( l a h ] l i o l . M a r . ( 2 ( X i i )4 2 : 1 3 7 1 5 7
Chemistry and physico-chemistryof phycocolloids
Marc LAHAYE
Institut National de Ia RechercheAgronomique,
Unité de Recherchesur les Polysaccharides,leurs Organisationset Inreractions,
Rue de la Géraudière,B.P. 71627,44316 Nantes cedex03, France.
e-m ail : Iahaye @nantes.inra..fr
Âbstract: A rcview on the chemistry, physico-chemistry and somc gel charactcristics of major phycocolioids is presented.
-fhcse
concern mainly agar, gclling carrageenans from red seaweeds, alginate frorn brown seaweeds and ulvans from green
seaweecis. Based on the available data, future research iùeas are proposcd to better defrne thc relationships betwccn the
diff'erent structural levels of these polysaccharides and their use âs rexturing agents.
Résumé : Une rcvuc de la chirnie, de la physico-chimie et dcs caractéristiqucs des gels des principaux phycocolloides est
presetttée. Blle concerne principalemcnt les agars et les carragénanes gé1ifrants des algues rouges, Ies alginates des algues
brunes et Lcs ulvanes des algr.resvertes. A partir des données disponibles, dcs domaines de recherche tuture sont proposés
poul mieux définir les relations entre les clifférents niveaux clc structure cle ccs polysaccharides et leurs utilisations conùrie
agenl.s tcxtrrra.nts.
Kel,patv4t'. seaweed, polysaccharides, chemistry, physico-chemistry.
Introduction
More than 3..5 million tons of seaweedsare han'ested
iurnuallyin the world and the rnain use of this biomassis for
lood consumption (-60 Vo) and for phycocolloid
productions (-40 7o, Jensen, 19931.Seaweedsare not
consumed as staple foods but rather for their particular
orgarrolcpticand I'trnctionalproperties arising lbr a good
parl from their cell-wall polysaccharides.The ability of
thcsc polynrcl-sto textureaqueoussolutionshas been used
Ior rnanv ycars and is at the basis of al industry using
seaweedsrs sourceof gelling, thickeningand stabilizing
agents- The purpose of thrs review is to summarize the
chcnrical and physico-chemicai basis ol thc textural
propcrticsof phycocolloids.The relationships
betweenthe
biology of seaweeds and the chemistry of the
polysaccharidcs(biosynthcsis,ccophysiological effects...)
will not be coveredin detailsmd informations can bt: ibund
in other r-ecentrcviews (Craigie, 1990,Cossor-r
et al., 1995).
The phycocolloidsconsideredare the industrially used agar,
carrageenanand alginatefrom led and brown seaweedsand
sonrc informations wiU also be presentedon ulvans liom
grcen se.aweeds.
General aspects on thc chcmistry
and physico-
chcmistryof texturingpolysaccharides
The basisof phycocolloidspropertiesrelics on the nature
and extent of intermolecular associations in orderod
I1u
r l s s û n r b l i e sf o r ' r i n g h y d r a t e d g e l
nctworks or on thcir interactionsby
enlanglcment of
random cO.il
p , ' l y . : r t . l r ai dr e . . 1 3 1 6 t t r t*o r i s c o u c
s , r l t t t j n t tI sI.r ca i m r r t t h i ss e c t i o ni s l o
strrnnrarizcgcneralprinciplesrelating
stntclurc to firnction tll' polysacchan cles.
Pyranose (tivc carbons a,nd onc
oxygen) sugar rings (primary
structure,Iiig. l) ale the rnainbuilding
units of phycocolloidsand theserings
adoptin generalthe mostenergetically
iavourablechair confonnationsnoted
4C1 u. lC4 clepen<iing
on the positions
ol (1.,1and C-l with respcct to the
planc O-5, C-2. Cl-3and C-5, the C-6
being outside lhe ring. On these
carbons, hyclrogen and hydroxyl
groups
are
linkcd
either
perpendrcularly to the plane and zue
then refcr to be in axial configuration
or in thc plane, in which casethey are
said to be equatorial. Linkages
between sugars in polysaccharides
occur through the hydroxyl group on
(l-l (anornericcarbon) with any of the
ofhcr fïee hyclroxyl prcsenton arrolher
sugar ring. l'he glycosidic linkage
formed can eithcr be cr if the OtI on C
I is on the opposiresidc of C-6 on thc
ring or [3 if they both ale on the same
side (t'ig. I ). This linkage gives
torsional dihedral angles g and ç
(sccondary structure, Fig. 2Â) which
alc rcsponsibJcfor the shape of the
polysaccharidc (tertiary structurc).
Anglc.sof'similar valuc all along the
polysaccharidc chain incluce an
ordcrccl r:onl'ornation of
the
ruracrornolecule,
othcrwise,it will bc a
rantlom coi[. Mcthyl ethcr, sulphate
hemiester, pyruvatc acetal, and
glycosrdcs cau substitute othcr
hyclroxylgroups.The mcthylenegroup
AI -(;AL I'OI-YSA(]CHARII)F]S
p - I t - g a l a c l o p y r a n o soeC ,
un,
l i
I Co
3,6-anhydro-cr-D-galactopyranose
'"*o-\-]--5--o"
HO---\-.--r/o\
HOzC
aC,
B-D-glucopyranuronic acid
ao"
û-L-Idopyranuronic acid aC,
aC,
B - o - m a n n o p y r a n u r o n i ca c i d
OH
c-t-rhamnopyranose lco
p - o - x y l o p y r a n o s ea C ,
Figure 1. Chemical structurc of the principal constitutive sugarsof phycocolloids.
Figure I. Stiucture chimicpredes principaux osesconstituantsdes phycocoltoides
at (l (r c;rr :rlso be replaced by a czrrboxylic acid group and
i n l h i r t c a s et h c s u g â ri s a u r o n i c a c i d ( F i g . 1 ) .
The gelling ability of phycocoiloids depcnds on thc
overal l shapegiven by the glycosidic linkage anglcs but also
o n t h e a b i l i t y o f t h c p o l y s a c c h a r i d e st o f c r r m i n t r a - a n d i n t c r r n o l c c u l a r p h y s i c a l l i n k a g c s ( l ' r y c l r o g c nb o n d s , e l e c t r o s t a t i c
lrrd
aC
3,6-anhydro-cr-t--galactopyranose
Vrn dcr Waa'ls interactions, Rccs, 19ti2). T'hese
i r : i s o c r i , r t r o na
src allcctcd by ncgative chârges originating
from carboxylic acid and-/or sulphate hcmicster groups
which tcnd to expenclthe polysacchzridesin water through
mutual electrostaticrepulsions,and./orby steric hindrance
due to olher groups(rnethy1,glycosylation...)substituting
for hydroxyls involved in intra-molecularor inter-molecular
hydrogen bonds. Co solutes such as salts modify the
solvaling ability of water and shift the equilibrium from
polymer-solventinteractionsto polymcr-self-associations
M. LATIAYE
l:r9
ùnd lead to gcl formation or prccipitation
( c e r r a ! l e o n a na, l g i n a t e s ,u l v a n s ) . I h c l o s s i n
\L/
OH
nrobiiity ol thc mucromoleculethrough the gel
\.-'t=--]-o.
fôrrriationor prccipitateis compcnsatecl
^
by thc
c n e r g c t i c a l i y f a v o u r a b l e n o n - c o v a l e n tb o n d s
fbrrrrltion. N'lmy of thcsc bonds arc in thernselvcs
weak ancl easily ltroken, but, acting coopcrzrtivcly,thcy stabilizc the lnt]sostructufc.
'l'hus,
a rnirrintumpolysaccharide
chain length is
fcquirc(i [br thc establishnent.of stable ordcred
self-associatedstructures.It is thereforeclear-that
the primary and secondary structuresincluding
B
thc rnolar weight reflecting the degree of
polymcrization of the polysaccharidesare basic
l)arametersfor their ovcrall properties.Irregular
cornpositron,glycosidic linkages or substitutions
contribute to increase the solubility of the
polysaccharides. Other
physico-chemical
C
conditions, such as lemperature, ionic strength
and pll also affect the fonrration and meiting of
the orderedstructurein breaking or preventingthe
fbrmation of the co-operativewezù.boncls.
The type of ordcred structure formcd by
polysaccharidcsdepends more on the glycosidic
11\ ^^/-\.\
/'\^.\.\f\
linkage gcornetry than on the nature of the
D
b u i l d i n gu n i t s ( R c e s e r a l . , 1 9 8 2 ) . 8 - 1 , 4
diequatorial glycosidic linkages such as those
found in ccliulose,mannan,chitin zLreparallei and
Figure 2. GJycosicliclinkage angles A, and the overall flat dbbon
aboutco-linearwith the sugarresidues.
They lead
B, buckled ribbon C, and hclical D shapesinduceti by tht: rcgular repctition
to flat ribbons able to pack into fibrillar assemblies
of these imglesalong polysaccharides.
(Fig 28) Diaxial parallel linkagesas found in
l'igure 2. Angles de la liaison glycosrdiqueA et confonnations en ruban
alginate,allow cavitiesthat can accommodateand
plat B, ruban torsadéC, ct hélice D induitespar )a répétition régulière de ces
stabilize ions ("egg box" structurcs) and form
anglesle long clc la chaine du polysaccharide.
ordercd structurcssuch as highly buckled ribbons
(Fig. 2C). Cilycosidic linkages not parallel or
predominantly dependent on thc molar sizc of thc
tliagonally oppositc intrcduce a regular "twist" to thc
polysaccharide
chainresultingin an helix usuallystabilized polysaccharide,is mcasured as the frâctional increase in
viscosityper unit concentralionunder conditir-rns
of extreme
by co-axial packing such as that found with agar and
diiution (without inter-chain interactions). This "intrinsic
carrageenans (Irig. 2D). The consequencesof such
viscosity" l4l is relatedto the molecuiar weight (M) of the
associationsare at the basis of the gelling properties of
polysaccharideby thc Mark-Houwink relationship:
phycocolloids.'['hc suprarnolecularorganizationsof the
polysaccharidcsgiving risc to the 3-dimensionaliryof ttre
III= Kwa
geJ nctwork (F'ig..3A, Reeset al., 1982) are stabilizedby
whele K and a valuesreflect the polysaccharidesshape in
'Junclion
zones"segnrentsinterspersed
the solvent. In case anionic polysaccharidessuch as
with disorderedor
"solubilizing"
sequences.The latter arise from structural
carrageenans,alginate and ulvan, polysaccharidescoil
inhomogcncrties disrupting the phycocolloids regular
dirnensiolrs arc aflected by ionic strength, electrostatic
gcolnetry. In case of carrageenanhelices, "super-helical repulsions ol chains in abscnce ol' adtled electrolyies
fibrc bundles" packings have also bcen proposcd where
.increasethe coil volumc. For snch polysacchirrides,the
"solubilizing
sequences"would bc absentor vcry short
intrinsic viscosity tlecreaseslinearly with 1/Il/2 ancl
(Vicbkc ct al., 1994, F'ig. 3B). When ordering of rhe
extrapolatesat intinite ionic strcngth to the intrinsic
polysaccliandesis r.iotachieved,the volunrc occupiedby
viscosity of the ecluivalentncutral polysaccharidc of thc
pol_ysaccharidc
coils in the solvent.leadsto an increasein thc
samc primary structure and degrcc of polymerizalion
s o l t r l i o n v r s c o s r t y ( M o l r i s , 1 9 9 2 ) ' f h i s v o l u m e , ( M o r r i s , 1 9 9 2 ) . T h c p r o p c r t i e so f p o l y s a c c h a r i d e si n
01
VA\--on
lrn'
UVVVVVVVVVV
A L C A I . P O I Y S A C C I I A I I . IT ) I i S
Chemicalstructurcand physico-chcmical
propertiesof phycocolloids
JUnCllOn ZOneS
With the above introductory remarks aimed ar defining thc
different importantfactorsinvolved in the physico-chernical
propert:ies
of phycocolloids,the rnain chemical and physicochemical characteristicsof water soluble gelling and/or
thickeningalgal polysacchaLrides
wili now be reviewed.
l. Galactans
Sourcesand extraction
B
Irigure 3. Schcrnatic rcpresentationsof'Junction zones" and
"solubilizing
sequences"in three-dimensionalgel networks accor,
ding to the model of Rees er al., (1982) A, and to the ,,super-helical bundles" of Viebke et al., (1994) Il.
Ii-igure 3. Représentatiouschématiquedes "z_onesde jonction,'
ct tlcs "séquetrcessolubilisantcs" dans le réseautri-dimcnsionnel
d r r m c r d è l et l c R e e s e t a 1 . , ( 1 9 8 2 ) A , c t d e s " s u p e r - h é l i c e s , ' d e
V i e b k e e t a l . . ( 1 9 9 4 )B .
solution
also tlepencl on concentration. Above a critical
concentration c*, the polysacchârides interact together by
physical entanglcment. Bclow c*, the polysacchzuidcs can
rnove freely with little interference with each other and the
viscosity is independent of shear ratc ("Newtonian
bchaviour"). Abovc c*, shoar rates will physically affect the
cntanglcd charns by more or less favouring their separation
("rcptation") frorn ouc another. At low shcar rate, the rate of
rlissociation and rc association of the interactions will
crquiliblatc and thc vrscosity will be rhe highest. At highcr
sheiu ratc, the viscosity decrease ("shear-thinning")
as the
rate of cnlanglenrent fornation is lower than the rate of de,
association. The liactional increase in viscosity linked to
polysaccharide
concentrations
belowc* is increased
by a
factrx ol about 2-5, consecutivc to a doubling of the
polysaccharidc concentration: above c* it increases by a
flctor ol aboul I0 (Molris, 1992).
lled seawcedsare wcll known sourcesof industrial gelling
and thrckeningccll-wall galactans,rcfcrrcd to as agar and
carrageenans. Gel.idium, Pteroclad.ia, Gelid.iella and
Gracilaria species constitute tlre main seaweed raw
materialsused for the production of agar and agaroseand,
now-a-days,most of tie carrageenanproductionsare from
farmed Eucheuma species (Stanley, 1987, Arnrisen &
Galatas, 1987). These polysaccharidesâre extracted from
dded algae by more or less hot alkaline solutions (100t20'C). After filtration, agar solutions are allowed to gel,
thcn de-wateredby pressing,dried and ground (Annisen,
1987) whereas carageenân in the filtered extract are
generaily precipitated by alcohol (Stanley, 1987).
Carrageenansolutions (kappa-carrageenan)can also be
gelled by addition of potassium ions, de-watered by
pressing,dried and ground (Stanley, 1987). Scrri-refinecl
carrâgeenans
zu'ealso producedby an alkaline heatment of
'Ihese
the seaweedsfollowed by thorough water rinsing.
treatments improve the gelling characteristicsof thc
carrageenanprcparationand rerrrovertrost of the proteins,
pigments an<i small metabolites. However, due to the
absenceof a ûue calTageenanextraction and of a liquidsolid separafion,such preparadonscontain other polymers
such as cellulosicmaterials(Hoffman et al., 1995).
Nomencllture und chentistry
Tbe naming of gelling algal galactans has recentJy been
undcr consideration by several authors (Craigie 1990,
Knudsenet al., 1994, Miller 1997). Originally, agar was
defined as polysaccheuides
with a basic backbonestructure
based on the repetition of alternating 1,3-linked B-I)
galaclosc and l,zl-linked 3,6-anhydro-cr,L-galactose.
This
disacchariderepeafing unit was namcd agarobiose or
neoagarobiose(Fig. 44, Araki 1966). Vcry carly on,
chenrical nrodiflcations of this structure by sulphatc
hemiesters, pyruvate acetal and,/or methyl ethers were
identified and the extent of substirution, pallicularly by
sulphateesters,Ied to tlie distinctionof gelling agaroseand
non-gcllingagaropectinrnolecules(Araki, 1966). It was
lattershownto be an oversimplisticview of the distribution
of the agiu"moleculeswith regar<I
to the existingcontinuurn
M I-^llAYIl
betwecn neutfal and highly
r,hlu'gcclgalactans (L)uckrvorth &
Y a p h e ,1 9 7 l ) a n d b e t w e e nl o w 1 o
rnethyl
highly
subsritutcd
rnoleculcs(Lahayc ct al., 1986).
'1'hus,
ergar-typepolysaccharides
wcre shown to have an intportant
intrâ- and inter-molecularhybrid
naturc, explaining in palt the
v ; r r i a l . i l i t yo f g e l l i n g p r o p e r t i e s
obscrved between different agarLrxtracts or fractions. For that
rcason,Craigie (i990) proposedto
refer
to
the
agar-type
polysaccharidcs
as
the
argarocolloids
and
then
to
clistinguish among them the geiling
a g a r a n t . ln o n - g e l l i n g a g a r o i d \ . T h e
term agarose, referring
t. the
^:^'
n l g n c s r g e t r r n g r r a c t l o n o^.:
I a
gâr
trsed firr biotechnological purposes
(Renn, 1984), has a precise
chcmical clelinition that is basicallt
and i<leally composed solely of
A
agarobiose
(
neoagarobiose
B
carrabios€
,1
oR2
neocarrabiose
Figure 4. chemical stmcture and names of the repeatingdisacchande units in agaroseA
and carrageenanI]. Rl= sulphateand R2 = hydroxyl: kappa-carrageenan;
Rl = R2 = sulphate:
iota_carraseenan.
Figure a. S[rrcture chimique et noms des unités disacc]raridiquesclerépétition rJansl'agar o s e A e t l e c a r r a g h é n a n e B - R l = s u l f a t e e t R 2 = h y d r o x y l : k a p p a - c a r r a g h é n a n e ;=I R
tl2=
sulfate: iota-carraghénane'
unsubstitutedagarobioserepeating
units (Fig. 4A). The basiccommon featurein agarcolloidsis
name these polysaccharidesand as a counterpa.rtto the new
their construction on the repetition of the 3-linked 0-Dterms proposed Ibr agarcolloids, Knutsen et al. (1994)
galactose alternating with the 4-linked c-L-galactose to
introduced the names "ca.rageenan" and "carageenose".
which Knutsen et al. (1994) has proposed the name
Theserefer to polysaccharidesbased on the repetition of 4
"agaralt". 'fhe agar
fraction is emiched in agarobiose linked cr -D-galactose alternating with 3-linked [3-Dlepeatingunits; that is, it containsmore 3,6-anhyrlo-<r-I_- galactoseand to those where the 4-linked sugar is a 3,6galactoso.The closely related galactan,carrageenan,based
anhydro-crD galactose.respectively.As for "agaran" these
on the carrabioseor neocatrabioserepeatingstructure(Fig.
new terms rue probabiy increasing the confusion around
4B), diffcrs frorn agalocolloids by the 4-linked o.-Dthesegalactans,but in fact reflect the complexit)' of properly
galactosereplacing for the 4-linked cr-L-galactoseand in
defining these polysaccharidcsin a way to conceal both
being usually rnore substitutedby suiphategroups.In fact,
chemical characteristicsand functional properties.
the nrrmberand position of the latter and the presenceof 4In this text, the term delined by Craigie (1990) will be
linked 3,6-anhydro-a-f)-galacrose
ttscd tbr agar-lype polysaccharidcs ald the old Greek
are at the basis of the l5
dillerrcnt disaccharidestructuresamong the 42 theorctical
symbol system for the carrageenan polysaccharides.
trnes (Stortz & Ccrezo, 1992) and to which Greek letters
Knutsen et al. (1994) proposed very useful shorthand
have ireen given ('l'able 1). 'l'his nomenclaturefollowed the
not.ationsto describethe different agar- and calrageenanoliginal naming of the carrageenanfrom Chondrus crispus
types repeatingstructures(-lable l). In these notations,the
cxtracts that precipitatc on addition of potassiumchloride
D and L letters refer to the 4-linked <r D- and o -l-(kappa-carragocnan)
liom that renraining soluble having a
galactose,r'espectively,DA and LA to the corresponding
low concentration clf 3,6-anhydrogalactose(lambdaanhydrogalactosederivatives,G to the 3-linked B-Dcarrageenan).Latter on, a third carrageenantype, gelling
galactose.Substituentgroups are definerl as M for methyl
with ciilcium ions was dcfincd as iota carrageenanand
cther, P lbr pyruvate,S for sulphate cstcr, l-heposition ol
gloupings of thc diflêrent carrageenandisaccharides which on thc sugar bcing given by the carbon numbcr.
'Ihese
rol)catingstnrcturesby tamilies accordingto the sulphation
new notationsare particularly hclpiul fbr c.lcscribing
(Clraigie,
were
proposed
1990,1àblc 1). However,
hybrid galactanshaving both agar and carragccnan-type
iratterns
suchnamingscannotaccountfor the highly complexhybrid
backbone characteristics.Such structures are found. 1br
rnrtureof thc polvsaccharidcssincc generallymore t}ranone
example, jn some extracts of (Jigartina skottsbergii, t
typc: ol'fepeating carrabiosestructure is fbund in the
carrageerophytc
havinga galactanfractionwith l,-galactosc
polysacr:harides.
With rcspcctto the difficulty of properly
(Oianciaet al.. i99ll; 1997),and in galactansploducedby
A I , ( J A I - I ) O I - Y S A C C }I A R I I ) E S
'lhl)le
l I)isacchar-idcrepeatingstrucluresof carrageenansa.
'l'ableau
L Structurc des unités disaccharidiquesdc répétition des cirnagénanesa.
I..1-linkedrr
1.4-linkedu
(ileek syrrbol
(i45c
(;.lS
G4S
(;45
(;4S
DA
DA2S
D6S
r (kappa)
r.(iota)
g (mu)
D2S
lJ (nU)
o (orrricron)
(;
DA
D6S
DA
D6S
Y (g,amma)
c'l (omega)
ù (psi)
G
(;65
G65
(;
(;
G25
G2S
G25
Gn2S
n2s,6s
D2S,65
DA2S
D2S,65
DA2S
DAS
D25
B (beta)
ô
c
À
0
(delta)
(alpha)
(lambda)
(theta)
kappa-lanrily
beta-family
lambda-family
E (xi)
n (pi)
r âdaptcdfrom Craigie (i990) and Knutsenet al. (1994), b refer to 1,3- ancl1,4- linked galacroseresidues,c letrers:G B Dgalactopyranosyl; D cr-D-galactopyranosyl;DA 3,6-anhydro-o-D-galactopyranosyl;
Lcr-L-galactopyranosyl;LA
3,6 anhydro <r.-L-galactopyranosyl;S sulphate;P pyruvateacetal;numberscorrespondto the carbon atom number on which
the substitutionis found.
()loitryteltis, LonLentaria, Gratel.oupia or Anath.eca (Nunn et
'l'akano
al., 197l;
et a.1.,1994; 1998; Usov & Ilarbakadze,
1978). For the dcscription of agar and camageenan-type
oligosacchalides,nr is added after the sugar symbol
positioned at the non-reducing end and nr' for the sugar
ncxt to it. Fbr the sugarat the reducingend, its letter symbol
r is cxtr:ndedin r cr, p and that of the sugarassociate<I
with
it in t.hedisacchariderepeatingstructLlreis extendedby r'.
For example I<appa-neocarratetraose
oligosaccharideis
symbolize<l as DAnr-(i4Snr'-DÂr'-G4Srcr,l3. This
rrorrrcnclature
has been iurther extendedto take into account
pfoportions of different substitutionsin galactans(Miller,
1997).Wrth thesc ncw notationsand proposednamingsin
minrl, lhe commercial polysaccharides
sold as agarose,
kappa-carragccnan,iota-carrageenanare hybrid structures
nrainly composetl of agarobiose(G-LA), carrageenose
4'-sull'ate(G4S-DA) anclcarrageenosc
2,4' disulfate(G4SDA2S) rcpeating structures. 'l'hc industriaL lambdacanageenanis usually a mixture of differentgalactansofthe
carragcclli.ln-type
unablcto gei.
'l'hc
basic chcmistry ol agal and carrageenan-typc
polysaccharitJes
has been the subjcct of several recent
revrews ((losson ct al., 199-5;Craigie, 1990; l-ahaye &
R o c h a s .l 9 9 l ; M r l l c r . 1 9 9 7 ;M u r a n o ,1 9 9 - 5P; i c u l c l l ,1 9 9 5 :
'I998)
Stanley, 1995, Usov,
and the lbllowing section will
brielly summarize some recent methods used for the
analysis of the primary and secondaryshuctures of these
galactans.
Sugar cornpositionalanalysis of red algal galactanshas
recently been markedly facilitated by introducing the acidstable reductant 4-methylmorpholine-boraneduring acid
hydrolysis and by optimizing methanolysisconditions.
These two depolymeriz-ationrnethods stabilize thc acidlabilc 3,6-anhydrogalactoseand derivatives into 3,6anhydrogalactitolor 3,6-anhydogalactosedimcthyl acetal
(Quemener& Lahaye, 1998; Stevenson& Irurneaux, 1991;
Usov, 1993) which until then could only be quantilicd by
colorimetry (Yaphc & Arsenault, 1965). It is now possible
to identify and quantify al'l the sugar componentsof algal
galactans by IIPLC techniques or by GC, after funher
chemical rlerivations (Quemener & L:rhaye, 1998;
Stcvcnson& Furrreaux,i991; Usov, 1993).The reductive
hydrolysis can also be used on uncxtracted algac, thus
tacilitating chemotaxononricstudies of unknown seaweccls
(LJsov& Kiochkova, 1992). Undcr mrlci conditions, both
reductivchydrolysisand mcthanolysisrncthodscan release
the basic clisaccharrderepeat units rvhich czur then bc
identifiedas ol'agarobioseor ciurabiosetype by IIPI-C or
M. I,AIIAYL
& liurneaux, 1995; Quorleneret al., 199-5;
CiC (Falshar.v
L,sov,I 998). Oncc the sugarshavebcenidentifiecl,
it can be
llcrccsslryto detcrrnini:thc absolr-rtc
sugarconfiguration(i.e.
L) or L enantionrcr).This can be dotcrrnineclby conversion
into glycosidesoi chiral alcohols
of lhc monosacchar-ides
(sce {Jsov, 1998; Takano ct al., 1993), by reductive
anrinationrvith chiral arnines(Caseset al., 1995) or by
oxidative hydrolysis followed by acctylationand conversion
into -çcc-butyl
esters(Erreact al., 1998)plior to GC or GCanalysis. It can alsosimply he achieved
massspectrornctry
by I)-galactoseoxidaseenzymatic kits on acid hydrolysate.
Sulphate and pymvic acid substituentgroups are usually
quantiliedby enzymatic,chemicalor spectloscopicmethods
((,'aceres
et al.. 1997;Chopin & Whalen, 1993;Craigie &
I-eigh, l97B; Matsuhùo & Ilivas, 1993; Rochas ct al.,
I986a).
I-inkage analysis and substitutinggroup locations have
been facilitated by improvementsbrought in the chemical
rnethylation rnethodsof sulphatedgalactâns(Stevenson&
Furneaux, 1991).Thc combinatronof both methylationand
rcductivehytlrolysis with GC-MS analysiscan rapidly give
inlormations on the substitution pattern of rcpeating
(Falshaw& F'urneaux,'l995). IR spectroscopy
clisaccharicles
is a convenient rrean to detelmine sulphate location and
(luânrity in algal galactans(Cacereset a1.,1997; Chopin &
Whalen, 1993; Matsuhiro & Rivas, 1993; Rochaset al-,
1986a; Sekkal & Legrand. 1993) and coupled to
rnicroscopy,it can provide inlbrmations on the location of
diflere:ntsulphatedpolysaccharidesin seaweeds(Sekkal et
ai., 1993; Fournet et zrl.. 1997). However, today thc most
powcrfirl û1eanto characteriz-ethe clremical structure of
rcgular algal galactansand their oligosaccharidesremains
nuclear magnetic resonance spectroscopy(Usov, 1984).
.Sevelalcarrageeuanand agar-typedisaccharidestructures
are now casily identifiedfrom their characteristicsctsofl3C
signals (Greel et al., 1985: Lahaye et al., 19t35;1989;
Rochas ct al., 1986b; Usov et al., 1980; 1983; Usov &
Shashkov;i985). With the irnprovementsin thc instruments
and thc developrnent of two-dirnensional cxperiments,
NMR spectroscopy is being incrcasingly used and the
clatabasoof both characteristicsl3C and lH signal chemical
shil'ts ol particular repeating structures is extending
(Cliiovitti et al., 1996; 1997;Falshawet al., 1996;Falshaw
& Furncaux,1998;1994;Knutscn& Grasdalen1992;Stortz
et at., 1994).Methodsusing computersirnulations
havealso
bcerrrplqplvsçd for the prediction or â-s an hclp in the
identitication of chcmical shift.sof agar and carrageenantype rcpcating structures (Stortz-& Cercz.o,1992; Miller,
1998). Solid statc l3C NUR spectroscopy
has also been
nsc.cito charactcrizcagtu'sand caJ'ragecnans
extracts,or ln
situ in algae(Rochas& Laha1,e,1989a;Saitôet al., 1990),
but this tcchnirpreis still waiting for irnprovements
to yield
hiuhc:rresolutionsDectra.
143
'l'he
salactanstructluesis oficn complcx owing in part t<r
thcir heterogeneoLlsnature. Fractionations can facilrtatc
structuralanalysis,but in many cast:s.thc c<tmplcxityis due
to thc intra molecular hybrid nature of the polysaccharidcs.
Several chemical approachescan bc used to isolatc
particular rcpeating sequences to crase their chemical
structure eluciilation and to scquencetheil distribution in
these galactans (Usov, 1998). However, by their high
substratespccificity,cnzymesrepresenttools of choice and
severalagarascs& cârrageenases
have been isolated (Potin
et al., 1999) and usecl successfuily to dcmonstrate such
hybrid nature of the algal galactans (Greer et al., 1984,
Greer & Yapbe, 1984a;b;c;Morrice et al., 1983; Usov &
Ivanova 1987). In particular,the use of kappa- and iotaca.rrageenases
reveaied that commercial kappa- and iotacarrageenans
were containing small amountsof the iota- or
kappa-carageenan-type
repeatingstructureseither in blocks
or as separate chains (Bellion ct a1., 1981; Rochas &
Heyraud,1981).
To complete the primary and secondary structure
analysesof poiysaccharides,
it is important to determinethe
mean number of rcpeating units, tlrat is, their average
rnolecularweight and the distribution of the moleculeswith
regardto their weight average(<M>W) and number average
(<M>N) rnolecular weight (polydispersity). Such values
correspondto <M>p = Xi NiMi / >i Ni and <M>qr = I1
wlMi / 11 wi = >i NiM;z / X1NlMi, where N; is the numbcr
of molecules and wi the weight of molecules having a
specific molecular weight M1- As stated earlier, thc
molecular weight of thc galactan is related with the gel
mechanicalproperties(Rochaset al., 1990; Stanley, 1995:
Tashiro et al., 1997a). These last years have seen the
developmcnt of chromatographictechnics coupled to
multiple or low angles lascr light scattering detcctors
nraking it possibleto determinemore easily the molecular
weight and molecular weight distribution of phycocolloids.
The average nrolecular '"veight of cornmercial ag;,rs is
between 80 000 and 140 000 g mol-l with polydispersity
lower than 1.7 (Rochas& Lahaye, 1989b)and from 176 000
to 420 000 g mol t in Graciktria agarswith polydispersrty
values bctween 1.9 to 3.3 (Murano, 1995). These values
agree with those obtaincd by sedimentation experiments
(llickson & Polson,1968; lhsh.iroct â1.,1997b).
Furthermore, the Mark-Houwink relationship fol agarose
molecular weight determinationby viscosity measuremcnt
"C in 0.75 M NaSCN
was establishcd
[r1l- 0.07Mt 72at 37
(I{ochas & Lahaye, 1989b).'l'hc rnolecular weight
determinedby IIPSEC and lascr light scatteringdetectionof
carragcenânsdemonstratca rlther high polydispcrsity
(MwiMn bctween 2.3 and 5.1) and a werght âverage
molecular wcight betwe.en300 000 and 600 000 g mol-l
(particularly lor À-carrageenan)(Lccaclrcux ct al., 198-5;
et al., 1991' Vannest
Sineh& Jacobson,1994;Slootmackers
t i i
4 l _ c A I _ t ) () L Y S A ( . . ' l j A R t | ) [ s
ùr ll., 1996; Vicbke ef al., 1995)rvhich arc also in goclrl
irgreeurcnt with vllucs obtaincd by sedimentation
(t{arding et al., 1997; Slootmackcrset al.,
clt:tcrlnination
I9()l). A Mark.f{ourvink reiationshiprelating inrr.insic
viscosityto molecularwcisht was given for K-canageenan
in 0.i N,I NaCl ar 2.5"(-'(Rochas ct al., 1990) [r1] =
l . l l x t 0 _ rM 0 . 9 s .
Oltcmical and biological .factors affecting the chemislry
of agar and carrageenans
Doviations [rom itjeal repeatingstructuresform agtuobiose,
cirrrabiose 4-sulphate or carrabiose 2,4-disulphate in
comrrercial agarosc,l<appa-and iota-carrageenann-rodify
f he geometry of the galactanmoleculesand thus affect their
gelling propertiesby introducing "solubilizing" sequcnces
(Rccr^et al., 1982). Among theserepeatingstructurcs,thosc
containing 1,4 linked galactose6 sulphateinstcadof 3,6anhydrogalactoscale often referred to as the "biological
prccursor" since enzymes have been isolatcd from algae
(Rees, l96l; Wbng & Craigie, 1978;Zinoun et al., 1997)
responsible for the conversion of 1,4-linked gar-lactose
6sulphatc to 3,6-mhvdrogalactose.Thus, these enzymesare
respousiblefor the conversionof "solubilizing" sequences
to "gelling" sequeuces.The expressionof such sulphate
elirninaseshas recentlybeenfollowed by massspcctrometry
ond l3C nru spectroscopythrough tlre fate o] l3C lub"l
incorporatcdin agarcolloids(Hemmingsonet al., 1996a;b;
llemrningson& Purneaux,1997).The NMR work clearly
showecl that the newl1, synthesized Gracilaria chilensis
agarocolloid is cnrichcd in L6S and then convertedin vivo
to au rgar mainly composedof agarobioserepeatingunits.
-flrcse
biosynthetic studies also stressed that agar
biosynthcsis is linked to starch metabolism, the L6S
conversion to [,^. is rncomplete,indicating some potcntial
physiological function of this sugar sulphate in the alga.
They nlso demonstratcdthe early synthesis of G6M
containing clisaccharidic structures although f'urther or
clilfcrenl rncthyl ether substitutionsmay also occur at some
laltcr stagesin older tissuesas inferred frorn the work of
(iraigic & Wcn (l9ti:l) on Gracilaria tickvchiae. A strong
alkali treatment of agarcolloids and canageenans also
convcrts thc L6S and D6S into LA and I)A sugar residues
(l'ercival & Mcl)owcll, 1967) and such a treatmentis
classicallyuscd in laboratories
(Craigie& Lcigh, 1978)anct
industlics (Arrrrisen& Galatas, 1987; Stanley, 1987) to
desuLphateancl inrprove the gelling propertiesof algal
gallctans. The kinetics of alkalinc cyclisationhas been
stuclieclin different galactansdernonstratinghigher ratesof
conversion whcn neither the 4-linked nor the 3 linkcd
galactosesarc sulphatcdon O-2 (Ciancia ct al., 1993;
Nosecla& (lclezo, 199-5).
Besidcs the growth status of the algae with rcgard to
polyslccharidc biosvnthesis,seawccdsbelonging to thc
Gigartinalesderrronstrate
rnarked vifiations in thc !]alactans
synthesizedaccordrngto their lif.c cyclc (Craigic, 1990).
Thus haploid gametophytes synlhesize carrageenals
belonging to lhe _kappa-family(kappa-. iota-, mu-, nucarrageenans;G4SDA, G4SDA2S, G4SD6S, G4SD2,6S)
whereas the diploid tetrasporophyteproduce carrageenans
belonging to the larnbda-family (delta-, Lambda-,epsilon-,
a$ha-, theta-carrageenan;
GD2,6S, G2SD2,6S, G2SD2S,
GDA2S, G2SDA2S). No such ploidy cffect has bccn
obscrvcdlbr agarophytes.
Confo rmation, p hy sico-chemical prop erties
and gel formation
The above review of thc chemistry of the agar anrl
carrageeljan-typepolysaccharides showed that chemical
substitutionscan affect in many ways the regularity of the
ordered structure of the galactans and thus their selfassociationsand propcrtiesin solution. The following part
surnmarisepresentdata on the conformation, the solution
and some gel propertiesofmore ideal polysaccharidessuch
âs agtu'ose,and cornmercialkappa- and iota-carrageenans.
A parose
Conformation
'l'he
molecular shapeof agarosehas been unravelledby Xray diffraction sfudies of films and fibres (Arnott et a1.,
1914a, Chandrasekaran,1998). lt is an oriented noncrystalline, doubie, half-staggered, left-handcd lrelix of
pitch 1.9nm (lengthof the repeatingunit h = 0.633 nm) with
a threefold symmetry ând made only of sugars in 4Cl
contbrmation.The inncr and outer dianrcterdimcnsionsare
0.42 and 1.36 nm ând the inner cavity can accommodate
watcr moleculeswhich mediate interchain hydrogen bonds
with the oxygen O-2 from the galactoseresidueand O-5 of
the anhydrogalactoscpositioncd toward the interior. Thcre
is no internal hydrogenbound stabiliziug thc doublc helix.
In agreenrentwith thesedata are the resultsobtainedby the
optical rotation studies of Shafer & Stevens(1995). It is
important to stressthat in the double helicai confomration
only the O-2 of tbc galactose,fàcing the inside of the hcliccs
is required for the stabilization of the systcm. Therefore,
substitutionby any chen-ricalgroup at that position would
prevcnt gelation.Other X-ray diffraction pattcrnshave been
obtained from films dried at 100 'C and favour extended
singlc helicesconformationsof pitch (h) ranging tiom 0.89
'l-hese
to 0.97 nrn (lroord& Atkins, 1989).
interpretaLions
are consistentwith the conformation of single strand
agarose deduced by molecular modelling studies using
agarobioseand neoagarobiosercpcating units as models
(Jimenez-Barbero
et a1.,1989) or the UV circular dichroic
data ol agaroscsolutions and dlied solutions (Arndt &
Stevens,1994). Othcr data confirming the existenceol
singlc r-igidagaroscextcndcdheliccs in thc sol-statccarne
M, t-ATtÀYE
i'r-onrrhc lorv angle neutron scattering ilicasurctnents
'l'his
(Ciuenetet rl.. 1993).
apparentdiscrepancyin the
corrfonnation of' agarose may be cxplained by the
obscrvationof the polysaccharicle
in two different statcsi.c.
the sol- and the gcl-state. L-r solution, agarosernay havc
crxtcndcdhclical shapcswhich contractto a widcr diameter
tlor-rblchcliccs as it gels. Furtherinsight of thc conformation
ol'agarose in thc gcl stateand pirrticuliuly to confirm or in
validatethe double intertwinedhelicalstructureversusthe
hclical diner is still wajting for berter resolved X-ray
patlenis.
untl someproperties
GeI.t'bnnntir-tn
Agarose gelatton involves the conversionof a fluctuating
extcudedhelical conformationin solutionto a rigid, ordered
clouble helical structureforming the junction zones of the
gel nctwork (Rees et al., 1982). 'lhe final gel fbrmation
occurs as a lwo steps mechzrnism: I) contraction and
doubl'ingof the helices formally refen-edto as the coil-helix
transition and 2) aggregationof helices. 'I'he gel sening
occnrs through t.hedemixion of the solution into polyrnerrich aindsolvent-rich rcgions and the extensionof gelation
by aggiomcration of the polymcr-rich dornains along
prcfcrential pathways of higher polyrner concentration.
,Sr.rchbehavii)urs are observed only at polysaccharide
concentration below 2t/o in solution. At higher
concentratlon.gclation is thought to happendirectly from
the homogeneoussolution (San Biagio et al., 1996).The
large difference between gel-setting and gel-fusion, also
callcd thermal hysteresis, is thought to arise fiorn thc:
lbrmation of large aggregates which remain stable at
ternperatluewe.ll above that of individual helicesforrnation
on cooling. Thc extent of hysteresis decreaseswith
incrcasingcontcnt of substituentgroups (Guiseley,1970;
Arnort et al., i97,1a)which is consisrcnr
with rhe inhibition
ot treliccs aggregation. The onset of gel formation on
cooling as deternlinedby mechanicalrneasurernentoccurs
at a lower temperature than the appearanceof the
confornrationaltransition measuredby optical rotation.
Such behaviourleflocls the requirementfor a certainamount
of hcliccs to createa continuousnetwork. Conversely,the
ge1l'usiondetenninedby n-rechanical
measurementsoccurs
ât a slightly lower tempcraturethân that of the total helixcoil transition(Mohamedet al., 1998). This temperature
is
particularly alfected by the way the gel was first obtained.
With rapid quenchingof an agarosesolutionfrotn 45 "C to
5 "(1,thc gcl-meltingtemperature
is about10 'C lower than
that frorn tJ'rcgel fom-redat a cooling-rateof l'(l min-l
(Mohamedct al., i998). F'urthennore,
the storagemodulus
(( i') wh ich is an t'.xpression
of the gel rrechanicalpropelties.
is also clccrcascd
rn fast qrienchedgels (âbout50ololower).
'lhcse
bchaviorrrsrcllcct thc bcttcr assemblyof hclices
aggrctation ltrrnrcd at lowcr cuoling rate of the agarose
solution and is associatedwith a higher gel turbidity
compared to that obtaincd by fast quenching. I'his
particular behaviour can be of practical interest in
tacilitating, for cxarnple,the rncorporationof agar in food
proccssings. fhe gel-setting tempcrature and the final
storagcmodulus ofthe gel also clependon the concentration
of agarosein solution.Once the confornrationalchangebas
occurred (obscrved by optical rotation), the ge1 develops
'l'he
with tirne.
temperatureat which the conformational
changebeginsdependson the agaroseconcentrationand the
rate of gel formation depends on both temperature and
polysaccharideconcentration(Mohamed et al., 1998).
The gelling properties are related to the primary and
secondarystructuresof the polysaccharides.Natural rnethyl
ether substitutions increasc thc gel settrng temperâture
(Guiseley, 1970) and the methyl on position 0-6 of the
3-linked B-D-galactoscis particularly responsiblefor this
bchaviour (-F-alshaw
et al., 1998). In contrast, chemical
mcthylation lowersthe gel-settingtemperatureas the results
of substitutionat O-2 of the 3-linked p-l)-galactoseresidues
thought to be involved in the stabilization of the double
helices through water-hydrogenbonds (Miller et al., 1994).
Double methylation with methyl ether substitutions at
position O-2 of the 4-linked 3,6-anhydro-cr-L-galactose
and
0-6 of the 3-linked B-D-galactosesignificantly increasethe
gel fusion temperature(112-113 "C tor Curdiea coriut:ea
and l2O-121 "C tbr C'. obesa which are nearly cornplctcly
doubly rnethylatedagars: Falshaw et al., 1998). Agarose
with high gelling temperaturesor agalosestlrat have been
chemrcâlly modified to lower their gelling and melting
temperatures are of a particular interest for specific
biotechnologicalapplications(Kirkpatrick et a1.,1993).
Details on agarose gel junction zones have been
investigatcd by various technics (microscopy, scattering
methodssuch as smali angle light scattering,small anglc Xray scatteringand small anglencutron scattering). By small
anglc nentron scattcring (SANS), Krucgcr et al. (1994)
observedthat the .junctionalnets âre mr-rrcswollen at low
agaroscconcentratiolls,which is cohelent with an increase
in pore size in the gel as the concentration is lowered
(Maalounet â1.,1998).A rninimaljunction zone thickness
of about 3..5-4.0nm has beenrepoiled and is close to a fibrc
diarreter of 5.0 nrn detelmined by chromatographic
methods (I-aurent. 1967) or observed by microscopy
(Dormoy & Candau, 199l; Whytock & Finch, 1991).
Melting of the gel obviously markedly affect the SANS
pattern, suggestingthe presenceof a widc distribution oI
with
particlesof relativelysmall sizewhich is in accordance
the rnodel of extended rods described by Guenet et al.,
(1993). If a fast quenching is used to gel the agarosc
solutjon, Lhen the nature of the intra-junctionalstrand
associationsare alTectedbut not leally the large scalc
stnrcturc.From this belraviour,Kruegeret al., (1994) infer
!6
AI-(IAI, POIYSACCHARII)ES
osr gcl rcprcscntsa randonldrstribufion Of compact
lhat a.r,ar
of arbitrary length but diffelirrg in
scif-similar.junctior.rs
t h i c k n e s s .W i t h i n t h i s g c l , a g a r o s ef i b r e s o f v a r y i n g
thicknessarepresentas randomlydistributcdclustelsof netlikc appearances,structured in 3D webs of dimension
betweerrone nn1(thicknessof individualsh'ands)
to several
lruncfi-cd
nnr.Such fibre buncllchctcrogencity
was suggested
by l)jabourov et zLl.(1989) by srnall angle X-ray scarrcring
rvho subdivided the populationsof fibre bundlesarbitrarily
to fit the data into two populations,one of 3.0 nm and the
olher ol' 9.0 nrn. The heterogeneousdistribution of fibre
bundles was also deducedfrom the birefringenceof agarose
gels cithel when an electric field is applicd or not
(Stellrvagen& Stcllwagen, 1995). This optical property
reflectsfhe oricntationof agaroscfibre bundlesor nricrogels
lractions within the macroscopic gel. These objects can
have sizes up to l0 micrometersdependingon the electric
tield strengthand the orientationchangescan be ineversible
if the applied field is too high or for a too long period of
'fhe
tirnc.
intrinsic birefringenceof agarosegel, which has
also been observedfor carrageenangels,is inhomogeneous.
It varies frorn gel to gel but appearsinherentto physical gels
and not to chcmical gels suchas polvacrylamide.In agarose,
thc oricntation of the bircfringent objects tbllows a
rnircroscopicalsinusoidalpattern. Thcsc objcctshavelengt}r
'fhe
scalesof millimetrcs.
fact thar the orientationof srnaller
parts of thc macroscopicgels can bc reversibly affectedby
electricfieldsis indicativeof the existenceof a hierarchyof
loosely connectedsubstructuresheld togetherby metastable
hydrogcn bonds (Steilwagen & Stellwagen, 1995). Thc
distr-ibution antl organization of the fibre bundles arc
importitnt with regard t.o application of agarose as a
chrornatographic and elecrrophorctic matrix since they
conclitiorr the gcl porosity. Pore siz-eslanging from about
200 nnr to ab()ut 2000 nm and their- proportion where
rcccntly rneasured by atornic force rnicroscopy (AFM,
Pcrnodetct al., 1997; Maaloun et al., 1998). This non
invasive mrthod does not rcquire specialprcparationof the
gel an<l therefore gives a picture of the "unperturbed" gel
allowrng the direct measurementof pore sizedistributionby
imagc analysis. It representsa recenta-lternative
to indirect
methods such as electron microscopy requiring sample
prcparations such as dehydration (Attwood et al., 1988;
Criess ct al., 1993).Using AFM, a direct rclationshipof
aga.rose
pore size was lbund with polymer concentrationand
with ionic strcnglh of the casting buffcr. An ernpirical law
rcrlatingporc siz-e(a) ard agaroseconcenûation(C) was
deduced (a-C tt.r,1,A relationship was also obscrved
betrvcenionic strerrgthof thc castingbulTeranrl porc size,
the lower the ionic strength. the lower the pore <irarnerer
(l\'[aalounct al., l99U).
lrlcctroendosmosisis one i[iportaut practicalpropcrty ol
agar()seusecias an clcctrophoresismatrix. It rs lelatr:dto lhe
presenceof rninor aûlounts oi chalged grcrups(sr.Llphate,
pyruvate) and is due to the migration of their counter ions
(cations) under thc application of an electnc field. This
rnigrationcreatesa flow of water molecule frorn thc zmode
toward thc cathode as a consequenceof which, neutral
moleculcs that would not migrate are pulied towârd the
cathode.
Agar gels loose water on ageing,a phenornenoncalled
syneresrs. This water exclusion is attributcd to the
contractionof the polymer network by a slow aggregationof
helices rcsulting in the decrease in interstitial space
available fol holding water. Syneresisis lower for agars
containing chargedgroups and is roughly inversely related
to the scluareof the agar concentralion(Stanley,1995).
Carrapeenan,ç
ConJormation
Similarly to agarose,kappa-, iota-carrageenanand partly
(furcellaran)orientedtbers
desulphatedkappa-carrageenan
yield X-ray tliffraction patternsinterpretedas representative
of threefold, righthanded double helices (Chandrasekaran,
1998; Picullel, 1995; Millane et al., 1989). The calcium
form of the iota-carrageenandouble helices are halfstaggeredwith a pitch of 2.6 nm and the DA2S and G4S
residuesare respectivelyin the lCo and 4C, conformations
(Arnott et ',il., 1974b). The two sulphate groups are oriented
towârd the ou$ide and that of the DA2S residue of two
adjacent helices are thought to be involved in iota.
carrageenangelation by caleium ions, forming ionic
interactions.The G4S residues are connected by six
interchainhydrogenbonds between 0-6 and O-2 per helix
turn. The inner and outer diameterswithout hydrogen atoms
are 0.34 and 1.42nm.
The fibers from the potassium form of kappa-carrageenal
ar-e less oriented, less crystalline and are less laterally
organized(Millane et al., 1988). The most reasonabielit of
tie X-ray dâtacorrespondsto a parallel double helix of pitch
2.5 nm (h = 0.833 nm) offset by a translationof 0.11 nm
along and a rotation of 28" from the half-staggeredposition.
'I'here
are only 3 liydr-ogenbonds betweenthe G4S residucs
and the overall diarnetersof the helix are 0.1 nm largcr than
those of thc iota-carrageenan.
Cairns ct al., (I991) rcinvestigatcd the X-ray diifraction pattern of kappacârrageenan and partly dcsulphated kappa-carragccnan
(furccllaran) in the presenceof glucomannan or various
galactornannans.
The authors did not observe intcraction
bctween the different polysaccharidcsalthough computer
modelling suggestedmutual polysaccharideconformational
change to optimise van der Waals and electl'ostatic
intcractions( lvaroskact al., I992). Suchmixcd gel systerrs
horvcver provided clearer X-r'ay pattcrns lor kappacirrrageenanwhich interprctalioncontradictsthe Millane ct
N4.LAHAYE
r l . , ( 1 9 U 8 )r n o ( l c l (. l a i r n sc t a l . . ( 1 9 9 1 )p r o p o s e da l o o s e l y
packcd rrcrrr:rtrc
lrcluidcrystallinearrayo1'doublchclicesof
ç,hich crornponent
châinsrotatcwitltoutan axial translation
l l , , n r t l r Ll'r r l l - ) t r t g c r c dp o s i t i o n .
L.anrbdacarragecnanrvoulcl form thrcetbld helices of
pitcrh2.52 nm. Inclicationsof lcli-handeclor righrhalded
heliccsstabiliz-cd
by hydrogenbonclsbetweenO-2 and O-3
across the (1->4) linkagc were obLaincdlr.om molccular
rrrodeLlingstudicsand tire rcplacementof the DA residuebir
l)65 or I)2,65 in the 4Ct conformalioninsteadof the lCa or
t h e p r e s e n c eo f G 2 5 w o u l d r e n d e r t h e d o u b l e - h e l i x
fbrnrationimpossible(Millane er at., 1989).
ConrpuTermodelling of kappa-, iota- and lambdacârragecnan stafting form crystallographic data or
calculated data for sulphated monosaccharides,yielded
models prcdicting a rnore flexible characterfor kappa- and
iota cârrageenansthan for lambda-carragcenan.
ln all cascs
the cornputeclstructurcs wcre in goocl agreementwith the
above proposedfrorn X ray diffraction of fibers (Urbani et
al., 199-1,
Lc Qucstelet al., 1995).At any rate,as for agarose
contbrrnation, there is still a debate on the fundarnental
tcrtrary and quâternary structures conformation of
carrageenaltsand particularly that of kappa-carrageenan.
l{ecent results from high performance size exclusion
chromatography coupled to laser light scattering
Ineasurementsshow the doubling of kappa- and iotacaffagcenan molecular weight on cooling the
chlomatographic solvent temperatlueor on increasing its
iorric:strength(Vir:bkcet al., 1995;Uedaet al., 1998;Hjercie
e'tal., l!)98). Whcther these are duplex of single helicesor
double intcrtwinedhelicesawait for further relinementof Xray rliffractionsor other physicaldata.
of tlansition at which the concentrarionof helices and coils
are equal ('fm) is invcrscly and lincarly rrlatcd to the
logarithmof thc t(xal cation concentration(CT, includrng
thc sulphatecountcrionsCc, with CT = Cs + 1Cc and Cs the
concentrationof addedcationsand y the activity coefficienr
'they
of the cation).
distinguishedtfuee groups of cations
with respect to their helix-promoting efficiencies : nonspecificmonovalentcatrons(Li+, Na+ and (CH,)nN+), nonspecilicdivalentcations(MgZ+,CaZ+,sr2f, Ba2+,Co2+and
7,nz+1and the specific monovalentcations (NH4+, K+, Cs+
and Rb+).No evidencehas yet beenobt?rinedto demonstratc
specific binding of the cations to the polysaccharidcin the
coil statebut. in conlrast, scveral studies have shown their
specitc binding to thc heiices even in non-aggregating
conditionsalthough the site of txation is still unknown
(Picuiell, 1995). Anions also affect the conformational
transition of kappa-carrageenan
in thc order F-<Cl-<NOr
<SCN-<I- which are in reverse order from the so-called
Hofmeister or lyotropic series promoting denaturation
(Nortonet al., 1984).
Furcellaranand partially desulphatedkappa-carrageenan
behave like kappa-carrageenan(Tanncr eI al., 1990, Zhang
et al., 1991, 199.1)except that thc temperaturcof transition
Tm is higher. The salt dependencyof the iota-carrageenan
has bcen also well documentedâxd shows a marked helixstabiliz.ingeffect of çn2+ (Piculell, 1995). J'he reported
small specificities to some monovalent cations is now
interpreted as arising from the small kappa-carageenan
(Piculell et al., 1987)which
proportionsin iota-carrageenan
also affect the rheological propertiesof the gel (Piculell ct
al., 1992)and thus, there would be no cation specificity for
the iota-ceLn'ageenan
ooil-helix transition.
As indicatcd above, therc is slill a dcbate on the
Gclformation
confbrmatiolrof carragecnans
in thc gel statebut most of the
As lor- agarosc,carrâgeenangelationis associatedwith a
informationsindicate the rapid formation of double helices
conforrnationaltransition going frorn randorn coils in
(Piculell, 1995).The rninirrum degreeof polynreriz.ationfor
scilrrtionto double-heliceson cooling and./oradding cations
both iota- and kappa-canageenansnecessaryto stabilize
(l)iculell, 1995). Single helices are also obtained with
double heUcesis around 100 as determinedby rrechanical
cenain saltssuchas NaI (Srnidsrod& Grasdalen,1984a),or
measuremenls(Hjerde et al., 1998).Once the double helices
(Uedaet al.. 1998).In case
at very low salt concentrations
are formed, they aggregate (Viebke et al., 1994) as
of kappa-carrageenana minimum of four disaccharide demonstrated by thc thermal hysteresis for kappa
repeat ullits was detcrmined as a minimurn to observe a
carrageenan.Such difference in gclling and melting
conf<rrrnll.ionaltransition by optical rotâtion (Rochaset al.,
temperaturesreflect^as for agarose,the temperatureof coil'fhe
1983).
transition of the chain ordering proceeds helix formation and that of the denaturation of helical
coopcratively and stârts liom an initiation site and then
aggregates,respectively.Such hysteresisdoes not exist for
extends along thc chains. Such transition dcpcnclson the
iota-canageenan(Piculell et aI., 1992) and for kappit
Lnolccularweightand thc concenfation(Piculcll,1995)and
carrageenanin Nal (Chronakiset al., 1996),a salt known t<r
is kinctically alïected by salt contenls and temperature promote kappa-carragcerransingle helices that do not
( N o r t o nc t a l . , 1 9 8 3 a , bA, u s t e ne t a l . , 1 9 8 5 ,1 9 8 8 ) . ' l ' h e ri es
aglgcgatc at low concentrations(Smidsrgd & Grasclalen,
a str-iking selectivity for cat.ions in kappa-carrageenan 1984a). These two bchavrorlrs indicate thc existence of
conlornralionaltransition u,hich howcvcr is not always
possiblc different gelling mechanisrnslbr kappa- ancliota
a s s o c i r r l e t<ol g c l l i r r m a t i o n( P i c u l e l l , 1 9 9 5 ) .R o c h a s&
carragccnans.As lor agarose,the observed turbidity and
( 1980)clcarly dcntonstrated
Ilin:rrrcftr
t.hûtthe tempcr.rture birefringcnccof kappa-carrageenan
and turcellarangcls are
A r . a i A l .P () L Y l i ^ c c t t A I t I [ ) u s
cvi(lcuceslix thc tbrrnationof olganrzecl,
orietrtedbundlcs. L. jrtponir:a ard Ascophl,l.luntrtodosunt,altl-roughfarmed L.
'['hc
hystcresisclisappcarsat low .saltconcentrationsfbr
japonica is bcing incrcasingly exploited. The mode of
kitppacarrageenanevidencingthe exlstcnccof conditions alginate extraction starts wilh the removal of undesirablc
rvc-reconfornrationaltransition occurs without aggregation smal) compoundsby acid washesand the transformationof
'Ihe
cven with spccific ions such as potassium(Rinaudo &
alginatein the cell wall into alginic acid.
alginic acid is
I{ochas, t9iil). By choosing appropriatesalts types and
then extracted, while convertcr-linto the soluble sodium
conccntrations, rlifl'erent kappa-catrageenan helices
fbrrrr by sodium carbonate or sodium hydroxicle
Irssociationsciut thus bc obtaincd : tiorn liquid netnatic neutralization.The insoluble algal residuesare removed by
crystalsusing scgrnentsof kappa-carragecnan
in Nal/CsI to
filtration, llotation or centrifugationand the soluble alginatc
clouhlchclical-r'odsanri super-helicalrods ir-rgels (piculell et
is precipitated either by convcrsion into alginic acid or
al., I 997)- Iota carrageenan
is unableto form liquid crystals calcium aiginate.The alginic acid is then convertedinto the
(lSorgstrcimet al., 1998)conforting differencesin molecular
tequired counterion by neutralisationwith the appropriate
irssociationbehavioursbetwoenkappaand iota-carrageenan. hydroxidc; the calciurn alginate is convertedto alginic acid
Keppa-carrageenan
gcl âppearsby elcctronmicroscopyas a
ând then neutralizedas above.The differencein the alginate
network of fibre bundles (l{ermarsson, 1989, Hermansson recovcry process clcpcnds on thc source and thus thc
ùr al., 1991,Sugiyamaer al., 1994.Borgstrômet al., 1996). chemistry of ttre alginate(McHugh, 1987).
ln contrast,iota-carragcenanunder salt free conditions and
Chemistry
in presenceof Lil appearsas linear extendetlor macrocyclic
(Stokire
structlrres
et al., 1993). T<ldate, no information is
Alginate consistsin a family of linear polysaccharidesbascd
available on the pores size and distribution formed in
on 1,4-linkedB-D-rlannuronicacid (M) and its C-5 epirner
can'ageerran
gels.
1,4-hnked o-L-guluronic acid (G) of varying proportions
Smidsr'pd& Grasdalen( 1984b)ar.rdRochaset al., (l990)
and distributions. Partial acid hydrolysis and enzymatic
dcmonstrateda <lependancyof kappa-carrageenan
storage degradation by alginate-lyases have demonstrated that
modulus (G') and the Young's modulus (E) on molecular
alginatecontainshomogeneousblocks of mannuronic acid
we:i.qht.
G'and D increaseup to an averaBemolecularwcight
and guluronic acid with regions where both acids alternate
of about 200 000 and then plateau,while the yield stressstill
more or lessregularly (Fig. 5).'l'he latter region cân contain
incr-case(Snridsr6d& Glasdalen,1984b, Ilochas et al..
some defective repetition rn having MMG or GGM
1990). The critical concentrationof kappa-carrageenan
to
structures interspersed. The chemical composition and
form a gel (c0) is in the order of the
conccnfation ol thc polysaccharideovcrlap
(c'r')in soh:tion(Rochasct al., 1990). As lbr
agalosc,lhc way thc kappa-carragccnan
gel is
folrrrcdlrasincidenceon the storagemoclulus A
G' (llcnlansson et al., l99l). C'is threetimes
highcr tbr a gel obtainedby coolingat 0.5 'C
-> 4)-o-L-GulA-(1 -> 4)<r-L-GulA- (1 -> 4)-o-r-GulA-(1 -> 4)-û-L-GulA-(1 ->
r n i n I I l r : r rr rt l . - i ( l r n i n
R r5
\-{-/ffi
B
2. Polyuronanes
-> 4)-Ê{-ManA-(1 -> 4)-lJ-D-ManA-(1 -> 4)-P-o-ManA-(1 -> 4)-P'D'ManA-(1
Alginare
Alginatc are the major maLricial cell wall
polysaccharidesof brown seaweeds(Kloareg
& Quatrano,l9u8) also lbunclin corallinered
algao(Okazakict al.. 1982,Usov et al., 1995)
anrl
prociucccl
by
sornc
bactclia
\
*\,-{\-'
((iacesa,
lgti8). lt is widely useclas a food gelling,
thickcning and stabilizingagent but has
vatious othel.tecirnrcll uses, particularlyin
t h c t c x t i l c i n d u s t r y ( M c F I u g h , 1 9 8 7 ) .T h e
c
*"R"^Æt'ff"
-> 4)-|J{-ManA-(1 -> 4){-L-GulA- (1 '> 4)-lJ-D-ManA-(1-> 4)-c-r-GulA-(1 ->
B and
Figure 5. Chernicalstfuctureof poly-guluronatc
A, poly-nrannuronate
inrlustr-ialalginatc production relies on thc
a l l e r n a l - i nngr a n n u r o n a t e - S l r l u r o nC
a t bc l o c k s e q u e n c e s .
frarvcslof wild populatiorlsof Macrocy.tti.\
I,'igure 5. Structurc chimique des séquencesde blocs de poly guluronate Â,
alterrrés(l
ltyrrlt rrt, LanLi.nrtriadigitata, L. hl,pt:rbnrea, poly nrannuronateB et mannurolrate-guluronate
M. LAIIAYE
strlcture of alginatehas alwaysbeendifficultto detcnnined
orving ro the lact that the uronic acid linkages are
particularlyresistantto acid h1'drolysis.
Most methodsfor'
compositionâlanalysisrely on colorimetricmeasurements
althorrghseveralother methodshavebeenproposed:l[PLC,
II{. circular dichroism,GLC, NMR. enzymes(Moe et al.,
lr)9-5).l3C andlH nrnr spectroscopy
is a powerful method
tbr detelmining the sequenceof the two uron.ic acids in
algurate(Crasdalenet al.. 1981, Grasdalcn,1983).From the
area of spccific signals the proportion of mannuronic and
gulurorric acid (M, G), the frequenciesof diads MM,
MG/GM, GG, as weli as thc triad cGG, MMM, cGM,
GMC, MMG and MGM can be neasured. Averagc block
lcngth citn thcn be calculatcd<N>C = F1;/Fy6 and <N>14
= FM/lrMG or by excluding single G and M units in average
to takc inlo accountirregularitiesin the alternatingsequence
clistlibution <N>G>l = GC - FUCtvt) / F66y
and
<N>M>i = (FM - ts-CprC)/ FMVC.
The composition and the distribution of alginate cliffer
among species,as well as between tissues.Old tissue antl
strpes are generally richer in polyc blocks which most
likely confer a high mechanicalresistanceof the algae to
environrnental stresses.The guluronic acid content and
distribution is under the control of activity of thc cnzyme
mannLlronanC-5 cpimerase(Hagen ROdde& l,arsen, 1997;
'[he
Skjâk-lJræket al., 1986).
diffcrent enzymesand gcnes
involvcd in the alginatebiosynthesis
are well studied,using
alginateproducingbacteria(Gacesa,1998).
Alginate averagemolecuiar weight <M>y7 vary between
170 000 to 1640 000 g mol-l dependingon the source and
thc mode of extraction (Martinsen et al., i991; Moe et al.,
1995).Generaliythe polydispersityindex (<M>w / <M>N)
is botween 1.4 - 2.6 lbr comrnercialpreparationand can
reach 5.6 for lab preparcd samples (Moe cr al., 1995).
Mark-Houwink constantshave bcen determinedto measure
the nrolccular weight of alginate fiom their intrinsic
viscosity in 0.1 M NaCl at 2-5"C (Martinscner aI., l99l).
'I'hc
K and a valuesdiffer frotn 7.3 x 10-sto 6.9 x 10-6and
0.92 to l.i3 for M- and G-rich alginate,respectivelyand
reflect the extendeclshapeof G-licl-ralginate.
ConJornnîion, pltysico-cltemical properties and gel
form.ation
Thc coniormation of alginate has been determined from
X-ray drtfraction pattcrnsobservedfrorn polyG and polyM
blocks of fibrc bundles (Chandrasekaran,
1998). PolyM
llbrcs arc cxtcnclc(l ribbon-like twofold helices (h =
0.52 rrrn) stlbilizcd by hydrogcnbonds betweenO-3 and
()--5(0.27 nrr) acrosscach glycosidicbridgc oxygen atom.
'l'hc
rrrannuronic
acid is in thc 4ct conlbrnation.ln packing
trvo ht:lictrs,a hyclrogcnbontl is fbrlncclbctwccnone of thc
carboxylateoxygcnsto O-3 ol a ncirhbouringchain of thc
sarnepolanty.Antipalellcichninsareconncctcdby ll-boncls
149
betwcen O-2 and O-5. PolyG fibles forrn fwofold hcliccs
(h = 0.435nm) with a 0.87 nm pitch. 'l'he guluronicacid is
in the lc4 conlbrmationand bccauseol thc 1,4-diaxial
linkages,thc helix is a buckledribbon stabilizedby H-bonds
between O-2 and one of the carboxylatc oxygens âcross
every glycosidiclinkages.When packedwith anothcrchain,
water connectsthe guluronatcresiduesvia O-2 and O-3 and
via O-5 and O-3. The pairwise assocjation of l-relicesis
thought to be mediatedby calcium jons irr the middle
resemblingan "egg box" (Grantet al., 1973).
Alginate is well known for its ion binding and
demr>nstrates
an affinity for alkaline eanh metals in lhe
order Mg << Ca < Sr < Ba. This selectivity is due to the
gr.rluronicacid blocks as the polyM and alternating
sequencesdo not demonstrateselectivity.The selectivity is
also dependenton the ionic composition of thc alginate gel
as specilicailnities show a strong cooperativity(Moe et al.,
1995).
Alginate is insolubleat pI1 below the mean pKa of their
constitutivc uronic acids (3.4-4.3) and its solubility also
dependson its composition, rnolecular weight and on thc
ionic strengthof the solution(Moe et al., 1995).If the pH of
an alginatesolution is slowly lowered below this value, an
acid gel is obtained.On fast acidificationof the solution,the
alginate precipitates.Alginate in solution is more stable at
pH around 6 9; acidic pH lead to hydrolysis while high pH
promote B-eliminationreactionsleading to the formation of
unsaturatednon-rcducing encls. Furthcrmore, alginate
stability is markedly affected by reducing agents such as
polyphenolics contaminants which also promote Felimination reactions. This is one of the reasons
formaldehyde is used during the industrial extraction of
alginate. This compound precipitates proteins and
polyphenolsin the algal residucs.
Alginate forms very viscous solutions dependingon the
concentration,molecularweight, cornpositionand sequcnce
of the polysaccharideand on the ionic strergth of the
'I'cmperature
solvent.
has littlc cllcct on viscosity.A gei or
precipitateis formed when a gel prornoting divalent cltion
is introduced to an alginate solution. The reaction is
instantaneousand leads 1o the formation of strongjuncrion
zoncsin the gel and consequently,it is difficult to obtain an
homogcneous gel. A dialysis and an intcrnal gelation
nethods are available to forrn hontogeneousgels under
controlledkinetics.In the dialysis rnethod,the gelling ion is
allowed to diffuse through the alginate solution. The
graclientof gelling ions in the gel is controlled by the
molecular-weight of the alginate and the conccntrationof
gelling and non gelling ions (SkJâk-BriEk
et al., 1989)but
cânnot totally be avoidcd. Calcium ion concentratton tn
alginatebeadsof variousconrpositionmadein diffcrentsalt
conditionswas nevertheless
shown to bc hrgherfrom the
cternal
o u t s i d et o t h ei n s i d eo f b c a d s( l h u e t a l . . 1 9 9 7 ) . ' 1 ' hi n
ALGAL PoI,'1S/\CCI IA RIDES
gelrtion rrrcthoduscsthc propcnyol thc slow dcgradation
of
D-!lucono,t\-lactonc((il)L) with calciurncarbonatcor Ca'lhc
h,L)lA c:omplcx.
slow tlcgradationof GDL acidifiesthc
solvcntwhich allowsthc rcleascof Ca [i-omthe carbonatcor
thc F.I)'lA (I)rageter al.. 1990).'l'hegellingkineticcan also
bc nroclulatcdby thc ionic-fbrm of thc alginatc (l)rager er
ai., 1998). Sodiurn ion demonstratesa higher.selective
binding thau potassium ion to alginate and thus can
somehorvretardsgelâtion by competingwith calcium. Once
fomed, the gel will show tendenciesto syner.esis,
that is
water will be excluded and t_hrsis related to the content of
added gclling ions, the uronic acid composition antl
sequenceof the alginate (Draget et al., 1990, Moe et al.,
1995). Calcium alginatc gel in solutionsof monovalentrons
swcils consequently to the displaccment of Ca by nongelling ions and even the polyG region demonstrateCarnonovalcnIcxchangein high rnonovalentsalt concentration
( W a n g& S p e n c e r1, 9 9 8 ) .
Alginale gel tblms libre bundies of thickness betwecn
I 3^2(r nm cotnposedof guluronic acid riclr sequencesand
pores ranging in size from 5 to 150 nm (Andrescnet al.,
1 9 7 7 ,V e l u r a j a& A t k i n s . 1 9 8 9 ) .
The gel mechanicalpropertiesrely on the concentration
of alginate, its content of guluronic acid and polyc
secluences
and the gellingion used(Skjâk-Bræket al., 1986,
.Srniclsrgd
& Crasdalen,1984b).'fhestoragemodulusof gel
rnacle by dialysis is dependent on molecular weight of
alginatcup to 80 000 g rnol-L(Moe et al., 1995,Smidsrod&
(lra.sdalcn.1984b).
In scveralulvan, rhamnoscis sulphatedon O-3 and somc
xyloselesiduesarc sulphatcdon O-2 (Lahayeet al., 1998:
Ray & Lahaye, 199-5b).Four repeat units have been
identifiedin ulvan (F'ig.6). Two aldobiuronicacids called
ulvanobiuronicacid 3-sulphatetype A and type If and noted
'I'hese
A3s and B3s in sholt.
are 1.4-linkcdp-D-glucuronrc
acid or cr-1,-iduronic acid linked to 1,4-linked
c r - l - - r h a l n n o s e3 - s u l p h a t e . r e s p e c t i v e l y . ' l w o o t h e r
disaccharidescalled ulvanobioseswere also idcntificd
where the uronic acids of A3s and I]3s are replaced by
A
JS
cfl
è4)- B-DGlcA{1è4)- ct-u-Rha3eulphab(1>
A
ufuanotiuronicacil Ssulphabtype
B,,
CH
>4)-
cr-L-ldoA{1>4)ufuanofjuronicacd
Ulvan
o.-L+lha3€ulphab{1è
3sulphab tYPe
B
Although qrccn seâ\\,ccdsbelonging to Ulvales are not
indr-rstriallyuscd as sourceof phycocolloids,thcy synthesize
water soluble polysacchalidesthat a-reable to lbrm weak
gcls ru prescnceof divalent cations,boric acid and at a
--o--ts
lightJy hasic pH (l1aug, 1976: Lahaye & Axelos, 1993;
Lahaye et al., 1996).IIlvan extractedfrom severaldifferent
)4)- [J-D-Xyl{1}4)- o-Lfiha3€ulphatell)
s p e c l c s a r c c o m p o s c d o f r h a m n o s e ,x y l o s e , g l u c o s e ,
acid 3sulPhate
ulvanob'rose
glucunrnic and iduronic acid and sulphate (Percival &
McDorvcll, 1967; Quemeneret al., 1997;Ray & Lahaye,
I 99-5a,Yarnamotoet al., 1980).As for alginate,the presence
ol'acid stableuronic acid linkagesieadsto difficulties in the
quantitative defcrrnination of the component sugars.Two
types o1'linkages ale particularly refractory to acid
hytlrolvsis in ulvan. theseare ttre aldobiuronic acitl i.e. the
CH
uronic acicl-aldoselinkage of the glucuronic acid-rhamnose
)4)- [i-DXyl2sulphab{1>4)cr-L-Rha3sulphat+{'l>
and the linkage betweoncontiguousglucuronicacid. The
ulvanobiose acid 2',3disulphate
lolrncr can be cleavedby the use of a glucuronidaseafter a
mild acid hydrolysis(Quemeneret al., 1997;Lahayeet al..
1999) but a quantrtativedegradationnethod ibr glucuronan
Figure 6. Chemical structurc,namcs and shorthandnolationsof
is still lacking.Irurthermore,ulvan containsalso the acid
repeatrngdisaccharidesfound in ulvan.
labile iduronicacid tbr which an easilyavailablestandaldis
Figure 6. Structurechiniiquc, noms et notations abrégéesdes
not avrilâblc to quantilativelytletermineits concentration. u n i t é sd c r é p é t i t i o nt l c i ' u l v a n e .
U.
M.I,AIIAYF,
1 , 4 l i n k c c l l i I ) - x y l o s e o r x l , l o s e 2 - s u l p h a t c( U 3 s a n d
U2s'3s,Lahayect al., 1998, I-ahaye,1998).Otherstructures
coutaining blanching of single glucuronicacid residue1o
O 2 oi. rharnnose3,sulphatein A3s (A2g3s) or conriguous
I ,zl linkccl 13-D-glucuronicacids whcre also observed
(l-ahayc & Ray, 1996; Lahaye et a1.,1997; 1999).As for
other phycocolloids, the isolation of an enzyme, an ulvanlyase,has greatly helped in the fine stmcturalelucidationof
ulvan. In particular, it allowed to observed the djflerent
proportionsand scquencepatternsexisting for the repeating
disacclraridcs (Lahaye et al., 1991 Lahaye, 1998).
Molecular weights of ulvan from JapaneseIJlva pertusa,
(/. t'ongLobataand Enteromorpha proliftra were determined
by scdimentationequilibrium to be 91 000, 820 000 and
320 000 g.mol I (Yamamotoet aL.,1980;Yamamoto,1980).
The rnolecularweight of E intestinalisulvan determinedby
light scattering experiments,ranged between 189 000 to
502 000 g.mol I dependingon the extractionconclitions(De
llcviers & I-eproux, 1993). Besides gelling proper(ics,
rrlvans from UIva sp. and Enteromorpha sp. develop low
viscous solutions : the intrinsic viscositiesat 20 "C in 0.lM
citrate buffer rangedbetween 51, 155 ml g-1 1o, U. pertusa,
U. r:onglobataand E. prolifera (Yamamotoet al., 1980) and
were 175 and 36 rnl g-l fot ulvan from U. Iactuca and
E. compressaat31 'C in 0.150 M NaCl (Lahaye& Jegou,
19 9 3 ) .
Conclusions
and perspectives
controlof theirbiosynthesis,
or by controllcd
modificatiol)s
d u r i n gt h c i rc x u ' a c t i ur n d p r o c e s s i n g .
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