoxic and antioxidant activity,
posed to remove other components before the although leading to less purified fucoidan,
extraction process. Alternatively, these unde- could recommend the simpler method for its
sirable compounds can be separated from the utilization at industrial scale (Wang et al.,
crude extract after the extraction process. The 2015). Alternatively, deoiling by sc-CO2 extrac-
algal biomass can be extracted during 1–24 h at tion (25–40 MPa, 40–45°C, 2–3 h) was pro-
25–80°C with 70–96 % ethanol to remove the posed (Quitain et al., 2013; Saravana et al.,
low molecular weight components, colored 2016). The yield and structural characteristics
matter, lipids and lipophilic pigments (chloro- of fucoidans was similar for extraction with
phylls, fucoxanthin, carotenes) (Sakai et al., 70% ethanol and for supercritical CO2 (55 MPa,
2002; Ponce et al., 2003; Lee et al., 2004; Kim 60°C, both with pure solvent and with 5% etha-
et al., 2007; You et al., 2010; Sokolova et al., nol as modifier), and except when using 5%
2011; Vasquez et al., 2012; Anastyuk et al., ethanol as co-solvent enabled obtaining highly
2014; Eluvakkal et al., 2014; Kumar et al., 2015; sulfated fucoidan with homogeneous composi-
Obluchinsksya et al., 2015; Wang et al., 2015; tions (Menshova et al., 2013). This technology
Baba et al., 2016; Liu et al., 2016; Shan et al., (25 MPa, 45°C) is useful for the extraction of
2016; Palanisamy et al., 2017). Alternatively valuable fractions, such as the carotenoid
(Ananthi et al., 2010; Costa et al., 2010; Dantas fucoxanthin (Sivagnanam et al., 2015). The
Magalhaes et al., 2011; Fernando et al., 2017) compounds removed in the deoiling stages,
or successively the removal of lipids and pig- fatty acids, phenolic compounds, fucoxanthin,
ments can be carried out with acetone (Silva also present desirable properties, i.e., antibac-
et al., 2005; Yang et al., 2008a; Imbs et al., terial, antioxidant, antihypertensive (Eluvakkal
2015), or with a mixture of acetone and etha- et al., 2014; Sivagnanam et al., 2015; Lorbeer
nol (Dinesh et al., 2016; Shan et al., 2016). et al., 2017). Compilation of pretreatment
Sequential extractions are also useful, i.e., with stages can be found (Hahn et al., 2012; García-
ethanol, acetone, and chloroform successively Vaquero et al., 2017).
(Zvyagintseva et al., 1999, 2003) or with petro-
leum ether and acetone (Chattopadhyay et al.,
44.2.4 Conventional Extraction
2010; Sinha et al., 2010).
A mixture of methanol/chloroform/water in The appropriate extraction method should be
proportion 4/2/1 (v/v/v) is useful to remove selected to increase the extraction yield and to
lipids, protein and colored pigments, and in contribute to the high biological activity of the
polysaccharide extract. The correct adjustment c onventional techniques used for fucoidan
of parameters, such as liquid to solid ratio, tem- extraction are summarized in Table 44.2, and in
perature, pH, extraction time and number of some reviews (Ale et al., 2013; García-Vaquero
stages, greatly influences the yield and compo- et al., 2017). Fucoidan presents a wide variation
sition of the resulting fucoidan, prevents the in the molecular weight, ranging from 21 to
possible structural alteration of the sulfated 1600 kDa due to species variations and differ-
polysaccharides (Hahn et al., 2012), and deter- ences in the extraction and purification meth-
mines the purification process. In order to effi- ods (Lee et al., 2004; Li et al., 2006; Rioux et al.,
ciently optimize the operational conditions, 2007; Rupérez et al., 2002). Lower molecular
experimental design can be a useful tool to weight algal sulfated polysaccharides can be
evaluate the single and combined effect of the prepared by further chemical, physical or enzy-
most influencing variables on the composition, matic means to obtain oligosaccharides with
structure and properties of the target solubi- more diverse bioactivities. The influence of the
lized products (Ale et al., 2012; Lorbeer et al., extraction methods and other processing stages
2015a; Hifney et al., 2016). Examples of on the molecular weight is also discussed.
Table 44.2 Conventional technologies applied for the extra of fucoidan from seaweeds.
Seaweed Conditions References
Aqueous
Chnoospora minima DW, 90–95°C, 3–4 h Fernando et al., 2017
Ecklonia cava DW, 70°C, 24 h Lee et al., 2012
Fucus vesiculosus DW, rt., 24 h Fujimura et al., 2000
Fucus vesiculosus DW, 80°C, 3 h Shan et al., 2016
Halimeda sp. DW, 90–95°C, 3–4 h Fernando et al., 2017
Hizikia fusiforme DW, 70°C Li et al., 2006
Hizikia fusiforme DW, 80°C, 3 h, 3 stages Wang et al., 2012
Saccharina japonica DW, 80°C, 4 h Zhang and Row, 2015
Sargassum wightii DW, 65°C, 1 h, 2 stages Kumar et al., 2015
Sargassum cristaefolium DW, 40°C, 15 min Wang et al., 2015
Sargassum pallidum DW, 25°C, 2 h, 3 stages Liu et al., 2016
Sargassum polycystum DW, 65°C, 1 h Palanisamy et al., 2017
Sargassum stenophyllum DW, rt., 12 h, 3 stages Duarte et al., 2001
Sargassum trichophyllum DW, reflux, 1 h, 3 stages Lee et al., 2011
Undaria pinnatifida DW, 65°C, 1 h, 2 stages Yang et al., 2008a
Alkaline
Ascophyllum nodosum 0.01 NaCl+1% CaCl2, rt., 70°C, Marais and Joseleau, 2001
2 stages
Adenocystis utricularis 2% CaCl2, rt., 7 h, reextraction 70°C Ponce et al., 2003
Fucus dischitus 2% CaCl2, 85°C, 5 h Cumashi et al., 2007
Fucus evanescens 2% CaCl2, 85°C, 5 h, 4 stages Bilan et al., 2002;
Cumashi et al., 2007
(Continued)
Table 44.2 (Continued)
Seaweed Conditions References
Fucus evanescens 2 % CaCl2, 60°C, 3 h Imbs et al., 2015

Fucus serratus 2% CaCl2, 85°C, 5 h Bilan et al., 2006;
Cumashi et al., 2007
Sargassum binderi 2% CaCl2, 85°C, 24 h, 6 stages Lim et al., 2014
Sargassum fusiforme DW, 100°C, 4 h, 8 stages Cong et al., 2016
Sargassum sp. CaCl2 (pH 8), 4°C, 3 h, 4 stages Baba et al., 2016
Undaria pinnatifida 2% CaCl2, 85°C, 5 h Mak et al., 2013
Sargassum stenophyllum 4 M KOH, 10 mg NaBH4, rt. Dias et al., 2008
Acid
Adenocystis utricularis HCl (pH 2), rt., 7 h, reextraction 70°C Ponce et al., 2003
Cladosiphon okamuranus 30% HCl, 100°C, 15 min Nagaoka et al., 1999
Dictyota dichotoma HCl (pH 2), rt., 7 h, reextraction 70°C (9 Rabanal et al., 2014
stages)
Fucus evanescens 0.1M HCl, 60°C, 2 h Menshova et al., 2013
Fucus evanescens 0.1M HCl (pH 2–3), 60°C, 3 h Imbs et al., 2015
Fucus vesiculosus 0.01M HCl or 0.001–0.1 M H2SO4, Pielesz et al., 2011
60–80°C
Laminaria cichorioides HCl (pH 2.0–2.3), 60°C, 3 h, 2 stages Anastyuk et al., 2010
Padina sp. 0.2N HCl, 70°C, 1 h, 2 stages Lim et al., 2014
Saccharina japonica HCl pH (2.5), 90°C, 1.5 h, 3 stages Guo et al., 2013
Saccharina japonica 0.1M HCl, 60°C, 2 h Menshova et al., 2013
Saccharina japonica 0.1M HCl, 80°C, 4 h Zhang and Row, 2015
Saccharina japonica 0.05M HCl, 25°C, 2 h Saravana et al., 2016
Sargassum binderi 0.2N HCl, 70°C, 1 h, 2 stages Lim et al., 2014
Sargassum fulvellum HCl (pH 2), 100°C, 1 h Kim et al., 2007
Sargassum mcclurei 0.1M HCl, 60°C, 2 h, 2 stages Thinh et al., 2013
Sargassum oligocystum 0.1M HCl, 60°C, 2 h Menshova et al., 2013
Sargassum sp. 0.03M HCl, 90°C, 4 h Ale et al., 2012
Sargassum sp. 0.15M HCl, 45°C, 3 h, 4 stages Baba et al., 2016
Sargassum sp. McIlvaine’s buffer (pH 4.0), 60°C, 3 h Hifney et al., 2016
Sargassum tenerrimum 0.1M HCl, 25–30°C, 6 h, 2 stages Sinha et al., 2010
Sargassum swartzii 0.05M HCl, rt., 24 h Dinesh et al., 2016
Sargassum polycystum 0.1N HCl, 95°C, 12 h, 3 stages Chotigeat et al., 2004
Undaria pinnatifida 0.15N HCl Lee et al., 2004
Undaria pinnatifida HCl (pH 2), rt., 24 h Song et al., 2015
Undaria pinnatifida 1% H2SO4, rt., 6 h Hemmingson et al., 2006
Undaria pinnatifida 0.2M HCl, 60°C, 1 h You et al., 2010
DW, distilled water; rt., room temperature.

44.2.4.1 Water Extraction for Sargassum stenophyllum (Dias et al., 2008).

Since fucoidans are polar and hydrophilic Imbs et al. (2015) reported that extraction with
water soluble macromolecules, water is an an aqueous calcium chloride solution provides
ideal solvent for extraction (Cong et al., 2016; a purified preparation of the Fucus evanescens
Shan et al., 2016; Wang et al., 2012) in innova- polysaccharides, with lower content of lami-
tive low-chemical processes (Foley et al., 2011). naran, uronic acids, and polyphenols than
The traditional method for polysaccharide those obtained by hot extraction, although the
extraction is based on hot extraction with tap presence of phlorotannins contributed to the
or with distilled water (single or double) at antioxidant properties of the crude polysac-
70–90°C during periods ranging from 2–6 h in charide fractions (Lim et al., 2014).
1–3 stages. The high temperatures, long times, Otherwise, the joint extraction of alginates
and low efficiency are the major negative requires their further selective precipitation.
aspects. The use of cold (room temperature) Addition of CaCl2 (1–4%) can be mixed to the
conditions is possible, but longer extraction hydrolysate and kept at 4°C (overnight) for
periods (24 h) would then be required removing alginate, as the calcium salt, which
(Fujimura et al., 2000). In order to improve the can be separated by filtration (Bilan et al.,
efficiency and purity, dilute alkaline and acidic 2004, 2006; Li et al., 2006; Yang et al., 2008a;
solution methods, as well as intensified tech- Chattopadhyay et al., 2010; Sinha et al., 2010;
niques have been proposed. When acidic or Ale et al., 2013; Deniaud-Bouët et al 2014;
alkaline extraction is performed, the extracts Baba et al., 2016; Dinesh et al., 2016; Saravana
need to be neutralized to prevent the degrada- et al., 2016) and then addition of ethanol
tion of the target polysaccharide. allows the crude fucoidan precipitation
(Vasquez et al., 2012; Zhang and Row, 2015;
44.2.4.2 Alkaline Extraction Hifney et al., 2016; Sanjeewa et al., 2017).
A key variable during extraction is pH, since Alginic acid can also be precipitated by ethanol
protons or hydroxide ions interfere with the (Sokolova et al., 2011).
hydrogen bonds between the polysaccharides,
releasing them into the solution and leading to 44.2.4.3 Acid Extraction
increased yield. Alginates are hydrocolloids Polysaccharides can be extracted from algae
found in brown seaweeds, they are constituted with dilute acids at room or slightly elevated
by consecutive mannuronate residues, consec- temperature, but can result in the partial cleav-
utive guluronate residues or alternate mannu- age of sulfate esters leading to acid-induced
ronate and guluronate residues and can gelate depolymerization. Since the bioactivity depends
in the presence of Ca2+ or Mn2+. Therefore, the on the molecular weight, the monosaccharide
use of CaCl2 solutions as extracting solvent composition and the sulfate content, careful
offers the advantage of selectively precipitating control of the operational conditions (extraction
alginates during extraction (Cong et al., 2016; pH, time, and temperature) is needed to achieve
Dinesh et al., 2016; García-Vaquero et al., 2017; maximum yields and to maintain the properties
Palanisamy et al., 2017). In a common protocol of the extracted polysaccharides.
a 2% aqueous solution of calcium chloride is Slightly acidic solutions of HCl are com-
used in one to five stages (Bilan et al., 2004, monly used, facilitating the extraction process
2006; Usov and Bilan, 2009; Baba et al., 2016). and allowing a reduction in the extraction tem-
Different concentrations and temperatures perature and/or time (Dinesh et al., 2016; Imbs
have been proposed as an alternative to obtain et al., 2015; Lorbeer et al., 2015b; Menshova
alginates, laminarin and fucoidan (Lim et al., et al., 2015). In some cases, this could be the
2014). Direct extraction with an alkaline treat- best solvent, i.e. 0.1 M HCl acid was more
ment at room temperature also proved suited suited than 0.05 M H2SO4 and more suited
than 0.03 M H3PO4 for fucoidan extraction polysaccharides began to decrease. Also the
from Laminaria japonica (Zhang and Row, number of stages can be optimized, reextrac-
2015). In other cases, the influence of the sol- tion for 1–9 times ensures complete fucoidan
vent is not determinant, i.e. the extraction removal (Rioux et al., 2007; Ale et al., 2012;
yields and characteristics of the fucoidans from Lim et al., 2014; Rabanal et al., 2014).
Adenocystis utricularis were similar when The molecular weight of fucoidan is signifi-
extracted with distilled water, 2% calcium chlo- cantly influenced by the acidic treatment con-
ride solution and diluted hydrochloric acid (pH ditions (Yang et al., 2008b). Fucoidan polymers
2) at room temperature and at 70°C (Ponce extracted from F. vesiculosus with HCl treat-
et al., 2003), also the crude fucoidan yields from ment at 35 and 70°C showed the molecular
Sargassum sp., extracted by HCl and CaCl2 weights of 1600 and 877 kDa, respectively
solution were similar (Baba et al., 2016). Other (Rioux et al., 2007; Rupérez et al., 2002).
acids could be used, i.e. 1% H2SO4 was selected The reported molecular weights of fucoidans
for Undaria pinnatifida (Hemmingson et al., are between 100-1,600 kDa. The hydrolysis
2006), A further advantage of the extraction into lower molecular weight oligomers occur-
with hot acid is the simultaneous precipitation ring under higher temperature and in mild
of alginates as alginic acid. The use of hot buffer acidic media is, in general, not desirable, but
(60°C, pH 4.0) extraction was efficient to obtain could be of interest to enhance some biological
a high Sargassum sp. fucoidan yield maintain- properties (Pielesz et al., 2011).
ing high sulfate contents. Additionally, protein- Fucoidan accounts for a relatively minor
aceous and phenolic compounds co-extracted component in brown seaweed biomass.
with crude fucoidan contributed to its antioxi- Conventional extraction processes presented
dant potential (Hifney et al., 2016). yields of 1.2–9.8 % (Duarte et al., 2001; Yang
The acid concentration is another factor et al., 2008a; Nakayasu et al., 2009; Foley et al.,
that can affect the extraction efficiency. The 2011; Sokolova et al., 2011; Huang et al., 2016),
most commonly used acid is HCl at 0.01–0.1 between 2-22 % for acid extraction (Kuda et al.,
M (Chotigeat et al., 2004; Lee et al., 2004; Ale 2002; Hemmingson et al., 2006; Foley et al.,
et al., 2012: Zhang and Row, 2015). Classical 2011; Ale et al., 2012; Rabanal et al., 2014) and
multi-step extraction processes using acid 4.9% for alkaline (Dias et al 2008). Therefore,
(0.2 M HCl) by elevated temperature and the utilization of sequential stages oriented to
extended time had a detrimental effect on the the successive separation of the constituents
yield as the structural integrity of the polymer according to a biorefinery concept would allow
was disrupted (Ale et al., 2012), and at higher an integral utilization of the resources.
concentration (0.35 M HCl) is difficult to A sequential extraction and purification of
maintain high fucose content (Zhang and fucoidan, phenolics, and alginate from F. vesic-
Row, 2015; Baba et al., 2016). Usually reaction ulosus using and acidic stage as part of the pro-
times in the range 1–5 h have been proposed. cess was reported. Different examples can be
Temperature is usually selected in the range found and some sequences appeared to per-
from 30 to 90°C. Shorter times and moderate form similarly for different brown seaweeds
temperatures (42°C, pH 1.0, 159 min) for the (Lorbeer et al., 2017). The thawed material
acid treatment of Ecklonia radiata, facili- kept at 20°C was contacted with distilled water,
tated the sequential extraction of fucoidans to obtain an exudate fraction, which was chro-
and alginates in an industrially relevant matographed to separate fucoidan fractions,
context (Lorbeer et al., 2015a). The amounts and the seaweeds were further extracted with
of fucoidan and laminarin extracted from acid (Beress et al., 1993). Fucus vesiculosus was
L. japonica increased until the temperature sequentially extracted with water at 22°C and
reached 80°C, and then the levels of the two 60°C, then with 0.1 M HCl and with 2 M KOH
at 37°C to extract alginate and fucoidan polysaccharides, alternative technologies have

(Rupérez et al., 2002). Sequential extraction of been developed and implemented in order to
S. tenerrimum with 0.1 M HCl at 25–30°C in reduce energy consumption, emissions, costs
2 stages, and extraction of the residue with 2% and to increase safety and products quality
K2CO3 at 40–60°C in 3 stages (Sinha et al., (Hahn et al., 2012; Chemat et al., 2017).
2010). Defatted algal fronds were successively Among them some physical techniques,
extracted with cold 0.4% HCl (20-25°C) and including pressurized solvents, ultrasound-
with hot water (60-70°C (Zvyagintseva et al., assisted extraction and microwave-assisted
2003), A. nodosum was extracted with 0.2 N extraction, as well as those using biotechno-
HCl and then with distilled water at 100°C logical aids, can be successfully proposed.
(Foley et al., 2011), Laminaria gurjanovae was Overviews of novel techniques to extract bio-
extracted with acid (0.1 M HCl) at room logically active compounds from seaweed,
temperature and then with water at 60°C including the comparative advantages and dis-
(Shevchenko et al., 2007), and Sargassum advantages are found in different works (Hahn
wightii, was successive extracted in alkaline, et al., 2012; Michalak and Chojnacka, 2014;
acid and neutral pH (Eluvakkal et al., 2014). García-Vaquero et al., 2017).
Defatted seaweeds were treated with 2% CaCl2
at 70°C in order to precipitate alginates and 44.2.5.1 Ultrasound-Assisted Extraction
to extract laminaran and fucoidan, further Ultrasound (US) waves are associated to sound
extracted with 0.01 M HCl at 70°C and the algi- at frequencies above those detected by the
nate was finally extracted with 3% Na2CO3 at human ear, in the range 20–100 MHz are used
70°C (Rioux et al., 2007). Acid extraction was in chemistry. The propagation of ultrasound
proposed after extraction of S. siliquosum with waves through an elastic medium induces a
boiling distilled water, the residue was sub- succession of compression and expansion (rar-
jected to extraction using 0.1 M HCl at room efaction) cycles, resulting in heating and cool-
temperature for 12 h and the residue was boiled ing, respectively. Compression and expansion
at 100°C for 1.5–3 h, and then with 1% NaHCO3 cause the cavitation phenomenon, leading to
at 65°C (Vasquez et al., 2012). Another complex production, growth and collapse of bubbles.
sequence was applied to Turbinaria conoides, During solid-liquid extraction, high shear
initially extracted with 0.1 M HCl at 30–35°C, forces in the media caused by cavitation at the
and the residue and further extraction with 3% vicinity of solid materials can lead to pitting
Na2CO3 at 45–50°C, the residue left after extrac- and erosion. The intense mixing effect gener-
tion with alkali was extracted with water at ated in the liquid medium, favored by the pres-
80°C (Chattopadhyay et al., 2010). A series of ence of surface defects, entrapped solid or
sequential extractions of Himanthalia elongata impurities, enhances the solute mass transfer
with deionized water, NaCl, and urea at room and the microbubbles aid in the disruption of
temperature and at 40°C was proposed for the cell walls. The beneficial effects of US on the
detailed characterization of polysaccharide solid-liquid extraction by ultrasound is associ-
fractions and the cell wall architecture in algae ated to combined physical and chemical mech-
(Deniaud-Bouët et al., 2014). anisms. Physical effects of ultrasound include
fragmentation, erosion, increased solvent
penetration into canals and pores of the
44.2.5 Alternative Extraction Procedures
solid matrix, sonoporation, local shear stress,
To overcome the drawbacks of conventional destruction of cell walls, and detexturation of
extraction methods, requiring long extraction biomass structures, intense shaking at a mac-
time, relatively high solvent consumption roturbulences and micromixing (streaming
and could cause thermal degradation of the and micro-streaming). Chemical effects are
mainly related to the presence of solvent vapor, to the medium. Temperature is highly rele-
high pressures and temperatures inside the vant since it determines the properties of the
bubble, that generate primary radicals suscep- solvent and the solute. Increased values
tible of forming other radicals and promoting enhance the solute solubility, but care is
redox reactions (Bendicho and Lavilla, 2013; needed in order to avoid degradation of ther-
Chemat et al., 2017). molabile compounds, undesirable reactions
Equipment for extraction purposes require and to control cavitation. An increase of tem-
baths or probes, and can be implemented both perature results in a decrease of both viscosity
at lab and at industrial scale (Chemat et al., and surface tension, and an increase of vapor
2017). Most studies at lab scale are performed pressure. A rise in vapor pressure reduces
in batch extraction, but also semicontinuous sonication effects since more solvent vapors
processing by percolation with forced circu- enter the bubble cavity, and bubble collapse
lation and periodic ultrasound treatment less violently. Sonochemical effects are
(Obluchinsksya et al., 2015) and continuous favored by low temperatures. Thermostatized
industrial operation were reported. Flow- baths or water and/or ice baths with probes
through sonication can be used both in labora- are used to keep a steady temperature
tory and industrial processes. The basic (Chemat et al., 2017; Lavilla and Bendicho,
elements of an ultrasonic bath are the tank, the 2017). Dissolved gases facilitate formation of
electronic generator and the ultrasonic source cavitation bubbles from gas (vapors) dissolved
or transducer, which converts mechanical in the liquid and act as nuclei for bubble. To
or electrical energy into ultrasonic energy. overcome the limitations associated to liquid
Ultrasonic baths operate at 20–100 kHz, and degassing during ultrasonication, inert gas
the frequency determines the cavitation and bubbling was proposed to control composi-
the energy released (Chemat et al., 2017; tion of cavitation bubbles, and the addition of
Lavilla and Bendicho, 2017). Probes include a glass beads to increase particle disruption
generator (usually 20 kHz), transducer and (Lavilla and Bendicho, 2017). Frequency has
horn, which amplifies and concentrate ultra- to be selected for each material, and its varia-
sound energy, enhancing the effectiveness tion can result in a more selective extraction.
compared to bath systems. The intensity of cavitation decreases with fre-
The major factors affecting the action of quency, because the cavitation bubbles need a
ultrasound include those referring to the delay to be initiated during the rarefaction
medium (particle size, liquid:solid ratio, tem- cycle. The size of the cavitation bubble has an
perature, presence of gases) and those of the effect on the efficiency on the extraction. The
equipment (power, frequency, shape and size, solid:liquid ratio and particle size of the mate-
and time of sonication) (Vinatoru et al., 1998; rial are relevant to the efficacy of extraction.
Chemat et al., 2017). The extraction yield usu- The extraction yields may vary also due to
ally increases with power up to a certain plant material’s structure, plasticity or com-
threshold. The power intensity, expressed per positional differences which will result in dif-
area (W/cm2) or per volume (W/cm3), is more ferent degrees of impacts from cavitation
suited for comparative and scalable purposes effects (Chemat et al., 2017). With the increase
(Lavilla and Bendicho, 2017). Ultrasonic in the pressure amplitude, bubble collapse
intensity, expressed as the energy transmitted more violently. Shape and size of extraction
per time and emitting surface, is correlated to vessel, dimensions and position of the extrac-
the amplitude of the transducer and to the tion vessel in relation to transducers, also the
pressure amplitude of sound wave. The effec- probe tip height as well as the material,
tive US power can be calculated from the tem- should be considered (Lavilla and Bendicho,
perature increase due to the heat dissipated 2017). Some recommendations relating vessel
geometry have been suggested, flat bottom 44.2.5.2 Microwave Assisted Extraction
vessel such as a conical flask in order to attain Microwaves are radiations with frequency
a minimum reflection of waves and minimum ranging from 300 MHz to 300 GHz, generated
thickness of the vessel to reduce attenuation. by an electric field and a magnetic field oscil-
Exposure time has a parallel effect on the deg- lating perpendicularly to each other. Most
radation of cells structures, but too prolonged commercial microwave equipment, including
time could lead to undesirable reactions domestic ovens, operate at 2450 MHz,
(Lavilla and Bendicho, 2017). whereas industrial equipment operate at 915
Ultrasound is considered a green technol- MHz. Microwave heating occurs by ionic con-
ogy offering selectivity, reproducibility, mild duction and dipole rotation. The phase differ-
operation conditions, reduced thermal gra- ence between the orientation of the alternate
dients, more effective mixing, resulting in field and that of the dipole and the resistance
enhanced extraction yield and rates, and a of the solution to migration of ions under
reduction in extraction time, equipment size, the influence of the changing electric field
energy and use of solvent. UAE has high causes friction responsible for heating
possibility of industrial application since it is (Galema, 1997; Lidström et al., 2001). The
cost-effective in comparison with other novel ability of a material to interact with electro-
extraction techniques, easily scaled up, with magnetic energy and the efficiency of micro-
the possibility of high level of automation wave heating depends on the dielectric
and combination with conventional and/or properties of the material and its ability to
innovative extraction technologies (Bendicho absorb microwave energy and to dissipate heat
et al., 2012; Song et al., 2015; García-Vaquero (Lidström et al., 2001).
et al., 2017). Whereas in conventional extraction pro-
Ultrasound assisted extraction was used to cesses heating is mainly transmitted through
obtain polysaccharides from algae (Wijesekara convection and conduction, in microwave
et al., 2011), phenolics and fucoidans from A. heating, energy is delivered through molecular
nodosum during acid extraction (Kadam et al., interaction and the temperature gradient is
2015a), and also fucoidans from Sargassum inverted respect to conventional heating and
polycystum by ultrasonic wave pretreatment both energy and mass transfer gradients occur
(amplitude 80%, 15 min) before acid extraction in the same direction, from inside to the out-
(Sugiono et al., 2014). Ultrasound exposure side of the solid matrix (Flórez-Fernández
during extraction in mild acid medium was et al., 2015). Therefore, both energy and mass
reported to affect the content of F. vesiculosus transfer are facilitated. In addition, the sudden
fucoidan, and the composition of sugars, sul- increase of temperature of the intracellular liq-
fate groups and polyphenols, as well as their uids, causing water evaporation and pressure
biological activity (Obluchinsksya et al., 2015). on the cell walls, renders the protoplasm more
Following ultrasonication, the average molec- permeable, solutes more accessible to solvent,
ular weight of fucoidans decreases (Flórez- and facilitates the release of intracellular con-
Fernández et al., 2017a) and the biological tents into the medium (Zhou and Liu, 2006;
properties were enhanced, as reported for the Michalak and Chojnacka, 2014). Disruption
anti-inflammatory potential of U. pinnatifida of weak hydrogen bonds, promoted by the
(Song et al., 2015) or the immunomodulatory dipole rotation of the molecules, facilitates
properties of Hizikia fusiforme (Ha et al., 2008). disruption of the solute-matrix interactions
The technology has also been used to breakdown (Teo et al., 2009).
already extracted polysaccharide fractions The main elements in the equipment are
(Flórez-Fernández et al., 2017b; García-Vaquero the magnetron to generate microwaves, the
et al., 2017). waveguide to propagate the microwave to the
applicator, where the sample is placed, and (Veggi et al., 2013). The increasing microwave
the circulator, to move forward the microwave power favors the penetration of solvent into
radiation (Mandal et al., 2007). Two configu- the matrix and the dissolution and recovery
rations (open and closed) are possible of solutes, thus leading to increased yields.
depending on the operation pressure. Open However, power has to be selected to maxi-
vessels work at atmospheric pressure and mize yields and selectivity of the desired com-
closed vessels operate with the solvent under ponents, without affecting their stability and
pressurized conditions. Whereas the maximal activity. Extraction time influences the extrac-
temperature in open vessels is defined by the tion yield with a beneficial effect up to a maxi-
water boiling point, in pressurized equipment mum and further it could lead to degradation.
higher temperatures can be reached and the Microwave power and irradiation time could
physicochemical properties of water can cor- be considered together for process optimiza-
respond to subcritical conditions (Lidström tion, and temperature is also related to the
et al., 2001). Therefore, open systems require extraction time and the irradiation power. An
milder conditions, offer the possibility of increase in extraction temperature can
adding/removing solvent during the process enhance the yields due to the increased diffu-
without requirements for cooling down or sivity of the solvent into the solid and to the
depressurization and need lower investment, desorption of solutes, but formation of by-
but need longer operation times, whereas products, decomposition and/or degradation
closed systems require less solvent and shorter of target compounds could occur. The opera-
operation times than in open vessel systems tion temperature highly influenced the out-
(Mandal et al., 2007; Chan et al., 2011). comes of the process, fucose was the main
Depending on the distribution of radiation, monosaccharide of A. nodosum fucoidan
operation can be performed in single mode, extracted at 90°C whereas glucuronic acid
when radiation is focused providing an homo- was the main monosaccharide of fucoidan
geneous distribution on a restricted zone or in extracted at 150°C (Yuan and Macquarrie,
multimode, when radiation is reflected in the 2015b). Under pressurized conditions shorter
walls and results in a random dispersion of times (30 min) can be operative (Rodriguez-
radiation (Chan et al., 2011; Mandal et al., Jasso et al., 2011), but open-vessel micro-
2007). Modified techniques can enhance the waves offer a highly efficient and controllable
microwave process performance, such as alternative to convective heating for the
incorporation of high pressure, vacuum, inert extraction of fucoidan. Short times were
gases or ultrasounds (Chan et al., 2011; Flórez- enough for the extraction of fucoidans from
Fernández et al., 2015). Operation in batch E. radiata under classical conditions (HCl
and semi-continuous of this scalable tech- pH 2, 60°C), since only modest gains in yield
nique (Chemat and Cravotto, 2013) have been were achieved beyond 6 min, due to undesir-
reported. able declines in fucose and sulfate content,
Particle size has the same influence as in increase in laminarin, and reductions in the
other extraction processes, based on facili- molecular weight of fucoidans (Lorbeer et al.,
tated mass transfer at the larger surface area 2015b).
of smaller particles, but also influences the This energy-assisted method requires lower
heating rate in relation to the penetration amounts of solvent and provides improved
depth (Veggi et al., 2013). Similarly, addi- extraction yields, shortens extraction time
tional considerations on the liquid:solid ratio and energy consumption with advantages of
based on the ability of water to absorb energy reduced equipment size, and can be per-
are needed in microwave assisted processes formed in compact processes with potential to
be scaled up (Mandal et al., 2007; Li et al., 44.2.5.3 Enzyme-Assisted Extraction

2013). However, the heat generated during Cell wall degradation improves the accessibil-
the extraction process may cause damage to ity of solvent to solutes found intra-cellularly
heat-sensitive compounds (Michalak and in cell cytoplasm, to those that are part of walls
Chojnacka, 2014). or retained by hydrogen or hydrophobic bond-
Microwave radiation, firstly proposed for ing, and not accessible with a solvent in rou-
aiding in the desulfation stage, was also used to tine extraction processes. Hydrolytic enzymes,
assist in depolymerization of crude fucoidan. used either alone or in mixtures, have the abil-
After 1 min of heating led to a removal of ity to degrade or disrupt cell walls and mem-
60–93 % of the original sulfate, and besides the branes more efficiently than other techniques,
loss of sulfate and a moderate depolymeriza- such as ultrasound, and have potential for aid-
tion, the integrity of the polysaccharides was ing in the extraction of seaweeds bioactives
not affected by this procedure (Navarro et al., (Wijesinghe and Jeon, 2012; Adalbjörnsson
2007). It has been suggested that during the and Jónsdóttir, 2006).
heat treatment of U. pinnatifida, rather than Various factors including the particle size,
polymeric degradation, occurred a disruption solid to water ratio, enzyme composition
of the secondary interactions between fucoidan and concentration, temperature, pH, and
polymers, which facilitated a better dissolu- hydrolysis time are key factors, some of them
tion. The MW markedly decreased from 23,600 cooperatively affecting the extraction perfor-
to 2400 kDa after 30 s, whereas only a slight mance. Multiactivity complexes or cocktails of
decrease to 1900 kDa occurred up to 90 s and to enzymes offer a broad spectrum of activities to
500 kDa after 120 s. The value from 30 s-micro- disrupt the cell wall, facilitating hydrolysis due
wave heating was much lower than the values to synergistic effects, although the optimal
(5200–5900 kDa) by boiling water for 15 min conditions could require a compromise selec-
(Yang et al., 2008b). However, microwave tion among the optimal for all formulations
assisted acid (0.01 N HCl) hydrolysis of (Siriwardhana et al., 2008). Polysaccharides
fucoidans from U. pinnatifida did not enhance are the major component of the algae and the
anticancer activity and showed low inhibition use of these carbohydrases seems promising,
if treated more than 90 s, whereas when hydro- but also proteases have been successfully
lyzed in boiling water for 5 min, fucoidans proposed. Brown seaweeds contain 5-15 %
significantly increased anticancer activity, sug- proteins, which can easily combine with phlo-
gesting that the anticancer activity of fucoidans rotannins and both carbohydrates and proteins
could be enhanced by lowering their Mw only are barriers for the extraction of antioxidant
when they are depolymerized by mild condi- compounds from seaweeds (Pérez et al., 2013,
tion (Yang et al., 2008a). Some studies used 2014; Siriwardhana et al., 2008). Hydrolysis of
MAE on previously extracted fucoidans from proteins produces peptides which enhance the
U. pinnatifida to produce more valuable low- bioactivity of the algal extract (Adalbjörnsson
molecular-weight products of around 5–30 and Jónsdóttir, 2015). The use of proteases
kDa (You et al., 2010) and 446.5–3.1 kDa (Li favored the concentration of sulfated sugars in
et al., 2013). Also, Flórez-Fernández et al., the crude extract, attaining comparable values
(2017b) found a significant decrease of the as those found with hot water and with ultra-
oligomeric content in the crude fucoidans sound assisted extraction (Rodrigues et al.,
extracted by subcritical water extraction 2015). Enzymatic degradation of sulfated poly-
(mildly acidic medium), and further subjected saccharides preserving the sulfate groups can
to posthydrolysis under microwave irradiation be achieved by hydrolases, fucoidanases, α-L-
at 100 and 600 W during 1 min. fucosidases and galactosidases (Jiao et al.,
2011). Since most commercially available for- facilitates the accessibility of solvent to solutes
mulations do not hydrolyze fucoidan, these and also the accessibility of the enzyme to the
products are appropriate for the extraction of substrate. Liquid:solid ratio during enzyme
sulfated polysaccharides (Hahn et al., 2012). digestion is useful to assist in the hydrolytic
Selection of appropriate hydrolytic activities or reaction, as well as in the mobility of the
optimal mixtures is determinant because enzymes and products. pH and temperature
enzymes may constitute one of the major costs influence the activity and maximum values are
of treatment. After selection of the suitable enzyme-dependent, being amyloglucosidases,
enzymes various process conditions can be agarases, proteases, cellulases, β-glucanases
defined in order to obtain the maximum recov- and xylanases the most used activities at opti-
ery of active components. The enzyme to sub- mum conditions of pH (3.8–8) and tempera-
strate ratio has to be optimized since this factor ture (40–60°C) (Heo et al., 2005, Wijesinghe
significantly affects the efficiency and costs. and Jeon, 2012; Rodrigues et al., 2015; Petigny
Values in the range 0.2–3.0 % have been usu- et al., 2013). Temperature affects the enzyme
ally proposed, although higher concentration activity and reaction rates up to an optimum,
of enzymes have also been found. Some exam- above which enzymes are denatured and bio-
ples of the application of enzyme assisted actives can be degraded. Prolonged incubation
extraction of fucoidans, with the selected oper- times can enhance the degradation of cell
ational conditions are shown in Table 44.3 and walls, the optimal values have been commonly
are found in other works (Ahn et al., 2003; Heo found at 3–12 h (Heo et al., 2005; Siriwardhana
et al., 2003 and 2005; Kim et a., 2006; et al., 2008).
Wijesinghe and Jeon, 2012; Rodrigues et al., Enzymatic assisted extraction is an eco-
2015, Charoensiddhi et al., 2016). The mechan- friendly water based technology free from chem-
ical pretreatment causing cell wall breakage, icals (Puri et al., 2012), allowing enhanced yields
Table 44.3 Some examples of the alternative technologies applied for the extraction of fucoidan
from seaweeds.
Conditions (liquid:solid ratio

Seaweed (mL/g), enzyme, T, pH, t) References
Enzyme-assisted extraction
Canistrocarpus 5 mL/g, protease, 60°C, pH 8.0, 24 h Camara et al., 2011
cervicornis
Dictyopteris delicatula 5 mL/g, protease, 60°C, pH 8.0, 18 h Costa et al., 2010;
Magalhaes et al., 2011
Dictyota sp. 5 mL/g, protease, 60°C, pH 8.0, 24 h Costa et al., 2010
Durvillaea antarctica 100 mL/g, cellulase, α-amylase, 50–60°C, Olivares-Molina and
pH 4.5–6, 17 h Fernández, 2016
Ecklonia cava 20 mL/g, amyloglucosidase, 60°C, pH 4.5, Athukorala et al., 2006;
12–24 h Kang et al., 2011
Ecklonia cava 100 mL/g, carbohydrases, proteases, Heo et al., 2005; Kim et al.,
40-60°C, pH 4.5–8.0, 12 h 2006
Ecklonia radiata 100 mL/g, carbohydrases, proteases, Charoensiddhi et al., 2016
50°C, pH 6, 24 h
Hizikia fusiformis 100 mL/g, protease, 50–60°C, Siriwardhana et al., 2004,
pH 6.5–8.0, 72 h 2008
Conditions (liquid:solid ratio

Seaweed (mL/g), enzyme, T, pH, t) References
Ishige okamurae 100 mL/g, protease, 40–60°C, pH 6–8, 12 h Heo and Jeon, 2008
Lessonia nigrescens 100 mL/g, cellulase, α-amylase, 50–60°C, Olivares-Molina and
Lobophora variegata 2 mL/g, protease, 60°C, pH 8.0, 24 h Paiva et al., 2011
Macrocystis pyrifera 100 mL/g, cellulase, α-amylase, 50–60°C, Olivares-Molina and
Padina gymnospora 5 mL/g, protease, 60°C, pH 8.0, 24 h Silva et al., 2005
Sargassum coreanum 60 mL/g, protease, pH 6, 50°C, 12 h Ko et al., 2012
Sargassum filipendula 5 mL/g, protease, 60°C, pH 8.0, 24 h Costa et al., 2010
Scytosiphon lomentaria 100 mL/g, carbohydrases, proteases, Ahn et al., 2004
40–60°C; pH 4.5–8.0, 12 h
Sargassum horneri 50–100 mL/g, carbohydrases, proteases, Heo et al., 2005; Sanjeewa
40–60°C, pH 4.5–8.0, 24 h et al., 2017
Spatoglossum schroederi 5 mL/g, protease, 60°C, pH 8.0, 24 h Costa et al., 2010
Microwave-assisted extraction
Ascophyllum nodosum 1. 120°C, 15 min Yuan and Macquarrie,
2. MW: 17.6 mL/g, 0.1 M HCl, 90°C, 15 min 2015a
Ecklonia cava HCl (pH 2), 30 mL/g, 60°C, 6 min Lorbeer et al., 2015b
Fucus vesiculosus DW 25 mL/g, 120 psi, 1 min Rodriguez-Jasso et al., 2011
Undaria pinnatifida DW, 125 mL/g, 600 W, 140°C, 1 min Quitain et al., 2013
Pressurized liquid extraction
Himanthalia elongata DW, 103 bar, 20 min Santoyo et al., 2011
Saccharina japonica 0.1N NaOH, 140°C, 50 bar, 5 min Saravana et al., 2016
Saccharina japonica DW, 120°C, 3 h Wang et al., 2008
Sargassum fulvellum DW, 120°C, 3 h Jo and Choi, 2014
Sargassum muticum DW, 8 mL/g, 170°C; 36min González-López et al., 2012;
Balboa et al., 2013, 2015
Undaria pinnatifida DW, 125 mL/g, 140°C, 10 min Quitain et al., 2013
Ultrasound assisted extraction
Ascophyllum nodosum 0.03M HCl, US probe 75.78 W/cm2, 0.42–8 h Kadam et al., 2015a and b
Fucus vesiculosus US bath (42 kHz, 335 W), 20 min/h, 35°C, 4 Obluchinsksya et al., 2015
h, pH 4
Sargassum muticum HWE 50°C, 24 h; 2. US bath, 400 W, 50/60 Rodrigues et al., 2015
Hz, 50°C, 60 min (US 10 min, pause 2 min)
Sargassum muticum US bath, 25°C, 150 W, 40 Hz, 30 min Flórez-Fernández et al., 2017
Sargassum polycystum US pretreatment, US bath, 80% amplitude, Sugiono et al., 2014
15 min
0.03M HCl, 81°C, 4 h
Undaria pinnatifida 0.1M HCl, US probe 80% amplitude, 6 h Song et al., 2015
DW, distilled water; US, ultrasound.

HWE, hot water extraction.
MW, microwave.
and quality of the extracts, and could conform provide knowledge about structural character-
an initial step of a biorefinery based process. The istics of fucoidans (Chattopadhyay et al., 2010;
milder conditions of temperature and pressure Ale et al. 2013; Deniaud-Bouët et al., 2014).
offer a promising alternative to conven- Enzyme assisted extraction is a popular
tional methods, conserving the bioactivity of green extraction method due to the increased
the fucoidans (Kulshreshtha et al., 2015). extraction efficiency compared to water
Additionally, the enzymes used are non-toxic extraction and nontoxic nature compared to
food grade and could be used in large scale. the organic solvent extraction methods. The
However, the industrial applications could be compounds are cheaper and safer to use as
limited by the high price of enzymes, and by the material in functional foods, cosmeceuticals or
need to develop efficient strategies to recycle nutraceuticals. The sulfate content, a param-
them (Hahn et al., 2012; Petigny et al., 2013; eter related with the antioxidant and biological
Michalak and Chojnacka, 2014). Crude prepara- properties, was maintained (Sanjeewa et al.,
tions are potential agents due to the importance 2017; Fernando et al., 2017). In some cases, the
of synergistic effects with minor activities. A incorporation of carbohydrases and proteases
number of studies on the enzyme assisted extrac- during the extraction process had little or no
tion of seaweeds can be found, in most of them impact on total sugar yield from Ecklonia
the objective was the extraction of antioxidant radiata, although the molecular weight profile
fractions, and total phenolics (Ahn et al., 2003; was reduced by 20–50% compared to control
Heo et al., 2005; Siriwardhana et al., 2008), total extractions (Charoensiddhi et al., 2016).
sugars (Rodrigues et al., 2015), and fucoidan Also, despite the lower yields, Sargassum sp.
fraction (Camara et al., 2011; Costa et al., 2011; fucoidan that had been extracted by 15 mg/g of
Dore et al., 2013; Castro et al., 2015). As an aver- papain enzyme solution showed lower content
age, algal crude extracts contained mainly carbo- of fucose than that extracted by 0.15 M HCl
hydrates (45%), proteins (30%), polyphenols or by alkaline (CaCl2). However, fucoidan
(20%), ash (10%), and lipids (1%). Most studies extracted by acidic solvents could result in the
reporting the enzyme-assisted extraction of simultaneous extraction of undesirable prod-
macroalgal material propose the use of commer- ucts such as alginic acid and metals (Baba
cial food-grade enzymes, mainly proteases and et al., 2016).
carbohydrases, developed for terrestrial plant
material for the extraction of polysaccharide 44.2.5.4 Subcritical Water Extraction
fractions with antioxidant activity and biological Subcritical water extraction (SWE) is also
activities (Charoensiddhi et al., 2016; Ahn et al., known as high-temperature water extraction,
2003; Heo et al., 2005; Ko et al., 2012). superheated water extraction, pressurized hot
Commercial proteases and carbohydrases water extraction, hot liquid water extraction or
significantly improved biomass yield by 2–3 pressurized low polarity water extraction
times over that achieved with water, and the (Conde et al., 2010). This process is a particular
extracts showed a variety of biological activi- case of pressurized liquid extraction pressur-
ties (Ahn et al., 2004; Heo et al., 2005a, b; ized fluid extraction (PFE), accelerated fluid
Athukorala et al., 2006; Kim et al., 2006; Ahn extraction (ASE), enhanced solvent extraction
et al., 2008; Wang et al., 2010; Dore et al., 2013; (ESE), and high pressure solvent extraction
Pérez et al., 2013 and 2014; Kulshreshtha et al., (HSPE), when water is used as solvent.
2015; Olivares-Molina and Fernández, 2016). The basics of this process is the application
Mild extraction techniques coupled with the of high pressure to maintain the solvent in
use of enzyme digestion was useful for the liquid phase in conditions of pressure and
exhaustive sequential extractions and further temperature between the boiling temperature
composition analyses of cell wall material to at normal conditions (100°C, 1 bar) and the
critical point (374°C and 221 bar). Under may possibly reduce the quantity of other
these conditions, a marked reduction can be impurities such as alginate and polyphenols
noticed in viscosity, density, and in the dielec- (Saravana et al., 2016).
tric constant ε, the value at 25°C and atmos- Temperature, pressure, extraction time and,
pheric pressure is 80, a high value reflecting in semi- and continuous operation, flow rate,
the presence of hydrogen bonds formed by are the most influencing variables. Viscosity
water molecules. As temperature rises, this and density decrease and water diffusivity
value decreases, at 250°C and 50 bar is 27, a increases with temperature; therefore, this var-
value similar to that of ethanol at 25°C and 1 iable has a marked effect on yield and selectiv-
bar. The unique properties of water at ele- ity. By selecting the temperature, water polarity
vated temperatures and pressures, particu- can be tuned and can solubilize more nonpolar
larly the ionic product, dielectric constant, molecules (Rodríguez-Meizoso et al., 2006),
and density, make it an interesting reaction also the formation of novel compounds with
medium, since the biopolymers can react in improved activity was observed (Plaza et al.,
very short residence times and with high rates 2010). Temperature is determinant in the
of conversion (Rogalinski et al., 2008). The release of compounds forming part of the cell
addition of carbon dioxide has also been pro- walls, but adequate selection has to be consid-
posed for the hydrolysis of terrestrial biomass, ered to limit degradation reactions (Garrote
since carbonic acid serves as a catalyst. et al., 2003; Rodríguez-Meizoso et al., 2006).
Sometimes this technique is denoted as auto- The higher extraction temperature enhances
hydrolysis, a term referring to the hydrolytic solute solubility and the water transport prop-
breakage of polymers, promoted by the higher erties are improved, diffusivity increases with
value of the ionic product of subcritical water temperature and also decreases the viscosity
(10–12), compared to that of water at ambient and surface tension.
conditions (10–14) (Garrote et al., 1999; Subcritical water extraction proved suitabil-
Brunner, 2009). ity for the solubilization and depolymerization
Operation at high pressure requires corro- of fucoidan fractions (Balboa et al., 2013), and
sion special materials. Most studies have been it can be an initial part of a biorefinery process
performed in batch mode at small scale, semi- (González-López et al., 2012; Balboa et al.,
continuous and continuous flow equipment 2015). The highest ethanol precipitation after
(Petigny et al., 2015) and shows possibility autohydrolysis of F. vesiculosus was obtained at
of scaling-up and industrial application 180°C for 20 min (Rodríguez-Jasso et al.,
(Rodríguez-Meizoso et al., 2006). 2013), however lower temperatures have
Compared to conventional extraction, this been reported for fucoidan yield, 170°C for
technique facilitates the extraction process, Sargassum muticum (Balboa et al., 2013) or
decreasing extraction time and solvent require- 140°C for Undaria pinnatifida (Quitain et al.,
ments, being more efficient for the extraction 2013). The extraction efficiency is usually
and depolymerization of polysaccharides higher than with conventional solvents
(Garrote et al., 1999; Parajó et al., 2004; Balboa (Wang et al. 2008; Ale et al., 2012). Pressurized
et al., 2013; Khuwijitjaru, 2016) and is per- liquid extraction significantly improved the
formed in an oxygen and light-free environ- extraction yields from Himanthalia elongata
ment (Santoyo et al., 2011). Another advantage fucoidans using an accelerated solvent extrac-
of pressurized water extraction has been tor, with water as solvent at 103 bar (Santoyo
reported in relation to the enhanced purity of et al., 2011), and the yield of crude fucoidan
the extracts, particularly in a reduction of pro- from Saccharina japonica under pressurized
tein contamination in crude fucoidans, lower conditions (5–100 bar), and different solvents
than that with formic acid and NaOH, and it (water, 0.1% NaOH, 0.1% formic acid, and 25,
50 or 70% ethanol); the maximum yield was 2013), interaction of microwaves and ultra-
attained at 140°C and 50 bar (NaOH) (Saravana sound to enhance the effects of high frequency
et al., 2016). electromagnetic fields of microwaves, and the
Hydrothermal treatment (<140°C) is a good cavitational energy associated with sono-
alternative for depolymerization of fucoidan chemistry, the simultaneous use of micro-
without causing desulfation (Morimoto et al., waves and ultrasound to combine the effects
2014). This alternative is more rapid than the of enhanced energy with improved matter
aqueous processing at room temperature for transportation (Leonelli and Mason, 2010), or
prolonged times (2 weeks) (Anastyuk et al., the ultrasonic treatment during enzyme
2014), which leads to desulfated low molecular hydrolysis to accelerate the hydrolysis of poly-
weight fractions (Menshova et al., 2015). saccharides (Yachmenev et al., 2007), based
Advantages and challenges of these innova- on the effects on the surface of the substrate,
tive techniques are summarized in Table 44.4. the disaggregation of the enzyme molecules
They can also be performed in combination: and the removal of products. The conditions
microwave heating in closed systems to per- should be chosen carefully to avoid enzyme
form hydrothermal treatments (Quitain et al., denaturation.
Table 44.4 Advantages and limitations of the conventional and emerging techniques for the extraction
of fucoidan.
Extraction technology Advantages Disadvantages
Conventional Simplicity Low yield and selectivity

Long extraction time
High temperatures
High energy consumption
Generation of impurities
Pressurized hot Increased solubility, high yield Cost of equipment
water Elevated temperature and pressure Degradation of thermolabile
Selective solubilization by modulating compounds
operation severity
Hydrolysis of polymeric fractions
Shorter extraction time
Enzymatic assisted Moderate temperature Slow process
extraction Selective breakage Limited enzyme recycling
Food-grade enzymes available Enzyme cost
Scalable
Ultrasound assisted Low cost, simplicity and selectivity Lack of uniformity in the
extraction Effective mixing vessel
Fast start-up and response Localized heating and cooling
Mild conditions, low temperature and time needed
Reproducibility, scalability Formation of radical species
Combinable with other processes Noise pollution
Microwave assisted Fast start-up, uniform heating High energy demand

extraction Compact equipment Degradation of thermolabile
High yields compounds
Less solvent, energy and time
44.3 Purificatio 1111
44.3 Purification extracts to further purification processes

as precipitation, liquid-liquid fractionation,
Extracts obtained by the extraction techniques membrane filtration and anionic, size-exclu-
reported above are complex mixtures com- sion or affinity chromatography, as reported in
posed mainly of polysaccharides, proteins the works shown in Table 44.5. Precipitation
and polyphenols. In order to obtain purified is a complementary process after extraction
fucoidan fractions it is necessary to subject the which allows a fast, cheap and simple separation
Table 44.5 Examples of purification technologies for fucoidan refining from seaweed extracts.
Seaweed/crude extract Fractionation processes References
Precipitation
Undaria pinnatifida / Cen., prec. (1% CaCl2, 4°C, overnight), cen., Yang et al., 2008a
aqueous prec. (etOH), filtr., etOH, wash. (acetone)
S. polycystum /aqueous Cen., CaCl2, prec. (etOH), filtr. Palanisamy et al., 2017
S. cristaefolium /aqueous Cen., prec. (etOH), cen. Wang et al., 2015
Sargassum stenophyllum / Fra. (10% cet-Br), cen., dissolved (DW), Duarte et al., 2001
aqueous addition NaCl 0.5 M, fra. (1-pentanol), dia.
(membranes 12-14 kDa), f-dr.
Sargassum wightii /aqueous Prec. (1% CaCl2, 4°C, overnight), cen., Kumar et al., 2015
fra. (30% etOH, 4°C, 4 h)
Sargassum sp. /aqueous Prec. (3% hydrate-CaCl2, etOH), cen., dr. Hifney et al., 2016
Chnoospora minima, Prec. (95% etOH, 4°C, 8 h), cen. Shanura et al., 2017
Halimeda discoidea,
Halimeda gracilis,
Chnoospora minima /
aqueous
Hizikia fusiforme /aqueous Prec. (etOH, CaCl2) Li et al., 2006
L. japonica /aqueous Prec. (etOH), wash. (acetone, ether), dr. (60°C) Zhang and Row, 2015
Sargassum sp. /acidic Prec. (etOH), cen., wash., dr. Baba et al., 2016
U. pinnatifida /acidic Prec. (etOH), cen., wash. (etOH, acetone), dr. You et al., 2010
Ecklonia radiata / Prec. (70% etOH), cen. Charoensiddhi et al.,
enzymatic 2016
S. horneri /enzymatic Prec. (etOH, 4°C, 24 h), cen., f-dr. Sanjeewa et a., 2017
L. japonica /aqueous Prec. (75% etOH), wash. x3 (anhydrous- Wang et al., 2009
etOH), dr. (80°C)
Liquid–liquid fractionation
S. pallidum /aqueous Frac. (different etOH concentration), wash. Liu et al., 2016
(acetone), dr. (60°C), depr. ×3 (Sevag method)
Lobophora variegata / Frac. (increasing acetone concentration) Paiva et al., 2011
enzymatic
Membrane filtration
S. mcclurei /acidic Dia. (5 kDa) Thinh et al., 2013
U. pinnatifida /ethanolic, Dia. (3.5 kDa), f-dr., dissolved (DW). Song et al., 2015
acidic
(Continued)
Saccharina gurjanovae Dia. by UF (5 kDa), f-dr. Shevchenko et al., 2015

F. evanescens, S. japonica, S. Dia. Menshova et al., 2013
oligocystum /acidic
Fucus vesiculosus /aqueous Dia., f-dr. Rupérez et al., 2002
Ion-exchange chromatography
Sargassum fusiforme / Prec. (CaCl2), chr. (DEAE-cellulose, Cong et al., 2016
aqueous Sephacryl S-300), dissolved (DW), prec.
(CaCl2), cen., dia., prec. (95% etOH), dr., chr.
(DEAE-cellulose), eluted (DW, aqueous-
NaCl, 0.3M NaOH)
Undaria pinnatifida /acidic Chr., dia. (UF-14 kDa), f-dr., dissolved (DW), Hemmingson et al., 2006
chr. (DEAE-Sepharose CL-6B), elution (DW,
1, 2, 4 M NaCl), dia., f-dr.
E. cava MF, UF (0.1 μm, 50 kDa) Fraction >50 kDa Kang et al., 2011
prec. (99.5% etOH), cen., chr. (DEAE-cellulose),
equilibrated (50 mM CH3COONa pH 5.0),
wash. (same buffer 0.2M NaCl), elution (linear
gradient same buffer 0.2–1.2M NaCl)
Fucus vesiculosus /aqueous Prec. (etOH), filtr., dissolved, cen., dr., chr Fujimura et al., 2000
(DEAE-cellulose).
Himanthalia elongata / Anion-exchange chr. (DEAE-Sepharose CL 6B, Deniaud-Bouët et al.,
enzymatic equilibrated (50 mM CH3COONa buffer pH 2014
5), dissolved (same buffer), eluted (buffers of
increasing NaCl concentrations; 1M NaOH),
dia., f-dr.
Coccophora langsdorfii / Chr. (DEAE-cellulose), equilibrated (0.04N Anastyuk et al., 2014
acidic HCl), elution (linear gradient 0–2M aqueous-
NaCl), dia. (UF), f-dr.
Gel or size-exclusion chromatography

Padina gymnospora Prec. (acetone), cen., dr. chr. (Sephadex G-75), Silva et al., 2005
elution (0.2M acetic acid)
Himanthalia elongata / Size-exclusion chr.: dissolved (DW), chr. Deniaud-Bouët et al.,
enzymatic (Superdex-200, 50 mM (NH4)2CO3 2014
(Superdex-30 or 200).
Affinity chromatography
Fucus vesiculosus Chr. amino-derivatized Sepabeads® EC-EA Hanh et al., 2016
with toluidine blue
Sequential
Dictyota dichotoma /acidic Pre. (cetrimide), dissolved (increasing NaCl Rabanal et al., 2014
solutions).
Sargassum sp. /CaCl2 Pre. (10% cet-Br, 4°C, overnight), filtr, cen., Baba et al., 2016
wash. (DW), stirred 20% ethanolic NaI,
overnight, rt (×4), wash. (etOH), dissolved
(DW), dia. (2000 Da), f-dr.
44.3 Purificatio 1113
Adenocystis utricularis / Prec. (2% CaCl, rt.+70°C), fra. (cetrimide), Ponce et al., 2003
CaCl2, acidic prec. (10% cet-Br, overnight), cen., dissolved
(0.5M NaCl, overnight), cen., fra.
(1-pentanol), dia., concentrated, f-dr.
Sargassum fulvellum /acidic Dia. (10–12 kDa), pre. (etOH), dissolved (DW), Kim et al., 2007
pre. (CPC), pre. (3M CaCl2), cen., dia., f-dr.
S. binderi /CaCl2 Prec. (10% cet-Br), cen., wash. (DW), stirred Lim et al., 2014
(20% ethanolic NaI, wash. (etOH), dissolved
(DW), dia. (2 kDa).
S. fulvellum /aqueous Prec. (acetone, 4°C, 24 h), cen., dr., dissolved Jo and Choi, 2014
(DW), filtr. (siliceous earth), dia, prec.
(etOH), wash., dr.
H. fusiforme /aqueous Dia., prec. (CaCl2, 4°C, overnight), cen., dial., Wang et al., 2012
concentrated, f-dr., ion-exchange chr.
(Q-Sepharose, elution (step gradient 0–3 M NaCl)
F. evanescens /acidic Dia. (3.5 kDa), pre. (etOH):CPS, anion- Imbs et al., 2014
exchange chr. (elution (DW), linear gradient
(DW-2M NaCl), dia., f-dr.
U. pinnatifida /acidic Cen., filtr., neutralization, UF, pre. (etOH), Lee et al., 2004
filtr., wash., dissolved (25 mM CH3COONa pH
5), anion-exchange chr. (DEAE-Toyopearl
650M, elution (25 mM CH3COONa pH 5,
linear gradient same buffer: 2M NaCl (1:1)). 1.
Dissolved (20 mM Tris-HCl pH 7.2), anion-
exchange chr. (Q-Sepharose FF, elution (same
buffer, linear gradient 20 mM Tris-HCl (pH
7.2):4M NaCl (1:1)). 2. Dia., f-dr., size-
exclusion chr. (Sephacryl S300 HR, elution (25
mM CH3COONa pH 5 0.1M NaCl).
U. pinnatifida /CaCl2 Prec. (20% ethanolic NaI, 72 h), cen., wash. Mak et al., 2013
(etOH), dissolved (DW), dia. (12–14 kDa),
f-dr., dissolved (Tris-HCl buffer 0.05 M, pH
7.4), ion exchange chr. (DEAE-Sephadex
A-25, equilibrated (Tris-HCl), elution (DW;
1M, 2M NaCl)
F. evanescens /CaCl2 Dia. (UF) (100 kDa), pre. (etOH), dissolved Imbs et al., 2014
(DW), anion-exchange chr. (DEAE-cellulose
in Cl– form), elution (DW), linear gradient
(DW:2M NaCl (1:1)), dia., f-dr.
E. cava /enzymatic Prec. (etOH), cen., dissolved (DW), prec. (4M CaCl2, Lee et al., 2012
etOH), cen., pre. (CPC), salts dissolved (3M CaCl2),
prec. (etOH), dissolved (DW), dia. (10-12 kDa, 4°C,
72 h), f-dr., dissolved (DW), ion-exchange chr.
(DEAE-cellulose, equilibrated (50mM CH3COONa
pH 5.0), wash. (same buffer 0.2M NaCl), eluted
(same buffer 0.2-1.2M NaCl), dia., f-dr.
cen., centrifugation; cet-Br., cetyltrimethylammonium bromide; chr., chromatography; CPC, cetylpyridinium

chloride; depr., deproteinization; dia., dialysis; dr., drying; DW, distilled water; etOH, ethanol; frac., fractionation;
f-dr., freeze-dried; filtr., filtration; MF, micro-filtration; prec., precipitation; rt., room temperature;
UF, ultrafiltration; wash., washing.
of fucoidan-rich fraction by addition of CaCl2 strength of the eluting solvent (Khan, 2012).
or ethanol and stirring (Palanisamy et al., There are two types of ion-exchange chroma-
2017). Liquid-liquid fractionation could be tography, anion and cation-exchange, depend-
used with similar advantages, but both need ing of the charge of the moieties present in gels
large solvent volume. or exchangers (Khan, 2012).
Since fucoidans present negative surface
44.3.1 Membrane Filtration charges because of the linked sulfate ester
groups, this technique is broadly used for
Membrane technology separates fractions from fucoidan purification (García-Vaquero et al.,
complex mixtures in base of the molecular weight 2016). Based on this idea, anion-exchange
of the target molecules by using membranes of chromatography permits a separation of
different molecular-weight-cut-off (MWCO). fucoidan fractions of different level of sulfa-
Extracts can be desalted by membrane technol- tion. DEAE-cellulose is one of the most used
ogy in dialysis-against-water mode, and fractions gels for this separation (Cong et al., 2016,
of different molecular weight can be obtained by Anastyuk et al., 2014, Kang et al., 2011).
working in filtration mode. Dialysis occurs by an
osmosis process in which salts migrate to the
water phase when in contact with the salted 44.3.3 Size-Exclusion Chromatography
extract through a salt and water-permeable mem- Size-exclusion chromatography (SEC) or gel
brane. Filtration separates the fractions by a phys- filtration allows the separation of molecules of
ical process in which only the molecules smaller different shape and bulkiness by passing the
than the size of the pores in the membrane can original mixture through a porous matrix of
pass. Main factors affecting the filtration are the particles, which are chemically and physically
nature of the extract and the liquid:solid ratio. inert and stable, and eluting with water, diluted
Membrane filtration shows suitability for dialysis acid or buffer solution at relatively low pres-
and filtration of aqueous-CaCl2 extracts of defat- sure (García-Vaquero et al., 2016). Among the
ted Saccharina gurjanovae to produce desalted most common matrixes for SEC fucoidan puri-
water-soluble polysaccharidic fractions (<5 kDa) fication, there are Sephadex (Silva et al., 2005),
(Shevchenko et al., 2015). Similar uses were Superdex (Deniaud-Bouët et al., 2014),
reported for fractionation of polysaccharidic Sepharose (Dinesh et al., 2016, Dore et al.,
extracts from S. mcclurei, U. pinnatifida, F. evane- 2013), and Shodex Asahipak (Anastyuk et al.,
scens, S. japonica, S. oligocystum and Fucus vesicu- 2012, Imbs et al., 2010). Some authors reported
losus (Song et al., 2015, Thinh et al., 2013, the use of SEC after ion-exchange chromatog-
Menshova et al., 2013, Rupérez et al., 2002). raphy because buffered samples do not inter-
fere in this separation (Mori and Barth, 2013).
For example, Lee et al. (2004) fractionated
44.3.2 Ion-Exchange Chromatography
an acidic extract from U. pinnatifida by
Ion exchange chromatography is one of the anion-exchange chromatography with DEAE-
most used separation techniques for purifica- Toyopearl 650M, and further refined the sam-
tion of molecules. The process is based on the ple by size-exclusion chromatography with
covalent binding formed between charged Sephacryl S300 HR.
moieties in the chromatographic gel and
the oppositely charged molecules present in
44.3.4 Affinity Chromatography
the extract (adsorption), and the subsequent
desorption by washing with a buffer solu- The technique of affinity chromatography is
tion of increased salt concentration. The pro- based on passing a mixture of molecules through
cess normally changes the pH and the ionic a stationary phase with specific affinities or
44.4 Conclusion 1115
ligands in which the target compounds bind in a fucoidan and the cationic dye. Zayed et al. (2016)
reversible way. Unlinked compounds are washed validated the previous methodology, and proved
out of the column and an elution solvent is used it as a fucoidan purification method that can
for purified fraction recovery (Nagy et al., 2017). process the crude extract with no precipitation
Since biological activities of molecules are nor- steps. In addition, the purification permits the
mally associated to specific 3D structure and production of various fucoidan quality in base
binding sites, this separation allows to refine on the incubation conditions with the immobi-
fractions by bioactivity or structure (García- lized thiazine toluidine blue O dye.
Vaquero et al., 2017; Yuguchi et al., 2016). Sometimes various types of purification
Lectins are sugar-binding proteins that have methodologies are suitable for fucoidan purifi-
been used for fucoidan purification as reported cation, but a series of their advantages and
by Hahn et al. (2016). limitations might be helpful to decide which
Dye-affinity chromatography is the most com- one is more appropriate for each application
mon kind of affinity chromatography. Hahn (Table 44.6).
et al. (2016) designed a new methodology able to
purify fucoidan from Fucus vesiculosus extracts
up to a higher purity level than that showed by 44.4 Conclusions
commercial fucoidan (95%, Sigma-Aldrich).
They modified amino-derivatized Sepabeads® Sulfated polysaccharides exhibit a number of
EC-EA with toluidine blue to enhance the strong bioactivities of high relevance for therapeutic
donor acceptor interaction between the anionic applications. Brown seaweed fucoidans are
Table 44.6 Advantages and limitations of the purification and fractionation techniques for the refining
of fucoidan.
Purification
technology Advantages Disadvantages References
Membrane Suitable for large volumes of High energy consumption García-Vaquero

filtration sample Excessive membrane fouling et al., 2017
Automated High water consumption
Scalable to industrial scale because of frequent washing or
Suitable to install in a replacement
sequential system of Cheaper than chromatography
decreasing MWCO
Chromatography:
Ionic High performance and High salt concentration of Acikara, 2013
resolution elution solvents
Relative low cost Competition of other charged
Easy to automate molecules can decrease the
Capacity for a high sample effectivity of purification of the
throughput target compounds
Size-exclusion Buffered samples from other Large volumes of solvent Mori and Barth,
(SEC) separation techniques could Low resolution compared with 2013
be directly applied to SEC other chromatographic
Help to desalt techniques
Affinity Highly specific for protein Sulfate content can interfere in Hanh et al.,
purification the affinity of fucose-lectins 2016
particularly interesting since seaweeds are highly relevant for providing reproducible and
highly available and renewable sources that purified products. Innovative methods, based
could provide these bioactives. However, the on more efficient mass and energy transfer as
preparation of homogeneous products, with well as cell wall disruption provide improve-
constant properties regarding activity and bio- ments over classical extraction procedures and
availability, has limitations derived from the also offer techno-economical benefits. In addi-
variability in the sources, but particularly, due tion, the complete utilization of the raw mate-
to processing, which can influence the degree rial is favoured with multipurpose process to
of polymerization and also the sulfation obtain different products with potential appli-
degree. The design of the optimal extraction, cations for nutraceutical, cosmetic and phar-
fractionation and purification sequence is maceutical industries.
References
Acikara, Ö. (2013). Ion-exchange chromatography (marine brown alga). Food and Chemical
and its applications. INTECH Open Access Toxicology, 48 (1), 187–92.
Publisher. Anastyuk, S.D., Shevchenko, N.M., Nazarenko,
Adalbjörnsson, B.V., Jónsdóttir, R. (2015). E.L., Imbs, T.I. et al. (2010) Structural analysis
Enzyme-enhanced extraction of antioxidant of a highly sulfated fucan from the brown alga
ingredients from algae. Methods in Molecular Laminaria cichorioides by tandem MALDI
Biology, 1308, 145–50. and ESI mass spectrometry. Carbohydrate
Ahn, C., Jeon, Y., Kang, D. et al. (2004) Free Research, 345 (15), 2206–12.
radical scavenging activity of enzymatic Anastyuk, S.D., Imbs, T.I., Shevchenko, N.M.,
extracts from a brown seaweed Scytosiphon Dmitrenok, P.S., Zvyagintseva, T.N., 2012.
lomentaria by electron spin resonance ESIMS analysis of fucoidan preparations from
spectrometry. Food Research International, 37 Costaria costata, extracted from alga at
(3), 253–58. different life-stages. Carbohydrate Polymers,
Ahn, G., Hwang, I., Park, E. et al. (2008) 90 (2), 993–1002.
Immunomodulatory effects of an enzymatic Anastyuk, S.D., Imbs, T.I., Dmitrenok, P.S.,
extract from Ecklonia cava on murine Zvyagintseva, T.N. (2014) Rapid mass
splenocytes. Marine Biotechnology, 10 (3), spectrometric analysis of a novel fucoidan,
278–89. extracted from the brown alga Coccophora
Ale, M.T., Meyer, A.S. (2013) Fucoidans from langsdorfii. The Scientific World Journal, 2014,
brown seaweeds: An update on structures, 1–9.
extraction techniques and use of enzymes as Athukorala, Y., Kim, K.N., Jeon, Y.J. (2006)
tools for structural elucidation. RSC Advances, Antiproliferative and antioxidant properties of
3 (22), 8131–41. an enzymatic hydrolysate from brown alga,
Ale, M.T., Mikkelsen, J., Meyer, A.S. (2012) Ecklonia cava. Food and Chemical Toxicology,
Designed optimization of a single-step 44 (7), 1065–74.
extraction of fucose-containing sulfated Baba, B.M., Mustapha, W.A.W., Joe, L.S.,
polysaccharides from Sargassum sp. Journal Ibrahim, K., Badri, K.H., Jumali, M.H.H. et al.
of Applied Phycology, 24 (4), 715–23. (2016) Effects of extraction solvent on fucose
Ananthi, S., Raghavendran, H.R., Sunil, A.G. content in fucoidan extracted from brown
et al. (2010) In vitro antioxidant and in vivo seaweed (Sargassum sp.) from Pulau
anti-inflammatory potential of crude Langkawi, Kedah, Malaysia. AIP Conference
polysaccharide from Turbinaria ornata Proceedings, 1784 (1), 030045.
Reference 1117
Balboa, E.M., Moure, A., Domínguez, H. (2015) seaweed Canistrocarpus cervicornis with
Valorization of Sargassum muticum biomass anticoagulant and antioxidant activities.
according to the biorefinery concept. Marine Marine Drugs, 9 (1), 124–38.
Drugs, 13 (6), 3745–60. Castro, L.S.E.P.W., Pinheiro, T.D., Castro, A.J.G.,
Balboa, E.M., Rivas, S., Moure, A., Domínguez, Santos, M.D.N., Soriano, E.M. Leite, E.L.
H., Parajó, J.C. (2013) Simultaneous extraction (2015) Potential anti-angiogenic,
and depolymerization of fucoidan from antiproliferative, antioxidant, and
Sargassum muticum in aqueous media. anticoagulant activity of anionic
Marine Drugs, 11 (11), 4612–27. polysaccharides, fucans, extracted from brown
Beress, A., Wassermann, O., Tahhan, S., Bruhn, algae Lobophora variegata. Journal of Applied
T., Beress, L., Kraiselburd, E.N. et al. (1993) A Phycology, 27, 1315–25.
new procedure for the isolation of anti-HIV Chan, C. H., Yusoff, R., Ngoh, G.C., Kung, F.W.L.
compounds (polysaccharides and polyphenols) Microwave-assisted extractions of active
from the marine alga Fucus vesiculosus. ingredients from plants. J. Chromatogr. A,
Journal of Natural Products, 56 (4), 478–88. 2011, 1218, 6213–6225.
Bendicho, C., De La Calle, I., Pena, F., Costas, Charoensiddhi, S., Lorbeer, A.J., Lahnstein, J.,
M., Cabaleiro, N., Lavilla, I. (2012) Bulone, V., Franco, C.M.M., Zhang, W. (2016)
Ultrasound-assisted pretreatment of solid Enzyme-assisted extraction of carbohydrates
samples in the context of green analytical from the brown alga Ecklonia radiata: Effect
chemistry. TrAC – Trends in Analytical of enzyme type, pH and buffer on sugar yield
Chemistry, 31, 50–60. and molecular weight profiles. Process
Bendicho, C., Lavilla, I. (2013) Extraction/ Biochemistry, 51 (10), 1503-10.
Ultrasound extractions, in Reference Module in Chattopadhyay, N., Ghosh, T., Sinha, S.,
Chemistry, Molecular Sciences and Chemical Chattopadhyay, K., Karmakar, P., Ray, B.
Engineering, (Ed.) Reedijk, J., Elsevier, (2010) Polysaccharides from Turbinaria
Amsterdam. conoides: Structural features and antioxidant
Bilan, M.I., Grachev, A.A., Ustuzhanina, N.E., capacity. Food Chemistry, 118 (3), 823–29.
Shashkov, A.S., Nifantiev, N.E., Usov, A.I. Chemat, F., Cravotto, G. (2013) Microwave-
(2002) Structure of a fucoidan from the brown assisted extraction for bioactive compounds:
seaweed Fucus evanescens C. Ag. Theory and practice. Food Engineering Series 4,
Carbohydrate Research, 337 (8), 719–30. Springer, New York.
Bilan, M.I., Grachev, A.A., Ustuzhanina, N.E., Chemat, F., Rombaut, N., Sicaire, A.G.,
Shashkov, A.S., Nifantiev, N.E., Usov, A.I. Meullemiestre, A., Fabiano-Tixier, A.S.,
(2004) A highly regular fraction of a fucoidan Abert-Vian, M. (2017) Ultrasound assisted
from the brown seaweed Fucus distichus L. extraction of food and natural products.
Carbohydrate Research, 339 (3), 511–17. Mechanisms, techniques, combinations,
Bilan, M.I., Grachev, A.A., Shashkov, A.S., protocols and applications. A review.
Nifantiev, N.E., Usov, A.I. (2006) Structure of a Ultrasonics Sonochemistry, 34, 540–60.
fucoidan from the brown seaweed Fucus serratus Chen, X., Xing, R., Yu, H., Liu, S., Li, P. (2012) A
L. Carbohydrate Research, 341 (2), 238–45. new extraction method of fucoidan from the
Brunner, G. (2009) Near critical and supercritical soaked water of brown seaweed (Laminaria
water. Part I. Hydrolytic and hydrothermal japonica). Desalination and Water Treatment,
processes. The Journal of Supercritical Fluids, 40 (1-3), 204–08.
47 (3), 373–81. Chotigeat, W., Tongsupa, S., Supamataya, K.,
Camara, R.B.G., Costa, L.S., Fidelis, G.P., Nobre, Phongdara, A. (2004) Effect of fucoidan on
L.T.D.B., Dantas-Santos, N., Cordeiro, S.L. disease resistance of Black Tiger Shrimp.
et al. (2011) Heterofucans from the brown Aquaculture, 233 (1-4), 23–30.
Conde, E., Moure, A., Domínguez, H. and Parajó Dore, C.M., Faustino Alves, M.G., Will, L.S. et al.
J.C. (2010) Separation, extraction and (2013) A sulfated polysaccharide, fucans,
concentration processes in the food, beverage, isolated from brown algae Sargassum vulgare
and nutraceutical industries, in Extraction of with anticoagulant, antithrombotic,
Natural Antioxidants from Plant Foods, antioxidant and anti-inflammatory effects.
(Ed. Rizvi, S.S.H.). Woodhead Publishing Carbohydrate Polymers, 91 (1), 467–75.
Limited. Padstow, Cornwall, United Kingdom, Duarte, M.E.R., Cardoso, M.A., Noseda, M.D.,
506–68. Cerezo, A.S. (2001) Structural studies on
Cong, Q., Chen, H., Liao, W., Xiao, F., Wang, P., fucoidans from the brown seaweed Sargassum
Qin, Y. et al. (2016) Structural characterization stenophyllum. Carbohydrate Research, 333 (4),
and effect on anti-angiogenic activity of a 281–93.
fucoidan from Sargassum fusiforme. Eluvakkal, T., Shanthi, N., Murugan, M.,
Carbohydrate Polymers, 136 (20), 899–07. Arunkumar, K. (2014) Extraction of
Costa, L.S., Fidelis, G.P., Cordeiro, S.L., Oliveira, antibacterial substances, galactofucoidan and
R.M., Sabry, D.A. et al. (2010) Biological alginate successively from the Gulf of Mannar
activities of sulfated polysaccharides from brown seaweed Sargassum wightii Greville ex
tropical seaweeds. Biomedicine & J. Agardh. Indian Journal of Natural Products
Pharmacotherapy, 64 (1), 21–8. and Resources, 5 (3), 249–57.
Costa, L.S., Fidelis, G.P., Telles, C.B.S., Dantas- Fernando, I.P.S., Sanjeewa, K.A., Samarakoon,
Santos, N., Camara, R.B.G., Cordeiro, S.L., K.W., Lee, W.W. et al. (2017) FTIR
Costa, M.S.S.P., Almeida-Lima, J., Melo- characterization and antioxidant activity of
Silveira, R.F., Oliveira, R.M., Albuquerque, water soluble crude polysaccharides of Sri
I.R.L., Andrade, G.P.V., Rocha, H.A.O. (2011) Lankan marine algae. Algae, 32 (1), 75–86.
Antioxidant and antiproliferative activities of Fitton, H.J., Stringer, N.D., Karpiniec, S.S. (2015)
heterofucans from the seaweed Sargassum Therapies from fucoidan: An update. Marine
filipendula. Marine Drugs, 9, 952–66. Drugs, 13, 5920–46.
Cumashi, A., Ushakova, N.A., Preobrazhenskaya, Flórez-Fernández, N., López-García, M.,
M.E., D’Incecco, A. et al. (2007) A comparative González-Muñoz, M.J., Vilariño, J.M.,
study of the anti-inflammatory, anticoagulant, Domínguez, H. (2017a) Ultrasound-assisted
antiangiogenic, and antiadhesive activities of extraction of fucoidan from Sargassum
nine different fucoidans from brown seaweeds. muticum. Journal of Applied Phycology, 29 (3),
Glycobiology, 17 (5), 541–52. 1553–61.
Deniaud-Bouët, E., Kervarec, N., Michel, G. et al. Flórez-Fernández, N., González-Muñoz, M.J.,
(2014) Chemical and enzymatic fractionation Domínguez, H. (2017b) Feasibility of
of cell walls from fucales: Insights into the posthydrolysis processing of hydrothermal
structure of the extracellular matrix of brown extracts from Sargassum muticum. Algal Res.,
algae. Annals of Botany, 114 (6), 1203–16. 27, 73–81.
Dias, P.F., Siqueira Jr., J.M., Maraschin, M., Foley, S.A., Szegezdi, E., Mulloy, B., Samali, A.,
Ferreira, A.G. et al. (2008) A polysaccharide Tuohy, M.G. (2011) An unfractionated
isolated from the brown seaweed Sargassum fucoidan from Ascophyllum nodosum:
stenophyllum exerts antivasculogenic effects Extraction, characterization, and apoptotic
evidenced by modified morphogenesis. effects in vitro. Journal of Natural Products, 74
Microvascular Research, 75 (1), 34–44. (9), 1851–61.
Dinesh, S., Menon, T., Hanna, L.E. et al. (2016). Fujimura, T., Shibuya, Y., Moriwaki, S.,
In vitro anti-HIV-1 activity of fucoidan from Tsukahara, K., Kitahara, T., Sano, T. et al.
Sargassum swartzii. International Journal of (2000) Fucoidan is the active component of
Biological Macromolecules, 82, 83–88. Fucus vesiculosus that promotes contraction of
Reference 1119
fibroblast-populated collagen gels. Biological from Undaria pinnatifida (Phaeophyta).

& Pharmaceutical Bulletin, 23 (10), 1180–84. Journal of Applied Phycology, 18 (2), 185–93.
Galema, S. A. Microwave chemistry. Chem. Soc. Heo, S., Park, P., Park, E., Kim, S., Jeon, Y. (2005)
Rev., 1997, 26, 233–238. Antioxidant activity of enzymatic extracts
García-Vaquero, M., Rajauria, G., O’Doherty, from a brown seaweed Ecklonia cava by
J.V., Sweeney, T. (2017) Polysaccharides from electron spin resonance spectrometry and
macroalgae: Recent advances, innovative comet assay. European Food Research and
technologies and challenges in extraction and Technology, 221 (1-2), 41–7.
purification. Food Research International, 99 Heo, S.J., Jeon, Y.J., Lee, J., Kim, H.T., Lee, K.W.
(3), 1011–20. (2003) Antioxidant effect of enzymatic
Garrote, G., Domínguez, H., Parajó, J.C. (1999) hydrolyzate from a Kelp, Ecklonia cava. Algae,
Hydrothermal processing of lignocellulosic 18 (4), 341–7.
materials. Holz als Roh und Werkstoff, 57 (3), Heo, S.J., Jeon, Y.J. (2008) Radical scavenging
191–02. capacity and cytoprotective effect of
Garrote, G., Cruz, J., Domínguez, H., Parajó, J.C. enzymatic digests of Ishige okamurae. Journal
(2003) Valorization of waste fractions from of Applied Phycology, 20, 1087–95.
autohydrolysis of selected lignocellulosic Hifney, A.F., Fawzy, M.A., Abdel-Gawad, K.M.,
materials. Journal of Chemical Technology & Gomaa, M. (2016) Industrial optimization of
Biotechnology, 78 (4), 392–98. fucoidan extraction from Sargassum sp. and
González-López, N., Moure, A., Domínguez, H., its potential antioxidant and emulsifying
Parajó, J.C. (2012) Valorization of chestnut activities. Food Hydrocolloids, 54, Part A,
husks by non-isothermal hydrolysis. Industrial 77–88.
Crops and Products, 36 (1), 172–76. Hou, Y., Wang, J., Jin, W., Zhang, H., Zhang, Q.
Guo, H., Liu, F., Jia, G., Zhang, W., Wu, F. (2013) (2012) Degradation of Laminaria japonica
Extraction optimization and analysis of fucoidan by hydrogen peroxide and
monosaccharide composition of fucoidan antioxidant activities of the degradation
from Saccharina japonica by capillary zone products of different molecular weights.
electrophoresis. Journal of Applied Phycology, Carbohydrate Polymers, 87 (1), 153–59.
25 (6), 1903–08. Huang, C., Wu, S., Yang, W., Kuan, A., Chen, C.
Ha, J., Kwon, M., Han, J., Jin, L., Jeong, H.S., (2016) Antioxidant activities of crude extracts
Choi, G. et al. (2008) Enhancement of of fucoidan extracted from Sargassum
immunomodulatory activities of low glaucescens by a compressional-puffing-
molecular weight fucoidan isolated from hydrothermal extraction process. Food
Hizikia fusiforme. Korean Journal of Food Chemistry, 197, Part B, 1121–29.
Science and Technology, 40 (5), 545–50. Imbs, T.I., Shevchenko, N.M., Sukhoverkhov,
Hahn, T., Lang, S., Ulber, R., Muffler, K. (2012) S.V., Semenova, T.L., Skriptsova, A.V.,
Novel procedures for the extraction of Zvyagintseva, T.N., 2009. Seasonal variations
fucoidan from brown algae. Process of the composition and structural
Biochemistry, 47 (12), 1691–98. characteristics of polysaccharides from the
Hahn, T., Zayed, A., Kovacheva, M., Stadtmüller, brown alga Costaria costata. Chemistry of
R., Lang, S., Muffler, K., Ulber, R. (2016) Dye Natural Compounds, 45 (6), 786–791.
affinity chromatography for fast and simple Imbs, T.I., Skriptsova, A.V., Zvyagintseva, T.N.
purification of fucoidan from marine brown (2015) Antioxidant activity of fucose-
algae. Engineering in Life Sciences, 16 (1), 78–87. containing sulfated polysaccharides obtained
Hemmingson, J.A., Falshaw, R., Furneaux, R.H., from Fucus evanescens by different extraction
Thompson, K. (2006) Structure and antiviral methods. Journal of Applied Phycology, 27 (1),
activity of the galactofucan sulfates extracted 545–53.
Jiao, G., Yu, G., Zhang, J., Ewart, H.S. (2011) Sargassum coreanum on induction of
Chemical structures and bioactivities of apoptosis in HL-60 tumor cells. Journal of
sulfated polysaccharides from marine algae. Applied Phycology, 24 (4), 675–84.
Marine Drugs, 9 (2), 196–223. Kuda, T., Taniguchi, E., Nishizawa, M., Araki, Y.
Jo, B.W., Choi, S.K. (2014) Degradation of (2002) Fate of water-soluble polysaccharides
fucoidans from Sargassum fulvellum and their in dried Chorda filum a brown alga during
biological activities. Carbohydrate Polymers, water washing. Journal of Food Composition
111, 822–29. and Analysis, 15 (1), 3–9.
Kadam, S.U., Tiwari, B.K., O’Connell, S., Kulshreshtha, G., Burlot, A.-S., Marty, C.,
O’Donnell, C.P. (2015a) Effect of ultrasound Critchley, A., Hafting, J., Bedoux, G.,
pretreatment on the extraction kinetics of Bourgougnon, N. (2015) Enzyme-assisted
bioactives from brown seaweed (Ascophyllum extraction of bioactive material from
nodosum). Separation Science and Technology, Chondrus crispus and Codium fragile and its
50 (5), 670–75. effect on Herpes simplex virus (HSV-1). Marine
Kadam, S.U., Tiwari, B.K., Smyth, T.J., O’Donnell, Drugs, 13 (1), 558–80.
C.P. (2015b) Optimization of ultrasound Kumar, T. V., Lakshmanasenthil, S.,
assisted extraction of bioactive components Geetharamani, D., Marudhupandi, T., Suja,
from brown seaweed Ascophyllum nodosum G., Suganya, P. (2015) Fucoidan-A α-d-
using response surface methodology. glucosidase inhibitor from Sargassum wightii
Ultrasonics Sonochemistry, 23, 308–316. with relevance to type 2 diabetes mellitus
Kang, S., Kim, K., Lee, S., Ahn, G., Cha, S., Kim, therapy. International Journal of Biological
A. et al. (2011) Anti-inflammatory activity of Macromolecules, 72, 1044–47.
polysaccharide purified from AMG-assistant Lavilla, I. and Bendicho, C. (2017) Fundamentals
extract of Ecklonia cava in LPS-stimulated of ultrasound-assisted extraction, in Water
RAW 264.7 macrophages. Carbohydrate Extraction of Bioactives, 1st edn. (Eds.)
Polymers, 85 (1), 80–85. Domínguez H., González-Muñoz, M.J.,
Khan, H.U. (2012). The role of ion exchange Elsevier, Amsterdam, pp.
chromatography in purification and Lee, J., Hayashi, K., Hashimoto, M., Nakano, T.,
characterization of molecules in ion exchange Hayashi, T. (2004) Novel antiviral fucoidan
technologies. Kilislioglu, A. (Ed.). InTech Open from sporophyll of Undaria pinnatifida
Ltd. (Mekabu). Chemical and Pharmaceutical
Khuwijitjaru, P. (2016) Utilization of plant-based Bulletin, 52 (9), 1091–94.
agricultural waste by subcritical water treatment. Lee, J., Takeshita, A., Hayashi, K., Hayashi, T.
Japan Journal of Food Engineering, 17 (2), 33–39. (2011) Structures and antiviral activities of
Kim, K., Heo, S., Song, C. B., Lee, J., Heo, M., polysaccharides from Sargassum trichophyllum.
Yeo, I. et al. (2006) Protective effect of Carbohydrate Polymers, 86 (2), 995–99.
Ecklonia cava enzymatic extracts on hydrogen Lee, S., Ko, C., Ahn, G., You, S., Kim, J., Heu,
peroxide-induced cell damage. Process M.S. et al. (2012) Molecular characteristics
Biochemistry, 41 (12), 2393–01. and anti-inflammatory activity of the fucoidan
Kim, S.-H., Choi, D.-S., Athukorala, Y., Jeon, extracted from Ecklonia cava. Carbohydrate
Y.-J., Senevirathne, M., Rha, C.K. (2007) Polymers, 89 (2), 599–06.
Antioxidant activity of sulfated Leonelli, C., Mason, T.J. (2010) Microwave and
polysaccharides isolated from Sargassum ultrasonic processing: Now a realistic option for
fulvellum. Journal of Food Science and industry. Chemical Engineering and Processing:
Nutrition, 12 (2), 65–73. Process Intensification, 49 (9), 885–900.
Ko, S.C., Lee, S.H., Ahn, G., Kim, K.N. et al. Li, B., Wei, X., Sun, J., Xu, S. (2006) Structural
(2012) Effect of enzyme-assisted extract of investigation of a fucoidan containing a
Reference 1121
fucose-free core from the brown seaweed, Magalhaes, K.D., Costa, L.S., Fidelis, G.P.,
Hizikia fusiforme. Carbohydrate Research, 341 Oliveira, R.M., Nobre, L.T., Dantas-Santos, N.
(9), 1135–46. et al. (2011) Anticoagulant, antioxidant and
Li, B., Liu, S., Xing, R., Li, K., Li, R., Qin, Y., antitumor activities of heterofucans from the
Wang, X., Wei, Z., Li, P. (2013) Degradation of seaweed Dictyopteris delicatula. International
sulfated polysaccharides from Enteromorpha Journal of Molecular Sciences, 12 (5), 3352–65.
prolifera and their antioxidant activities. Mak, W., Hamid, N., Liu, T., Lu, J., White, W.
Carbohydrate Polymers, 92 (2) 1991–1996. (2013) Fucoidan from New Zealand Undaria
Lidström, P., Tierney, J., Wathey, B., Westman, J. pinnatifida: Monthly variations and
Microwave assisted organic synthesis—a determination of antioxidant activities.
review. Tetrahedron, 2001, 57, 45, 9225–9283. Carbohydrate Polymers, 95 (1), 606–14.
Lim, S.J., Aida, W.M.W., Maskat, M.Y., Mamot, Mandal, V., Mohan, Y., Hemalatha, S. (2007).
S., Ropien, J., Mohd, D.M. (2014) Isolation and Microwave assisted extraction – An innovative
antioxidant capacity of fucoidan from selected and promising extraction tool for medicinal
Malaysian seaweeds. Food Hydrocolloids, 42, plant research. Pharmacognosy Reviews, 1, 7–18.
280–88. Marais, M.F., Joseleau, J.P. (2001) A fucoidan
Liu, L., Heinrich, M., Myers, S., Dworjanyn, S.A. fraction from Ascophyllum nodosum.
(2012) Towards a better understanding of Carbohydrate Research, 336 (2), 155–59.
medicinal uses of the brown seaweed Menshova, R., Lepeshkin, F., Ermakova, S.,
Sargassum in traditional Chinese medicine: A Pokrovskii, O., Zvyagintseva, T. (2013) Effect
phytochemical and pharmacological review. of pretreatment conditions of brown algae by
Journal of Ethnopharmacology, 142 (3), supercritical fluids on yield and structural
591–19. characteristics of fucoidans. Chemistry of
Liu, X., Liu, B., Wei, X., Sun, Z., Wang, C. (2016) Natural Compounds, 48 (6), 923–26.
Extraction, fractionation, and chemical Menshova, R.V., Anastyuk, S.D., Ermakova, S.P.,
characterisation of fucoidans from the brown Shevchenko, N.M., Isakov, V. I., Zvyagintseva,
seaweed Sargassum pallidum. Czech Journal T.N. (2015) Structure and anticancer activity
of Food Sciences, 34 (5), 406–13. in vitro of sulfated galactofucan from brown
Lorbeer, A., Lahnstein, J., Fincher, G., Su, P., alga Alaria angusta. Carbohydrate Polymers,
Zhang, W. (2015b) Kinetics of conventional 132, 118–25.
and microwave-assisted fucoidan Michalak, I., Chojnacka, K. (2014) Algal extracts:
extractions from the brown alga, Ecklonia Technology and advances. Engineering in Life
radiata. Journal of Applied Phycology, 27 Sciences, 14 (6), 581–91.
(5), 2079–87. Mori, S., Barth, H. (2013). Size exclusion
Lorbeer, A.J., Charoensiddhi, S., Lahnstein, J., chromatography. Springer Science & Business
Lars, C., Franco, C.M., Bulone, V. et al. (2017) Media.
Sequential extraction and characterization of Morimoto, M., Takatori, M., Hayashi, T., Mori,
fucoidans and alginates from Ecklonia D., Takashima, O., Yoshida, S. et al. (2014)
radiata, Macrocystis pyrifera, Durvillaea Depolymerization of sulfated polysaccharides
potatorum, and Seirococcus axillaris. Journal under hydrothermal conditions. Carbohydrate
of Applied Phycology, 29 (3), 1515–26. Research, 384, 56–60.
Lorbeer, A.J., Lahnstein, J., Bulone, V., Nguyen, Morya, V., Kim, J., Kim, E. (2012) Algal
T., Zhang, W. (2015a) Multiple-response fucoidan: Structural and size-dependent
optimization of the acidic treatment of the bioactivities and their perspectives. Applied
brown alga Ecklonia radiata for the sequential Microbiology and Biotechnology, 93 (1), 71–82.
extraction of fucoidan and alginate. Nagaoka, M., Shibata, H., Kimura-Takagi, I. et al.
Bioresource Technology, 197, 302–09. (1999) Structural study of fucoidan from
Cladosiphon okamuranus TOKIDA. enzyme and ultrasound assisted process. XIX

Glycoconjugate Journal, 16 (1), 19–26. Portuguese-Galician Congress, Vigo
Nagy, G., Peng, T., Pohl, N.L.B., 2017. Recent liquid (Pontevedra), Spain. 13–15 November.
chromatographic approaches and developments Petigny, L., Özel, M.Z., Périno, S., Wajsman, J.,
for the separation and purification of Chemat, F. (2015) Water as green solvent for
carbohydrates. Analytical Methods, 9, 3579–93. extraction of natural products, in Green
Nakayasu S., Soegima R., Yamaguchi K., Oda T. Extraction of Natural Products, (Eds. Chemat,
(2009) Biological activities of fucose- F., Strube, J.), Wiley-VCH Verlag GmbH & Co.
containing polysaccharide ascophyllan KGaA, 237–63.
isolated from the brown alga Ascophyllum Pielesz, A., Biniaś, W., Paluch, J. (2011) Mild acid
nodosum. Bioscience, Biotechnology and hydrolysis of fucoidan: Characterization by
Biochemistry, 73 (4), 961–64. electrophoresis and FT-raman spectroscopy.
Navarro, D.A., Flores, M.L., Stortz, C.A. (2007) Carbohydrate Research, 346 (13), 1937–44.
Microwave-assisted desulfation of sulfated Plaza, M., Amigo-Benavent, M., del Castillo,
polysaccharides. Carbohydrate Polymers, 69 M.D., Ibáñez, E., Herrero, M. (2010)
(4), 742–47. Neoformation of antioxidants in glycation
Obluchinsksya, E., Makarova, M., Pozharitskaya, O., model systems treated under subcritical water
Shikov, A. (2015) Effects of ultrasound treatment extraction conditions. Food Research
on the chemical composition and anticoagulant International, 43(4), 1123–9.
properties of dry Fucus extract. Pharmaceutical Pomin, V.H. (2012) Fucanomics and
Chemistry Journal, 49 (3), 183–86. galactanomics: Marine distribution, medicinal
Olivares-Molina, A., Fernández, K. (2016) impact, conceptions, and challenges. Marine
Comparison of different extraction techniques Drugs, 10 (4), 793–11.
for obtaining extracts from brown seaweeds Ponce, N.M., Pujol, C.A., Damonte, E.B., Flores,
and their potential effects as angiotensin L., Stortz, C.A. (2003) Fucoidans from the
I-converting enzyme (ACE) inhibitors. Journal brown seaweed Adenocystis utricularis:
of Applied Phycology, 28, 1295–02. Extraction methods, antiviral activity and
Paiva, A.A.O., Castro, A.J., Nascimento, M.S. structural studies. Carbohydrate Research, 338
et al. (2011) Antioxidant and anti- (2), 153–65.
inflammatory effect of polysaccharides from Puri, M., Sharma, D., Barrow, C.J. (2012)
Lobophora variegata on zymosan-induced Enzyme-assisted extraction of bioactives from
arthritis in rats. International plants. Trends in Biotechnology, 30, 37–44.
Immunopharmacology, 11 (9), 1241–50. Quitain, A.T., Kai, T., Sasaki, M., Goto, M. (2013)
Palanisamy, S., Vinosha, M., Marudhupandi, T., Microwave–hydrothermal extraction and
Rajasekar, P., Prabhu, N.M. (2017) Isolation of degradation of fucoidan from supercritical
fucoidan from Sargassum polycystum brown carbon dioxide deoiled Undaria pinnatifida.
algae: Structural characterization, in vitro Industrial & Engineering Chemistry Research,
antioxidant and anticancer activity. 52 (23), 7940–46.
International Journal of Biological Rabanal, M., Ponce, N.M., Navarro, D.A.,
Macromolecules, 102, 405–12. Gómez, R.M., Stortz, C.A. (2014) The system
Pérez, L., Conde, E., Domínguez, H. (2014) of fucoidans from the brown seaweed Dictyota
Microwave hydrodiffusion and gravity dichotoma: Chemical analysis and antiviral
processing of Sargassum muticum. Process activity. Carbohydrate Polymers, 101, 804–11.
Biochemistry, 49, 981–8. Rioux, L., Turgeon, S.L., Beaulieu, M. (2007)
Pérez, L., Parada, M., Balboa, E., Conde, E., Characterization of polysaccharides extracted
Moure, A., Domínguez, H. (2013) Aqueous from brown seaweeds. Carbohydrate Polymers,
extraction of Sargassum muticum using 69 (3), 530–37.
Reference 1123
Rodrigues, D., Sousa, S., Silva, A., Amorim, M., digest of brown seaweed Sargassum horneri
Pereira, L., et al. (2015) Impact of enzyme- in RAW 264.7 cells. Nutrition Research and
and ultrasound-assisted extraction methods Practice, 11 (1), 3–10.
on biological properties of red, brown, and Santoyo, S., Plaza, M., Jaime, L., Ibáñez, E.,
green seaweeds from the Central West Coast Reglero, G., Señorans, J. (2011) Pressurized
of Portugal. Journal of Agricultural and Food liquids as an alternative green process to
Chemistry, 63 (12), 3177–88. extract antiviral agents from the edible
Rodriguez-Jasso, R.M., Mussatto, S.I., Pastrana, seaweed Himanthalia elongata. Journal of
L., Aguilar, C.N., Teixeira, J.A. (2011) Applied Phycology, 23 (5), 909–17.
Microwave-assisted extraction of sulfated Saravana, P.S., Cho, Y., Park, Y., Woo, H., Chun, B.
polysaccharides (fucoidan) from brown (2016) Structural, antioxidant, and emulsifying
seaweed. Carbohydrate Polymers, 86 (3), activities of fucoidan from Saccharina japonica
1137–44. using pressurized liquid extraction.
Rodríguez-Jasso, R.M., Mussatto, S.I., Pastrana, Carbohydrate Polymers, 153, 518–25.
L., Aguilar, C.N., Teixeira, J.A. (2013) Shan, X., Liu, X., Hao, J., Cai, C., Fan, F., Dun, Y.
Extraction of sulfated polysaccharides by et al. (2016) In vitro and in vivo hypoglycemic
autohydrolysis of brown seaweed Fucus effects of brown algal fucoidans. International
vesiculosus. Journal of Applied Phycology, 25 Journal of Biological Macromolecules, 82,
(1), 31–9. 249–55.
Rodríguez-Meizoso, I., Marin, F.R., Herrero, M., Shevchenko, N., Anastyuk, S., Gerasimenko, N.,
Señorans, F.J., Reglero, G., Cifuentes, A., Dmitrenok, P., Isakov, V., Zvyagintseva, T.
Ibáñez, E. (2006) Subcritical water extraction (2007) Polysaccharide and lipid composition
of nutraceuticals with antioxidant activity of the brown seaweed Laminaria gurjanovae.
from oregano. Chemical and functional Russian Journal of Bioorganic Chemistry, 33
characterization. Journal of Pharmaceutical (1), 88–98.
and Biomedical Analysis, 41 (5), 1560–65. Silva, T., Alves, L., De Queiroz, K., Santos, M.,
Rogalinski, T., Liu, K., Albrecht, T., Brunner, G. Marques, C., Chavante, S.F. et al. (2005) Partial
(2008) Hydrolysis kinetics of biopolymers in characterization and anticoagulant activity of a
subcritical water. The Journal of Supercritical heterofucan from the brown seaweed Padina
Fluids, 46 (3), 335–41. gymnospora. Brazilian Journal of Medical and
Rupérez, P., Ahrazem, O., Leal, J.A. (2002) Biological Research, 38 (4), 523–33.
Potential antioxidant capacity of sulfated Fernando, I.P.F., Asanka, K.K.S., Samarakoon,
polysaccharides from the edible marine brown K.W., Lee, W.W., Kim, H.-S., Kim, E.-A.,
seaweed Fucus vesiculosus. Journal of Gunasekara, U.K., Abeytunga, D.T.U.,
Agricultural and Food Chemistry, 50 (4), 840–45. Nanayakkara, C., De Silva, E.D., Lee, H.-S.,
Sakai, T., Kimura, H., Kato, I. (2002) A marine Jeon, Y.-J. (2017) FTIR characterization and
strain of flavobacteriaceae utilizes brown antioxidant activity of water soluble crude
seaweed fucoidan. Marine Biotechnology, 4 (4), polysaccharides of Sri Lankan marine algae.
399–05. Algae, 32 (1), 75–86.
Sakai, T., Ishizuka, K., Kato, I. (2003) Isolation Sinha, S., Astani, A., Ghosh, T., Schnitzler, P.,
and characterization of a fucoidan-degrading Ray, B. (2010) Polysaccharides from
marine bacterium. Marine Biotechnology, 5 Sargassum tenerrimum: Structural features,
(5), 409–16. chemical modification and anti-viral activity.
Sanjeewa, K.K.A., Fernando, I.P.S., Kim, E., Phytochemistry, 71 (2), 235–42.
Ahn, G., Jee, Y., Jeon, Y. (2017) Anti- Siriwardhana, N., Kim, K.-N., Lee, K.-W., Kim,
inflammatory activity of a sulfated S.-H., Ha, J.-H., Song, C.B., Lee, J.-B., Yeon, Y.J.
polysaccharide isolated from an enzymatic (2008) Optimisation of hydrophilic
antioxidant extraction from Hizikia fusiformis Usov, A.I., Bilan, M.I. (2009) Fucoidans-sulfated
by integrating treatments of enzymes, heat polysaccharides of brown algae. Russian
and pH control. International Journal of Food Chemical Reviews, 78 (8), 785–99.
Science and Technology, 43, 587–96. Vasquez, R.D., Ramos, J.D.A., Bernal, S.D. (2012)
Sivagnanam, S.P., Yin, S., Choi, J.H., Park, Y.B., Chemopreventive properties of sulfated
Woo, H.C. (2015) Biological properties of polysaccharide extracts from Sargassum
fucoxanthin in oil recovered from two brown siliquosum JG Agardh (Sargassaceae).
seaweeds using supercritical CO2 extraction. International Journal of Pharma and Bio
Marine Drugs, 13 (6), 3422–42. Sciences, 3, 333–45.
Sokolova, R., Ermakova, S., Awada, S., Veggi, P. C., Martinez, J., Meireles, M. A. A.
Zvyagintseva, T., Kanaan, H. (2011) (2013). Fundamentals of microwave
Composition, structural characteristics, and extraction. In “Microwave-assisted extraction
antitumor properties of polysaccharides from for bioactive compounds: Theory and
the brown algae Dictyopteris polypodioides and practice”. Chapter 2, Ed. Chemat F. and
Sargassum sp. Chemistry of Natural Cravotto G. Food Engineering Series 4,
Compounds, 47 (3), 329–34. Springer.
Song, K., Ha, S.J., Lee, J., Kim, S., Kim, Y.H., Wang, J., Zhang, Q.B., Zhang, Z.S., Li, Z. (2008)
Kim, Y. et al. (2015) High yield ultrasonication Antioxidant activity of sulfated polysaccharide
extraction method for Undaria pinnatifida fractions extracted from Laminaria japonica.
sporophyll and its anti-inflammatory International Journal of Biological
properties associated with AP-1 pathway Macromolecules 42 (2), 127–32.
suppression. LWT-Food Science and Wang, J., Liu, L., Zhang, Q., Zhang, Z., Qi, H.,
Technology, 64 (2), 1315–22. Li, P. (2009) Synthesized oversulphated,
Sugiono, Widjanarko, S. B., Adi Soehono, L. acetylated and benzoylated derivatives of
2014. Extraction optimization by response fucoidan extracted from Laminaria
surface methodology and characterization of japonica and their potential antioxidant
fucoidan from brown seaweed Sargassum activity in vitro. Food Chemistry, 114 (4),
polycystum. International Journal of 1285–90.
ChemTech Research, 6, 195–205. Wang, P., Zhao, X., Lv, Y., Liu, Y., Lang, Y., Wu, J.
Synytsya, A., Kim, W., Kim, S., Pohl, R., et al. (2012) Analysis of structural
Synytsya, A., Kvasnička, F. et al. (2010) heterogeneity of fucoidan from Hizikia
Structure and antitumour activity of fucoidan fusiforme by ES-CID-MS/MS. Carbohydrate
isolated from sporophyll of Korean brown Polymers, 90 (1), 602–07.
seaweed Undaria pinnatifida. Carbohydrate Wang, C., Wu, T., Hsieh, S., Tsai, Y., Yeh, C.,
Polymers, 81 (1), 41–8. Huang, C. (2015) Antioxidant activity and
Teo, C.C., Tan, S.N., Yong, J.W.H., Hew, C.S., growth inhibition of human colon cancer
Ong, E.S. “Validation of green-solvent cells by crude and purified fucoidan
extraction combined with chromatographic preparations extracted from Sargassum
chemical fingerprint to evaluate quality of Stevia cristaefolium. Journal of Food and Drug
rebaudiana Bertoni. J. Sep. Sci., 2009, 32, 4, Analysis, 23 (4), 766–77.
613–622. Wijesekara, I., Pangestuti, R., Kim, S. (2011)
Thinh, P.D., Menshova, R.V., Ermakova, S.P., Biological activities and potential health
Anastyuk, S.D., Ly, B.M., Zvyagintseva, T.N. benefits of sulfated polysaccharides derived
(2013) Structural characteristics and from marine algae. Carbohydrate Polymers, 84
anticancer activity of fucoidan from the (1), 14–21.
brown alga Sargassum mcclurei. Marine Wijesinghe, W.A.J.P., Jeon, Y.J. (2012) Enzyme-
Drugs, 11 (5), 1456–76. assistant extraction (EAE) of bioactive
Reference 1125
components: A useful approach for recovery Yuan, Y., Macquarrie, D. J. (2015a) Microwave
of industrially important metabolites from assisted step-by-step process for the
seaweeds: A review. Fitoterapia, 83 (1), 6–12. production of fucoidan, alginate sodium,
Yachmenev, V.G., Condon, B.D., Lambert, A.H. sugars and biochar from Ascophyllum
(2007) Intensification of enzymatic reactions nodosum through a biorefinery concept.
in heterogeneous systems by low intensity, Bioresource Technology, 198, 819–27.
uniform sonication: New road to “Green Yuguchi, Y., Ly, B.M., Van Nguyen, B., Van,
Chemistry” in Industrial Application of T.T.T., Thuy, T.T.T. (2016) Study on branched
Enzymes on Carbohydrate-Based Material structure-physiological activity relationship
(Eds. Eggleston, G., Vercellotti, J.R.) ACS of fucoidan. Chemistry Letters, 45 (7), 840–42.
Symposium Series, 972, 137–56. Zayed, A., Muffler, K., Hahn, T., Rupp, S.,
Yang, C., Chung, D., Shin, I., Lee, H., Kim, J., Finkelmeier, D., Burger-Kentischer, A., Ulber,
Lee, Y. et al. (2008a) Effects of molecular R. (2016) Physicochemical and biological
weight and hydrolysis conditions on characterization of fucoidan from Fucus
anticancer activity of fucoidans from vesiculosus purified by dye affinity
sporophyll of Undaria pinnatifida. chromatography. Marine Drugs, 14 (4), 79.
International Journal of Biological Zhang, H., Row, K.H. (2015) Extraction and
Macromolecules, 43 (5), 433–37. separation of polysaccharides from Laminaria
Yang, C., Chung, D., You, S. (2008b) japonica by size-exclusion chromatography.
Determination of physicochemical properties Journal of Chromatographic Science, 53 (4),
of sulphated fucans from sporophyll of 498–02.
Undaria pinnatifida using light scattering Zvyagintseva, T.N., Shevchenko, N.M.,
technique. Food Chemistry, 111 (2), 503–07. Popivnich, I.B. et al. (1999) A new procedure
You, S., Yang, C., Lee, H., Lee, B. (2010) Molecular for the separation of water-soluble
characteristics of partially hydrolyzed fucoidans polysaccharides from brown seaweeds.
from sporophyll of Undaria pinnatifida and Carbohydrate Research, 322, (1-2), 32–9.
their in vitro anticancer activity. Food Chemistry, Zvyagintseva, T.N., Shevchenko, N.M., Chizhov,
119 (2), 554–59. A.O. et al. (2003) Water-soluble
Yuan, Y., Macquarrie, D. (2015b) Microwave polysaccharides of some far-eastern brown
assisted extraction of sulfated polysaccharides seaweeds. Distribution, structure, and their
(fucoidan) from Ascophyllum nodosum and its dependence on the developmental conditions.
antioxidant activity. Carbohydrate Polymers, Journal of Experimental Marine Biology and
129, 101–07. Ecology, 294 (1), 1–13.

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1093

Extraction and Purification of Fucoidan from Marine Sources

44.1 ­Introduction et al., 2017), antitumoral (Yang et al., 2008a;

separation and concentration operations.

Extraneous materials, moisture 44.2.2 Sample Conditioning

Seaweed Conditions References

Table 44.2 (Continued)

Seaweed Conditions References

Fucus evanescens 2 % CaCl2, 60°C, 3 h Imbs et al., 2015

DW, distilled water; rt., room temperature.

44.2.4.1 Water Extraction for Sargassum stenophyllum (Dias et al., 2008).

at 37°C to extract alginate and fucoidan polysaccharides, alternative technologies have

be scaled up (Mandal et al., 2007; Li et al., 44.2.5.3 Enzyme-Assisted Extraction

Conditions (liquid:solid ratio

Table 44.3 (Continued)

Conditions (liquid:solid ratio

DW, distilled water; US, ultrasound.

Extraction technology Advantages Disadvantages

Conventional Simplicity Low yield and selectivity

Microwave assisted Fast start-up, uniform heating High energy demand

44.3 ­Purification extracts to further purification processes

Seaweed/crude extract Fractionation processes References

Table 44.5 (Continued)

Seaweed/crude extract Fractionation processes References

Saccharina gurjanovae Dia. by UF (5 kDa), f-dr. Shevchenko et al., 2015

Gel or size-exclusion chromatography

Table 44.5 (Continued)

Seaweed/crude extract Fractionation processes References

cen., centrifugation; cet-Br., cetyltrimethylammonium bromide; chr., chromatography; CPC, cetylpyridinium

Membrane Suitable for large volumes of High energy consumption García-Vaquero

fibroblast-populated collagen gels. Biological from Undaria pinnatifida (Phaeophyta).

Cladosiphon okamuranus TOKIDA. enzyme and ultrasound assisted process. XIX

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44.1 Introduction et al., 2017), antitumoral (Yang et al., 2008a;

44.3 Purification extracts to further purification processes