J Appl Phycol (2016) 28:533–543
DOI 10.1007/s10811-015-0563-z
Chemical characterization and quantification of the brown algal
storage compound laminarin — A new methodological approach
Angelika Graiff 1 & Wolfgang Ruth 2 & Udo Kragl 2 & Ulf Karsten 1
Received: 4 July 2014 / Revised and accepted: 6 March 2015 / Published online: 27 March 2015
# Springer Science+Business Media Dordrecht 2015
Abstract The polysaccharide laminarin (β-1,3-glucan) is
used as a long-term carbon storage compound in brown algae.
This chemical storage form of carbon enables perennial brown
algae in seasonally fluctuating ecosystems to uncouple growth
from photosynthesis, i.e., most of these plants grow as season-
al anticipators in winter based on remobilization of laminarin,
while in summer, growth typically ceased to fill up the storage
pool. Because of this high ecological relevance, a reliable and
precise method for determination and quantification of lami-
narin is needed. Therefore, a simple, efficient, cold water ex-
traction method coupled to a new quantitative liquid
chromatography-mass spectrometrical method (LC-MS) was
developed. Laminarin was determined in 9 out of 12 brown
algal species, and its expected typical molar mass distribution
of 2000–7000 Da was confirmed. Furthermore, laminarin
consisted of a complex mixture of different chemical forms,
since 15 chemical laminarin species with distinct molecular
weights were measured in 9 species of brown algae.
Differences in chain length and number of laminarin species
seem to be species specific and hence may indicate some
chemotaxonomic value. Laminarin concentrations in the algal
tissues ranged from 0.03 to 0.86 % dry weight (DW). The
direct chemical characterization and quantification of laminar-
in by LC-MS represents a powerful method to verify the
* Angelika Graiff
angelika.graiff@uni-rostock.de
1
Institute of Biological Sciences, Applied Ecology and Phycology,
University of Rostock, Albert-Einstein-Straße 3b,
18059 Rostock, Germany
2
Institute of Chemistry, Analytical and Technical Chemistry,
University of Rostock, Albert-Einstein-Straße 3a,
18059 Rostock, Germany
biochemical and ecological importance of laminarin for
brown algae.
Keywords Kelps . LC-MS . Mannitol . Seasonal growth .
Carbon storage
Introduction
Brown algae (Phaeophyceae) commonly dominate intertidal
and subtidal rocky shores and provide particularly as kelp
community much of the three-dimensional structure, habitat,
and food for associated organisms (Dayton 1985; Hurd 2000;
Pérez-Matus et al. 2007). Due to their high productivity and
biomass, they represent important links in marine nutrient and
carbon cycles (Dunton and Schell 1987; Duggins et al. 1989;
Fredriksen 2003).
Brown algae exhibit a very unique central and storage car-
bon metabolism (Michel et al. 2010). The photoassimilate D-
fructose 6-phosphate is not used by these algae to produce
sucrose as in higher plants but is rather converted into the
polyol D-mannitol (Stenhouse 1844; Yamaguchi et al.
1966). Brown algae lack or contain only very low concentra-
tions of free sugars such as glucose, fructose, or sucrose
(Yamaguchi et al. 1966). These free sugars are supposed to
undergo immediate biochemical conversion into D-mannitol
and polysaccharides.
The long-term carbon storage polysaccharide in brown al-
gae is laminarin (β-1,3-glucan), which chemically markedly
differs from storage products of most other living organisms
that typically use glycogen or starch (α-1,4-glucans).
Laminarin was first described by Schmiedeberg (1885), who
isolated it from Laminariaceae. It consists of chains of β-1,3′-
linked glucose units with occasional β-1,6′-linkages (Percival
and Ross 1951; Beattie et al. 1961). These β-1,6′-linkages are
534
present in a linear chain of β-1,3′-linked glucose residues and
mainly as interchain linkages which lead to a ramification of
the molecule (Peat et al. 1958; Annan et al. 1965). The
branching factor determines water solubility; the higher the
branching content, the higher the solubility in cold water
(Annan et al. 1965). Therefore, laminarin was found in
water-soluble and water-insoluble form in different
Laminaria species (Laminaria digitata and Laminaria
hyperborea) (Black 1950). The degree of polymerization of
water-soluble laminarin is of about 20–25 glucose units
(Black et al. 1951; Beattie et al. 1961; Read et al. 1996).
Furthermore, this polysaccharide is polydisperse, consisting
of a minor G-series with polymers containing only glucose
residues, and a more abundant M-series with glucans termi-
nated with a 1-linked D-mannitol residue (Anderson et al.
1958; Read et al. 1996). Mannitol ranges from 2.4 to 3.7 %
of both soluble and insoluble laminarin (Fleming et al. 1966).
Consequently, the chemical variation in this storage molecule
is based on the number of β-1,6′-linkages, the degree of
branching, and the presence or absence of terminal mannitol
residues (Craigie 1974). However, the biosynthetic pathway
of laminarin itself as well as of that which connects mannitol
and laminarin is so far unknown. Michel et al. (2010) started
to identify several genes in the Ectocarpus genome which are
likely to be involved in laminarin metabolism and constructed
a putative pathway for carbon storage.
Based on experiments with radioactive carbon, Yamaguchi
et al. (1966) concluded that mannitol and laminarin are bio-
chemically interchangeable storage compounds in brown al-
gae. Additionally, over the course of a year, brown algae shift
their major storage compounds from mannitol in spring to
laminarin in autumn (Dethier and Williams 2009). Mannitol
is remobilized and translocated in perennial species of brown
algae from mature storage tissues to supply the rapidly grow-
ing meristematic parts with carbon for biomass formation
(Schmitz et al. 1972; Schmitz and Lobban 1976; e.g.,
reviewed by Gómez and Huovinen 2012). In kelps, special
sieve tube cells serve for long-distance transport between the
different tissues (Lobban and Harrison 1994; Raven 2003).
Laminarin shows not only quantitative changes but also chem-
ical variations. These chemical variations are depending on
alga species, tissue type, reproductive conditions, and season-
al growth and environmental factors (Read et al. 1996;
Chizhov et al. 1998; Rioux et al. 2010).
Stored photosynthates are of particular ecological rele-
vance for perennial brown algae because they often allow
the uncoupling of growth from photosynthesis, which is a
crucial adaptation to strong seasonally varying ecosystems
such as the polar regions. Here, many kelps remobilize the
stored carbon for the development of new blades during the
dark season to be morphologically and physiologically
Bready^ when the sunlight returns. These so-called seasonal
anticipators fix and store new carbon during summer without
J Appl Phycol (2016) 28:533–543
growth (Wiencke et al. 2009; Gómez and Huovinen 2012). A
high variability in seasonal patterns of the laminarin pool has
been described for different brown algae from various habitats
(Chapman and Craigie 1978; Gorham and Lewey 1984;
Gómez and Wiencke 1998; Dethier and Williams 2009).
Almost all studies measuring laminarin from algal material
use specific kinds of extraction methods, hydrolysis condi-
tions, and tests for glucose as equivalent units for laminarin.
The extraction methods applied are soaking dried algal mate-
rial overnight in sodium hydroxide and homogenization in a
tissue grinder, after the addition of hydrochloric acid (Dethier
and Williams 2009), or the extraction of laminarin using eth-
anol (20 %) at 75 °C for 2–3 h (Gómez and Wiencke 1998).
Another method is the direct extraction of laminarin from
dried algal material with sulfuric or hydrochloric acid and
use of the supernatant after neutralization for determination
of laminarin (Black et al. 1951; Gorham and Lewey 1984;
Devillé et al. 2004). According to the patent by Yvin et al.
(1999), the supernatant was treated with absolute ethanol to
precipitate laminarin. Especially, Gorham and Lewey (1984)
believed that most of the measured glucose units are derived
from laminarin and that cellulose was not hydrolyzed under
their conditions used. Furthermore, hot acidic hydrolysis (HCl
or H2SO4) of ethanol extracts (Chapman and Craigie 1977;
Gómez and Wiencke 1998) or enzymatic hydrolysis (Johnston
et al. 1977; Devillé et al. 2004) of laminarin was applied.
Laminarin quantification was undertaken, either indirectly af-
ter hydrolysis by measuring the cleaved glucose units with
enzymatic assays (Lloyd and Whelan 1969; Gómez and
Wiencke 1998), or with the anthrone reagent method (Yemm
and Willis 1954), both calibrated against glucose standards.
To our knowledge, so far, no intercalibration nor any test for
reproducibility or sensitivity of the different analysis methods
has been undertaken to ensure that these analytical approaches
are comparable. In preliminary analyses, we tested different
extraction methods (with ethanol 20 or 60 % for 3 h at 80 °C),
hydrolysis conditions (alcoholic extracts hydrolyzed with 1 N
HCl for 1 h at 100 °C or laminarin directly hydrolyzed with
0.5 M H2SO4 for 4 h at 100 °C), and two different quantitative
enzymatic assays for glucose (after Lloyd and Whelan 1969,
as well as after Gómez and Wiencke 1998) with purified com-
mercial laminarin (Sigma Chemical) and received an unex-
pectedly broad range of quantitative data. Based on these re-
sults, we had the impression that the applied methods are not
adequate for qualitative and quantitative laminarin analysis.
Therefore, we reviewed the known literature on laminarin
quantification concerning the applied methodological ap-
proaches. There is a broad variety of laminarin concentrations
reported, ranging from 0.1 to 1.3 % dry weight (DW) in Fucus
serratus (Kremer 1975), 0.3 % DW in Sargassum muticum
(Gorham and Lewey 1984), 1–15 % DW in Ascoseira
mirabilis (Gómez and Wiencke 1998), up to 10–20 % DW
in Fucus distichus (Dethier and Williams 2009), and even
J Appl Phycol (2016) 28:533–543
30 % DW in L. hyperborea (Jensen and Haug 1956). While
this huge range in laminarin quantities may depend on the
algal species, and physiological and environmental condi-
tions, it is also possible that the analytical approach has a
strong effect.
Therefore, in the present study, we tested different extrac-
tion methods for laminarin from different brown algae and
used purified commercial laminarin as a reference. We devel-
oped a new analytical liquid chromatography-mass spectro-
metric method for identification, characterization, and quanti-
fication of laminarin from brown algal tissue.
Material and methods
Algae material and sampling sites
Single individuals of L. hyperborea, L. digitata, Saccharina
latissima, F. serratus, Fucus vesiculosus, Fucus spiralis,
Himanthalia elongata, Cystoseira tamariscifolia, Pelvetia
canaliculata, Ascophyllum nodosum, Halidrys siliquosa, and
Dictyota dichotoma were collected in autumn (18 November
2013) during spring lowtide from the shore of Finavarra,
County Clare, west coast of Ireland (53° 09′ 25″ N, 09° 06′
58″ W). For further analyses, Saccharina latissima thallus
material was sampled in autumn (10 October 2014) during
low tide from the infralittoral fringe at the southeastern part
of the island of Helgoland (North Sea, Germany; 54° 10′ 39″
N, 7° 53′ 36″ E). After sampling, the different algae were
immediately transported to the lab, lyophilized, and sent to
the University of Rostock.
Extraction methods for laminarin and extraction
efficiency from algal material
To test the suitability of different solvents (methanol, ethanol,
ethanol/water (1:1, v/v), ethyl acetate, tetrahydrofuran (THF),
and ultrapure water) for the solubility of the different chemical
laminarin species, commercial laminarin from L. digitata
(Sigma L9634, Lot: SLBG0687V, CAS: 9008-22-4) was used
and analyzed using liquid chromatography-mass spectrometry
(LC-MS) analysis (LTQ Velos Pro ion trap spectrometer with
Accela HPLC, Thermo Scientific). Laminarin species were
separated on a Kinetex column (2.6 μm C18, 150×3 mm).
The mobile phase was 90 % ultrapure water and 10 % aceto-
nitrile, run isocratically at a flow rate of 0.2 mL min−1. MS
was working in ESI negative ion mode in a mass range of
100–4000 amu. Additionally, a second purified commercial
laminarin substance (Alfa Aesar J66193, Lot: K04Z011,
CAS: 9008-22-4) was tested.
Hot and cold extraction methods for laminarin from lyoph-
ilized brown alga material were tested. Hot extraction follow-
ed a standardized microwave extraction method for plant
535
components in ultrapure water as solvent. The conditions for
microwave extraction were 20 % of 400 W (MarsXpress 5
CEM) at a rate of 5 °C min−1 to 37 °C for 5 min. For cold
extraction, samples were ground to <1 mm grain size with an
analytical mill (Ika MF 10 Basic). The algal material (~1.5 g
DW) was extracted in ultrapure water or THF (8 mL) on a
shaker (250 rpm) for 5 h. After the addition of surplus ultra-
pure water or THF (4 mL) and shaking manually, 1 mL of the
sample was filter-centrifuged (45 μm) at 14,000 rpm
(HettichMikro 22 R). The slightly viscous supernatant was
free of suspended material and converted into a microvial
(300 μL) for further analysis.
The extraction yield of laminarin from Saccharina
latissima thallus material was studied using three subsequent
steps of cold water extraction (6 mL) from the same algal
material (0.2 g) according to the method described above.
The obtained extracts were analyzed using LC-MS analysis.
Hydrolysis of laminarin
For comparison of the LC-MS analysis with standard proce-
dures which quantify laminarin depending on acidic hydroly-
sis and subsequent HPLC measurement of resulting glucose
concentrations, commercial laminarin (20 mg) was hydro-
lyzed in 3 mL of 0.5 M sulphuric acid for 15 min as well as
for 180 min at 121 °C. Three independent acidic hydrolysates
were analyzed for glucose concentration using ion exclusion
chromatography (Knauer HPLC box Model-96, HPLC
interface box 1996, Knauer) equipped with a KnauerEurokat
H column (10 μm, 300×4 mm) and operated at 65 °C using a
0.006 M sulphuric acid mobile phase at 1.2 mL min−1.
Concentrations of glucose were determined using
EuroChrom 2000 software (Knauer) by comparison to glu-
cose standards.
Characterization and quantification of laminarin
with LC-MS
Laminarin was identified and quantified as well as its distinc-
tive molar mass distribution was characterized using LC-MS
analysis. Therefore, different laminarin concentrations (0.7,
0.375, 0.14, 0.07, and 0.014 mg mL−1) were analyzed.
Laminarin was quantified by comparison of the retention time
and peak area with standard solutions (calibration range
0.375–0.07, 0.7–0.07, and 0.14–0.014 mg mL−1) using
Xcalibur software. Additionally, to the full-scan measure-
ments of the LC-MS, a special simultaneous zoom scan mea-
surement was used to identify charge states of laminarin to
calculate the molar mass distribution. Molar mass was deter-
mined at the maximum of the statistical molar mass distribu-
tion. After identification and characterization, the commercial
laminarin was used as reference. Furthermore, laminarin from
water and THF extracts of 12 brown algae was identified and
536
quantified by comparison with reference laminarin solutions
and, if present, its molar mass distribution was characterized
using LC-MS analysis.
Determination of glucose, mannitol, carbon, and nitrogen
content
Glucose contents were determined after extraction using high-
performance liquid chromatography (HPLC). Extracted sam-
ples were analyzed in an HPLC (SmartLine, Knauer GmbH)
equipped with a SUPELCOGEL Ca column (30×7.8 mm
without precolumn) and RI-detector (S2300 PDA S2800).
Water was used as eluent at a flow rate of 0.8 mL min−1 at
75 °C. Glucose was quantified by comparison of the retention
time and peak area with standard solutions using ChromGate
software.
Mannitol was extracted from three subsamples of 10–
20 mg powdered alga material (L. hyperborea, L. digitata,
S. latissima, F. serratus, F. vesiculosus, F. spiralis,
H. elongata, P. canaliculata, A. nodosum, H. siliquosa)
and quantified, following the HPLC method described by
Karsten et al. (1991).
For analyzing carbon and nitrogen contents, dried algal
material was ground to powder and three subsamples of
2 mg from each alga thallus were loaded and packed into tin
cartridges (6×6×12 mm). The packages were combusted at
950 °C and the absolute contents of C and N were automati-
cally quantified in an elemental analyzer (ElementarVario EL
III, Germany) using acetanilide as standard according to
Verardo et al. (1990).
Results
Comparison of solvents and extraction methods
for laminarin
Solubility and detectability of purified laminarin from
L. digitata markedly differed between solvents (Table 1).
Additionally, the number of detectable chemical laminarin
species varied between the solvents. THF, as well as ultrapure
water, showed four different laminarin species, and one of
these appeared to be a branched molecule. In ethanol/water
(1:1, v/v), only three different laminarin species were found.
Additionally, the signal was lower compared to extracts in
water and THF. Laminarin was insoluble in methanol, ethanol,
and ethyl acetate as no detectable signals in the LC-MS mea-
surement were apparent. The second commercial laminarin
substance showed no detectable chemical laminarin species
by LC-MS; however, the signals indicated that the substance
consisted of disaccharides.
Comparison of hot and cold extraction for laminarin from
lyophilized alga material revealed that hot extraction was not
J Appl Phycol (2016) 28:533–543
Table 1 Solubility, number of branched laminarin molecules, and the
number of laminarin species of commercial purified laminarin from
Laminaria digitata analyzed in different solvents
Solvent Solubility Branched Number of
molecules laminarin species
methanol − − −
ethanol − − −
ethanol/water (1:1, v/v) + + 3
ethyl acetate − − −
tetrahydrofuran (THF) + + 4
ultrapure water + + 4
A plus (+) indicates solubility of laminarin in the particular solvent as well
as presence of branched laminarin molecules. A minus (−) implies that
laminarin was not soluble in the solvent
suitable because of the high viscosity of the extracts, as well as
low or no signals of laminarin in the LC-MS measurements.
Additional analyses of extracts gained after three subsequent
extraction steps of laminarin from S. latissima thallus
material with cold water showed that the first step extracted
mainly unbranched short polysaccharide chains, the second
mainly branched long polysaccharide chains, and the third
no laminarin at all. However, the mass spectra of the extracted
polysaccharides differed from the commercial laminarin.
Furthermore, the first extraction provided a much higher
quantity of polysaccharides compared to the second extraction
step. Therefore, based on the complete signal values of the
LC-MS analyses, it was possible to estimate that the first ex-
traction step extracted already 90 % of all polysaccharide spe-
cies from the algal material. The second extraction 10 % and
the third showed no signal for laminarin.
Analytic tracing of laminarin after acidic hydrolysis
The hydrolysate of commercial laminarin after acidic hydro-
lysis for 15 min at 121 °C showed in the ion exclusion chro-
matography a conspicuous signal for oligomer carbohydrate
units next to the actual glucose signal, as well as a distinct
sulfuric acid signal. According to 5000 Da as the mean molar
mass of laminarin, the degree of the hydrolysis was only 15 %
referred to an averaged calculated glucose quantity based on
unbranched laminarin. However, the precise calculation is dif-
ficult because the exact ramification degree and polydispersity
of the laminarin molecules can only be estimated. The acidic
hydrolysis after 180 min at 121 °C for estimating the com-
pleteness of the hydrolysis revealed a degree of 67 % under
these conditions. The chromatogram showed no oligomer car-
bohydrate units anymore but distinct peaks for glucose and
sulfuric acid. However, the transparent color of the hydroly-
sate turned into a slight brownish discoloration after 180 min
indicating different chemical processes resulting finally in py-
rolysis of laminarin.
J Appl Phycol (2016) 28:533–543
Characterization of laminarin
Commercial laminarin from L. digitata in different concentra-
tions was identified by its retention time of 3.70 min (Fig. 1)
and its distinctive mass spectra of different chain lengths. This
method allowed us to certainly quantify laminarin also in very
low concentrations (<0.014 mg mL−1). Due to the applied spe-
cial simultaneous zoom scan measurement, laminarin was iden-
tified as doubly charged. Based on this evidence, the typical
molar mass distribution of 2000–7000 Da could be confirmed
for the commercial laminarin by LC-MS analysis.
After the distinctive chemical characterization of the com-
mercial laminarin, it was possible to identify this storage com-
pound in water extracts of L. hyperborea, L. digitata,
S. latissima, F. serratus, F. vesiculosus, F. spiralis,
H. elongata, P. canaliculata, and D. dichotoma by
comparison of the retention time (3.67–3.75 min, Fig. 1) and
the distinctive mass spectra. Laminarin from L. hyperborea
consisted of the longest glucose chains in comparison to lam-
inarin from L. digitata consisting of the shortest chains
(Fig. 2). Furthermore, there were differences in chain length
and number of chemical laminarin species depending on each
alga species. Fifteen different laminarin species were identi-
fied from the nine brown algal species containing this poly-
saccharide (Table 2). For example, in F. serratus and
F. vesiculosus, four different laminarin species with molar
masses ranging from 2600 to 4400 Da were present. In con-
trast, for S. latissima only, one short laminarin species
with a low molecular weight of 2300 Da was detected
(Table 2). A laminarin species with a molecular weight of
Fig. 1 Chromatograms of commercial laminarin from Laminaria
digitata as reference showing a distinctive single laminarin peak at a
retention time of 3.70 min. The chromatogram of Cystoseira
537
3200 Da was found in five of the nine algae tested. Algal
species containing laminarin possessed one branched laminar-
in species as detected by the mass spectra (Table 2).
In contrast, no laminarin could be determined in extracts of
A. nodosum, H. siliquosa, and C. tamariscifolia (Fig. 1).
Surprisingly, compared to the THF extracts of reference
laminarin, the THF extracts of all 12 algal samples
contained no detectable laminarin.
Chemical composition of the different algae
The kelp L. hyperborea contained high concentrations of man-
nitol and laminarin in the meristematic blade (343.41 ±
26.88 mg g−1 DW mannitol, 8.58±0.09 mg g−1 DW laminarin,
mean±SD) which concentrations were similar to those found in
L. digitata (241.81 ± 3.17 mg g−1 DW mannitol, 7.51 ±
0.07 mg g−1 DW laminarin). In contrast, the kelp Saccharina
latissima accumulated 270.63±15.12 mg g−1 DW mannitol but
only 0.91±0.12 mg g−1 DW laminarin in the whole blade. The
storage of mannitol as well as laminarin in the thalli and mer-
istematic tips of the fucoid species F. serratus (132.75±
5.92 mg g−1 DW mannitol, 3.82±0.93 mg g−1 DW laminarin)
and F. vesiculosus (200.61±7.52 mg g−1 DW mannitol, 2.56±
0.19 mg g−1 DW laminarin) was considerably lower compared
to the Laminaria species. F. spiralis contained only traces of
laminarin (0.04±0.19 mg g−1 DW) which were similar to lam-
inarin contents in H. elongata, P. canaliculata, and
D. dichotoma (Table 3). Additionally, even the mannitol con-
t e n t o f F. s p i r a l i s w a s s i m i l a r t o H . e l o n g a t a ,
P. canaliculata, A. nodosum, and H. siliquosa compared
tamariscifolia reveals no laminarin in contrast to Saccharina latissima
showing a clear laminarin peak at 3.70 min
538 J Appl Phycol (2016) 28:533–543

Fig. 2 LC-MS mass spectra of extracted laminarin from Laminaria hyperborea and Laminaria digitata showing different chain lengths

to the other Fucus species. While no laminarin was detected
in C. tamariscifolia, A. nodosum, and H. siliquosa, they
contained mannitol in considerable amounts (Table 3).
Only traces of free glucose were found in L. digitata
(0.21 mg g −1 DW) and F. spiralis (0.11 mg g −1 DW)
meristematic blades. However, C. tamariscifolia which did not
contain any laminarin showed a higher concentration of glucose
(0.47 mg g−1 DW) compared to L. digitata and F. spiralis.
Moreover, D. dichotoma exhibited the highest concentration of
glucose (1.28 mg g−1 DW) which was almost double the
J Appl Phycol (2016) 28:533–543 539

Table 2 Composition of laminarin extracted from different brown alga and the development of a suitable method for extraction of
species
this polysaccharide. For these purposes, a commercially puri-
Number of Molar masses of fied laminarin substance was tested, and distinctive chemical
laminarin species laminarin species (Da) features were identified.
The comparison of different solvents revealed that the com-
Laminaria hyperborea 2 3200, 4300
mercial laminarin from L. digitata was soluble and detectable
Laminaria digitata 3 2100, 2600, 3200
in ultrapure water as well as in THF without losing quantity
Saccharina latissima 1 2300
like in ethanol/water solutions. This is in contrast to Black
Fucus serratus 4 2600, 3200, 3400, 4400
(1950) who found that a more water-soluble form of laminarin
Fucus vesiculosus 4 2600, 3200, 3600, 4400
is best extracted from L. digitata in aqueous solution after the
Fucus spiralis 3 2400, 3500, 3800
addition of ethanol. Additionally, this author suggested the
Himanthalia elongata 2 2800, 4000 presence of a water-soluble form of laminarin in L. digitata
Pelvetia canaliculata 3 3200, 3600, 3900 and a water-insoluble form in L. hyperborea. With the com-
Dictyota dichotoma 1 3000 mercial laminarin we could confirm a water-soluble and
Molar mass was determined at the maximum of the statistical molar mass
water-insoluble form of this macromolecule based on the ap-
distribution. In all brown alga species, branched laminarin molecules plication of water versus THF as extraction solvents. In our
were detectable brown algal samples, however, this was not the case as THF
did not yield any of this storage compound. There are only
concentration of laminarin (0.66 mg g−1 DW) stored in this alga few options to explain these data: First, there is indeed no
(Table 3). No free glucose was found in L. hyperborea, water-insoluble laminarin present in the brown algae tested,
Saccharina latissima, F. serratus, F. vesiculosus, H. elongata, or second, there may be interactions of this laminarin fraction
P. canaliculata, A. nodosum, and H. siliquosa. with the complex matrix of cell wall components hindering
Carbon and nitrogen content did not strongly differ be- the extraction with THF. The latter case may completely ham-
tween the different brown algal species. However, per extraction yield from natural algal material in contrast to
L. digitata and S. latissima showed lower C/N ratios com- the good solubility of purified commercial laminarin in THF.
pared to the other species (Table 3). The commercial laminarin was extracted from L. digitata by
Sigma Chemical (Sigma-Aldrich Chimie S.a.r.l.) who provid-
ed laminarin in a lyophilized powder. However, this company
Discussion is not able to provide any information about the extraction
procedure and purity of the laminarin product. Hence, it is
The aim of this study was the chemical characterization and not certified as a standard. It might be reasonable to assume
quantification of laminarin in different brown algal species that this commercially available laminarin is a product being

Table 3 Mannitol, glucose, laminarin, carbon, and nitrogen content (% DW) as well as C/N ratio of 12 different alga species sampled in fall 2013 at the
coast of Ireland

Tissue Mannitol Glucose Laminarin C N C/N ratio

(% DW)

Laminaria hyperborea Meristematic blade 34.34±2.69 - 0.86±0.01 35.50±0.23 1.45±0.01 24.52±0.15

Laminaria digitata Meristematic blade 24.18±0.32 0.02 0.75±0.01 29.85±0.06 2.21±0.07 13.54±0.44
Saccharina latissima Whole blade 27.06±1.51 - 0.09±0.01 29.38±0.12 1.96±0.06 15.05±0.53
Fucus serratus Blade incl. Meristematic tips 13.27±0.59 - 0.38±0.09 34.04±1.35 1.17±0.12 29.51±4.11
Fucus vesiculosus Blade incl. Meristematic tips 20.06±0.75 - 0.26±0.02 34.87±0.46 1.00±0.18 35.82±6.64
Fucus spiralis Blade incl. Meristematic tips 9.18±0.78 0.01 0.04±0.001 32.39±0.55 1.25±0.10 25.99±2.14
Himanthalia elongata Reproductive sporophyte 4.57±0.53 - 0.05±0.01 30.69±0.47 1.16±0.05 26.42±0.89
Cystoseira tamariscifolia Whole n.a. 0.05 - n.a. n.a. n.a.
Pelvetia canaliculata Whole 4.79±0.15 - 0.03±0.001 34.17±0.33 1.06±0.02 32.38±0.67
Ascophyllum nodosum Middle part and tips 9.47±0.48 - - 35.16±0.32 0.93±0.05 37.98±2.44
Halidrys siliquosa Whole 11.53±1.39 - - 36.40±1.09 1.22±0.15 30.23±3.82
Dictyota dichotoma Whole n.a. 0.1 0.07±0.002 n.a. n.a. n.a.

Minus (−) indicates the absence of glucose and or laminarin in the samples. Insufficient available biomass of Cystoseira tamariscifolia and Dictyota
dichotoma for mannitol, carbon, and nitrogen analyses is indicated by n.a. Values are expressed as mean±SD of three subsamples from a single
individual per species
540
obtained from algal material by chemical or enzymatic hydro-
lysis according to the US patent for use of laminarin in cos-
metics and pharmacology (Yvin et al. 1999). However, the
applied extraction methods cannot chemically modify lami-
narin. Another applied method for the isolation of laminarin is
a preparative HPLC which is particularly costly. Despite all
this, the distribution and quantity of different laminarin spe-
cies (ramification degree and chain length) in the commercial-
ly available laminarin substance can vary depending on envi-
ronmental and individual conditions of the harvested
L. digitata. Nevertheless, our LC-MS measurements demon-
strated clearly that the commercial laminarin consists of four
different laminarin species showing the typical molar mass
distribution of 2000–7000 Da.
Results of cold water extraction of laminarin from algal
material proved to be successful as laminarin could be detect-
ed and quantified by LC-MS analysis. The three-step extrac-
tion with cold water from Saccharina latissima thallus mate-
rial indicated that the first and second extraction yielded al-
ready 100 % of available polysaccharides and in the third
extraction step no laminarin could be detected. Moreover, a
quantification depending on the commercially available lam-
inarin from L. digitata was problematic because of the
species-specific variability as well as number of the chemical
structures of laminarin. This points also to a great challenge
working with natural substances as a standard compound for
calibration because the chemical structure and quantity are
depending on the biological source and environmental factors.
The resulting aspect of laminarin variability has to be consid-
ered in all applied analytical approaches. According to our
experiences, LC-MS measurements provide the most precise
and reliable data as they detect the different chemical struc-
tures of laminarin.
Presence, quantity, and number of chemical laminarin spe-
cies varied between the 12 analyzed brown algal species. Our
results showed that laminarin occurs in the frond of
L. hyperborea and L. digitata and to a lesser extent in
S. latissima, F. serratus, and F. vesiculosus, while
F. s p i r a l i s , H . e l o n g a t a , P. c a n a l i c u l a t a , a n d
D. dichotoma contained only traces of this storage compound.
These general findings are in accordance with the early studies
of Black (1949, 1950).
The different studies quantifying laminarin from alga ma-
terial used numerous methodological approaches (see, e.g.,
Black 1950; Johnston et al. 1977; Gagné et al. 1982; Gómez
and Wiencke 1998; Dethier and Williams 2009). Laminarin
was often quantified as total sugar equivalents after acidic
hydrolysis of the complete algal biomass (Black et al. 1951;
Gorham and Lewey 1984; Devillé et al. 2004). This, however,
will most probably result in a strong overestimation of lami-
narin, since acidic hydrolysis will cleave all sugars or sugar
residues present in the biomass such as, for example, glyco-
lipids, glycoproteins, and celluloses. Furthermore, we showed
J Appl Phycol (2016) 28:533–543
that the acidic hydrolysis of pure commercial laminarin is
incomplete and hence not quantitative because a larger
amount of oligomer carbohydrate units than single glucose
units was received under the widely applied hydrolysis con-
ditions (e.g., according to Schiener et al. 2015). The received
concentration of glucose from laminarin varied depending on
the hydrolysis duration, and the hydrolysis of polysaccharides
is leading to uncontrolled cleavage of sugar units (Templeton
et al. 2012). In addition, the occasional reduction of free
sugars to sugar alcohols due to hydrolysis conditions can re-
sult in varying and inaccurate results for quantification
purposes.
In fronds of L. hyperborea collected in autumn from the
Norwegian coast, 25 % DW mannitol and 30 % DW laminarin
have been reported (Jensen and Haug 1956). Our results re-
veal in autumn fronds of L. hyperborea from the coast of
Ireland 34 % DW mannitol, which is similar to the values of
Jensen and Haug (1956), but only 0.86 % DW laminarin. In
comparison to laminarin and mannitol contents published in
other studies, we always found substantially lower laminarin
but similar mannitol contents in the analyzed brown algae.
The obvious differences in laminarin quantities may in prin-
ciple depend on the algal species, the season, and other envi-
ronmental factors, but we are confident that the applied ex-
traction methods and hydrolysis conditions led in most of the
earlier studies to a strong quantitative overestimation of
laminarin.
In addition to different quantities of this storage polysac-
charide, laminarin represents a mixture of chemical structures
with species-specific compositions (Read et al. 1996; Chizhov
et al. 1998; Rioux et al. 2009, 2010). We characterized 15
chemical laminarin species with distinctive different molecu-
lar weights from 9 species of brown algae. Differences in
chain length and number of laminarin species seem to depend
on algal species. Especially, F. vesiculosus and F. serratus
contained four laminarin species with quite similar molecular
weights. This may indicate more similar biosynthetic path-
ways for laminarin synthesis from genetically closer related
species compared to other species. Similarly, the linear sugar
alcohol altritol was found in six genera of the order Fucales,
and five of these genera are closely related according to mo-
lecular phylogenetic and other data (Wright et al 1987; Raven
et al. 2001). For this reason, it seems possible that also differ-
ent laminarin species show taxon-specific patterns and may
provide further information as chemotaxonomic markers. In
the present study, we proposed a new LC-MS method as an-
alytical tool to carefully chemically identify, characterize, and
finally quantify laminarin in extracts from different brown
algal species to overcome the methodological problems de-
scribed. Furthermore, this method provides information across
a range of molecular sizes present in the polydisperse popula-
tion of laminarin molecules, which may be used as a chemical
fingerprinting for different algal species, life cycle stages, or
J Appl Phycol (2016) 28:533–543
growth season. The data illustrate the power of modern LC-
MS technology for defining and quantifying variable natural
mixtures of laminarin.
The carbon and nitrogen content of the analyzed algae was
similar to those reported for other brown algae (e.g., Surif and
Raven 1990; Lehvo et al. 2001). Furthermore, we found only
traces of free glucose in the algal material, which is in agree-
ment with Yamaguchi et al. (1966).
Accumulation of storage compounds and translocation of
photosynthates have a high ecological importance for peren-
nial brown algae as these compounds substantially support
rapid growth of special meristems like developing reproduc-
tive thallus parts. In F. vesiculosus, the maxima of mannitol
reserves were found in the receptacles, while the highest lam-
inarin concentrations were detected in basal thallus parts
(Küppers and Kremer 1978). In Macrocystis pyrifera, a down-
ward transport of photosynthates from the apical,
photosynthesizing parts is mediated to the basal parts of the
kelp for carbon storage in autumn (Lobban 1978). Therefore,
the laminarin content is subject to marked seasonal variation
(Black 1948, 1949, 1950). A relationship between seasonal
growth, storage, and nitrogen availability has been shown
for F. vesiculosus in the Baltic proper (Lehvo et al. 2001). In
late spring and summer, biosynthesis and storage of reserve
compounds occur when light conditions for photosynthesis
are optimal but nutrients are limiting (Hatcher et al. 1977).
In this period, F. vesiculosus uses stored nitrogen for rapid
growth. However, during the dark and cold winter,
F. vesiculosus survives and grows slightly because of stored
carbohydrates (Lehvo et al. 2001). In contrast, L. hyperborea
(Lüning 1968), M. pyrifera (Wheeler and North 1981; Gerard
1982), Laminaria longicruris (Chapman and Craigie 1977;
1978), and Saccharina latissima (Black 1950) showed intense
biomass formation during winter and early spring when nitro-
gen is not limiting. In these kelps, internal organic carbon
increases in summer as carbon assimilation exceeds carbon
utilization and reserve carbohydrates are built up. In detail,
mannitol and, to a minor degree, laminarin reach their highest
concentrations at the time when growth starts to decrease in
summer (Chapman and Craigie 1978; Gagné et al. 1982;
Jensen et al. 1985). Saccharina latissima (Black 1950) accu-
mulates both mannitol and laminarin in summer in contrast to
the kelp Saccorhiza polyschides (Jensen et al. 1985) which
only accumulates mannitol. Both species, however, subse-
quently remobilize mannitol during winter to almost complete
absence. This degradation of storage carbohydrates, which are
built up in summer, supplies the energy requirements for
growth during high nutrient availability in winter and early
spring when photosynthesis ceases due to light limitation
(Lüning et al. 1973; Chapman and Craigie 1977). Especially,
the Arctic kelp Laminaria solidungula grows under sea ice in
absolute darkness in winter powered by laminarin and manni-
tol synthesized during the previous summer season (Dunton
541
and Schell 1986; Dunton and Jodwalis 1988). A general bio-
chemical pattern in perennial brown algae of the Northern
Hemisphere appears to be that mannitol and laminarin are
accumulated mainly during summer months resulting in max-
imum contents by late summer and early autumn, followed by
strong decline during winter to support winter growth (Black
1950; Lüning 1979). In Antarctic Desmarestiales and
Ascoseirales, an increase of mannitol during spring and sum-
mer results in depletion of laminarin, suggesting that these
compounds support demands during lamina elongation
(Drew and Hastings 1992; Gómez and Wiencke 1998;
Wiencke et al. 2009). These algae seem to suffer a photosyn-
thetic carbon deficit during the growth period, which may be
compensated by reutilization of storage carbohydrates
(Gómez and Wiencke 1998).
In conclusion, the presented cold extraction of this storage
polysaccharide from brown algae is a very simple method and
suitable for further exact quantitative determination of these
complex compounds. The obtained extract from dried algal
material can be analyzed directly by LC-MS. The direct char-
acterization and quantification of laminarin by LC-MS is a
helpful approach of modern biochemical analytics to verify
the importance of laminarin for brown algae.
Acknowledgments We gratefully thank Udo Nitschke and Andreas
Wagner for collection and provision of the algal material, and Juliane
Müller and Henrike Pfefferkorn for their invaluable help in the lab. This
research was funded by the Project BIOACID Phase II of the German
Federal Ministry of Education and Research (BMBF; FKZ 03F0655L).
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