frcnris in Iioorl Scicncc &
fcchnoloqv l.l (l(X)l) 7.f 9l
SCIENCE
I FOOD
T,TECHNOLOGY
n
Kevtew
tg and 13c
high
resolutionNMR
spectroscopyof
carrageenans:
applicationin
researchand
rndustry
.
I
F. van de Veldux,T,l
S.H.Knutsenr$
A.l. Usov,ll
H.S.Rollemafand A.S.Cerezol
: * '' ' " . s
ts a gencric name for a family of linear,srrlfatcdgajactans,
obtained by extractionfrom certain speciesof rcd sc;rweecls
( R h o d o p h y t a )S. i n c e n a t u r a l c a r r a g e e n a n .sr r e m i x t u r e s0 f
different sulfated polysaccharides,
their con-rpositiondiffers
from batc-hto batch. Therefore,the quantitativeanalysisof
carrageenan batches is of greatest importance for both
i n g r e d i e n ts u p p l r e r sa n d f o o d i n d u s t r i e st o d e l r v e ra c o n s t a n t c o n s u m e rp r o d u c t a n d t o d e v e l o p n e w a p p l i c a t i o n s
based on their unique intrinsicproperties.NowaclaysNMR
spectroscopyis one of the standard toois for the determi_
nation <tf the chemic.alstrLlctureof c_arrageenan
sarnpres.
This review gives an overview of NMR-spectroscopy(both
1 H -a n d l r C - N M R )
a s a p o w e r f u lt o o l f o r t h e q u a l i t a t i v ea n c l
q u a n t i t a t i v ea n a l y s i so f c a r r a g e e n a ns a m p l e s .I n a c l d i t i o n
to tables containing cl'remicalshift data for both rl-1-ancl
1:rC-spectra,
cletails about sample preparation, sele<_tive
degradationand fractionationtechniquesare inclucied
O 2002 ElsevierScrenceLtd. All rrehtsreserved.
I
Introduction
Carrageenans represent one of the major texturising
rngredients in the food industr-y. 'l-hey are natural
ingredients, which are used for clecadesin food applicarNIZO food research,product
tions and regaded as save (Van de Velde & De Ruiter.
Technology
Department,PO Box20,6710BAEde,The Nethertanàs
2002). Carrageenan is a generic name lor a farnily of
(Tel.:1 31-3-18-659-582;fax:
* 31-318-65G400.;e-mail:
polysaccharicles,obtained by extraction frorn certain
Fred.van.de.Velde(â)n izo.nl)
speciesof red seaweeds(Rl'rodophyta). During the last
rWageningenCentrefor Food
f'cw years, the total carrageenan market showed a grow
Sciences,pO Box552,
rzite oT' 3o pcr year reaching an estirnated worldwiclc
6700 AN Wageningen,The Netherlands
SMATFORSK,
sales of' US$ 310 million in 2000. -fhc dairy sccl.or
NorwegianFood ResearchInstitute,
ilccounts for a large part of the carrageenanapplications
O s l o v e i e n1 , N - 1 4 3 0Â s , N o r w a y
in food products, such as frozen desserts.chocolate ntilk.
i N.D-ZelinskyInstitute
of Organic Chemistry,Russian
cottage cheese,and whippcd creant. In aclclition to this,
Academy of Sciences,Leniskiiprospect47, Mor.o*
carrageenansarc uscd in various non-dairy {bod proclLrcts,
119991,Russia
such as instant products, .jellics. pet foocls, sauces,lrncl
!lUniversidad
d e B u e n o sA i r e s ,F a c u l t a dd e C i e n c i a s
n o n - f o o d p r o d r . r c t s .s u c h a s p h a r n t a c e L r t i c alli i r n t u l a E x a c t a ys N a t u r a l e sD, e p a r t a m e n t od e
euimica
t t o n s , c o s n r et t c s a n c lo i l w e l l d r i l l i n g f i L r i c(ll r r r e s o n .2 0 0 0 ;
O r g a n i c a ,P a b e l l ô n2 , C i u d a d U n i v e r s i t a r i a1 4 2 8 ,
-fhcrkclscn.
1 9 9 3 .V a n c l c V c l c l e& D c l { L r i t e r ,2 0 0 2 ) . t n
B u c n o sA i r e s ,A r g e n t i n a
g e n c r a l .L - a r r a g c c n a n
s e r v c sA s a g c l l i n g . s t a b r l i s i n ga r r r l
\ t \ t ( ) \ i l \ l r r r r l t l i r tlrr:r : t ' r r t .
S i n c c n u t r r n r l c a r - r - t r g e c n l rlrrrrsc n t i r t r t r c s o l ' i l i l l c r - c n t
( , l r , t r i e c n , r l st ( ' l ) l ( , \ ( , n l
r r n t , o l t l t t , r r r , r j o rt t , x t u r i s l r r l t g t ( . _
s r r la
l l c t l p 6 l ) ' s u c c h u r i r l c st h. c i 1 c 9 r . r r 1 ' rtpi tsrir r c i l l c r . sl l . t r r t r
t l i r n t s i l l l r t , f o o r l i n c l L t : t r , l, l.r t , r , , , r r c
n , r t l r , t lr n ! r l r l r c t t l s ,
b l t l c l r t o b l r l c l t . [ ' l t c r c l i r l c .t l t e ( l u l l l ) t i l i l t i \ c i r n l r l v s r s
t t ' l r rl rr , t r l L i s i ' rl l r r l c i , i r l r , l, l l o r l , r l r l r l
r , t l i l l s ( , r r r , r r 1 t . r , n ; r r r o l ' c l r r l l r g c c r l i n b l r l c l r c si s t r l ' g l e l r l c s t i n t l - r o t - t l u t c e
lirr.
h o t l t i n g t c t l i c r rslr r p p l i e rl sr r r r li i r o r l i n t l r r s t r . itc( s) ( l ù l i \ c r .
\ r ' l l ( : l ) r l t ( l I t ! 1 , t l t l l r ( ) i , | l \l l
l{l(1n,..
i t c ( ) n s t l i l t ic ( ) n s u n l c t I' r , r t l t r c l , r n r l I i , , 1 lr' el o 1 t 1 1 1 1 1
; l
I
' l
i
, , '
i , ,
[ . v , r r rr / t ' l ' r , / r i r . r ' l a l . l I r e r x l s i n I r x t c ] - S r i c n c . r , &I t , L h r x t l o t y
1 | (2(X))) / I 9)
'O:S
OH
-*-'--__)
enzyme
,o\
DA
DA
G4S
G4S
D6S
Mu-carrageenan
Kappa-carrageenan
OH
-...---_----_>
enzyme
.o.'1
d' ^
Fp
S--\
o-^Y.o.
f-[o-a''
,*T"
G4S
D2S,65
DA2S
lota-carrageena
n
H
OH
-------------)
l
9
(
S--\
P
H
l
G2S
D2S.65
Lambda-carrageenan
Fig'
-l'
I
o
o. /-l'"-7'
'/o\./1-\,'-\,.^\/
oso.G2S
-
J"o.DA2S
Theta-carrageenan
S c h e m a t i c r e p r e s e n t a t i o no f t h e r J i f f e r e n st t r u c t u r e s o f t h e r e p e a t j n gd i m e r i c
u n i t s o f c a r r a g e e n a n sT. h e I e t t e r c o d e s r e f e r t o t h e
n o m e n c l a t u r e o f K n u t s e ne t a / . ( i 9 9 4 ) .
applicationsbascdon their unique intrinsicproperties.
Nowadays NMR spcctroscopy(both 'H- and FCNMR) is one of the standardtools for the determina_
tion of the chemicalstructureof carrageenansamples.
Thc NM R spcctroscopyof carrageenansis useclin
industry and for research with different approaches
correspondingto differentultimategoalsor interests.In
the Iirst casethis techniqueis seenas a scrceningtool to
detcrrninethe possiblecommercialor inclustrialvalueof
raw extracts obtained frorn still unexploiteclrcd sea_
wecclsanclalso usedto control the quality of carragce_
nan products and to study the influenceof processrng
paramcters,such as temperiltureand pt.l, on the struc_
ture. A dillcrent approachis useclwhen the intercstis
centredin the systemof carragecnans
biosynthesisecl
by
t h es c a w c e d
o r i n t h e d e t e r m i n a t i oonf t h c h n c s t r u c r u r e
o f ' a c a r r a g c c l l t no r a c a r r i l g e c n afnl - a c t i o nT. h i s r e v i e w
g r v c sa n o v c r v i c wo f -N M l i - s p c c t r o s c o pay s a p o w c r l i r l
t o o l f o r -t h e u n a l y s i so l ' c u r n r q c c n asna r n p l e s .
S t r u c t u r eo f c . r r r a g e e n . r n s
( ' r r r r i r g c e t i l n si l r e l l l l r r l i l y o l ' u ' l t c r s o l u b l c .
lincltr..
'Iltcv
s r r i l l r t e rgl t r l l c t u n s .
t r r . cc o n t l - r o s c or ll ' l l t e r - n l r t i n q
l - l i r r k e r ll l - t) g i t l i l c t ( ) l ) \ 'nn(t) s c( ( i - L r n i t s r) r n c 4
l _ ln
i kcrl ry-
o-galactopyranose
(D-units) or 4-linked3,6-anhydro-crtt-galactopyranose
(DA-units),forming the disaccharide
repeatingunit of carrageenans
(seeFig. l). The letter
codesin Fig. I refer to the nomenclaturedevelopedby
Knutsen, Myslabodski,Larsen, and Usov (1994) (see
Box l). The sulfatedgalactansare classifiedaccording
to the presenceof the 3,6-anhydro-bridgeon the 4linkedgalactoseresidueand the positionand numberof
sulfategroups.For commcrcialcarrageenanthe sulfate
content falls within the range from 22 to 38% (w/w).
Commercial(food-grade)carrageenanshave a weight
averagemolecularrnass(M*) ranging from 400 to 600
kDa. Besidcsgalactoscand sulfate,other carbohydrate
residues(for exantplexylose,glucoseand uronic aci<ls)
and substituents
(lor examplemethylethcrsanclpyruvate
groups)can bc presentin carrageenan
prcp;irations.
Since
n u t u r u l lc a r r a g c e n a n
i s a r n i x t u r eo l n o n h o n r o l o g o u s
p o l y s a c c h a r i c l el hse. t c r m d i s l r c c h a r i c lrce p c a t i n gL r n i t
r e f è r st o t h e i c l c a l i s esdt r u c l u r c .
-f
he ntostcolnlnorltypes ol carrirgccnâIl
are traclition-l'hc
l t l l 1 i, < l c n t i f r cbr lv a ( i r - c e kp r e l i x .
t h r e cc o n t n r c r c i u l
l l o s t n n p o r t l l l l t c t r r r i t g c c l l l r r lsr r e c l r l l c t lt - . r - . l r r t r l) , - c l r r r i l g e t : n l l l ) .I l r e c o r r c s l - r o n r l t n gl t . l l ) A ( i n s p i r c c l n l r n r c s
l r r r c l I e l t c r c o t l c s u r c c i r r r : r l r c c l l ( ) s2c. ' 1 'c l i s r r l l l t t c( ( i 4 S -
llor l. Nonrcncl:rture
of carrtgeenans
Traclitionally carrilgecnansare identilicd by a Greek prelix, indicating the lnrjor component of the sarnple.This nomenclatureis i:sed universallyin tracle,sciencc,and legislation.However,this systemis not suitableto describemore complex
polymersunambiguously.
To describemore complexstructures,Knutsen,Myslaboclski,Lilrsen,and Usov (1994)proposedan
alternativenomcnclaturefor carrageenanand agar. This letter code based nomcnclature(seeTable 1) is founcledon the
worldwicleacceptedIUPAC nomenclature(McNaught, 1997)and allows a systematicclescriptionof complex polymer moleThe structure basedon a perfect alternatingsequenceof B-(l+3)-linked l-galactopyranoseresiduesand cr-(l+4)-linked
cr-rles.
3.6-anhydro-o-galactopyranose
or a-(l+4)-linked o-galactopyranoseresiducsis calted the 'ideal' structure. Based on the
Knutsen-nomenclature,the names and letter codesof the dimeric structuresof x, r, and l-carrageenan are carrageenose4'sulfate (G4S-DA), carragcenose2,4'-disullate(G4S-DA2S),and carragccnan2,6,2'-trisulfate(G2S-D2S,6S),respectively.
This letter code nomenclatureis gaining importance in scientificpublications dealing with carrageenansancl agars. Most
authors describingcotnplex structuresadopt this nomenclature.In addition to the notalion (S) for ihe sulfate estér distribu,
tion, methyi ethers(M), pyruvate acetal (P), and glycosylunits such as xylose (X) are introduced to describecarrageenanswith
diffcrcnt substituents(Miller, 1998).Agars cliller from carrageenansas they have the l-configuration for the 4-linked galactose
residue.Incidentally this configuration is observedin carrageenans(Stoiz & Cerezo,2000)and in the correspondingletter
codesthe letter D is replacedby the letter r.
Table
-f.
Alternative letter code nomenclature of the different carrageenans, as developed by Knutsen et at.(1994)
etler code
Found in carrageenans
IUPAC name'
[i
Not foundo
3 - l i n k e dB - o - g a l a c t o p y r a n o s e
4-linked u-o-galactopyranose
4-linked 3,6-anhydro-a-D-galactopyranose
Sulfate ester (O-SOt)
3-linked p-o-galactopyranose 2-sulfate
x,p
r,r,)",p,v,0
1,0
K/T,P,V
3 - l i n k c d P - D - g l a c t o p y r a n o s4
e- s u l f a t e
4 - l i n k e d3 , 6 - a n h y d r o - c r - o - g a l a c t o p y r a n o2s -es u l f
4-linked o-o-galactopyranose 2,6-disulfate
4-linked cr-o-galactopyranose6-sulfate
r,0
2S,6S
S
À,v
Ir
l n t e r n a t i o n a lU n i o n o f P u r e a n d A p p l i e d C h e m i s t r yn o m e n c l a t u r e a s r e c o m m e n d e d i n i 9 9 6 ( M c N a u g h t ,1 9 9 2 ) .
Not found in natural occurring carr:rgeenans,but can be founcl in desulfate<Jcarrageenan samples.
DA2S). curnrgecnose4'-sulfate (G.ttj-DA), and carrageen u n 2 . 6 , 2 ' - t r i s u l { a t c( G 2 S - D 2 S , 6 5 ) . B e s i d e st l t e s e t h r e e
mlUor carragecnantypcs, trvo other types,called 1t_and vcarnrgceuân (letter code C;4S-l)2S,65 and G4S_t)6S,
respcctively),a[c oflen cncountered in comntercial carrageenan saruplcs.p- And v-carrageenanare thc biological
prccLlrsorsof rcspectivcly r- ancl r-carrageenan The dif_
fcrent types of car-rageenanare oblaitrccl front difT'ercnt
spccics of the [Lhoclophytl. r-Cun.agccnan is pr.cc l o n r i n a n t l yo b l l r i n e db y c x t r a c t i o no f ' t h e t r o p i c a ls e : r w c c t j
Krt1t1ttt1.tlt.1'L'tt.;
ulyurc:ii, knorvn in tr-ac.lcas E'ut,lrcuntu
( o t I ( ) | t i i( o r s i r r r p l yc o t t o n i i ) ( R u c l o l p h , 2 0 0 0 ) .E t t c h c r r n r r t
tlt'ttticLtlttItrtrr
(lr-lrdc narrrc [.,ttt'ltatrtrta
,s:piottosuttt
or slntp l y s l ) i l t o s u n t )i s t h e n t r i n s p e c i c sl i r r t h c p r o r l L r c t i o no l '
t c i r r r i r l t c c n i l n .l r o r t h c p r o t l u c t i o r r o f ' t h e r c - i r n d L - c l r r l ' l g L r c n l u ln. t i x t r r r - cos1 ' p l l n t s o l b o t h s c r L l r l s t l r g c so l ' s c a -[
u c e c l s o l t h c l r b o r c s p e t i e : l l r e u s c r l . I r e s e : t n c c c . l rsr c
L t s t r r l l cv r t r - t rt te t l t i i t l r l r l k l r l il r t r l c r l r t c r lt c n t l l c f i l t u t . ct (s)
t l t n s l o r n t t h c b r t r l o g t e lPr lI ' c c u l ' s ( )1l 'rs-l.t r t r vl c l l l l l g c c n l l t
i n t o ç r r r r t Il c l t l l l l g ù ù n i u t(ss c cb c l o * ) . ) , ( , l l r . l r g e c n . r rns
t ) l r t 1 1 i 1 1I çr r, r1r nr i r l l i ' r ' c r rstP c e i e sI r . o n ti l t c ( i i . q t r t . I i r.tttnt d
( l t r t r t t tl r :
l t c l t r ' t i t .I l t c s P o l i , P l lt\i t . I l l r n t s r r l ' l l t c s es c l r
GLriry, 1982),whereasthe gametophytic plants produce a
t</t-hybrid type of carrageeltillt.These r/r-hybricl carragecnansconsist of a rnixed chain containing both r- and
r-units (Van de Veldc, Pcppelman.Rollema, & Tromp.
200 l) ancl range from almost pure r-carragcenan to
altnost pure K-carrageenan(Bixler, 1996). Thc procluct i o n o f ' À - c a r r a g e e n a ni n ' r p l i e st h e s e l e c t i o no l - s a m p l e si n
the sporophyte stage while the extractior"rcan bc carricd
o t r t w i ( h h o t w i l l e r a s t h e c y c l i z a t i o n .i n a l k a l i n e r n c d i u n r ,
to O-carrageenanis clifllcLrlt(C'iancra, Nosccla. Matuler v i c z ,& ( l c r e z o . 1 9 9 3 )a n c l t h i s p r o d u c t h l r sc s s c n t i a l l yt l i c
s a n r ep r o p c r t i e sa s ) , - c a r n r g c c n a (nG l i c k s m a n . 1 9 8 3 ) .
r - A n c l l - c a r r a g e e n u l ta r c g e l f o r n r i n g c l r r r u - c i c c n a n s .
r v h c r c u s) - - c u r n r q c e r l r ni s l l h i c k u t e r . I ' h c r l i l l c r c r r e ci r r
r h e o J o g i c l rbl c h t r v i o u t b c t v u c c r r - u n d r - c u r r i l g e c n i u io n
o r t e s i c l cl t n r l l - c u r n r u c e n i t n o n t h c o t h c r s i t l c r c s L r l t s
l i o n r t h c l j r c l t h l t t t h c I ) Â - t r n i t so l - t h c s c l l i n s o n c s l t : l r c
t l t e l ( ' * - c o n l i r n t l r t i o nl r n t i t h c l ) - L r r r i t isn i - c l r r - n r g c c r u r n
t l t t r t o t . [ . | t e r ( ' 1 - c o r r l i r r n l r t r o on l t l t e . l . 6 - l r n l r l r l l o - u u l t l l i c t o p v l l t t t o s Lv rl n i t si r r t l r r r t lK - c i r f n r ! r e c n ilrrnl l o u s l r
I t c l t r ' l t: le c r r r r i l ltu s t l r r eul rr ' . \ \ l t i r l t r s t s s e n l i : rl]i r r '1h t ' g r e l
'l
l i r t r r r i t t l" ) l ( ) P t r t i c \ l r c r u r t L r r i P
l l f c c r . l l s ( )(r)sl r l r r r r l
\\r'ù(ls i)lrrLlrrtei. LlluitgL,ùniln (\lt(
h (llllll!lr'CIlllrl. r'-
lrtrtjlcts. Wcst.
&
linri
lr
(lU'litlfcl)iirl.
il|L' :rl\() lt()n-
7(t
+C'l-conuclling culrlgccnans with thc D-units in thc
l b r n u r t i o n . O c c u r r e n c eo 1 ' d i s a c c h a l i c iuen i t s w i t h o u t t h e
3 , 6 - a n l i y c l r or i r t g a n c l h a v i n g a a C 1 - c o n f b r m a t i o nc u u s c s
"kinks"
i n l l t e r c g u l a r c h a i n a n d p r c v e n t st h e f o r r n a t i o n
o f h c l i c a l s t r a n d s a n d b y c o n s c q u e n c ep r e v c n t s g e l a t i o n
of tlre carragcenan. Irt vivo, r- and K-carrageenan are
formed ellzynlatically licln.r their precursors, by a sulfohydrolase (Wong & Craigie, 1978, De Ruitcr e/ a/.,
2000). The usual presence of considerable amounts of
precursor units in commercial carragcenan prcparations
has a strong negative effcct on the functional (e.g. gelling) propertics (Hansen, Larsen, & Grsndal, 2000; Van
de Velde et ul., in press). Therefore, in industrial processing, prior to use crucle carrageenan is submitted to
an alkalinc treatment or alkaline extraction, catalysing
(Therkelsen, 1993;
the cyclisation reaction with OH
Van de Velde & De Ruiter, 2002) (Table l).
Analysis of carrageenans
At present, there is still lack ol- adequate analytical
techniqucs to determine the amounts, the polydispersity,
and the pr-rrity of carrageenans in food products and raw
matcrials. Different techniques and approaches use<Ifor
this type of analysis are, for example colorimetry and
immunoassays, FIPLC and electrophoresis (Roberts &
Qucmener, 1999). To reveal the (detailed) molecular
structure of carrageenans,a chemical analysis is mostly
donc on isolated and purified carrageenan samples. [n
the beginning, chernical rnodification and degradation
nrethocls were time-consuming and teclious analytical
techniclues.In the mid 1970s a rcal boost was given by
the ir.rtrocluction of NMR spectroscopy. Nowadays
N M R s p e c t r o s c o p y( b o t h I H - a n d r ] C - N M R ) i s o n e o f
thc standard tools lor thc deterrnination of the chemical
strLrctureof carragcenan samples(Roberts & Quemener,
1 9 9 9 , U s o v , 1 9 9 8 ) .l n i i d d i t i o n t o N M R s p e c t r o s c o p y ,
othcr analytical techniques, such as sullâte content
analysis, nronosaccharidc composition analysis (Jol,
Neiss, Pcnninkhof, Rudolph, & l)e Ruiter, 1999) and
m et h y l a t i o n a n a l y s i s( F a l s h a w , I l i x l e r , & J o h n d r o , 2 0 0 1 ,
F a l s h a w & F u r n c a u x , 1 9 9 4 )a r e a p p l i e d .
NMR spectroscopyof the maior carrageenan types
pritlr trl
satnplesare soniclttecl
Itr gcncral,cltrrâgccnall
are carrccordingthe spcctraand the Nlvll{ exçrerttncnts
to reducethc viscosityof
ried out at clevatedternperature
tl.resolution (high viscosityresultsin line broaclening).
lrC isotope,
Due to the low natural abundanceof thc
rrC-NMR
high
prepared
at
relatively
are
samplcsfor
lHto
D2O)
compared
(5
in
w/w
l0%
concentrations
N M R s a m p l e s( 0 . 5 - 1 . 0 %w / w i n D 2 O ) . O v e r v i c w so f
the lfl and 13C-NMR spectraof the most in.rportant
Figs.2 and
carrageenan
typesare shown in respectively,
3. Both the lH- and llC-NMR spectra reveal that
"pure"
samplesare rare. Thc r- and r-carcarrageenan
rageenansamplesmay containextraneousunits of r- and
which is quite well known
respectively,
K-carrageenan
for carrageenansextracted from K. olvarezii and E.
tlenticulatum.Sampiescontaining precursor carrageeof
nans are alwayshybrids containinglow percentages
the precursorunits.
t 3 C - N M Rs p e c t r o s c o p y
l3C-NMR spectraof highly regular
Proton-decoupled
the spectraofcorresponcling
red algalgalactansresernble
Sincethe pairs of diads G-D
substituteddisaccharides.
and G-L, as well as G-DA and G-LA, are diastereoisomeric,they give different spectra,the differences
being especiallynoticeablefor the anomeric carbon
(Table2). This observationrnacleit possible
resonances
to recommendthe I3C-NMR spectroscopyas the simof the
plest tool for distinguishingthe polysaccharidcs
Yaphe,&
agar and carragccnangroups (Bhattacharjee,
H a m e r ,1 9 7 8 Y
; aroyskyS
, h a s h k o v&, U s o v , 1 9 7 7 ) .
r- and r-carragecThe trC-NM R spectraof gelforn.ring
nan were studied,using model syntheticmonosaccharide
derivatives(Usov, 1984) or oligomeric polysaccharide
fragments (Greer, Rochas, & Yaphe, 1985; Rochas,
Rinaudo,& Vincendon,1983),and interpreted,as indi'Iable
was concated in
3. Later on this ir.rterpretation
f i r m e d b y v a r i o u s2 D N M R e x p e r i m e n t (sC h i o v i t t ic l
were
al., \997; Falshawet al., 1996).Theseassignments
successfullyuseclto identify the correspondingpoly-
T a b l e 2 . ' 3 C - N M Rc h e m i c a l s h i f t s o f b a s i c a g a r and carrageenan structures
Diad
Unit
(, I
(,
c-1
I
(, lA
(,
| ..\
( , 1 )
t 0J . /
I( ) ( ) . ( )
c-2
700
()().+
I ( ) 2. 1
i o.)
(lfj j
( r ( )( )
lo.t lJ
, ) t ,i
(r(l J
,i( ) {r
(,
l )\
l
C h c m i c a l s h i f t s( p p m )
'rl i
o )
Rei.
c-.]
c-4
c-s
c-6
tJI0
7't.o
6tt.()
7',.t,
7)2,
( rl . l
8))
t J ( )I.
()B ll
7 [ \( )
/'l 1)
()5 fl
i J ){ . 1
(,{r I
. ' (I . l
.ili ( J
79 l
77 .t
,'t\ i
L . r l r ; i , v t 'Y
, aphe ;ttrclRrtt lras t'l()fi5)
( ) 1)
t)1.1
(r').1
l , r l r , r 1 , t 'Y, , r p l r t ' ,V t t ' t , r n r l R o t l t a s I l ( ) l J ( i )
{ )I i
( r l l
l l s r r r , .\ ' , r r o t s k \ ' .. i l ) ( l s l r , r ' l r l i o \ ' I l ( ) l i o l
I i . . o r , r r r l \ l r , r : l r l . o vi l r r t jr ,
(r lJ
L v a n r / r ' V e ' k i e .r , l a l . t I r c n t l s i n f t t o t t S r r c n r t ' &
ii
t\^
\
I
\__/^,
)
\
\_=-_
'
[-]r^
/t _ /^
,\
56
5.4
i\^
5.2
/,
5.0
4.8
t^, )
- Jf,^,À.,".
[^
4.6
4.4
(ppm)
42
4.0
3.8
3.6
3.4
32
F i g . 2 . r t l - N M R s p e < : t r ao f t h e m a j o r c a r r a g c c n a nt y p e s { r o r n b o t t o m t ( ) t o p : K c a r r a g e e n a nf r o m K a p p a p h y c u sa l v a r e z i i( w i t h p e r m i s s i o n
o f [ l s e v i e r a d o p t c t l f r o t n V a n t l e V e l d e r , t a / . , 2 0 0 1 ) ;r c a r r a g e e n a n f r o m É u c h e u m a d e n t i c u l a t u m( w i t h p e r m i s s i o n o f F l s e v i e ra d o p t e d
f r o r r V a n d e V e r l d ee t a / . , 2 0 0 ' l ) ;v / r - h y b r i d c a r r a g e e n d nc o n t . r i n i n g2 5 0 Â v - c a r r a g e e n a n( s p e c t r u mp u b l i s h e dw i t h p e r m i s s i o no f C P K e l c o ) ;
À-r'arrageenan
f r o m / r d a e a u n d u l o s a( w i t h p e r m i s s i o no f E l s e v j e a
r d o p t e c lf r o m S t o r z e t a / . , 1 9 9 4 ) .
, . r j C - N M R c h c m i c a l s h i f t s { o r t h e m a i n s t r u c t u r a l u n i t s r - r fc o m m c r c i a l c a r r . r g e c n à n s
Ir.,Ot"
( arrageenan
Unit
C h c m i <a l s h i f t s{ p p m }
,
(,'1S
I)A
(,.1\
I ) A ]\
(,15
I ) . l s{ ) s
'è-s
Rel.
c-r
a-t
ai
c-4
It ) 2 5
.
( ) 5 ].
()
()().
696
789
i9)
7,11
/t\ l
7:l IJ
768
61 l
(,1').5
t l s o v a n c lS h , r s l r k o(vl ( l B 5 )
1 0 22
')t I
b() J
/5 0
i6.t\
7/ t\
7)2
/8J
7.1t\
/ /.o
(rl J
(,() fl
t l s o v a r r r i\ l r a s l r k r i vl l ( l t j 5 )
I0J,I
'll{,
7)t
7)1\
il)5
6.1.1
i.t )
( r l l
I , r J s l r , r ,r lvn ( l I r r ] ( , , r r \ i l ' ) ( ] 4
tl{) l
( tt\ i'
()ul
/ ' . 1l l
c6
I
I' v:rrr</r,yek/('et.tl.i Itt:rt]sin ftxttl 5rir.nrr.& Itrhnol<tgy ll()0(-)2) il
92
^*-lil[,[l,,,L,
-*JL-,\ --*
L
L ]
- * 1
105
T
- t
100
|
|
r
95
r
'
r
I - r - - - - . T '
90
r
- t
r -
B0
r
r
f - -
75
.-lL__
70
65
(ppm)
F i g .3 . r r C - N M R s p e c t r a o f t h c m a j o r ( a r r a g e e n a nt y p e s f r o m b ( ) t t o m t ( ) t o p : K c a r r a g c c n a nf r o r n K ; t S t p a p h y c uasl v ; t r e z i(i w i t h p c r m i s s i o n
o f E l s e - ' v i e. irt rl o p t e r l f r o m V a n d c V e l d e e t a / . , 2 0 0 ' l ) ; r - c a r r a g e e n a nf r o m l l u c h c u r n a t l e n t i c u l a t u m( w i t h p e r m i s s i o n o f l : l s e v i e ra c l o p t e d
l r o r n V a n t l c V e l c l e r - t a i . . 2 0 0 . 1 )v; i r h y b r i c lc a r r a g c c n a nc o n t a i n i n g 2 5 o l ov - c a r r a g e e n a n( s p c c t r u m p u b l i s h e d w i t h p e r m i s s i o no f ( - P K e l c o ) ;
) ' - c a r r a g e e n a nf r t i m l l c l . r e at t n d u l o s a( w i t h p e r m i s s r o no f L l s e v r e ar c l o p t e df r c . r mS t o r z r : t a / . ,. 1 9 9 4 ) .
saccharidesisolatcd from new sources(Usov & Shashkov,
much later (Ciancia, Matulewicz, Finch, & Cerezo, 1993,
l9lt-5).Thc rvell-rcsolvccl
spectrlrnlof-À-carrageenan
Storz r,/ ut.,1994) (Table 4).
[G2SD2S,6S1,,
rvas obtained n.ruchlater (Falsharv& I.urneaux,
It slrould bc notccl that the biological precursors
l99zl; Storz. Ilrtcott, Clhcrniak, & Clcrczo. 1994) cluc to
mcntionecl above and rnany other possible c:rrrurgecnun
s o t l t e t c c h t t i c l r lc l i f i i c u l t i e sw
. hich are usually explainecl
d i u c l sa r e u s u a l l y f i r u n d o n l y i l s c o r n p o n e n t so f ' h y b r i c l
by thc liigh visctlsitY ol- À-carragcenan solulions.
p o l y m e r i c n r o l c c u l c s( s e e t h e s p c c t r r o 1 ' p - a n c l v - c a r r n r A s s i s n l l l e t t to l ' l t l t o t t t c t - i rcc s o n a n c c si l ) t h c s p c c t n ro f t l i c
l l c c n l l t c o n t l r i n i t r gs a r n p l e si n l i i g s . 2 l t n d I l n c l I { e l . s .
llitlltlgiclrll)fcCtlrSors1t-lttltlr'-cltI.t.iIgCCIlllllrvltsltllrclcllv(]llllcill
|}ellitlrl.l}r-iglrrlcl.I)rtrtttc.lrntlI]ocick(|98]).rvIlcrcltst|tcsignlr|sintlrcs1-tcctr:ttlI'srtchIlybr.irlcllr-r.ltu
ctlttl1lIctcitltcr1lt-ctlLtitrtltl1.tltcscsl]cctrltrr,ltsprtllisllc
L r ' , t r t r / r , W r / r i c c l a / . r l r c n r / - s i n f o o d - S r . i c r xr ' & I t ' < h n o h t g y l 3 ( 2 0 0 2 ) 7 - l 9 )
r:IC-NMR chcmical
Table 4-
shifts for the most common
Carragcenan
f'I
c-1
c-2
c-3
c-4
c-5
c-6
C
D2S
102.7
947
69.6
75 . 4
81.9
78.1
66.9
78.3
7 5 .)
77 1
6 16
70.0
F a l s h a wc r a / . ( 1 9 9 6 )
C
DA
102.7
94.7
69.7
70.3
80.5
79.5
66.5
78.3
7\ . 5
77.O
61.1
69.7
L i a o ,K r a f t ,M u n r o , C r a i k , & B a c i c ( 1 9 9 3 )
( , 2S
100.3
95.6
77.6
71.7
77.2
77 1
678
79.5
71.7
77.0
61.1
69.8
F a l s h a wa n c l F u r n e a u x( - 1 9 9 , 1 )
102.7
947
69.4
701
80.3
79.5
66.0
78.5
72.9
76.9
67.2
69.1
U s o v a n d S h a s h k o v( 1 9 8 5 )
DA
D4S,6S
DA
1 0 28
95.3
69.6
69.9
78.9
79.5
74.O
78.9
72.7
77.O
68.2
69.6
Liaoel a/.(1996)
C4S
D6S
105.3
98.2
70.7
68.9
7B.B
70.7
73.8
79.5
75.4
68.9
61.9
68.4
Storz et al. (1994);Ciancia et a/. (1993)
C4S
D2S,6S
105.3
98.B
70.7
76.8
80.4
68.9
73.8
79.5
75 . 4
68.9
61.9
68.4
Crancia et al. 11993);Storz et al. fi9941
DA2S
(;6S
{?)
càrrageenan slructural unils encountered in hybrid molecules
C h e m i c a l s h i t t s( p p m )
arranged in blocks, e.g. the given diad has the neighbours
of the same structure on its right and left. Evidently, this
assumption may not be valid for many real polysaccharides, and hence, the signal assignment for minor
diads suggestedin the literature [see,for example Ciancia et al- (1993)] should be regarded only as tentative.
Most carrageenansdiffer only in 3,6-anhyclrogalactose
content, degree of sulfation and positions of sulfate
groups. An attempt was made to calculate the l3C-NMR
spectra ofmany possible carrageenan structures,including thosc, which were not isolated at that time from
natural sources, using spectral features of the known
polysaccharides (Storz & Cerezo, 1992). Flowevcr, the
experimental spectra of a- ancl l"-carrageenan,showed
insuilicient coincidence with the predicted values. Evidently the amount and specific position of charged sulfate
groups may result in conlormational changes of polysaccharide chains, which are not taken into consideration by aclditiveschernesin calculationsof chemicalshifts.
1H-NMRspectroscopy
The IFI NMR spectra
of monomeric methyl B-ugalactopyranosicle and methyl 3,6-anhydro,cr-o-galact o p y r a n o s i d eh a v e i n i t i a l l y b e c n u s e d a s t h e b a s i so f t h e
interplctation cl{'the proton spectra of the gelling r- and
r - c a r m g e e n a n( W c l t i , 1 9 7 7 ) .L a t e r o n t h e s ea s s i g n n t e n t s
I t"0," t.
I
in *"* .r'".i."i shifts
of ,i" o-ono-"rifp-ron,.nl
c a r r a g e e n a n s( C i a n c i a e t . r 1 , ,1 9 9 3 ;S t o r z e t a l . , " 1 9 9 4 )
a.,r..rg""n"n
Monosarcharirlc
C h c n r i c a l5 f i i f 1{ p p m )
lK
l)Âl\
f),.\
5jl
5|
ii
l)r5,(,s
I
l
lr
I ) . 1\ ( , \
I)(r\
' ,r ( )
i
i
r
have beenconfirmedby the spectraofthe neocarrabiose
(Knutsen& Grasdalen,1992a).For the
oligosaccharides
biologicalprecursorunits, v- and p-carrageenan,
only
the cr-anomericprotons were assigned(Ciancia et al.,
1993;Storz et al., 1994),due to the complexityof the
hybrid samplescontainingtheseprecursors.The viscosity of l"-carrageenan
samplesallowsonly the assignment
of the a-anomericprotonsin a pure sample(Storzet al-,
1994).Nowadays,quantificationof differentcarrageenan types in a sample by IH-NMR spectroscopyis
protons (Dbasedon the resonances
of the cr-anomeric
and DA-units) in the region from 5.1 to 5.7 ppm (see
Table 5). The signalsfor the B-anomericprotons (Gunits) are lcsssuitablefor either identificationor quantificationpurposes.
NMR spectroscopyof minor substituentsin
carrageenans
Several carrageenanshave additional substituents,
'Ihus,
which may be identifiedby NMR spectroscopy.
a
small amount of 3-linked 6-O-methyl-o-galactose
residues was found in K-carrageenanfrom Kappophycu.t
ulvarezii(Bellionet al., 1983)and in severalothcr poly(Chiovitti et al., 1998).Theseresiduesgive
saccharides
specificsignalsfcrrOMe at 59.0,for the substitutedC-6
a t 7 1 . 8 ,a n d f o r t h e n e i g l i b o u r i n gC - 5 a t 7 3 . 3 p p n i i n
l r C l - N M R . S r n a l la m o u n t so f t e r m i n a lx y l o s er c s i c l u e s
w e r c a l s o d e t e c t e di n m a n y c a r r a g e c n i i n sb,u t t h c i r
l o c a t i o nu s u a l l vr e r n a i n su n c e r t a i n .l n t h e c r t s eo f a n
u n u s u a l ' p c , l y r a c c h a r i c[lG
c 4 , 6 S - I ) A ] , ,f i o m P h r t t e h t (orpusp(pero(drlos (Liao cl ul., 1996)NMI{ spectrltl
d a t a i n c l i c a t c dt h e u t t i r c h n r e not f x y l o s e r c s i t l u c sa l
p o s i t i o nJ o l ' s o n r c , 1 - l i n k e.dr - t ) - g a l a c t o sr c s i ( l u c sI.n
r-cirrflrgcclutrt
fl'ont fur'/rctuttutlcttl i ttr/alrriitthis Irttrr.tos l t c c h l r r i cpl cr - o b l b l yo c c u p i c sp o s i t i o n( ro 1 ' 3 - l i n k c [t ]l - t r q r r l l r c t o rscr sr i r l r r c( [s) s o vr r n t iK r t r t t s c rLt .r n p u b l i s l r c t l ) .
l . \ , , r / )( / ( ' \ . i , / r / r ' r ' l . r / . , ri r l i r r i s i n l r x t r l - S r ' l t , r r rJr l l c t i r r r o / o L y l j t . ! ) 0 1 ) i l
1)
Table 6.
t)'l
' 'C-NMR <:lremical
shi{ts for several pyruvated carrageenans
C h c n r i c . r ls h i f t s ( p p n r )
P o l y s a c c h a r i d eb a c k b o n e
c-2
c-l
cP
f)A
cP
t)A2S
c-3
t0'1..1
()\.2
102.0
91..1
6().2
75.2
76 . 7
77.9
Pyruvate acetal
c-4
c-s
c-6
676
76.1
66.7
67.5
78.4
667
77 2
65.6
700
Methyl
Carboxyl
;l
F a l s h a r ,avn d F u r n e a u re t . t / .( ' 1 9 (
25.5
Pyruvic acid is il conttrlon componcnt of many colrplex carrageenâns.It forms a cyclic acetal at positions 4
aud 6 of 3-linked galactose residucs and may be founcl
in diads rclated to cr.-and 0-carrageenans.This substituent can be identified by characteristic signals of its
carbons together rvith specific substitution effectson the
corresponding carbon atoms of 3-linked D-galactose
(Clhiovitti t,r ul., 1998 Falshaw & Furneaux, 1995)(Table
6 ) . T h e p y r u v i c a c i d k e t a l s a r e a l s o d e t e c t c di n t h e ' H NMR spectra by methyl proton resonanceswith a chemical shift of 1.45 pprn (Chiovitt;t et al., 1997).This signal
does not ovcrlap with other protolt resonancesin these
carbohydratc derivativcs. In addition, specific substition
effects on the cl.remicalshifts of the other protons are
o b s e r v e ciin t h c r H - N M I { s p e c t r a( C h i o v i t t i e t t t l . , 1 9 9 7 ) .
According to the structure ol sullated galactans, rcd
algae are traditionally divided into agarophytes and
carrageenophytes. FIowever, galactan sulfates of intermccliatestnlcture, rvhich may be termed as n/l-hybrids,
havc bcen founri recently in rnany species.They seem to
be characteristic to rcpresentatives ol Cryptonemiales
and Rl'roclyrnenialcs,n.ray be isolated from several
spccicsof Cerantialcs, lncl surprisingly are also present
as minor componcnts in ntany speciesof Gigartinales
"classica["
known as
carrageenan sources (Miller,
1997). Thcse o/r--hybrids are no l'nofc carrageenans,
a n d t h c i r s t r u c t u r a l a n a l y s i sw i l l n o t b e c l i s c u s s e d
here,
b u t i t s h o r r k l b e n o t e d t h a t t h e i r ' r C - N M R s p e c t r e ar r e
rather complex and, as a rule, can not be interpretcrl
tunambi guously withoLrt aclditional eviclenccobtained by
c l i e m i c a lm c t h o d s ( l r s t e v c z ,C i a n c i a , & C c r e z o , 2 0 0 1 ) .
NMR spectroscopy of contaminants and additives in
carrageenan samples
( ' a r r l tg c e n t r np r c p t r r a ito n s n t a y c o n t u in a c l n t i xt u r e s o f
tn'o dillcrcnt origins. Sonte rif then are coniponentsof
101.7
1 75 . 9
Chiovitti et al. (1998)
Resirlue
C h em i c a l s h i f t s ( p p m )
c-1
\ n r \ .l r, . r
I t r . r , r l7 l ) l l l L r r o p r r , t r o : t ,
7 l ) l l l L r r( ) l l \ l , l l l ( ) ! ( '
l i I I 1 t L il i, , 1 L r r , r r , , . . L
1 0 { l. r
(.-2
t)t
i' I t)
l{)l )
I
led algal biomass, which were not removed in the carrageenan isolation procedure (Rudolph, 2000; Van de
V e l d e & D e R r . r i t e r2, 0 0 2 ) .O t h e r c o m p o u n d s ( i n o r g a n i c
salts, sucrose, galactomannans) can be added by manufacturers to irnprove and/or control some functional
propcrties of carrageenan samples (solubility, viscosity,
g e l s t r e n g t h ,e t c . ) . S m a l l c o n t a m i n a t i o n s a n d a d d i t i v e s ,
such as inorganic salts and sugars, can be removed by
dialysis, prior to recording the spectra.
trloridean starch, a branched ( 1 4,I 6)-cr-p-glucan
structurally related to plant amylopectins and animal
glycogens, is a reserve polysaccharide of red seaweeds.
It is soluble in water and can accompany carragcenans
in the extraction and prccipitation steps.Apart from the
detection of glucose in an acid hydrolysate, the presence
of floridean starch can be confirmcd by the well-known
set of signals of 4-linked a-t-glucopyranose residues in
the rrC-NMR spectrum (seeTable 7) (Colson, Jcnnings,
& S m i t h , 1 9 7 4 ;K n u t s e n & G r a s d a l e n . 1 9 8 7 ) .I n t h e ' l l '
NMR spectra, floridean starch is detected by the signal
of the anomeric proton of the a(l+4)-linked t>-glucopyranosyl at 5.37 ppm (Knutsen & Grasdalen, 1987).
The resonance of the anomeric proton of the a(l +6)linkcd o-glucopyranosyl appears at 4.98 ppm. Floridean
starch can be removed by starch degrading enzymcs or
by any procedure ol' separation of acid ancl neutral
polysaccharides.
Galactans of the agar group may be present in currag e c n â n s ,w h c n n ' r i x c da l g a l p o p u l a t i o n s c o n t a i n i n g b o t h
agarophytes and carrageenophytes arc used for carrageenan manulacture. As in the case of o/r--hybrids,
r . r n a m b i g u o u isd e n t i l i c a l i o r ro f a g a r - l i k e s t r u c t u r c s m a y
r e q u i r c c h e r l i c a l e v i d e n c et o c o n r p l e l n e n tt h e r r C - N M I I
s p e c t r - a lc l l t a . S o r n c r c c l a l g a e c o n t a i t r w a t e r - s o l u b l e
s r , rnl ft e c l x y l o r n a n n a n s a n d n eu t n r l x y l a n s , b r - r t t h e s e
p o l y s l r c c h a r i t l e sa r c u s L r a l l ya b s e n t f r o n r t h e c o l r n r o n
I . r b l c 7 . ' ' ( N M R t l r t ' r n i c a ls h i [ fs o f , u r y l o s e . r n r l r u < r o s c
Subsllrrcc
l
Rc{.
c-3
c-4
, t I
; | .l
.-ll ',
' . 1 , :
t , l . , r , /
/ ( l ( l
{)I()
l } ( ) ( l (! ' 1 . r / I l r ) l J l l
. - l l i
I ) ' r:
c-6
/
l ' r - l
I e ch n o / r x y I i ( 2 0 0 2 ); ' 3 9 2
II
l
il
1
I
il
r{i\
I
ilil,,|1,,,\
Hil
.A/\,/[^
il
|,
il
1t
56
E , À
 a
J Z
l\
I
i
( ^
J.U
À o
{.O
i1/l
iii\
1 l
r
ni,
u
44
42
(ppm)
-
t
40
, l'\
38
,_t
tu
),
,,o
|,,
(0.50.4r
t'viw, 600 r\4|lz. 56"( ).
aI l s p c c t r . l
c i t r t . a g c c n o p h y t ei rsn t l o r r l l u c c i d e n t u l l yr n a y b e f i t L r n di r r
c r u l s 1 ' r c c i cosf ' t h c o r . c l c rN e n r a l i u l c s . r r C - ! - À
c l r r n l g 0 c n l u l s .I L c r l t r l g l l x 1 ' J l r r rl sr r c l r b u n r l i r n l i r r r - c l . r c o l ' b o t h t y p c s o f ' p o l v s u c c h l r r i c l e s\ \ ' c r c p L r h l i s h c t l
s c n t l r t l \ c so l l l t c o r r l c l s N c n l r l r t r l c st r n t l I r i r l n t i r r - i u l c s . ( K o l c n t l er . . [ ' u j o l . I ) u r n o r t t c . N { i t t t r ] c i v t c z .& ( ' c t ' c z o .
t I t c l c l r s s L r l l l r l c r\ \l l ( ) l n i u l n i t n sr r c r - cl i r r r n t i( ) l l l v u r s e \ , l 9 ( ) 7 : K o r " l c . I I i t s c h . S l l t s h k o r , . I J s o r ' .& Y r t r o t s k t ' .
f . van tlc Vr./rir.t,I ;tl.I Irt'nds in f otxl -S<
it,nrt & 7c<hrrokrgy j3 2002) 73 92
Mh
I
105
100
95
90
85
(ppm)
r
B0
r
r
r
I
75
r
r
r
r
I
70
r
-
r
r
i
65
Fi8'5.rr(NMRs[)0ctrar;ftht.nlajclr(]arr.lg(}en(lnvariants,sLlcr()Se
alv.trt,t ti
( 1 0 % rw i w , 1 5 0M H z , t ] O " C ) .
1 9 8 0 ;[ J s o v & I ) o b k i n l , 1 9 9 l ) a n d n t i r y b e u s c ( lf o r t h c i r
iclentilication.
A r l t l i t i v c s s u c h i t s s u c r ( ) s cu n ( l g l u c o s eu r c o l l c l l u s e ( l
t o l i l l u s t t l r c ' ,i s c o s i t y o f ' t h c c o n t n r c r c i a lc t r r r a g c c n a n
p r c p a r a t i o n s . S L r c r o s ei n t e r t c r c s w i t h c o l o r i n t c t l i c p r o c c c l u r c so f ' c l c t e r r n i n a t i o no f c i r r r a g e e n a n sb a s c c lo n s p c c i f i c c o l o r r c l r c t i o n s o l ' 3 . ( r - l r n l i y t l r o g l r l l c t o s c r. r n u c
f l ' u c t o s cu n t l - 1 . ( r - r r n h y t l l o s r r l u c t obsech a r , c s i n t i l u r l l r i n
R I
t h e s cp l o c c c l u r c(sA n t l e l s o nc t l l o r v t l e .1 9 7 4 ;Y a p h c &
A r s c n o u l t .l 1 ) 6 - 5 I)n. u c k l i t i o nt.h e a n o r n e r i sc i g n a lo l ' a r > - g l u c o p y r a n olsccs i d u ci n t h e l r C - N M R s p e c t r u r no f
(llock, Pedcrsen,
sLlcrosc
& Pedersen,
1984)may overlap
w i t h t h a t o f ' D A 2 S r e s i d u eo f r - c a r r a g e e n aaut a b o u t 9 2
ppm (sceFig. 5 and Tr"rrcluois,
Acquistapace,
Arce-Vera,
& Welti, 1996).Ncverthelcss,the prescnceo[ sucrose
cletectedby its other signalsin
cau be unar.nbiguously
the rrC-NMR spectrulnof the mixture (Table7). 'Ihese
a d d i t i v e sd o n o t d i s t u r bt h e N M R a n a l y s i sa n d c a n i n
generiil be detecteclbcsidesthe diffcrent carrageenan
fomrs (see Figs. 4 ancl 5). Only the resonanceof the
anomericproton of glucoseoverlapswith the resonance
of [-carrageenanin 'tl-NMR. If necessary,
glucoseand
sucrosctogethcrwith inorganicsaltscan be removedby
d i a l y s i so r e t h a n o lp r e c i p i t a t i o n .
The gelling propertiesof carrageenans
can be considerably improved by addition of several galactomannansor mannans of some higher plants (Imeson,
2000; Therkelscn, 1993).NMR studieswere used to
elucidatethe possible modes of interaction betwcen
polysaccharidecomponentsin these blends (Rochas.
Taravel,& Turquois, 1990).Sincethe 'rC-NMR spectra
of galactomannans
are well-known(Grasdalen& painter, 1980),the correspondingspectraof mixed preparations recorded at clevated tcmperatures(above the
meltingpointsof gels)can be usedto detectthe presence
of galactontannanadclitivesand to calculatethcir content relative to carragecnan.In tH-NMR spectrathe
anomericproton of the cr-o-galactopyranose
is observed
at -5 pprn ancl that of the B-r>-mannopyranose
at 4.8
ppm (Grasdalen
& P a i n t e r ,1 9 8 0 ) .
Analysis and quantification of the composition of
carrageenanblends by NMR spectroscopy
I n i n d u s t r i a la p p l i c a t i o n st h e c o m p o s i t i o no f c a r r a gt:cnanblenclsis vcry important with respectto the
desireclfunctionality.The type of carragcenanpresent
ln it commcrcialpreparationanclpossibleadditivescan
to a largecxtent clcterminethe functionalpropertiesof
t h c f i r r l r lp r o t l t r r ' t C
. i r r r : r g e c r l rpr r c p l r r t r t i o n sm. i x t u r c s
of carragcenirns
and commcrcialcarrageenan
blendscan
c l u a l i t a t i vl ye a n c l q u a n t i t a t i v c l yb e c h a r a c t e r i s c tbl y
N M R s p c c t r o s c o p yN.M R t e c h n i q u cas l l o r vt h e i d e r i t i fication of vurious carrageenanfirrms iincl the determinution of the rnolar ratrosand the contentof the
i n c l i v i c l u aclo n r p o n c l l t si n t h e m i x t u r e . B o t l - rr l l - a n d
l r C ' - N M I I s p c c t r o s c o payr e a p p l i c a b l eI b r t h e s ep u r p o s c s .' l l - N M R h a s t h c a d v a n t a g eo f a r e l a t i v c l yh i g h
s c n s i t i v i t yt.H - N M R s p e c t r ao f s a m p l e w
s ith low carrageenirnconcentration(0.5 I .0o/"wlw) can be recordcd
i n i r c o u p l eo f r n i n u t e sT. h e a n a l y s i so f t h e ' t l - N M R
spectrais based on the position and intensity of the
resonances
ol the a-anomericprotons of the repcating
u r . r i t( s e eT a b l e 5 ) . ' r C - N M R h a s t h e a d v a n t a g eo f ' a
high information content. Due to the high chemical
shift dispersion,in a lIC-NMR spectrumeach carbon
atom of the repeatingunit of a carrageenanvariant
givesrise to one singlesignaland the variouscarrageenan variants show unique and characteristicpatterns
(seeTables3 and 4). Quantitativedeterminationof the
compositiono[ a mixture of carrageenans
can be based
on the intensityof the resonances
of the anomericcarbonso[both ringsofthe repeatingunit. A disadvantage
of r3C-NMR is its low sensitivity.For the recordingof
l3C-NMR spectrasampleswith high carrageenan
concentration(7 l0% w/w) have to be used.Even then a
rIC-NMR experimenttakes approximatelyl2 l8 h to
reacha reasonable
signalto noiseratio (Si,[.
In most casestH-NMR will sufficefor the characterisation of a carrageenan
blend. ln caseswhere initially
I H - N M R i s n o t c o n c l u s i vaen d a d d i t i o n aild e n t i { i c a t i o n
is required, r3C-NMR spcctroscopycan be applied.
Adclitivessuch as sucroseand glucosewhicli are often
usedto adjust the viscosityof the carrageenan
preparations,do not disturb the NMR analysisand can in general be detectedin addition to the differentcarrageenan
forn.rs(see Figs. 4 and 5). Only the resonanceof the
anomericproton of glucoseoverlapswith the resonance
o f p - c a r r a g e e n ai nn r H - N M R .
Quantitativeapplicationsof NMR techniquesrequire
a n u m b e r o f s p c c i a lp r e c a u t i o n a r ym e a s u r e sI .t i s o f
paramount irnportanceto ensure that the inter-pulse
delay amountsto at least4-5 tirnesthe longcstI1 value
of the resonanccs
uscd for thc analysis.Table 8 shows
11 valuesfor a number of relevantcomponcnts.It is
s e e nt h a t f o r I F I - N M R o f s a m p l e sc o n t a i n i n gm o n o - o r
cli-saccharicles
relativclylong intcr-pulsedelayshave to
b e a p p l i e d .O n t h e o t h e r h a n d r r C - N M I { s p e c t r ao f
carragccnans
can be rccordcdr-rsing
relativelyshortinterp u l s ec l e l a y sA. n a c l d i t i o n nclo n t p l i c a t i n fga c t o rf o r l r f l N M R i s a n o s s i b l ev a r i a t i o no f t h e N O E c l l - c c tfso r t h c
t '
T . r b l o t l . l o n g i t u r l i n . r l r e l . r r . r t i o nl i m e s o f a n u m b e r o f l : r ( l - a n d I H - N M R r e s o n a n c e so f x - c a r r a g e e n a n , t - c a r r a g c e n a n ,s u c r o s e a n d
S
] lucost'
Conrpounrl
Resonances
F r c r l u e n c y( M H z )
i
K,rnil
I r
t (ill,lll{'r'l(ll
,tnrlI(,ll,rii(,('l,ll
,
:1,',:,'
:l.l
r
\ r , i l . ' l , , r r ,n , , , t l , , , i t ,
I I N \ 1 R : 7 , t l l ( ) l l ( ' r J (l ) l o l ( ) n s
'l
I \,\1R: ,rll
'lI \\1li
. r r , , n r , , (i l ) r r l r ) r l
I, (s)
,{)(}
(r00
{)o()
( }. 1 , r
(rll{)
(,
It
1:rC-and tll-NMR spectroscopy
Sample 1
Sample 2
ttc-NMR
t''c-NMR
'H.NMR
4.9
80.6
11.4
0
3.4
5B
73 . 8
16.9
0
3.5
K
,14.8
t
2 12
238
0
10.3
p+v
À
Uniclcntificd
40
2i.2
25.5
o
11.]
Sample 3
'H-NMR
37.2
267
20.6
9
6.6
36.6
23.3
21.8
14.7
3.9
S a m p l e 1 : n e u t r a l l y e x t r a c t c d c a r r a g e e n a nf r o m S a r c o t h a / i a
c r i s p a t aS
. a m p l e 2 : n e u t r a l l ye x t r a ( : t e dc a r r a g e e n a nf r o m É u c h e u m a t l e n t i c u l a t u m . S a m p l e - J :c o m m e r c i a l c a r r a g e e n a ns a m p l e ,c a l l e d l , - c a r r a g e e n a n .
differentcarbons.Floweverin the caseof carrageenans
all carbon atoms of the repeatingunit haveone proton
attached.Basedon this one would expectlittle variance
in the NOE effectfor the different carbons.Experiments
with a mixture of kappa- and iota-carrageenan
using
continuous composite pulse decoupling and gated
decouplingproduced apart from S/N, the same result
for the intensity ratio of the anomeric carbon resonances(Rollema,unpublished).
Table 9 illustratesthat reasonable
agreementis obtained
betweenresultsobtainedby lH- and I3C-NMR spectro-
scopywith respectto the determinationof the quantitative
compositionof carrageenanmixtures.It should be mentioned that the relativelylow S/N of r3C-NMR spectra
implicatesthat minor componentscan either not be detected or if their signalsaredetectable,the contentdetermined
by t3C-NMR will havea limited accuracy.
Finally if use is made of an appropriateinternal or
external reference,using rll-NMR spectroscopythe
molar ratios and the absolutecontentsof carrageenan
forms and addedmono- or di-saccharides
can be determined in a singleexperiment.
G4Snr,, .
H-l
G4SBH_l
Ânrs'4
I o,*,-o
*o'-.lill^;,ilo'"n
i,".,
G4saH-4
A*H- t A.ÊtI-l ^nrH-l G4SnrII-4
\,.
G6olt-t
\ tt
\ '.'.\ t
l
G
D.lililtilii/1ri
_ill[jt-___I iuu.\_iJUHlqLJrUll^
tJl
LJ
I I L1 . ,) ,
A
i
l
r
l
l
f
l
i
l
t
,
__-JtiLlï__
J\ruJlill|ltlîJu|]lrtirt
_r
il
lli'l
.J
5..1
l
5.0
4.7
l(l(ll,tl
B
\G"'n.,
"l:ri-s
4.1
85
tH-NMR spectroscopy of carragecnan
fragments
K n o * l c d g c l r b o r r t t h c r l i l l c r en t o l i g o s a c c h a r i t l c si r r
tlic dillercnt ll.uctir-rnscun be uscd tcl cleclucc the
s t r u c t u r c o l ' t h c o r i g i n a l c a r r a g e e n a ns a m p l e . I r ' r t h i s
f l u g m c n t u n a l y s i s a p p r o a c l t , r l l - N M R s p e c t r o s c o p yi s
r u s e dl b r i t s l r i g h s e n s i t i v i t y l n d p o s s i b i l i t i c st o r e s o l v e
the finc stnlctul'c of carragecnan oligosaccharides.
Carrageenan oligosaccharidesare prepared by chemical
or cuzymatic hydrolysis. The higher ancl lower molecular urass fiactions are separated by precipitetion
mcthods ancl flrther fractionated by gel permeation
chrornatography (GPC) or size exclusion chrom:rtography (SEC), scc Box 2. Iligh field 'H-NMR spectroscopy is than used to elucidate the detailed
molecular structure of the different di-, tetra-, and
oligosaccharides.
NMR-elucidatcdsequcnce information
A sulfation of a certair.rcarbon will influence not only
t h e c h e m i c a l s h i ft o n i t s a t t a c h e d p r o t o n b u t a l s o t h e
protons on other ring carbon atonts and even neigh-
b o u r i r r gs u g u r .r . c s i c l u c ls l.r c r c l b r et h c i d c n t i t y o l ' t l r c
l t c t L u tcl r t l n r q c c n l t rnc s i c l r r ac s r v c l l l r s s ( ) n r es c ( l u c n c c
i n l b r m a t i o nc a n b c o t r t a i n e c M
l . i n i n r a l s c c l u c n cien l b r t n a t i o nl o r t h e p a r t l yd e s u l l i i t ecda r r a g e c n ufni o m a l k a l i
treatcd l-urt'ellaria ltrntbric'aliswas obtainecl by 400
M H z r f l - N M R s p e c t r o s c o p yd, o c u n . r e n t c db y a r . r
approximately0.02 ppm upfield shilt of the rcsonancc
o l ' A l r - G c o u r p a r e dt o A 1 y 1G 4 S ( K n u t s c n , M y s l a b o d s k i , & G r a s d a l e n ,1 9 9 0 ) .S e q u e n t i a il n f o r r n a t i o n
r e g a r d i n gt h e d i s t r i b u t i o no f O - m e t h y g
l r o u p s( G 4 M ) i n
agar fi'om Porph,vraunthilicalishad mr"rcl.r
earlier been
demonstrated
with 90 MIIz rrC- NMR spectroscopy
( 3 6 0 M t l z f o r r H ) ( M o r r i c e ,M c l e a n , L o n g , & W i l l i a m s o n ,1 9 8 3 )E
. v e na t a v e r y l o w f i e l ds t r e n g t ho l 2 . 3 5
T, the effectof a desulfatedreducingcnd neighbourof
3,6 anhydrogalactose
i.e. DAç; C (94.6 ppm) as comparedto DAcr G4S (95.2ppm) (Knulsen& Grasdalen,
1987) were noticed. Howevcr it is likcly that some
sequence
inforn-ration
beyondthis might be obt:rinedby
observingthe anornericrcgioncharacteristic
for both the
3-linkedand 4-linkedresiduesin rvellrcsolvedsDectraat
Box 2. Preparationof carrageenanfragments
Depolymerization
Chemical depolymerrsation
In gencral a slight degradationof the molecular weight will reducethe viscosityin a sampleand henceimprove its NMRspectrumespeciallycluc to lcssline broadening.This can be achievedby non-specificmetho<lssuch as ultrasonic clegratlation
(Van cleVelcleet o1.,2001)and acid hydrolysis(Caram-Lelham,Sun<lelof,
& Andersson,1995;Rochas& Ileyraud, lDSt;. fne
latter is dillcult to control sinceselectivedegradationol 3,6-anhydrounits (DA2S or DA) and removal of sulfategroups is
most likely to occur. A strategy based on autohydrolysis(Storz & Cerczo, 1991)has been <Jeveloped
with quite piomising
resultsbut hirs so far not beenusedby many workers.Another depolymerisation
tool, which mighf be usefulin combination
with NMR, is oxidative radical depolymerisation.Treatment of a polysaccharidesolution with ferrous ions plus ascorbicacid
at rooln temperaturewill induce random depolymerisation(Hjerde, Kristiansen,Stokke, Smidsrsd,& Christensen,1994;
Yamada et al., 1997).The use of chemical fragmentation must be done vcry carefully, taken into account the possibility of
lateral reactions concomitant with the lytic ones, i.e. autohydrolysiswill produce the splitting ol the a-(3,6-anhydro)galactosidic
linkagesflankedby sulfategroups (Ciancia,Matulewicz,Storz, & Cerezo,lgil) bua also will producc tÉe
hydrolysisol the p-galactose2-su[fate,as a result of the loss of this sulfategroup the resonanceof the a-galactose2,6-sulfates
will displacedfrom æ92.0ppm ro x94.'7ppm (Noseda,1994;Noseda& ceiezo- 1993).
Enzymaticdepolymerisation
As for polysaccharidcs
in general,the useof specilicenzymesis an ultimatetool for structuralanalysisof carrageenans.
By
sclccting a suitable cnzylne, i.e. carrageenase,well-defincd oligosaccharicles
with charactcristicstructural elenrentscan bc
obtainedin reasonablehigh quantities(Bellionet at., 1983;Greer, Shomer,Golclstein,& Yaphc, 1984;Rochas& Ileyrautl,
l9iil). On thc othcr hand molcculescnrichedwith uncommonirrcgulariticsor norl crrrxgecnlnconstituentsn-rightbe retained
anclsrthseqttently
isolatctlby a properfiactionationtechniclue
(seebelow).In this way. crypticstructures,that otherrvisewould
bc rllaskccl
bclorvthc noiselcvcl in the lrC-NMR spcctracan be idcntified.Carrageenascs
arc crrcio-p-galactanascs,
clcavingthe
intcrttalB-(I '4) linkageswith sulfatcsubstitutionpattern relateclspecificity,
reviewcclby De Ruiter & Ruclolph(1997).OligtlsltccIlaritlcsol.tlroltcocarrabiclsoserics(I)A(;l1),,
rcgioltstltatpoSSCSSStrIlcturalregtrlariticso|accrtairrlcngtIl-I}y|arthetlrtlstpoprrlar
bctlbtltiIrcrlIl.tltlrscvcrelbactcria'IlhltssLlcccssftrllybccnproduced
:\lrscrr.I 9()-l).
e ( ) n r r ! ' r c t i l il lL\ rl r i l i r l r l ci r r r. . o L i t i r\ rt lcu c { u r cl r n l r ] v s i s .
Fractionation
5 r ' p a r a t i o n b a s e c lo n s o l u [ r i l i t yi n e t h ; r r r o l
are nevcr occurringstructurallypr.rrebr-rtwith a varying ratio of characteristicstructuralelernenlsas nrcnClarrageenans
tioned in Fig. l. ln orclcr to isolatespecificstructuralmotives occurringin low quantity the use ofspccific carragcenases
combined'uvithsonrefractionationprocedureis requircd.I;or the subsequent
analysisNMR-spectroscopyis the nost preferred
tool (seebclow). Adding alcohol to an aqucoushyclrolysatchas beenuscd to prccipitatethc high polyrncricwciglrt fraction,
often containinga high content of irregularstructuressuch as 6-sulfatedprecursorunits or 3,6-anhydrounits with "rvrong"
sulfate substitution on itsclf or its r-reighbouring
residues.The term enzymeresistantfraction (ERF') was introduced lor sucl.r
fractions(llellion et al.,1983).
ln principle it is possibleto produce pure oligosaccharides
of a certain type, i.e. (DA2S G4S)" or (DA-G4S),, provided such
occur in separatemolcculesor in long blocks.At this point it must be noted that if irregularitiesalong the carrascquences
gccrlanchain occurcloseto the linkagesubjectedto enzymatichydrolysis,but not so closeto hinderan enzymaticattack on the
actual beta I 4 linkagc,oligosaccharides
carryingboth precursoror unsulfated3-linkcdunits might be producedand hence
solubilisedin thc 6O 80% ethanolfraction(Knutsen& Grasdalen,1992b).So lar thereis no literatureclescribing
enzymeswith
capability to spccificallyrcmov<:all correspondingsugar units from a certain carrageenansample.
Nevertheless,alcohol precipitation might be used for a rough estimateof the âpparenrkappa-contentin different samples
aftcr a trcatmcnt with r-carragecnase(Fig. 9). The resultsof such a fractionation combined with the grosscomposition of the
different major structural elementsas obtained with 1H-NMR, some indication of the occurrenceof sequencesof a certain
length(block size)could be obtained.As opposedto precipitation,leachingofa previouslydried hydrolysateinto increasingly
water diluted cthanol solutions was shown to give more definedfractions of oligosaccharide,which easilycan be subjectedto
NMR analysis(Knutsenet ol., 1995).
t00
*l
-If
.9
il
601
s
EC
AP
FLHI
\\
rnr
FLCS
f
E. mur
iota
Ethanolsotuble
Fig. 9.
T h c a p p a r c n t ( D A - G 4 S ) , c h a r a c t e r o f s o n r e c â r r a g e e n a n sa s e s t i m a t e c l: r f t e r t h e d i g e s t i o n w i t h r - c a r r a g c c n a s c . ' f h c c o n t c n t
i s e s l i m a t e d a s t h e p e r c e n t a g e o f o l i g o s a c c h a r i d e sa s c o m p a r e d t o t h e a l c o h o l i n s o l u b l e l r a c t i o n . E R F w a s e s t i m a t e d g r a v imetically afier prccipitation in 7070 ethanol, dialysis and lyophilization. Wherc EC: a commercial extract f'ron Koltpuphycu.s
u l v a r c z i i ( L I ' f E X 1 7 1 5 0 5 ) ,A P : a c o m m e r c i a l c x t r a c t l r o n t F u r c e l l l r i a l w n b r i c a l i s ( L I T E X A P 6 4 l ) , F L H I : t h c K C l - i n s o l u b l e
f l ' a c t i o n o f a h o t w a t c r e x t r a c t [ - r o n tF u r t e l l u r i u l u n b r i c u l i . r , F L C S : t h e K C l - s o l u b l e f r a c t i o n o f a c o l d - w a t e r e x t r a c t o f l \ r t c ! lurilluttlbrit'tth.ç,[lG:thcliotwatcrcxtracto|Ettt'huttltttPe|utinac.EM:1hehotwaterextractof
a c o r r t m c r c i a l c x t r a c t f r o m E u t h e w n u t l e r L t i c u l a t w n( S I G M A ) . L l n p u b l i s h e d r e s u l t s f r o m ( K n u t s e n , 1 9 9 2 ) .
S e r p a r a t i oI n
r a s c co] n ( l r r ( ) n t a t o s r a l ) h y
l\sis.scltttcclttot1lltttlgrltllIticstc1.lisItcctlcd.
(\{cl-c:rrr&WtIIilltllsstlrl.l97())itntl[}iil(ieltl'pc1|]ll(cl.illIS(Ktttrtsctl&(ir.lrstilrlcIl.
L v . r r rc / t ' V e k / r , t ' t i l . 1 [ r e n d s i n f o o < t S r . l e , n c r ' &T a c h n o k r y l y1 3 ( ) 0 0 i ' t 1 3 9 2
T a b l e 1 0 . D i a g n o s l i c r H - N M Rc h e m i c a l s h i f t d a t a f o r c a r r a g e e n a no l i g o s a c c h a r i r l e so f t h e n e o c a r r a b i o s et y p e
Prolon
rCcrH--l
lcp H-1
JC F]-.i
sc H-.1
rca H-4
lcp il-4
rc fl-4
rA+Ccr ll-.1
2^+cB H-l
1A H-j
6A |.l'1
)A H-2
4A l1-2
6A I1-2
2A H-5
2A
H-4
,A H-3
oA
f1-4
6A H-4
4A H-5
OA H-5
1A tJ-3
6A H-3
(DA-G4S)2hexa
(DA-G4S)3tetra
DA-G-DA-G45 tetra
(DA2S-G4S)2tetra
5 320
4 654
.1.660
5 320
,1.653
4.658
5.320
4.650
4.618
. 5 . 3 45 . 3 1 4 7G 4.68
4.67 4.68
4.899
4.4840
4.855
5.121
5..104
5.085
4.897
4.837
4.1)9
5.119
5 101
.5065
5.00 '1.96
4.94-4.90
4.96 4.92
5.29
4.144
4 . 10 3
4.143
4.O48
4.86
4.69
4.648
4.606
4.535
4.483
4.667
4.592
4.529
4.502
(4.66)
4.409
4.423
4.48
4.352
4.367
(4.69)
4.658
.1.901
4.835
4.855
5.120
5.103
5 . 10 l
5.084
4.142
4.138
1.102
4.650
4.607
4.535
4 607
4.479
4.650
4.409
4.529
4.352
).t/
5.25
\4.66)
4.69
4.55
F o r n u m b e r i n g o f t h e d i f f e r e n tu n i t s t h e c o u n t i n g s t a r t sa t t h e r e d u c i n g e n d w i t h a n u p p e r c a s en u m b e r . I n t h e f a r l e f t c o l u m n t h e s u l f a t e
p o s i t i o ni s n o t g i v e n b u t c a n b e f o u n d i n t h e o t h e r c o l u m n s .T h e t e t r a s a c c h a r i d e( D A 2 S - C 4 S )w
2 a s r e c o r c l e da t 3 0 ' C a t 3 0 0 M H z ( K n u t s e n
e t a / , 2 0 0 1 ) , a n d t h e r e s t w e r e r e c o r d e d a t 3 0 ' C a t 5 0 0 M H z ( K n u t s e n& C r a s d a l e n , 1 9 9 2 a )w i t h a c e t o n e i n D 2 O ( ô 2 . 2 2 5 )a s i n t e r n a l
r e f e r e n c e .C o n c e n t r a t i o nd e p e n d e n t s h i f t v a r i a t i o n sw e r e r e c o r d e d f o r t h e o l i e o s a c c h a r i c l eTs h
. iswas most oronounced for the C4S units
i n D A 2 S - C 4 S - t y p eo l i g o s a c c h a r i d e sT.h i s i s c J e n o t e da s a r a n g e w h e n t h e c l i f f e i e n c ew a s e x c e e d i n g0 . 0 2 p p m . T h e h i g h e r s h i f t v a l u e s w e r e
q]ygpfec"rded for the mos
mples (Sletmoen, 2000).
relativelyhigh lieltl (>11.7 T). To achievea proper
resolutionof the Cl signalsthe polysaccharides
shoul<i
be subjectedto a previousdepolymerisation
(seeBox 2),
conservingthe sulfateand 3,6-anhydroresiclues
and the
spectramust be subjectedto somestatisticalanalysesas
done for alginates(Grasdalen,1983).
obtained from oligosaccharidesare more resolved and
some very useful diagnosticlH-signalscan be found.
lH-NMR assignmentsand coupling constantsof ditetra- and hexasaccharides
of the DA-G4S type, and
the effect of desulfationare presentedelsewhere(Knutsen & Grasdalen, 1992a).Assignments of the similar
regionfor (DA2S G4S)" or iota-typeoligosaccharides
is
S p e c i ailn f o r m a t i o no b t a i n e db r ys p e c t r o s c o p i c
given elsewhere(Knutsen et al., 2001).In practice some
a n a l y s i so f o l i g o s a c c h a r i d e s
selectedpeaksin the spectralregion from approximately
The additional information obtained from specrra
4.3 ppm to 5.3 ppm can be used to characterise(DA
rccordedfrorn oligosaccharide
samplesas comparedto
G4S)" or kappa-typeoligosaccharides
with respectto sulpolymcric san'rples(Van de Velde er c/., 2001; Welti.
lation and chain length.The effectof a desulfationof the
1977)is relateclto resolvcdresonances
from reducing
reducingend resiclueis demonstratedby Fig. 6 showing
anclnon-reducingend residues.
This is only achievedas
this selectedI H-spectralregionof tetrasaccharides
purified
long as the levcl of rcsidualwater is minimisedby repefiom a K-carrageenase
hydrolysateof Kappaphycus
alvarated frcezeclrying in D2O. In generalby applying carezii an<lFttrcellariucarrageenan.Some usefulresonanccs
rageenases
and a suitablefractionation,the rcsulting
lor neocarrabioseoligosaccharidesand somc observed
'fables
clligosaccharide
spectraarc lesscomplexdue to the presplittingdue to spin-spincouplingare givenin
l0
emincnce
o f n c o c a r r a b i o st yc p eo l i g o s a c c h a r i d egsi v, i n g
and I I, respectively.
The co-existence
of oligosuccharides
n o u r c c l u c i n ge n c l J , 6 a n h y d r o g a l a c t o saen d j _ l i n k e c i
with eithçr DA G + or DA G4S-+ type non redlrcing
g:rlactosc
on the rcclucinscnclrcspcctively.
Furtherrnore
cntl sequcnce
co-occLrrring
in a SIIC {raction(Bio Gcl P4
s o l n eo f t h e l L { - r e s o n l r n coc fs a D A - u n i t n e i g h b o u r i n g
s c eK n u t s c r &
r ( l r a s d a l e n1. 9 9 2 bo) l a F u r c c l l a r i l - e a n u t h c l - l t n k c c lr c d u c i n ge t r t lu n i t w i l l b e s p l i t d l l e t o t h e
gcenanhyclrolysitte
is clcntonstratcd
by thc rcsonanccs
of
i t n o n l c r i cc q u i l i b r i u r t ta n r l n l r v b c t r s c cal s c l i l r g n o s t t e H 5 ( ' ' 4 4 l p p n r )a n c lH 3 ( - , 1 . 3 - 5
p p r n )o f t h e l ) A - u n i t
p c l t k si n h i g h l i c l t l s l ) c c t r uA. l t h o u g hi n s o r u cc a s e sl i ) r
i n l f r g .7 . ' I ' h c l l c k o l r c c l L r c i negl l d r c s i c l u cisn a c l r l i t i o n
p o l v r n le s p e c t r -l lrrl c n ' r c s o n r r n c n
c st l y b c c l s i l y â s s i l r n c ( l
t o l ) r t : s c n co
c l ' t h e - ( i . 1 S a 1 1r rt : s o n a l t c er t 5 . 3 ? p p n t
b v t h c i r c- h a l l c t c r i s t icco r r p l i n cu ( ) n s t i u r l s . ' f hse1 - r c c t r u ( l c n ) o l ' l s t n l ttch c p L r r i t vr v i t h r c s p c c tl o n c o c u r n r b i o s e
r H - N M R s p c c t r a o f c a r r a g e e n a no l i g o s a c c h a r i d e s
I T a b l c 1 1 . S o n r e o { t h e c h a r a c t e r i s t i cs p l i t t i n g sd u e t o s p i n - s p i n c o u p l i n g s i n
I
r,,ot,,n
ft1
lil
lt2
H]
t tr'
H5
In residue/sequence
S p l i t t i n g( H z )
Contributing couplinB
' C a S a n dC 4 S B
-(,.1Scr
-(,,1Sand C.lSB
-C4Scr
-Cu
t)A
DA2S
DA2S
DA
DA
DA
DA
79
l9
-t3
1.1
2.5
2.4
2.3
5.9
Jr:
lt.t
l;,c
t;;
I t.q
lt,z
h,t
lt.t
)t,t
5.4
1.9
Broad
)t,t
lt.s
Combinations
A h y p h e n d e n o t e s t h e r e m a i n i n gc h a i n w h e r e a sC r e e k l e t t e r ss p e c i f yt h e a n o m e r i c f o r m . P o s i t i o n si n t h e c h a i n s d o o n l y a f f e c t s h i f t v a l u e .
T h e s i g n a l sf o r l - 1 5o f 3 , 6 a n h y d r o g a l a c t o s eu n i t s a p p e a r a s b r o a d p e a k s .F o r a c t u a l s h i f t p o s i t i o n ss e e T a b l e ' 1 0 a n d f o r a l l c o u p l i n g c o n . a l u e sl o r D A 2 S u n p u b l i s h e d .
s t a n t s s e e K n u t s e n & C r a s d a l e n( 1 9 9 2 a )V
type oligosaccharides and can be used to evaluate
enzyme specificity (Knutsen & Grasdalen, 1992b). It
should be noted that by comparing the area of above
mentioned resolvedsignalsfrom the non-reducingend 3,6
anhydrogalactosewith the resonancesfor all Hl of all 3,6
anhydrogalactosc units (-5.1 ppm) the degree of polylnerisation can be estimated.
To rninimise the obscuring of certain resonances in
the spectra of the oligosaccharidesby residual water, the
temperature must be set to an appropriate value. At
room tcmperature the residual water peak is occurring
in tire region of H4 of G4S (i.e. 4.8 ppm) units whereas
at elcvated temperature this signals shifts upfield to
about 4.2 ppm. An alternative approach is selective
irracliation of the water peak by a pre saturation techn i q u c o r a p p l y i n g a d o u b l e p u l s e s e q u c n c et a k i n g i n t o
account the relative long relaxation time ol small n.roleculcs such as water. Uowever, such techniques might
influcnce the areas of neighbor"rringrcsonances(unpublished results). Temperature induced shift in some resonances night occur even for small oligosaccharides.In
geucral, the eflècts were largest for the protons attached
to carbons involvecl in the glycoside linkagcs, smallcr
for thc protons attacheclto their neighbouring c:rrbons
'Ihis
itncl very slnall for the rnost distant ones.
is dramatically dcn.ron:itratedfbr the spectrlrntof thc oligosacchariclc rrrixtLrreoriginating I'ron f'ur tellurkl-carrageenan (F ig.
7 ) . F i n a l l y t h e t e m p c r a t u r ei n d u c e d s h i f i s a r e i l l u s t r a t e d
f i r r t h e p r r r e t e t r a s u c c h a r i c lD
c A G4S DA G4S in Fig.
l J . l t i s c v i c l e n tt h u t a r n o r e c l e t i r i l c (sl p c c t r a li n f o r n r a t i o t r
is obtainerl lrt 25 Cl coutpured to 90"C ancl thal ntctrc
r c s ( ) u u n c c cs o l l c s c c a t t h c c l c v a t c c lt c n r p c n r t u r e .
Outlook .rnd perspectives
l l r t ' r o l c o i N N l R s l ) ( ' (t r ( ) s ( ) [ ) yo i r i r r r a g t ' t : n ; t nr sn
i r r r l L trsvt , r rr r l rt ' s r ' , r rl rt
I : o l i r r r l r r s t n l l p r r r p o s c N i \ 4I { s l t c c t l l r l l e o b t u i n c t l
u i t l r o L r ll l r l t l r c r 'p r r l i l i c l r t i o nt r r r t lo r l l i r c t i o n l r t i o ror l ' t l r c
sample, as primarily the overall composition is rmporl3C- and
tant. Resonancesin the anomeric region of the
rH-NMR spectra reflect the main diad components,
which determine the type of the possible major carrageenansin the sample. It is important to remember that
a pair of correlated resonancesdetermines a diad. The
spectrum is a supcrposition of the resonances of these
diads and, therefore, represents only an average conlposition of the structural units present in the sample,
without any indication whether these units are prescnt
in the san'rernolecule or in diffcrent ones (see for cxample Van de Velde et o1.,2001). When substituents occur
that not affect characteristic chemical shift pattcrn of
the annomeric region, for l3C or IFI-NMR spectroscopy, they might be identified by typical resonancesrn
othcr regions of the spectra.
'H-NMR spectroscopy with all its advantages, such
as low sample concentrations and short analysis tirttesin
particular is suitable for thc cluantification of'thc clifferent carrageenan types. The u-anomeric protons of the
major carrageenanstypes (r, t, I, p ar"rdv) give signals
in the region 5.1 5.7 ppm, which are easily separated in
a high-field instrument. Routine rheological analysis of
the raw extract completes the commercial-value analysis
of the original or alkali-treated carrageenan.
In summary, thc NMR spectra(ltl and lrCl of rlrw
extracts givc good identillcation of thc major type of
cilrrageenansproducecl by the seaweedwhen comparccl
w i t h s p c c t r a o f ' " r n o d e l " c a r r â g e e n a n s( ' f u r q u o i s c t a / . ,
-Ihe
with enhanced rcso1996).
availability of ecyuiprr.rcnt
I u t i o n , r v i l l p r o m o l e t h e r H - N M l L s p e c l r o s c o p yf o r t h i s
l u s ce v c r rr n o r c . I t i s p o s s i b l ct h a t t h c s p c c t r as h o r v t t t i t t t r t '
"kinks"
o r t r i t c c s i g n r t l so r i g i n a t i n g f ' r - o t nI r o n - p r c c r - t r s o r
( i . e . n o n - s u l l u t e c lz - r r - g i L l t t c t o s ct t r t i t s ) r v h i c h w i l h o t r t
" t 1 , ' 1 - r c " c a r n t g c c t t a nc l l l t t t t o t l t t l l t l c i t s
o1changing thc
r l t c o l o s i c r rbl c h i t v i o t t r .
A c l r l l è r ' e nrtt 1 - l p r o l t ci sh L t s c t rl v h e l to t l c l o c t t s s c so l l t h c
:iYStL'lll tll' citl'l-itlcctllrtts [liosYtrtlresrsctl
bv
lhc
sclincctl
v , u r ( k , V ( , / ( i ( ' t ' l . r i . i ' l r t , r r r l r r n f o o r l S t i c r /rtr,tr.h&l o l o g y l l ( 2 ( 1 0 ) ) 7 1( ) )
R_Â,CtS-ÂrH.{-G.tsr
( R - A H - l - c - R ' , A n r r r " ,- ( i ! S - R ' )
(R-^I.l -(i{s0. R-Att_
t - ( , { S .R ' )
R-ÂH-lG4Sc
25"C
----
A --_JL
0ttlt-
L{ln-.,,,t.rr,r)
l
Anr,t,,-(i{S'R
R-(;.^r{,,t-cds-R.
90"c
B
5.3
5.0
4.3
4.6
ppm
t i g . 7 . ( A ) r H - r e g i o n o f t h e 5 0 0 M H z - s p e c t r ao f a m i x t u r e o f p a r t l y d e s u l f a t e dn e o c a r r b i o s et y p e o l i g o s a c c h a r i d e fsr o m f u r c e i l a r i a - c a r r a g e e n a n r e c o r d e d a t 2 5 " C . O n l y s e l e c t e da s s i g n m e n t sa r e d e n o t e d o n t h e f i g u r e .( B )F o r c o m p a r i s o na s p e c t r u r no f t h e s a m e s a m p l e r e c o r d e d a t
9 O " C i s i n c l u d e d . R i s i n d i c a t i n ga r e m a i n i n g p a r t o f a n o l i g o s a c c h a r i d ec h a i n t o w a r d s t h e n o n - r e d u c i n ge n d a n d R ' t o w a r d s t h e r e d u c i n g
e n d . P r e s e n t e do n l y i n ( K n u t s e n ,1 9 9 2 ) .
{ G . l S n r r - , .G . t S 0 1 1 -À r , , , r )
1,
lnr,,-.,
f,
An"H-J
G4SH ..rGatutt
' \ l
It,i
--*/t-5.3
5.0
4.7
Ppm
4.2
3.9
1.6
,,
o r l n t l l c d c t e l u r i n a t i o no f t h e f i u c s t r L r c t r r roel ' r l c a r r l g c e n a no f t r c l t r r â g c c n a nl l ' u c t i o n .' f h e i d en t i l i c u t i o n t t f u
cil rrirgccnlrrt nti rt rrrc plotlircotl b1,'a scirt cerl rc(lLur cs iul
'l
c l l t b o n r t cp r ( ) c c s so f ' c r t l t r c t i o n a n t l l r - l r c t i o n u t i o n .l r e
c o t r p o s i t i o nt r r r r rl r c l r lo l ' t h c l l - t r c t i t r nosb t l r r n c riln t l i c u t e
U i c p t c s c n e co l t r r i r r o rc ( ) l l t p ( ) l l c n l sl l. t c t l i s l t c r s i o ror l
s t r u c t u r rs: i n t h e s y s t c n -rrr n r l t l r e s t r c n g t l t o [ ' t h e I t t t c r l c t i o n o f ' t h c s e r n o l e c r r l c si n t h e s c a r v c e c l i s s u e . S L r h l ' r i r c t r o n r r t i o n ,w h c r ) n c c c s s a r y , q i v e s t l t c c l i l l t r - c n t
"prcccs"
o l ' a . j i u s u l v1 ' r u z z l ct.l t c s o l t t t i o r t o l ' u h i c h r i ' i l l
s h o r . vl r r ro v c n r l l l ) i c t u r c( ) l ' t l r ec l l f r l g c c n a ns y s t c n rs v l l
t l r e s i s e t lb y t l r c s c r r r v c c c lI .t i ( h c s t L t t l vt r l t l t i s 1 ' t v t t . l c .
()o
lrt'ncls in / oorl .Sr'lcnce/i kr hnokrgy I I (2O02)/ I ,)2
N M I { s p e c t r o s c o ppyl u y sa n i m p o r t a n tb u t n o t a l l w a y s
a c o n c l u s i v er o l e a s i n t h e i n d L r s t r i aal p p r o a c h .T h e
ntain dl'awbackof this n.rcthodology
in the detcrmination of fine structuralcletailsin carragecnans
is due to
the con.rplexity
of thcsepolyrners,their molecularinteractionsar.rdtheir high molecularweight.Due to its low
sensitivity,l3C-NMR spectroscopycân not <Jetect
low
percentages
of minor components.Negativeresultsdo
not exclude the presenceof minor components,for
examplethe presenceof a-l-galactoseand/or 3,6-anhydro a-r--galactose
and the correspondingo/l-hybri<is
and/or agarans (Estevezet al., 2001). The IH-NMR
spectraof cornplexpolysaccharides
usuallyshow broad
resonances,
sometrmes
overlappingand difficultto inte_
grate. In the case of carrageenansthe diads can be
recognised
by the resonances
of the a-anomericprotons
(Feldman,Storz,Vigna, & Cerezo,1994).
A new approach to evaluate r3C-NMR spectroscopicaldata for the structural analysisof red algal
galactans was suggestedrecently (Miller & Blunt,
2000a,b).This approachis bascdon the availabilityof a
large number of r3C-NMR spectra of different well
characterisedgalactans.The '3C-NMR spectrurnof a
polysaccharidewith an unknown structureis assigne<l
by a mathematicalprocedurethat determinesthe bestfit
betweenthe experimentalspectrumand the elementsof
the databaseof spectraof characterised
galactans.In
spite of rather complicatedmathernaticalterminology
used,in principlethe approachseemsto be fruitful, but
has some marked experimentallimitations,since it is
difïicult to carry out desulfationand n.rethylation
steps
quantitatively.Incompletechemicalmodificationsmay,
of course,resultin undesirablecomplicationof spectra.
Nevertheless,
severalexamplesoI successful
application
of this approach to the elucidation of structuresof
complex galactans, including o/l-hybrids (Miller,
2001a;Miller & Blunt, 2000c)and an unusualcarrageenan (Miller,200lb),are found in the literature.
Most ol theseproblernscan be overcomeby selective
modificationof the molecules(i.e. desulfation)or by
selective,
if not specific,
fragmentationof the carrageenan
-f
molecule. hese fragmentations can be carried out
throughenzymaticor chcmicalmethocls(seeBox 2). By
clegrading
carrageenans
with structure-specific
enzymesa
sericsof sulfatcdoligosaccharides
of'known structureis
formcd.Flowever.sincecarrageenan
moleculcsin general
are hybridsand also may contain "abnormal" units
intcrspcrscdin thc backbonc sontc larger fiagments,
resistantto cnzyrnolysis
anclwith a sizeclepcncling
ttn in
rvhich scrlucrrccthcy occrtrwill be enriched.Thcse are
s u r t a b l cs a n t p l c sf i r r N l v tI { a n a l y s i su s t h e y c o n c e n t r a t e
l h c s eu n r r s t u rnl r i n o r r l c t a i l s .I I o w e v c r t h i s r n i g h t b e a
c l ' r l l l c n g t n ut i r s k . s i n c c c o r r - r p l e n
x r o l c c u i c ss t r t t n q l y
c l c v i l r t i nlg' r ' o r rur r c q u l a r - s c ( l u c nnct ics h to c c u r -c,s p c c i u l l y
I t tc i r r r i r g e c l l uptr c c r r r s (r)irc hn t o l e c r r l cIsr .rs r r c lct l t s c st h c
l u p p l i c t r t i oonl r l l - N \ l I l s p c c t r o s c o l tuy,.i l lr - c s Lirtlrtl r i s h c r
scnsitivity,urore reliableintegrationof the signalsand
h e n c e t h c b e s t p o s s i b l eq r , r a n t i t a t i vrec s u l t s .I n m o s t
cascssomecor"nplernentary
rnethocls
lcrriclentification
of
constituents
in the samplelike rnethylationanalysis(Falshawel a|.,2001 Falshaw& Furneaux,1994)or reductive
hydrolysis(Jol, Neiss,Penninkhof,Rudolf, & DeRuiter,
1999)or the uset3CNMR or 2D-NMR techniques
must
be performedsimultaneously.Finally if high resolvedl3CNMR spectracan be obtained from such fractions some
crucial insightinto the sequentialnature ofa carrageenan
and its biosynthesis
might be obtained.
Acknowledgements
The authors gratefully acknowledge Dr. Brian
Rudolph (CPKelco,Lille Skensved,Denmark) for providing the v-carrageenan
sample.Dr. Henk A. Schols
(Wageningen University and Wageningen Centre for
Food Sciences,
The Netherlands)and Dr. Gerhard A.
de Ruiter (NIZO food research,The Netherlands)are
acknowledgedfor their helpful discussionsand critical
readingof the manuscript.
References
Anderson, W., & Bowtle, W. (1974\.Determination of rotac a r r a g e e n a nw i t h 2 - t h i o b a r b i t u r i ca c i d . A n a l y s t ,9 9 , 1 7 8 1 8 3 .
BarbeyronT
, . , F l a m e n t ,D . , M i c h e l , C . , P o t i n ,P . ,& K l o a r e g ,B . ( 2 0 0 1 ) .
T h e s u l p h a t e d - g a l a c t ah
ny d r o l a s e sa
, g a r a s e sa n d c a r r a g e e n a s e s :
structural biology and molecular evolution, Cahiers c/c Biologic
M a r i n e .4 2 . 1 6 9 1 8 3 .
B e l l i o n ,C . , B r i g a n d ,C . , P r o m e ,J , - C . ,& B o c i e k ,D . W . S . ( 1 9 S 3 ) .
l d e n t i f i c a t i o ne t c a r a c t é r i s a t i o nd e s p r é c u r s e u r sb i o l o g i q u e sd e s
c a r r a g h é n a n e sp a r s p e c t r o s c o p i ed e R M N - l 3 C . C a r b o h y d r a t e
R e s e a r c h1.1 9 , 3 1 4 8 .
. :rC-NMR
B h a t t a c h a r j e eS
, . S . ,Y a p h e ,W . , & H a m e r , C . K . ( 1 9 7 8 ) 1
s p e c t r o s c o p i ca n a l y s i so f a g a r , k a p p a - c a r r a g e e n a na n d i o t a carrageenan. CarbohydrateResearch,60, C1 C3.
. e c e n td e v e l o p m e n t si n m a n u f a c t u r i n ga n d
B i x l e r ,H . J . ( 1 9 9 6 ) R
marketing carrageenan.Llydrobiolog,ia,3261327,35 57.
. arbon-l3 nucrear
B o c k , K . , P e d e r s e nC
, . , & P e d e r s e nH
, . ( 1 9 8 4 )C
m a g n e t i c r e s o n a n c ed a t a f o r o l i g o s a c c h a r i d e sA. d v a n c c st n
Carbohydrate Chemistry and Biochemistry,42, 193*225.
. reC a r a m - L e l h a mN
, . , S u n d e l o f ,L . O . , & A n d e r s s o n ,T . ( 1 9 9 5 ) P
p r a t i v e s e p a r a t i o no f o l i g o s a c c h a r i d e fsr o m k a p p a - c a r r a g e e n a n ,
s o d i t r m h y a l u r o n a t e ,a n d d e x t r a n b y S u p e r d e x( I M ) 3 0 p r e p
grade. CarbohydrateResearch,273, 71-76.
C h i o v i t t i ,A . , [ 3 a c i c A
, . , C r a i k , D . , . , M u n r o , S . L . A . , K r a f t ,C . L , &
L i a o ,M . L . f 9 9 7 ) . C e l l - w a l lp o l v s a c c h a r i d e sf r o m A u s t r a l i a nr e d
a l g a e o f t h e f a m i l y S o l i e r i a c e a e( C i g a r t i n a l e sR, h o d o p h y t a ) :
n o v e l , h i g h l y p y r u v a t e d c a r r a g e e n a n sf r o m t h e g c n u s
Callophyr:us.Carbohydrate Rcscarch,299, 229 243.
C h i o v i t t i ,A . , B a c i c ,A . , C r a i k ,D . j . , K r a f t ,C . 1 . , L i a o ,M . - 1 . ,F a l s h a w ,
R . , & F u r n c ; r u xR
, . l l . ( ' 1 9 9 8 )A p y r L r v a t c ccJa r r a g e e n a nf r o n r
A L . r s t r a l i asnp e c i r n e n so f t h c r c c l a l g ; t5 a c r o n c r r ; .tr' i l i l o r r n e .
( a r b r y ' t y t l r a LI {er ' s c l r rh , ) 9 9 , 2 ) 9 2 4 7 .
C ' l r i o v r t iA, , B a < r t :A, . , C r a i k ,I ) . 1 . ,M u n r o , S . L A . , K r a f t ,( , . 1 . ,&
I t a o , M . - l ( 1 ( ) 9 t il)a. r r a g c t : n , r n w
s i t h c : o r n p l esxu l ) s t i t u t i { 'pnJ t
t o rn s f r o r r r u l ; r k , r uo { t h r : g c n r r sI r y t l : r r xl o n i r t n t(. ' a r l x t l t y r ! r a t t '
R t ' s i , a r/rr , \ ) . 5 ,) , 1\ ) 5 2 .
( i r r r rt a , \ 1 . ,\ 1 , r t L r l t ' r v i cNz1. ( . , I r n c l t ,[ ' . ,& ( e r t , z o ,^ S i l ( ) 9 i )
[ ) t ' t t ,t tr n , r t r o n( ] l t l l ( 's t r r l l(r l r ( , s{ ) i ( y \ t ( ) (r r l ) t ( ( , t l , t ! l ( , e l t , l n s
l , ( ) n r( ; i t , r / 1 r f ls, lro l l s l r I r q rlir y r r r c t l t v l , r t i O r r , i l t , i l V s r sN, llnr l(R
l
s l ) ( ' l(r ( ) ! (( ) l ) v ( , t r l x t l t y r l r t l t ' l i r , s r 'h, r, r,rl i l i , , l - 1I l . l l j
L r ' , t r t r . / r ,V c l r / i ' r ' t t l , i I r t n r l s i n f o o c l - S r i e n r r , . \ l e L h t x t k t y y
( - r , r n r a , N l . N l , r t L r l e w i c zN,. 1(.. . , S t o r t z ,a _ ., , \ . .& C - c r t , z or,\ . S . ( 1 9 9 . 1 ) .
R o o n r t e n r [ ) ( ' r n t L r r tl 'o, r v f i t ' l t l ' r ( N M R s l t c rt r . t o f d c g r a c l e ccl . r r r a g { ' ( : n a n sl ). a r t l l . ( ) n t h e s p e . c i f i c i toyf t h e a L r t o h y c l r o l y sri cs a c t i o n i n k a p p a / i o t a a n c l n r u / r r u s t r u c t u r e s ./ n k , r n a t j o n a /J o u r n aol l
Bioktg,icalMacronro/ccti/es,1.], 337 3lO.
C i a n c i a ,M . , N o s e d a ,N 1 .D . , M a t u l e w i c z ,M . C . , & C e r e z o ,A . S .
( 1 9 9 3 )A
. l k a l i - r n o c J i f i c a t i o nf c a r r a g e e n a n sm
: e c h a n i s ma n d
k i n e t i r : si n t h e k . t p r p a / i o t a -m, L l / n L ra- n d l a m b d a - s e r i e sC. a d r o hydràte Polymeri, 20, 95-98.
P,. ,J e n n i n g sl,l . j . , & S m i t h ,l . C . P . 0 9 7 4 . C o m p o s r r r o n ,
C<rls<rn
s e q u e n c e ,a n d c o n f o r m a t i o n o f p o l y m e r s a n d o l i g o m e r so f
g l u c o s e a s r e v e a l b y c a r b o n - 1 3n u r : l e a rm a g n e t i c r e s o n a n c e .
Jortrnitlol the Amerir:an (hemistry Society,96,B0S1 B087.
, . , R u d o l p h ,8 . , C e n i c o t , S . ,K l o a r e g ,8 . ,
D e R u i t e r r(,, . A . , R i c h a r c l O
P c n n i n k h o f ,8 . , a n d P o t i n , P . ( 2 0 0 0 )S r r l f o h y c l r o l a s ecso, r r e s p o n c l i n ga m i n o a c i d a n d n u c l e o t i d es e q u e n c e s ,s u l f o h y d r o l a s e
p r e p a r a t i o n s p, r o c e s s e sa, n d p r o d u c t s t h e r c o f . W o r l d p a t e n t
a p p r l i c a t i o nW
: O0068395
. a r r a g e e n a nb i o t e c h n o l o g y .
D e R u i t e r ,C . 4 . , & R u d o l p h ,B . ( 1 9 9 7 )C
lrends in Food Sclence & Technology,S, 3B9*395.
. L-Catacran
E s t c v e z J, . M . , C i a n c i a ,M . , & C e r e z o ,A . 5 . ( 2 0 0 1 ) D
h y b r i d s a n d a g a r a n sf r o m g a m e t o p h y t e so f t h e r e d s e a w e e d
Cymnogongrus torulosus. Carbohydrate Research,331,27 .41.
. t r u c t u r ea n d p e r f o r F a l s h a w ,R . ,B i x l e r ,l l . J . ,& J o h n d r o ,K . ( 2 0 0 1 ) S
m a n c e o f c o m m e r c i a l k a p p a - 2c a r r a g e e n a ne x t r a c t sl . S t r u c t u r e
analysis.Food Hydrocolloids, 15, 441 452.
. a r r a g e e n a nf r o m t h e t e t r a F a l s h a w ,R . ,& F u r n e a u x ,R . ( 1 9 9 4 )C
sporic stage of Cigartrnadccipiens (Cigartinaceae,Rhodophyta).
( arbcthycl
ratetResearch,252, 171-,182.
. a r r a g e e n a n sf r o m t h e
F a l s h a w ,R . ,& F u r n e a u x ,R . H . ( 1 9 9 5 )C
tetrasporic stages of Cigartinaclavtferaand Crgartinaa/veata
(Cigartinaceae,Rhoclophyta).Carbohydrate Researcl't,
276,
155 165.
F a l s h a w ,R . ,F u r n e a u x ,R . l l . , W o n g , H . , L i a o ,M . - 1 . ,B a c i c ,A . , &
. t r u c t u r a la n a l y s i so f c a r r a g e e n a n s
C h a n d r k r a c h a n gS
, . ( 1 9 9 6 )S
f r o m B u r n c s ea n c l I h a i s a m p l e so f C a t e n e / / an r p a eZ a n a r r J i n i .
Carbohydrate Rescarch,285, 81 99.
F e l c i m a nS
, . C . , S t o r t z ,C . A . , V i g n a , M . S . , & C e r e z o ,A . S . ( 1 9 9 4 ) .
C a r r a g c e n a n si n t h e " s t e r i l e "s t a g ef r o m C i g a r t i n as k o t t s b e r g i i .
Analescle Ia Asoci.trkyt ()uimira Argentina,82, 117 124.
Cli<:ksman,M. (1981).tood flydrocctlloids,Vol.2: SeaweedÉxtracts
( p p . U 9 ) .B o c . rR a t o n : C R C P r e s sI n c .
. i g h f i e l d ,1 H - n . m . r s. p e c r r o s c o p yo f a l g i n a t e :
C r a s d a l e n ,1 1 .( 1 9 8 3 ) H
S e q r r e n t i asl t r u c t u r e a n d l i n k a g ec o n f o r m a t i o n s .C a r b o h y c l r a t e
R e s e a r c h1
, 18,255 260.
C r a s d a l e n ,H . , & P a i n t e r ,f . J . ( 1 9 U 0 )N. M R s t u d i e so f c o m p o s i r i o n
a n d s e q u e n c ei n l e . g u n t e - s e e6di a l a c t o m a n n a n sC. a r b o h y d r a t e
R e s e a r c h , 8 15,9 6 6 .
C r e r : r ,C . .W . , R o c h a s ,C . , & Y a p h e ,W . ( . ' l 9 8 5 )l .o t a - c a r r a g c t : r r a r r
o l i g o s . r c c h a r i r l eass m o d c l c o m p o r r n c l sf o r s t r u c t L r r aal n : r l y s i so f
i o t a - c a r r ; t g e e n ab
n y r r C - N M Rs p e c t r o s c o p y B
. otanlc.M
r a r i n a ,2 8 ,
9 11.
C r c c r , C . W . , S h o m c r , 1 . ,C o l < l s t e i nM
, . 8.,& yaphe, W. (1984),
 r t , r l V ' tr 'l l { , l i l , r H e { . n ,t r r, r' n l/ / \ l } | { , , m
1 U \ (i / ( ) r n I \l r y r r r i n g
k a p p a - . t n r l i o t . l ( , r r r a g e c n a s e ,sr n d B C - N . M . R . s l ) e c l r o s L o p y .
./29,
-196
( arlxtltyrlrato
R c s r ' , r rhr,
189
l l a n s c n ,J . l 1 .I,a r s e n ,H . , & ( , r o n d a l ,I ( 2 0 t X ) C
) .a r r a g e e n a n
( orn[)os r t i o n s, U t r ln t 0 t h o r l sf o r t l t t ' i rp r o c l utri o n . l l S p . t t e r r at p p l i c a t r o n :
r , 9 '5l
L l 5 6 , t X1
l l j r r r l r ' ,1 , K r r s t i , r n s t :In ,S . ,S t o k k e l, l I , S n r i r l s r u x( j),. , &
( l r r i s t l n s u nt,] . t . ( 1 9 9 , 1() .o n f o r r n a t r o rnk , 1 r c , n < 1rt l,er rpto l y r n c r i / . t t r ( ) ikl i n ( t t rsi .O f P o l 1 , i , r, rl rt a r i c l t :sst U c l r c b
r ly v i s cO s i t ,
r n r . , l \ t 1 t ( , n l ( , l(t ,l 1
s t I x t h , t I r a l lL, ,o l y r r t l r s),, 1 ,) ( 1 5 ) 7 5 .
l n l ( ' \ o r ) , , , \l ) i - 1 0 ( l ( () ,)u r , r r 1 r ' r , r r ,l rnn( , ( ) i , h r li 1 r sf,. l ) r \ \ N i l l i a r n s
t I t l s ] / / , u r r rl io r r kt l l l . t l t r x , r / / o r ri/l.t p l j / 1 0 2 ) ( ; r n r l r r r r l g t , :
\ \ ' o r c JrJi ' , rlr I , L rllir. lr r n ! lI t r l
l r l . ( N \ r , : r I ( , I , t , r r r r r I l r l ]l . R r t r l o l J lrJl r. , \ I ) ( , R t r i t t(,,r .z \
| ) (20O) ll
92
9t
( 1 9 9 9 )A
. novt:l high-Perrfonnan
e (a n i o n - r - x c l r a r r grt h r o n t a t r i g r . r p l r i t :m c t h o c l f o r t h r : a n a l y s i so f c . t r r a g e c n a n s; r n t l a g . t r s
( o n t a i f) i r . r !3l , 6 - a n h y d r o g a l at () s e .^ n a 4 4 l . i t l B t o c h t ' t n i s t r y2, 6 8 ,
213 )22.
K n u t s e n ,S . H .( 1 9 9 2 ) l.s o l a t i o na n d a n a l y s i so f r e d a l g . r lg a l a c t a n s .
P h D t h c s i s ,[ J n i v e r i s yo f T r o n c l h e i m T
, r o n c l h e i m ,N o r w a y
K n u t s e n ,S . l l . , & C r a s d a l e n H
, . ( 1 ! l U 7 )C. h a r a c t e r i z a t i o no f w a t e r e x t r a c t a b l ep o l y s a c c h a r i d efsr o r n N o r w e g i a nf u r c e l l a r i a
) . ( C i g a r t i n a l e sR, h o d o p h y c e a e )b y l R a n d
l u m b r i c a l i s( H u c l s L
NMR spectroscopy. BotanlcaMarina, 30, 497 -5O5.
K n u t s e n ,S . H . , & C r a s d a l e n l,- 1 (. 1 9 9 2 a )T. h e u s e o f n e o c a r r a b i o s e
o l i g o s a c c h a r i r J ewsi t h d l f f e r e n tl e n g t h a n d s u l p h a t e s u b s t i t u t i o n
a s m o < J e cl o m p o u n d s f o r r H - N M R s p e c t r o s c o p y .C a r b o h y t l r a t e
Re.search,
229, 233 244.
. n a l y s i so f c a r r a g e e n a n sb y
K n u t s e n ,S . H . , & C r a s d a l e nH
, . ( 1 9 9 2 b )A
enzymic degradationg
, e l f i l t r a t i o na n d r H N M R s p e c t r o s c o p y .
Carbohyrlrate Polyme rs, 19, 199-210.
K n u t s e n ,S . H . , M u r a n o , 8 . , D ' A m a t o , M . , T o f f a n i n ,R . , R i z z o ,R . ,&
. o d i f i e d p r o c e d u r e sf o r e x t r a c r i o n a n d
P a o l c t t i ,S . ( 1 9 9 5 )M
a n a l y s i so f c a r r a g e e n a na p p l i e d t o t h e r e d a l g a t l y p n e a
muscilormis. lournal ol Applied Phycology,7,565 57L.
. harK n u t s e n ,S . H . , M y s l a b o d s k iD
, . E . ,& C r a s d a l e n ,l i . ( 1 9 9 0 ) C
a c t e r i z a t i o no f c a r r a g e e n a nf r a c t i o n sf r o m N o r w e g i a n F u r c e l l a r i a
lumbri<:alis(Huds) L. by rH-NMR spectroscopy. Carbohydrate
Research,206, 367-372.
K n u t s e n ,S . t l . , M y s l a b o d s k i D
, . E . ,L a r s e n ,8 . , & U s o v , A . l . 1 1 9 9 4 ) .
A m o d i f i e d s y s t e m o f n o m e n c l a t u r ef o r r e d a l g a l g a l a c t a n s .
BotanicaMarina, 37, 163-169.
K n u t s e n , S . H . , S l e t m o e n ,M . , K r i s t e n s e n T
, . , B a r b e y r o n ,T . ,
K l o a r e g , 8 . , & P o t i n , P . ( 2 0 0 1 ) .A r a p i d m e t h o d f o r t h e
s e p a r a t i o n a n d a n a l y s i so f c a r r a g e e n a no l i g o s a c c h a r i d e s
released by iota- and kappa-carrageenase.Carbohydrate
Resear<:h,3i1, 101 106.
K o l e n d e r ,A . A . , P u j o l ,C . A . , D a m o n t e , E . 8 . , M a t u l e w i c z , M - C . , &
Cerezo, A. S. (1997\.The system of sulfated alpha-(1-> 3)-linked
D-mannans from the red seaweed Nothogenia /â.çtlgiala:
struct u r e s , a n t i h e r p e t i ca n d a n t i c o a g u l a n tp r o p e r t i e s .C a r b o h y r l r a t e
R e s e a r c h),0 4 , 5 3 6 0 .
K o v a c ,P . , l - l i t s c h ,1 . ,S h a s h k o v A
, . S . , U s o v , A . 1 . ,& Y a r o t s k y ,S . V .
( 1 9 8 0 ) r. r C N M R s p e c t r ao f x y l o - o l i g o s a c c h a r i d easn d t h e i r
a p p l i c a t i o nt o t h e e l u c i d a t i o no f x y l a n s t r u c t u r e -( a r b o h y d r a t e
Re.search,85, 177 185.
L a h a y e ,M . , Y a p h e , W , & R o c h a s ,C . ( 1 9 S 5 )l.r C N M R s p e c t r a l
a n a l y s i so f s u l f a t e da n d d e s u l f a t e dp o l y s a c c h a r i d e so f t h e a g a r
type. Carbohydrate Research,143, 240-245.
L a h a y e ,M . , Y a p h e ,W . , V i e t , M . T . P . , & R o c h a s ,C . ( 1 9 S 9 )1. ' ' CN M R
s p e c t r o s c o p i ci n v e s t i g a t i o no f m e t h y l a t e d a n d c h a r g e d a g a r o s e
oligosaccharidea
s n d p o l y s a c c h a r i d e sC. a r b o h y r J r a l e
Re.search,
190, 249 -265.
L i a o ,M . - 1 . ,C h i o v i t t i ,, 4 . ,M u n r o , S . L . A . , C r a i k , D . J . ,K r a f t ,C . T . , &
Bacir:A
, . ( ' 1 9 9 6 )S. u l f a t e dg a l a c t a n sf r o m A u s t r a l i a ns p c c i m e n s
crf the red alga Phace/c.rcarpLts
pepero(arpos ((,igartinales,
Rhoclophyt;r).Carltohyr)rateRc.se'arch,
296, 2l7 247.
L i a o ,M . 1 . , K r a f t ,C . - 1 . ,M u n r o , S . L . A . , C r a i k , D . J . ,& B a r : i c ,A .
( . , | 9 9 3B
) .e t a / k a p p ( r - c a r r a g c x t n aansse v i d e n c e f o r c o n t i n L i e d
s e p a r aito r r o f t h c f a m i l i e sD i r - r a n e m a t a r : e aaen r l S . tr co c J i : reca r :
(CrgartirraleR
s ,h o d o p h y t a ) .J o u r n a rl l P h y c o l o g y 2
, 9, B.\3 814.
. ,rrr.rgecnan
M c ( a n c l l e r s fs., 1 . ,\ , V e s t j, A . , & C r r i r y , , \ 1 .D . ( 1 9 t t 2 )C
p a t t { . r r r 9i n t h e ( , i g a r t i n a c e n eB. i o (h { ' r n i cr / S y s l c n r a t .rsca r r r l
F c o k r y y ,1 0 , 2 7 5 ) t l 4
( -;-r. r n ; r e t ' t : n ; i s c
N ' l t l e a n , , \ 1 .\ ' V . ,& W i l l i a r l s o n , f . B . ( 1 9 7 9 )I.< a J > ; >
. / ) r ' J l r l o L r r n ; ttll
t r ( r n ri ) s t r r t / o r r r r ) / r ,(1i l5r r . t g ( , ( , 1 ) ( ) v olfu, ft o
B t o rl t t , r r t t s l t y(,) J , i 5 t ' i t 1 .
l o r t t ' n cl . i t L r r io' i r ; t r l r o l t l , r i r , r t c s
N 1 cN , r r r g l r lr.\ l ) . ( ' l ( ) ( ) / N
( t r l x t l t y r l r , t l /Li ,c s r , , r r r l)tt,) , ' , 1 t ) ) .
N , 1hi cr , l ,( , . . l l , r n r c r t ,f ) l l , i r l r l r - r o nI. . V c r n o t , I . K l o , r r e qI,l , &
l ) i t l t ' I r t ' r (t )] 1 ] { ) l ) OI rl 1 r t t ' : s t t ,l t)tt l r ] l r 1 , l l t (r )tryt s l , t l l t z , t l t,et t t r l
tt)
/ . r i r r r / t , 1 1 , / r i t , t ,. lr / , i I r t . r r r i si n I t x t c l - 5 r r c r r r r ' &/ i , r h r r o / o g y l' l t ) l X ) ) . ) 7 ]
I ) r e l r l r r , l r y X r l y a n , r l l , s r (s) t t h c t ' ( - . t r r . l g a ' c n a sf reo r n
; 1 / l c i o r n o r , tksr r l i s . , ' \ r t a( - r l , s t , r / i ( ) , q f J I ) l r f t , r . \ tD, t l i oBni t l o g i t , t l
( r y . s l a / i o g r . r P /.r5y6, ,76 6 7 6 t l .
M i l l C r ,l . J . ( . 1 9 9 7 )i .l r e ' < : h e n r O t . r x O n o n sl i iCg n i f i c . t n c e
of the w.tters o l L r b l er c ( l a l g , r lp o l y s a c c h a r i c l e s/ l.c c e r r tR e s e a r c hD e : v e l o p /))('/rlsin Phytot hemtslry, /, 'r3.1 565.
N l i l l e r ,l . l . ( 1 9 9 8 ) l.h e s t r u c t u r e o f a p y r u v y l a t e d c a r r a g e e n a n
e x t r ù ( t e d f r o r n S l e ' n o g r a r n û )len f e r r u / p t a s c l e t e r m i n e db y
' t C N N 1 Rs p e c t r o s c o p y .
B ( ) t a n i c .M
r a r i n a ,4 ' 1 , . 1 0 5 - 3 1 5 .
M i l l e r , l . J . ( 2 0 0 1 a )T. h e s t r u c t u r e o f t h e p o l y s a c c h a r i d eÏ r o m
f l y r n e n o t l a d i as a n g u i n e at h r o u g h r r C N M R s p e c t r o s c o p y .
IJotanit.rMarina, 44, 245 251.
M i l l c r , l . J .( 2 0 0 1 b )T. h e s t r u c t u r eo f t h e c a r r . r p i e e n aenx t r a c t e df r o m
t h e t c t r a s p o r o p h y t i cf o r r n o f . S t e n o g r a m m ei n t e t r L t p t aB. c t t a n i c a
M.trina, '14, 583-587.
. c w C - l 3 N M R m e r h o d sf o r
r \ ' l i l l e rL, J . ,& B l r r n t ,I . W . ( 2 0 0 0 a )N
d e t e r m i n i n gt h e s t r u c t u r e o f a l g a l p o l y s a c c h a r i d e sP. a r t 1 . T h e
c f f c c t o f s u b s t i t u t i o no n t h e c h e m i c a l s h i f t so f s i m p l ed i a d
galactans. Ilotanlca Marina, 43, 219 25O.
. ew C-13 NMR methodsfor
M i l l e r ,L J . ,& B l u n t , J . W . ( 2 0 0 0 b ) N
d e t e r m i n i n gt h e s t r u c t u r e o f a l g a l p o l y s a c c h a r i d e sP. a r t 2 .
C a l a c t a n sc o n s i s t i n go f m i x e d i l i a d s .B o t a n l c aM a r i n a ,4 3 ,
251 261.
) .e w C - 1 1 N M R m e t h o d s f o r
M i l l e r ,l . J . ,& B l u n t , J . W . ( 2 0 O O c N
d e t e r m i n i n gt h e s t r u c t u r e o f a l g a l p o l y s a c c h a r i d e sp. a r t 3 . T h e
structrrre of the polysaccharide from Clac:lhymenia
oblongifolia.
IJotanlcaMarina, 4,1,263 271.
M o r r i c c , L . M . , M c l e a n , M . W . , L o n g , W . F . , & W i l l i a m s o n ,F . B .
( i 9 8 3 ) .P o r p h y r a np r i m a r y s t r u c t u r e .A n i n v e s t i g a t i o nu s i n g
B-agaraseI from P.seudornonas
at/anticaand llC NMR specrros c o p y .É u r o p e a nl o u r n a l o f B i o c h e m i s t r y , 6 T 36 8 4 .
. o l i s s a c a r i t l e ossu l f a t a d o si s o l a d o sd a f a s e
N o s e c l aM
, .D. (1994)P
t e t r a s p o r o f i t i c ac l e C i g a r t i n as k o t t s b e r g l lp. h D t h e s i s ,U n i v c r s i d a c j eF e r d e r adl o P a r a n a ,B r a s i l
. o o m t e m p e r a t u r e ,l o w f i l e d
N o s e c l aM
, . D . , & C e r e z o ,A . S . ( 1 9 9 3 ) R
'rC NMR spectra
o f d e g r a d c d c a r r a g e e n a n sp. a r t l l l . A u t o h y d r o l y s i so f a l a m b d a c a r r a g e e n i l na n d o f i t s a l k a i l - t r e a t e d
deriv.rtive.interrnaliona/JoLtrnalol BiologicalMacromolecules,15,
1 7 71 U 1 .
O s t g a a r d ,K . , W a n g e n , B . F . , K n u t s e n ,S . H . , & A a s e n ,l . M . ( 1 9 9 3 ) .
L a r g e - s c a l cp r o d u c t i o n a n d p u r l f i c a t i o no f k a p p a - c a r r a g e e r r a s e
frcrm P.sc,uclontr)ni-s
carraéleenovctra
for applications in seaweed
biotechnology. Inzyme an<lMicrobial Tec-hnc;logy,
15, 326-j33.
P o t i n , P . ,R i < - h a r dC, . , B a r b e y r o n ,T . , H e n r i s s a t 8
, .,Cey, C.,petillot,
Y . , F o r e r s t , 8 .D, i d e b e r g ,O . , R o c h a s ,C . , & K l o a r e g ,B . ( 1 9 9 5 ) p
. roc e s s r n ga n c l h y d r o l y t i c m e <h a n i s m o f t h e c g k A - e n c o d e dk a p p a carragecnase\of Aile.romonascàrra1eenovora.Eurctpeanlournat
o l B k t c h a n t i s t r y2,2 8 , 9 7 1 9 7 5 .
R o b e r t s ,M . A . , & Q u e m e n e r , B . ( 1 9 9 9 ) M
. e a s u r e m e n to f c a r r a g e e n a r r si n f o o d : c h a l l e n g e sp, r o g r e s s a
, n d t r e n c j si n a n a l y s i s I. r e n r l s
i n f c t o r l S cf t ' r r c r ' & I c r h n o l o g y , 1 0 , l f r g T 8 l .
R o c h a s (, - . ,& l l c y r a r r < A
1 ,. ( 1 9 8 1 )A. c i d a n r l e n z y m i ch y < l r o l y s iosf
k a p p a - c a r r , t g e e n a nl l.r l y m c r t s u l l t , t i n.,5 ,1 7 2 1 .
R o r : h . r s(,- . , R i n a L r d oM
, . , & V i r r c e n c k r nM
, . ( 1 9 U J )S. p e ( : t r ( ) j ( o p i (
( h : i r . l c t { ' r i z , t t l o lnn c l c r i n f o r n r . r t i o ror f o l i e o k a p p a c a r r a g e e n a n s .
/ n t t ' ir r , r t r r r r rJ, ro/ ur n a l o l B i o l c t gai rl M a r r o n t o / t , t . u /se, 5 , 1 1 1 - 1 15 .
R o rh a s ,( . , I , i r , r v e lL, - R . ,& I r r r c l u o i sL. ( 1 9 9 0 )N. r \ ' 1 sRt u d i e so f
\ y n e r r r s t i ( l < a p p . r , t r r ù g c e r ' l J nr : . r r o i tg a l . r ct o r l a n n a n g t l s .
l n l t ' r n a t i o n . ll r u r n a l o f l ] K i l o ! . i c , t l4 . t r o m o k r - l k , . s ,/ 2 , . J 5 . J ] 5 8 .
R u c l o l p l lrl,. ( 2 ( ) ( X )\ )t ' , r r v t ' t , pr lr o r l L r c tr :t ' t 1a l g a c{ ) f e c o n ( , r n i (\ t g ,
n r l i c a r r r rl'n. R . I r \ 1 . i r t i n[ ., I ) . ( ' a r t t ' r ,I r \ 1 .l ) , t v i s &
, C J .f l i r l t
( [ r l s) . À . / , r r i r,rrrr' r r / r . s l t r r ' , r 1lr:,,rr L x ! lrsr I l , t n t l l x r t kf p 1 r 5. - 5
l 529).
I a r ( , r s l t r ,| 5 r \ I i ' r l r r r o r r i rI , L r l r l r s h l (t -go n r P , r n yI .n r.
\ i t , l l r o r ' n \ 1 l - 1 0 ( ) l )\ t) r L t rl u r . t l u l \ ' ( , \ l i e . i t t oor f) o l q o s a r( l t . u t ( l c s
r r s r r rrrql r ,t li i t i , l t t o Ji tl \ 1 R , r r r r\ 1
l S r \ s t L r r lovl r , r r r , r t t , r , n i. li trrrl
\ l ) { ' (r j( ( ( l | ( l t l ( ' ( ' l l , l \ (\ ,1( , r s t I rt l I , . i s ,, \ p , I rL r l t L r rt ,l rnl i r I r : r l 1, r l
Nott,rt
.\s \rrrrr,n
92
. , & ( t ' r o z o , . S . ( 1 1 ) 9 ' 1l l)i.g h
S t o r t z ,C ; \ . , B a r . o r tC
, . [ . . ( ' l r e r r r r a kR
f i e l d N N ' l Rs p e c t r o s . o p yo f . y s t { ) c a r [ ) i c. t t r d t c ' t r . r s p o r t t :' a r r a
g É r e n a r lfso r r n i r i d a e au n t l u l o s a .( a r b o l r y c / r a t cR c s t ' . l r r . h2,6 1 ,
\17 ))6.
S t o r t z ,C . A . , & C e r e z o ,A . S . ( . 1 9 9 1 )R. o o m - t e r n p e r a t u r el,o w - f i e l d
C - 1 3 N M R s p e c t r ao f c l e g r a d e dk a p p a - i o t a - c a r r a g e e n a n s .
Inlernational|ournal ol BiologicalMacronole<:ules,13,
101 104.
Stortz, C. A., & Cerezo,A. S. (1992).The rrC NMR spectroscopyof
c a r r a g e e n a n sc:a l c u l a t i o no f c h e m i c a l s h i f t sa n d c o m p u t e r - a i d e d
structural determination. Carbohydrate Polymers,1B, 237-242.
Stortz, C. A., & Cerezo, A. S. (2000).Novel findings in carrageenans,
agaroids and "hybrid" red seawccd galactans. Current foy;ir:s
Phytochernistry,4, 121 133.
. a r r a g e e n a n I. n R . L . W h i s t l e r , & J .
T h e r k e l s e nC
, . H . ( 1 9 9 3 )C
N. BeMiller (Eds.),/ndustrialCums: Polysaccharitlesand their
D e r i v a t i v e s( p p . 1 a 5 - 1 8 0 ) .S a n D i e g o : A c a d e m i c P r e s sl n c .
T u r q u o i s ,T . , A c q u i s t a p a c e S
, . ,A r c e - V e r a , F . , & W e l t i , D . f l . ( 1 9 9 6 ) .
C o m p o s i t a o no f c a r r a g e e n a nb l e n d s i n f e r r e d f r o m I r C N M R
and infrared spectroscopy. CarbcthydratePolymers, 31,
269-27A.
. M R s p e c t r o s c o p yo f r e d s e a w e e dp o l y U s o v , A . l . ( 1 9 B 4 )N
s a c c h a r i d e sa: g a r s ,c a r r a g e e n a n sa, n d x y l a n s .B o l a n i c aM a r i n a ,
27,189-202.
U s o v , A . l . ( 1 9 9 8 ) .S t r u c t u r a l a n a l y s i s o f r e d s e a w e e d g a l a c t a n s
of agar and carrageenan groups. Food flydrocolloicls, 12,
301 308.
U s o v , A . 1 . ,& D o b k i n a , l . M . ( 1 9 9 1 ) .P o l y s a c c h a r i d eos f a l g a e . 4 3 .
N e u t r a l x y l a n a n d s u l f a t e dx y l o m a n n a n f r o m t h e r e d a l g a t i a g o r a
valida. BioorganiceskaiaKhimija, 17, 1051-1058.
U s o v , A . 1 . ,& S h a s h k o v A
, . S . ( 1 9 8 5 ) .P o l y s a c c h a r i d eos f a l g a e .3 4 .
Detection of iota-carrageenan in Phyllophora brorJiaei(Turn.)
l r C - N M R s p e c t r o s c o p y .B o t a n i . a
J . A g .( R h o d o p h y t a )u s i n g
Marina, 28, 367 373.
U s o v , A . 1 . ,Y a r o t s k y ,S . V . , & S h a s h k o v ,A . S . ( 1 9 8 0 ) r. r C - N M R
spectroscopy of red algal galactans. Biopolymers, 19,977-.990.
V a n d e V e l d e , F . ,& D e R u i t e r ,C . A . ( 2 0 0 2 ) .C a r r a g e e n a nI.n
A . S t e i n b û c h e lS
, . D e B a e t s&
, E . i . V a r r D a m m e ( t d s . ) ,B i o p o l y m e r s
(Vol. 6) Polysac<:haride
{rom Eukaryotes
ll P<tlysaccharicles
( p p . 2 a 5 2 7 4 ) .W e i n h e i m : W i l e y - V C H .
V a n d e V e l d e , F . ,P e p p e l m a n ,l i . A . , R o l l e m a ,H . S . , & T r o n r p ,R . t l .
( 2 0 0 1 )O
. n t h e s t r u c t u r eo f k a p p a / i o t a - h y b r i dc a r r a g e e n a n s .
Carbc:hydrateResearr:h,331, 271 2Bf .
V a n d e V e l d e , F . ,R o l l e m a ,1 1 . S .C, r i n b e r g , N . V . , B u r o v aL, V .
C r i n b e r g ,V . Y a . ,& T r o m p , R . H .( i n p r e s s ) .C o i l - h e l i xt r a n s i t i o n
of iota-carrageenan
as a function of chain regularity.
tsictpolymers
. a r r a g e e n a n sP. . r r t ' 1 2 .T h e . 1 0 0M H z p r o t o n m a g W e l t i , D . ( 1 9 7 7 )C
n e t i c r e s o n a n c es p e c t r ao f m e t l r y l b c t a - t t - g a l a c t o p y r a n o s i d e ,
m e t h y l 3 , 6 - a n h y d r o - a / p h a - o ' g a l a c t o p y r a n o s i r. lreg,a r o s e ,
k a p p a - c : r r r a g c r : n aann, d s c 8 m e n t s o f i o t a c a r r a g e e n a n
(M),
ancl agarose sulfate. /orrrrraiol (.henti<';ilRese'ar<h
3.56t1-35U7.
. u l f o h y d r o l a s ea ( t i v i t y a n d
W o n g , K . F . , & C r a i g i e ,i . S . ( 1 9 7 8 ) S
( : a r r a g € r € - ' nbai o
n s y n t h e s i si n C h o n r l r u s r r l s p u . s( R l - r o d o p l ' r y < : i : . r e ) .
PlantPhysiology,61, 663 666.
Y a p h e , W , & r \ r s c n a L r l t(,, . P . ( l 9 6 5 ) . I n r p r o v r : cr.ol s o r ri n o l
r i n ù tj o l r o f f r u c t o s o ,a r r r l
r e a g c n tf o r t h e ( l e t o m
) , 6 ' i v r i h y r l r o g a l t; or (s e i n p o l y s ; r t< - l r a r i c l e,s4. n , r i y l rari
I i u " l r r r t t , t r/,J l . l t i l t j
, . , 5 , r i t o ,L , \ , V a t , r n . r l r1t '.,,t J t l r i y a r r a ,l l , &
Y , l n , r t i ; r f, . , ( ) g , r n r o A
N , r k r u a w , aY,. ( 1 9 ( 1 7l 1) r. r ' p l r a t i o n. r n r l: r n t i - l l V , r tt i v i t y o f l o w
n r o k ' rL r l , rrrv t ' r g l rtt. r r a l l ( ' ( , n . l n s , i n lt l r t ' i rs L ral ftc r l r l t ' ri r , , r lv e s
( , t t l x t l t y , t l t , IL' locl 1 . r n c r s1 ,2 ,r I ' i ' r
Y , r r r r t s l < 1\./5, \ l r ; r s l r l < o. v\ .\ & l l s o v , . \ | l l t ) i ; \ r \ r , r l ; s i o
sl
' { N \ ' 1 R' 1 x ' tl r ; r ( ) i \ r ) n r ( 'r c r l . r ' , r * , i ' r ' rgl , r l , rtr, r n s l i l t r t r t ' , t n t
r r ' . / , , r 7k,/rr r r r r 1 1i , r |, | i 5 I I i 7 .