F om he seaweeds’ca ageenan composi ion o he hyb id ca ageenans’
hyd ogel elas ici y: Iden i ica ion o a ela ionship based on he con en in
io a-ca ageenan
Loic Hilliou
a,*
, Izabel C is ina F ei as Mo aes
b
, Ped o Lúcio Almeida
c,d
a
Ins i u e o Polyme s and Composi es, Uni e si y o Minho, Campus de Azu ´
em, 4800-058, Guima ˜
aes, Po ugal
b
Uni e sidade de S˜
ao Paulo (USP), Depa amen o de Engenha ia de Alimen os, Pi assununga, S˜
ao Paulo, 13635-900, B azil
c
Mechanical Enginee ing Depa men and UnIRE, Ins i u o Supe io de Engenha ia de Lisboa, Ins i u o Poli ´
ecnico de Lisboa, 1959-007, Lisbon, Po ugal
d
I3N-CENIMAT, Ma e ials Science Depa men , School o Science and Technology, NOVA Uni e si y Lisbon, 2829-516, Capa ica, Po ugal
ARTICLE INFO
Keywo ds:
Ca ageenophy es
Hyb id ca ageenan
Io a ca ageenan
Solid-s a e NMR
Hyd ogel
S ain ha dening
Rheology
ABSTRACT
The ca ageenan composi ion o 12 comme cial seaweeds used in he p oduc ion o hyb id ca ageenans (HC) is
es ima ed om he analysis o solid-s a e NMR spec a and is sys ema ically compa ed wi h he chemical
s uc u e o HC ex ac ed in ho wa e .
1
H NMR showed ha he isola ed HC con ain om 5 o 80 mol% kappa-
ca ageenan, om 90 o 10 mol% io a-ca ageenan, while om 8 o 23 mol% biological p ecu so s a e p esen in
he copolyme ’s chain. A good co ela ion is ound be ween he io a-ca ageenan con en in he seaweeds and in
he ex ac ed HC. The shea elas ic modulus o 1 w % HC gels in he p esence o 0.1 M KCl is signi ican ly weake
and gels o m a lowe empe a u es when he io a-con en in he seaweed is la ge han he kappa-con en .
Howe e , hese weake gels show s ain ha dening. A cu -o o 30 mol.% o io a-ca ageenan in he poly-
saccha ide is iden i ied ha sepa a es s ain so ening s i e gels om s ain ha dening so e gels. O e all, he
se o da a sugges s ha he ca ageenan gel elas ici y is uled by he sel -assembly o io a-ca ageenan moie ies
in he HC, and HC gel elas ici y can be di ec ly linked o he io a-ca ageenan composi ion o seaweeds.
1. In oduc ion
Va ious amilies o ed seaweeds, belonging o he Giga inales
o de , bio syn hesize in hei cell walls a amily o sulpha ed linea
polysaccha ides known as ca ageenans. These na u al polyme s ind
comme cial applica ions as gelling agen s, iscosi y enhance s o
o mula ion s abilize s mainly in he ood indus y (Piculell, 2006) and
mo e ecen ly in he pha maceu ical sec o (Pacheco-Qui o, Ruiz-Ca o,
&Veiga, 2020). The ca ageenan indus y classi ies hese poly-
saccha ides by hei iscoelas ic p ope ies in wa e : he
kappa-ca ageenans which o m s ong bu b i le hyd ogels, he
io a-ca ageenans which p oduces less elas ic gels and he non-gelling
lambda-ca ageenans. The chemical s uc u e o hese linea poly-
elec oly es is howe e mo e complex han his applica ion-d i en
classi ica ion.
Ca ageenans a e be e desc ibed as andom block copolyme s
(Guibe e al., 2008; an de Velde e al., 2005;Hilliou, 2021) wi h blocks
composed o di e en sequences o disaccha ide uni s consis ing o
3-linked β-D-galac opy anose (G-uni s) and 4-linked
α
-D-galac opy -
anose (D-uni s) o 4-linked 3,6-anhyd o-
α
-D-galac opy anose (DA-u-
ni s). The mos na u ally occu ing disaccha ide uni s a e p esen ed in
Fig. 1, which akes on boa d he nomencla u e in oduced by Knu sen,
Myslabodski, La sen, &Uso , 1994. The a ie y o block ypes, leng hs
and dis ibu ion on he ca ageenan chain s ems om he na u al
complexi y o hei biosyn hesis in he cell walls o he seaweeds. The
ca ageenan chemical s uc u es span om homopolyme s, such as
kappa-ca ageenans made o 100% o 93 mol.% o G4S-DA (he e
labelled as κ) (Rochas, Rinaudo, &Land y, 1989; an de Velde e al.,
2005), o highly he e ogeneous polyme s, called hyb id ca ageenans,
made o blocks o κ, blocks o G4S-DA2S (he e labelled as
ι
), blocks o
G4S-D2S (
μ
) and blocks o G4S-D2S,6S (
ν
) all andomly dis ibu ed on
he chain. Ca ageenan chemical he e ogenei y is speci ic o a ou h
class o indus ially ele an ca ageenan, he kappa-2 o weak kappa,
which a e made o 20–50 mol.% o
ι
, he es being κ(Bixle , 1996;
Villanue a, Mendoza, Rod igueza, Rome o, &Mon a˜
no, 2004),
endowing his ca ageenan wi h gel p ope ies in e media e be ween
* Co esponding au ho .
E-mail add ess: [email p o ec ed] (L. Hilliou).
Con en s lis s a ailable a ScienceDi ec
Food Hyd ocolloids
jou nal homepage: www.else ie .com/loca e/ oodhyd
h ps://doi.o g/10.1016/j. oodhyd.2024.111007
Recei ed 29 Oc obe 2024; Recei ed in e ised o m 10 Decembe 2024; Accep ed 18 Decembe 2024
Food Hyd ocolloids 162 (2025) 111007
A ailable online 20 Decembe 2024
0268-005X/© 2024 The Au ho s. Published by Else ie L d. This is an open access a icle unde he CC BY license (
h p://c ea i ecommons.o g/licenses/by/4.0/ ).
kappa- and io a-ca ageenans.
The mono onic inc ease in he hyd ogel elas ici y wi h he κmol.%
con en in hyb id ca ageenans only made up o κand
ι
blocks, is e-
po ed o a la ge se o ca ageenans in a seminal s udy by an de Velde
e al., 2005. Such ela ionship is ye o be es ablished o he mo e
he e ogeneous hyb id ca ageenans con aining all disaccha ide uni s
p esen ed in Fig. 1 (Souza e al., 2023, K aiem, Ben Yahia, Aschi, &
Hilliou 2023). Indeed, he mac omolecula models ela ing he ca a-
geenans hyd ogel mic os uc u es o hei elas ic p ope ies ha e
ecen ly been ques ioned (Hilliou, 2021), as well as he coil- o-helix
ansi ion p e equisi e o gel o ma ion (Wes be y, Rio, Wa e land, &
Williams, 2024).
The na u e o he ela ionship be ween he ca ageenans chemical
s uc u e and i s gel elas ici y is hus ye un esol ed despi e he ex en o
esea ch on kappa- and io a-ca ageenan homopolyme s which suppo s
hei indus ial use. We aim he e o ex end he scope o he ela ionship,
wi h he possibili y o ind a link be ween he ca ageenan’s composi ion
o comme cial seaweeds and he gel p ope ies o he polysaccha ides
om which hese a e ex ac ed. The iden i ica ion o such a link will
boos he ca ageenan indus y which sou ces he biomass om cul i-
a ed and wild seaweeds speci ically selec ed o he ex ac ion o a
single ype o ca ageenan (Bixle , 1996). The sc eening o algae o
hei polysaccha ide composi ion can ely on well-es ablished in a ed
spec oscopy echniques di ec ly pe o med on d ied samples (Chopin &
Whalen, 1993;G´
omez-O d´
o˜
nez &Rup´
e ez, 2011;Pe ei a, Amado,
C i chley, an de Velde, &Ribei o-Cla o, 2009). Howe e , in a ed
spec oscopy does no p o ide quan i a i e es ima es o e.g. κo
ι
con-
en s in he seaweeds. Solid-s a e NMR spec oscopy is mo e sensi i e
bu has i ually no been used o a quan i a i e chemical analysis o
algal ma e ials (Ghassemi e al., 2021;Rochas &Lahaye, 1989).
High-Resolu ion C oss-Pola iza ion Magic Angle Spinning High Powe
Decoupling
13
C NMR spec oscopy (HR CP-MAS-HPD
13
C NMR) o
g inded d ied seaweeds was ecen ly pe o med o quan i a i ely assess
he ca ageenan composi ion (Aze edo, To es, Almeida, &Hilliou,
2022) and also o add ess he e ec s o aquacul u e condi ions on he
ca ageenan g ow h in he cul i a ed algal ma e ial (Aze edo e al.,
2015). Bu no co ela ion was ound be ween he seaweed’s ca ageenan
composi ion and he chemical s uc u es o he ex ac ed ca ageenans
no hei gel p ope ies. Two issues may ha e led o un esol ed e-
la ionships. Fi s ly, he se o s udied seaweeds p oduced ca ageenans
con aining 70 o 40 mol.% κ. This is a oo small window o iden i y a
ela ionship wi h he gel elas ic p ope ies, as hese a e di icul o access
by o a ional heome y, in pa icula in he ange 50 o 40 κmol.% ( an
de Velde e al., 2005). Secondly, an e hanol p ecipi a ion was used o
eco e he ca ageenans, which migh ha e esul ed in a ac iona ion
o he ca ageenans ac ually con ained in he seaweeds.
He e, he ange o chemical composi ion o ca ageenans is ex ended
by s udying 12 comme cial seaweeds used in he indus y o p oduce
kappa-, io a- and a ious kappa-2-ca ageenans. Whole ca ageenan
ex ac s we e isola ed om he seaweeds, hus no e hanol p ecipi a ion
was used. The heological cha ac e iza ion o ca ageenan gels was
ex ended o he la ge de o ma ion egime which has been o e looked in
he li e a u e (Hilliou, 2021;Hilliou, Wilhelm, Yamanoi, &Gonçal es,
2009; an de Velde, 2008). Finally, aking on boa d he colloidal gels
heo ies which showed ha he elas ici y is uled by he mechanically
weake s uc u al uni (Shih, Shih, Kim, Liu, &Aksay, 1990), we sys-
ema ically laid down he gel heological cha ac e is ics as a unc ion o
he
ι
mol.% con en . The objec i e o such da a ea men is o show ha
he
ι
mol.% con en will be mo e e ec i e han he κmol.% con en
employed ea lie ( an de Velde e al., 2005) and will con ibu e o
iden i y a aluable chemical s uc u e- unc ion ela ionship which is
s ill c i ically missing o ca ageenan gels (Cha da e al., 2024).
2. Expe imen al
2.1. Seaweeds
12 comme cial d ied seaweeds dona ed by Ca gill we e used as
ecei ed o ca ageenan ex ac ion. These seaweeds a e labelled om A
o L and belong o he Spinosum (A o D) and Co onii (I o L) genus
( ade names) o he indus ial p oduc ion o he io a- and kappa-
ca ageenans, whe eas seaweeds E o H belong o he Giga inaceae
amily o he p oduc ion o hyb id ca ageenans. Fo HR CP-MAS-HPD
13
C NMR spec oscopy, seaweeds we e c ushed in o powde s using a
co ee g inde , esul ing in seaweed pa icles wi h sizes below 0.5 mm.
2.2. Ca ageenan ex ac ion
12 ypes o ca ageenans we e ob ained om he 12 comme cial
seaweeds. The ex ac ion was pe o med by soaking he d ied whole
halli (1.5 g) in 100 mL dis illed wa e a 80 ◦C o 2 h. Then he sus-
pension was homogenized in a blende and he esul ing slu ies we e
Fig. 1. Chemical s uc u es o he disaccha ides uni s mos ly ound in gelling ca ageenans: κ(G4S-DA),
ι
(G4S-DA2S),
μ
(G4S-D6S) and
ν
(G4S-D2S,6S). Black
a oms indica e hose connec ed o o he disaccha ide uni s, whe eas g ey, ed, whi e and yellow a oms indica e ca bon, oxygen, hyd ogen and sulphu a oms,
espec i ely. Numbe s close o he ca bon a oms indica e he
13
C NMR chemical shi s in ppm ( an de Velde e al., 2004) o he peaks assigned o he co e-
sponding ca bon.
L. Hilliou e al. Food Hyd ocolloids 162 (2025) 111007
2
u he hea ed a 90 ◦C o 1 h. Solid/liquid sepa a ion was pe o med by
cen i uging he ho slu ies a 8000 pm o 10 min. The esul ing su-
pe na an was eco e ed, cas in o plas ic cups and d ied in an ai
en ila ed o en a 50 ◦C o e nigh . The esul ing ilm samples o ca -
ageenans we e inally s o ed in plas ic bags in a idge un il u he
analysis wi hin 1 mon h.
2.3. NMR spec oscopy
The expe imen al p o ocol used o pe o m High-Resolu ion C oss-
Pola iza ion Magic Angle Spinning High Powe Decoupling
13
C NMR
spec oscopy (HR CP-MAS-HPD
13
C NMR) has been de ailed elsewhe e
(Aze edo e al., 2022). In b ie , algal powde s we e loaded in CP-MAS
ubes and spec a we e eco ded a oom empe a u e and 500 MHz
wi h a B uke AVANCE III spec ome e . MAS was pe o med a 10 kHz,
and 1000 scans we e accumula ed o eco d a spec um. The con ac
ime o c oss-pola iza ion was 1500
μ
s and a a iable amp ampli ude
pulse was used. A spinal composi e pulse sequence was used o he
high-powe p o on decoupling.
P o on NMR spec a we e eco ded on 1 w % ca ageenan solu ions
in D
2
O. P io o measu emen s, solu ions p epa ed by s i ing ho
ca ageenan suspensions a 80 ◦C o 1 h we e ul a sonica ed du ing
hou s using a DCG-300H ba h (MCR L d., Holon, Is ael), un il he is-
cosi y o solu ions signi ican ly d opped o a oid NMR line b oadening.
The solu ions we e hen loaded in NMR ubes and spec a eco ded wi h
a B uke A ance III spec ome e (Bille ica, MA, USA) a 400 MHz and a
70 ◦C. D
2
O signals we e used as chemical shi e e ence, which likely
causes sligh a ia ions wi h espec o he chemical shi s e e enced
elsewhe e o ca ageenans ( an de Velde e al., 2004, Pe ei a, &Roll-
ema, 2004).
2.4. Ca ageenans’molecula mass and sal composi ion
Size exclusion ch oma og aphy (Wa e s 600 appa a us, wi h a Wa-
e s 2410 di e en ial e ac i e index de ec o , Wa e s Po ugal, Lisboa,
Po ugal) was pe o med wi h ca ageenan solu ions (0.1 w %) p epa ed
in 0.1 M NaCl a 90 ◦C. Ho solu ions we e injec ed in a PolySep-GFC-P
Linea column (Phenomenex, Alcobendas, Spain), and measu emen s
we e pe o med a 40 ◦C. The column was p e iously calib a ed wi h
pullulan ha ing molecula masses anging om 6300 o 642 000 g/mol.
All measu emen s we e conduc ed in duplica e.
The con en s in po assium, sodium and calcium o ex ac ed ca a-
geenans we e measu ed by Induc i ely Coupled Plasma-A omic Emis-
sion Spec oscopy (Ul ima, Ho iba Jobin-Y on, F ance). Ca ageenan
ilm samples we e dissol ed in ho wa e ( ypically 0.1 g in 100 mL) a
80 ◦C, and solu ions we e loaded in he ICP-AES au osample . The
calib a ion was pe o med wi h solu ions con aining a single elemen ,
bu wi h a ying concen a ions.
2.5. Gels p epa a ion and heological cha ac e iza ion
Ca ageenan gels we e p epa ed in 0.1 M KCl wi h a polysaccha ide
concen a ion o 1 w %. Unde such sal and ionic s eng h, gels wi h
su icien elas ici y o be s udied by o a ional heome y, a e p oduced
o all ex ac ed polysaccha ides, in con as o 1 w % ca ageenan in 1
M NaCl (Mo aes &Hilliou, 2024). Ho ca ageenan solu ions we e
loaded a 85 ◦C in he pa allel pla e geome y o a s ess con olled
o a ional heome e (MCR 302, An on Paa , G az, Aus ia). The pla es
we e p e-hea ed a 85 ◦C, and he im o he shea ing geome y was
co e ed wi h dodecane o limi wa e loss. A empe a u e quench om
85 ◦C o 25 ◦C was pe o med wi hin 1 h while bo h elas ic (G
′
) and loss
(G
″
) moduli we e eco ded by applying a small ampli ude oscilla o y
shea wi h a equency o 1 Hz and an ampli ude o 0.01 %. The la e is
oo small o gene a e la ge enough s ess when he samples a e in he
liquid phase a highe empe a u es, bu ensu es ha no s ain-induced
s uc u e is se ing du ing he liquid- o-solid ansi ion. The no mal
o ce was main ained a 0 N du ing cooling by allowing he gap o
accommoda e o he olume change du ing he liquid- o-solid ansi-
ion. This s a egy a oids any a e ac du ing he measu emen o G
′
and
G
″
h ough he phase ansi ion (Mao, Di oux, &Snab e, 2016). Finally,
he gels we e equilib a ed du ing 30 min a 25 ◦C be o e pe o ming a
s ain sweep om 0.01% o 500% a 1 Hz, o assess he la ge de o ma-
ion beha iou o he gels.
3. Resul s and discussion
3.1. Ca ageenan composi ion o seaweeds
The HR CP-MAS-HPD
13
C NMR spec a o ep esen a i e seaweeds
a e displayed in Fig. 2. All o he seaweeds showed quali a i ely simila
spec a including he b oad peaks in he 175 ppm egion and be ween 40
and 20 ppm, which a e indica i e o py u a es, lipids and pigmen s
p esen in seaweeds (Rochas &Lahaye, 1989), see Fig. S1 in Supple-
men a y In o ma ion.
Focusing on he chemical shi s egions whe e signals assigned o
ca bons om he disaccha ide uni s illus a ed in Fig. 1 a e expec ed,
di e ences in he in ensi ies and b oadening o lines in he 110-90 ppm
egion a e e iden in Fig. 2. In his egion, 3 NMR lines con ibu e o he
spec a which a e assigned o ca bon numbe 1 (C1) o G4S (107-104
ppm), o D6S and D2S,6S (100 ppm), and o DA and DA2S (97-94 ppm),
acco ding o he chemical shi s lis ed o he ca ageenans om he
kappa amily ( an de Velde e al., 2004).
Mo e di e ences a e seen in he egion 80-65 ppm, which is due o
he con ibu ions o all o he ca bons om all disaccha ide uni s dis-
played in Fig. 1, esul ing in 5 lines. In con as o his, he peak a 60
ppm is nea ly iden ical o all seaweeds as only C6 om G4S con ibu es
o his signal. The i ing o each spec um y(x) wi h he ollowing
equa ion based on he addi ion o 9 Lo en zians L
i
was shown o p o ide
a sa is ac o y quan i a i e es ima e o he disaccha ide uni s κ,
ι
,
μ
and
ν
con ained in he seaweeds (Aze edo e al., 2015;Aze edo e al., 2022):
y=y0+ (a+b+c+d)L1(x1) + (c+d)L2(x2) + (a+b)L3(x3)
+ (2b+c+2d)L4(x4) + (4a+2b+c+d)L5(x5) + (2a+2b
+2c+d)L6(x6) + (a+d)L7(x7) + (2a+3b+5c+4d)L8(x8)
+ (a+b+c+d)L9(x9)
(1)
In equa ion (1), he Lo en zians L
i
(x
i
)a e gi en by
Li(xi) = 2
π
w
4(x−xi)2+w2(2)
whe e wis he wid h o he Lo en zians (wis aken as a cons an
Fig. 2. HR CP-MAS-HPD
13
C NMR spec a o h ee g inded d ied seaweeds
labelled B, H and J. The in ensi ies o NMR lines a e e ically shi ed o allow
o he s acking o all spec a in he igu e and acili a e he compa ison.
L. Hilliou e al. Food Hyd ocolloids 162 (2025) 111007
3
assuming o simplici y ha all ca bons in he ca ageenan disaccha ide
uni s show iden ical molecula dynamics and wi ness iden ical c oss
pola iza ion e ec s o neighbou ing p o ons), and x
i
is he posi ion (in
he ppm scale x) o he Lo en zian line L
i
. The ac o s mul iplying he
di e en L
i
in equa ion (1) esul om he ela i e con en s a,b,cand
din he ype o disaccha ide uni s (κ,
ι
,
μ
and
ν
, espec i ely) mul iplied
by he numbe o ca bons o ha disaccha ide uni ha con ibu e o he
line L
i
. Thus, only 4 pa ame e s (a,b,cand d) a e i ed in equa ion (1) o
each spec um, whe eas he posi ions x
i
o he 9 Lo en zians L
i
a e ixed
du ing he i ing bu wi hin he chosen ange o ppm assigned o each
line (see espec i e anges in Table 2 o (Aze edo e al., 2022)). Fig. 3
illus a es he i ing o he spec um measu ed wi h seaweed A o
equa ion (1). Equa ion (1) en ails a se o hypo hesis de ailed elsewhe e
(Aze edo e al., 2022). The main assump ion is ha he 9 lines a e
con ibu ed by 4 ca ageenan disaccha ide uni s. The e o e, con ibu-
ions om o he ypes o polysaccha ides o o he non-ca ageenan
compounds, which could show up a ound 100 and 60 ppm, a e no
aken in o accoun in equa ion (1). As such, he compu ed line in Fig. 3
does no ep oduce some de ails o he expe imen al spec um. Ne e -
heless, he i s did con e ge o all expe imen al spec a and e u ned R
2
alues be e han 0.9 in all cases, in spi e o he noise inhe en o he HR
CP-MAS-HPD 13C NMR spec a.
The esul om he i ing o equa ion (1) o all spec a eco ded wi h
he seaweed samples is p esen ed in Fig. 4. I displays he ca ageenan
composi ion (in κ,
ι
,
μ
and
ν
) o all seaweeds ob ained by he compu-
a ion o pa ame e s a,b,cand d om he i ing p ocedu e. The quali y
o he i s is mi o ed in he e o ba s compu ed om he i s o each
disaccha ide uni .
This ca ageenan analysis indica es ha he seaweeds con ain all
ypes o disaccha ide uni s om he kappa amily, bu in di e en p o-
po ions, a ying om seaweeds ich in
ι
o seaweeds ich in κ.
Seaweeds E o H show mo e he e ogeneous composi ions in disac-
cha ide uni s and a e hus used o he ex ac ion o kappa-2 ca a-
geenans, in con as o o he seaweeds indus ially used o he
ex ac ion o io a- (seaweeds A o D) o kappa-ca ageenans (seaweeds I
o L). In e es ingly, all seaweeds con ain a signi ican p opo ion o
ca ageenan biological p ecu so s
μ
and
ν
. In pa icula , seaweed H is
made o mo e han 50 mol.% o
μ
disaccha ide uni s which a e he
biological p ecu so s o κdisaccha ide uni s (Chopin &Whalen, 1993;
Piculell, 2006).
3.2. Cha ac e iza ion o ex ac ed ca ageenans
The chemical s uc u e o he 12 ca ageenans ex ac ed om he
seaweeds is p esen ed in Fig. 5. The ela i e con en s in κ,
ι
,
μ
and
ν
a e
compu ed om he a ios o he peak a ea o he signal assigned o he
anome ic p o on o a speci ic disaccha ide uni s o e he o al a ea o he
ou peaks assigned o all anome ic p o ons om he kappa-ca ageenan
amily. Examples o p o on NMR spec a o selec ed ca ageenans can be
ound in Fig. S2. NMR signals which a e no assigned o ca ageenans
we e ound in some spec a. Fo ins ance, a peak a 5.28 ppm was ound
in all ex ac s om he Spinosum, wi h la ge in ensi y o sample D (see
Fig. S3), and o a lesse ex en in all ex ac s om he Co onii. Simila ly,
a peak a 5.02 was ound o sample I, whe eas peaks co esponding o
py u ic acids and o he compounds we e ound up ield in he spec a o
all ca ageenans bu hose om Co onii (see inse o Fig. S3). These
signals can be caused by low molecula weigh compounds such as
suga s o pigmen s and sal s which a e no emo ed by he ex ac ion
ou e employed he e. As such, hese compounds, which we e no u he
analysed, can impac he gelling p ope ies epo ed he e, o a leas
a ec he e ec i e ca ageenan concen a ions used in he heological
s udy.
The chemical s uc u es o all ex ac s a e quali a i ely eminiscen
om he ca ageenan composi ion o seaweeds, as is e idenced when
compa ing Figs. 4 and 5. Whe eas ca ageenan A is essen ially a io a-
ca ageenan con aining small amoun s (below 10 mol.%) o
μ
and
ν
disaccha ide uni s, all o he ca ageenans exhibi mo e he e ogeneous
s uc u es, om polyme chains ich in
ι
, o mo e hyb id s uc u es
con aining all κ,
ι
,
μ
and
ν
uni s and chains which a e iche in κ.
None heless, 8 ca ageenans ( om E o L) con ain 10–30 mol.% o
ι
as
well as signi ican amoun s o
μ
and
ν
. As such, none o hese ca a-
geenans quali y o he indus ial de ini ion o kappa-2 (Bixle , 1996;
Fig. 3. HR CP-MAS-HPD
13
C NMR spec um o seaweed A (symbols) and i o
equa ion (1) o he spec um ( ed hick line) esul ing o he addi ion o he 9
Lo en zian lines ( hin g een lines).
Fig. 4. Ca ageenan composi ion (in mol.% o κ,
ι
,
μ
and
ν
) o all
es ed seaweeds.
Fig. 5. Disaccha ide uni s composi ion (in mol.%) o he 12 ex ac ed ca a-
geenans assessed om he quan i a i e analysis o hei p o on NMR spec a.
L. Hilliou e al. Food Hyd ocolloids 162 (2025) 111007
4
Villanue a e al., 2004) which is based on he con en s in κand
ι
.
Ca ageenan p ecu so s
μ
and
ν
a e usually con e ed du ing he alka-
line ex ac ion o ca ageenans, which was a oided in he p esen s udy
o minimize he ca ageenan physical and chemical changes occu ing
du ing ex ac ion.
Table 1 summa izes he molecula mass dis ibu ions o all ex ac ed
ca ageenans, as well as hei composi ion in elemen s whose chlo ine
sal s a e known o acili a e he agg ega ion o ca ageenan helices and
hei consecu i e gel se ing.
All ca ageenan possess molecula masses be ween 200 kDa and 2 ×
10
6
kDa, which is in ag eemen wi h he molecula masses epo ed o
samples ex ac ed om a ious ca ageenophy es (Piculell, 2006; an de
Velde e al., 2005). All M
w
a e abo e he c i ical molecula mass M
c
iden i ied by Rochas, Rinaudo, and Land y (1990) o
kappa-ca ageenans. Abo e M
c
=180 kDa, kappa-ca ageenan gel
p ope ies do no depend on M
w
. No e howe e ha ano he s udy
showed ha wo hyb id ca ageenans wi h iden ical chemical s uc u e
bu wi h di e en M
w
>M
c
showed e y di e en gel p ope ies (Souza,
Hilliou, Bas os, &Gonçal es, 2011). The molecula mass dis ibu ions
PDI o he ca ageenan lis ed in Table 1 a e mos ly a ound 2, bu ca -
ageenans E and K show much la ge PDI, which sugges s ha some
polysaccha ides in hese samples may show M
w
below M
c.
Un o u-
na ely, he e is a lack o epo s abou he e ec o molecula mass
dis ibu ions, and pa icula ly he ole o smalle chains, on he gelling
p ope ies o ca ageenans (Rochas e al., 1990;Souza e al., 2011). As
such, and assuming ha he alue o M
c
holds ue o he ca ageenans
epo ed in Table 1, one can conjec u e ha he molecula masses o he
ca ageenan samples s udied he e will no a ec he gelling p ope ies.
The elemen al composi ions epo ed in Table 1 sugges ha ca a-
geenan samples do no p esen signi ican amoun s o calcium. Howe e ,
ca ageenans ich in κo in
ι
a e ex ac ed wi h mo e po assium and
sodium han he mo e hyb id ca ageenans. This highe con en in ca -
ions canno howe e be co ela ed wi h he o al amoun o mo e
sulpha ed disaccha ide uni s as ca ageenans B o H show a ound 20
mol.% o
μ
and
ν
uni s. Since dis illed wa e was used du ing ex ac ion,
hese ca ions o igina e om he seaweeds. I has been ecen ly showed
ha sal s o med wi h he ca ions ca ied by Chond us c ispus seaweeds
can a ec he eco e y o mo e o less sulpha ed ca ageenans (Baha i
e al., 2021). Howe e , u he ex ac ion s udy will be needed o
elucida e he o igin o he leaching o mo e ca ions wi h less he e oge-
neous ca ageenans.
The chemical cha ac e is ics displayed in Fig. 5 and Table 1 do no
allow o de ailing he a ia ion in he ca ageenan chemical composi-
ions om chains o chains wi hin each ex ac . Dedica ed cha ac e -
iza ions a e ac iona ion o enzyma ic hyd olysis as pe o med
elsewhe e would be equi ed (Rochas e al., 1989;Guibe e al., 2008),
which a e ou o he scope o he p esen s udy. Ca ageenans wi h la ge
PDI such as sample K could ac ually be mix u es o nea ly
homopolyme s made o kappa-ca ageenan o io a-ca ageenan disac-
cha ide uni s (Rochas e al., 1989), ins ead o mo e hyb id copolyme s.
Howe e , i has been shown ha ca ageenans om he comme cial
seaweeds selec ed in he p esen s udy, especially om he Giga-
inaceae, a e a he copolyme s as no sepa a ion o kappa- and
io a-ca ageenan homopolyme s could be seen ( an de Velde e al.,
2005), whe eas ca ageenans om Co onii and Spinosum a e essen-
ially homopolyme s ( an de Velde, 2008).
3.3. Can we link he ca ageenan composi ion o seaweeds wi h he
chemical s uc u e and gelling p ope ies o ex ac ed ca ageenans?
3.3.1. Chemical analyses
Fig. 6 compa es he quan i a i e chemical in o ma ion e ie ed
om he solid-s a e NMR spec a wi h hose measu ed by p o on NMR,
ocusing on disaccha ide uni s κand
ι
, which a e esponsible o he
gelling capabili ies o ca ageenans om he kappa–ca ageenan amily,
in opposi ion o
μ
and
ν
uni s which a e iewed as de ec s (Hilliou, 2021;
Piculell, 2006; an de Velde e al., 2005).
The igu e shows ha ca ageenans ich in κo
ι
a e ex ac ed om
seaweeds ha con ain la ge amoun s o co esponding disaccha ide
uni s. The co ela ion is less sa is ac o y o mo e hyb id ca ageenans,
since da a d i away om he line ha sugges s a pe ec linea ela-
ionship be ween he wo se s o da a. Pea son co ela ion analyses o
he wo se s o da a e u n a Pea son’s coe icien o 0.865 (p- alue o
2.8 ×10
−4
) o bo h κ-and
ι
-con en s. Howe e , i e o ba s a e
conside ed in he linea co ela ion analysis (using e o ba s as weigh s
in a linea i o he da a), a be e esul is ob ained o
ι
disaccha ide
uni s (coe icien o de e mina ion R
2
=0.91) han o κdisaccha ide
uni s (coe icien o de e mina ion R
2
=0.79). Such di e ence is na u-
ally caused by he la ge e o ba s associa ed wi h he solid-s a e NMR
analysis o κ-con en s in he seaweeds (see Fig. 6). Mo eo e ,
ι
-con en s
span a wide ange o alues ( om 0 o 90 mol.%) han κcon en s ( om
1 o 77 mol.%). As such, he con en in
ι
disaccha ide uni s wo ks be e
in linking he ca ageenan composi ion o seaweeds o he chemical
s uc u e o he ex ac ed ca ageenans.
No co ela ion was ound be ween he con en s in
μ
and
ν
uni s o he
seaweeds and in espec i e ca ageenans (see Fig. S4). This is inhe en o
he la ge e o s associa ed wi h he quan i ica ion o hese disaccha ide,
as hei small amoun s in bo h seaweeds and ca ageenans a e close o
he limi o de ec ion o NMR spec oscopy. Also, as men ioned abo e,
o he low molecula weigh compounds ex ac ed oge he wi h he
ca ageenans, and he p esence o o he compounds in he seaweeds
con ibu ing o signals o e lapping hose assigned o
μ
and
ν
in he solid
Table 1
Weigh a e aged molecula mass (M
w
), polydispe si y o he molecula mass
dis ibu ion (PDI) and elemen al composi ion in po assium (K), sodium (Na) and
calcium (Ca) o all ex ac ed ca ageenans.
Ca ageenan
sample
Mw (10
6
g/
mol)
PDI K (w .
%)
Na (w .
%)
Ca (w .
%)
A 1.453 1.85 7.25 3.36 0.29
B 0.728 1.76 12.1 6.1 0.66
C 1.173 2.10 14.63 9.08 0.34
D 1.047 2.18 5.67 2.82 0.27
E 0.816 4.25 2.28 6.12 0.37
F 0.234 2.29 2.69 6.61 0.38
G 0.591 2.19 1.93 5.24 0.32
H 0.856 2.08 1.92 5.87 0.35
I 1.395 2.53 10.3 2.82 0.31
J 0.412 3.84 11.7 3.4 0.58
K 1.380 5.39 19 6.63 0.24
L 1.266 2.90 5.04 1.27 0.19
Fig. 6. F ac ions o κand
ι
con ained in he ex ac ed ca ageenans e sus he κ
and
ι
con en s in he seaweeds. The line is a guide o he eyes o e alua e a
possible linea co ela ion be ween he wo se s o da a.
L. Hilliou e al. Food Hyd ocolloids 162 (2025) 111007
5
s a e NMR spec a, can al oge he be esponsible o he lack o co e-
la ion. In addi ion,
μ
and
ν
uni s can be con e ed in o κand
ι
uni s
du ing ca ageenan ex ac ion. As such, all ca ageenan gelling p op-
e ies will now be laid down as a unc ion o he
ι
con en s in he
seaweeds.
3.3.2. Gelling p ope ies
The se ing o he ca ageenan gels du ing he cooling o ho 1 w %
solu ions in 0.1 M KCl is shown in Fig. 7. These sal condi ions we e
chosen o sc een any e ec om he sal s b ough by he ca ageenans
( aking he sample wi h he la ges amoun o po assium K in Tables 1
and 1 g o his ca ageenan in 100 mL o 0.1 M KCl will b ing 0.24 g o
sal , which is esidual when compa ed wi h he nea ly 4 g o K b ough
by KCl), while gels wi h su icien elas ici y could be o med in he
heome e . Howe e , he empe a u e dependence o he s o age
modulus G
′
is a he noisy a highe empe a u es and ende s di icul a
clea es ima e o he empe a u e T
on
o he onse o gel se ing based o
he ise in he elas ici y G
′
o he cooling solu ion. Simila di icul ies
we e epo ed o he heological s udy o a se o hyb id ca ageenans
wi h κ ac ions below 50 mol.%, whe e no empe a u e o he gel
ansi ion de ined as he empe a u e whe e G’=G
″
could be measu ed
( an de Velde e al., 2005).
To bypass he low shea s ess gene a ed by he gelling unde an
oscilla o y s ain o 0.01 %, T
on
is he e in e ed om he empe a u e
dependence o he gap which is se by main aining he no mal o ce o 0
N. This is illus a ed in Fig. 7b o selec ed ca ageenan gels. A highe
empe a u e, he gap g ows linea ly wi h he cooling, as expec ed om
he he mal expansion o he shea ing geome y. A he sol-gel ansi-
ion, he sample sh inks ( hus exe ing a nega i e no mal o ce on he
pla es) and he gap is dec eased o main ain he no mal o ce o 0 N, as
well as a con ac be ween he sample and he shea ing pla e. The em-
pe a u e a which he he mal dependence o he gap is no mo e linea is
aken he e as T
on
.Fig. 7b indica es ha sample B does no o m a gel
wi hin he es ed empe a u e ange since he gap only exhibi s a linea
he mal expansion. This can be a ibu ed o he ac ha his ca a-
geenan p esen s he lowes amoun o
ι
wi hin he se o
ι
– ich samples
(see Fig. 5). In addi ion, ca ageenan B does no con ain enough κ
disaccha ide uni s sensi i e enough o KCl o p omo e he o ma ion o a
ne wo k wi h su icien elas ici y o gene a e a measu able s ess unde a
0.01 % s ain (Piculell, Nilsson, &Muh beck, 1992). In con as o his,
sample A o ms a u bid and weak gel, as he p esence o only 5 mol.% o
κdisaccha ide uni s weakens he io a-ca ageenan ne wo k as shown in
Piculell e al., 1992.
Fig. 8 p esen s a selec ion o s ain sweep es s pe o med on equil-
ib a ed gels. The LAOS beha iou o all es ed ca ageenan gels is shown
in Fig. S5.
In addi ion o he measu emen o he elas ici y G
0
o he gels de ined
he e as he alue o G
′
measu ed in he linea egime, wo ypes o la ge
ampli ude oscilla o y shea (LAOS) beha iou can be in e ed om he
cu es. The gel o med wi h ca ageenan I shows a la ge elas ici y G
0
and an ab up loss o elas ici y a s ains o he o de o 10 %. In con as
o his, gelled ca ageenans A and E sus ain much la ge s ains as G
′
inc eases wi h he applied s ain, passes h ough a maximum and
e en ually d ops a la ge s ain alues. The inc ease in elas ici y, i.e. he
s ain ha dening, was shown o be e e sible when consecu i e s ain
sweeps a e applied wi hin his egime o s ains, in con as o he
i e e sible d op o elas ici y exhibi ed by sample I (Mo aes &Hilliou,
2024). S ain ha dening is he hallma k o ib illa ne wo ks (Meng &
Te en je , 2017;S o m, MacKin osh, Lubensky, &Janney, 2005),
including pec in (Ca illo, MacKin osh, &Dob ynin, 2013) and aga ose
(Be ula e al., 2019) gels. O e all, he da a epo ed in Fig. 8 a e in
quali a i e ag eemen wi h heo e ical p edic ions o ne wo ks made o
ib e-like s uc u es. No e he e ha colloidal gels desc ibed as a ac al
ne wo k o clus e s made o s ings o pa icles show s ain ha dening
when s ings’elas ici y is weake han he elas ici y o con ac s be ween
clus e s (Gisle , Ball, &Wei z, 1999). Indeed, he s ain ha dening o
gels o med wi h di e en concen a ions o a hyb id ca ageenan wi h a
chemical s uc u e equi alen o samples E o H, was success ully
a ionalized by he heo y o ac al colloidal gels. The ac al dimen-
sion compu ed om he i ing o he heo y o he s ain dependen
shea modulus nicely ma ched he ac al dimension measu ed om he
analysis o mic oscopic images o he gels’s uc u es (Hilliou e al.,
2009). Ano he possibili y o explaining he s ain ha dening comes
om a od and coils model de ised o gela in gels (G oo , Bo , &
Ag e o , 1996): loose ca ageenan chains no in ol ed in od-like ag-
g ega es could s e ch unde de o ma ion and gi e he epo ed s ain
ha dening. This model has been indeed es ed wi h some success o a
hyb id ca ageenan gel o med in NaCl (Hilliou, 2021).
3.3.3. Hyb id ca ageenan gel p ope ies ela e wi h he con en o
io a–ca ageenan in he seaweeds
Ha ing all seaweeds’ca ageenan composi ions and he ca ageenan
gels p ope ies a hand, i is na u al o ques ion abou he exis ence o a
ela ionship, which could unde pin biomechanical di e ences in ca -
ageenophy es’cell walls, bu also p edic he gel p ope ies o he
ca ageenan wi hou ca ying ou any ex ac ion. An a emp o add ess
his ela ion is p oposed in Fig. 9a whe e all hyb id ca ageenan gels’
linea and nonlinea he mo- heological p ope ies a e plo ed as a
unc ion o he
ι
–con en in he seaweeds.
Fig. 9a es ablishes a ela ionship be ween he linea elas ic
Fig. 7. E olu ion o he s o age modulus G’(a) and he gap o he heome e (b) du ing he cooling o ho ca ageenan solu ions: sample A (squa es), sample B (black
do s), sample H (up iangles) and sample J (down iangles). The a ow in (b) indica es he empe a u e T
on
.
L. Hilliou e al. Food Hyd ocolloids 162 (2025) 111007
6
p ope ies o he hyb id ca ageenan gels and he
ι
–con en in he sea-
weeds: seaweeds wi h la ge
ι
–con en s deli e less elas ic gels, as ex-
pec ed o hyb id ca ageenan gels (Bixle , 1996;Hilliou, 2021;Piculell,
2006; an de Velde, 2005) including hose made om polysaccha ides
wi h signi ican amoun s o biological p ecu so s (Hughes, Leona di, &
Geno ese, 2023). No e ha such ela ionship is much less con incing i
he compa ison is ca ied ou wi h he κ–con en , as he e a e gaps along
he κ–con en scale and simila κ–con en gi e gels wi h e y di e en
elas ici ies, see Fig. S6. The gel elas icmoduli G
0
o he
ι
– ich hyb id
ca ageenans in Fig. 9 a e smalle han 116 Pa, he alue measu ed wi h
a comme cial io a-ca ageenan in simila gelling condi ions (Mo aes &
Hilliou, 2024). The same holds ue wi h κ– ich hyb id ca ageenans
which show moduli smalle han 21 kPa, he modulus measu ed wi h a
comme cial kappa-ca ageenan (Mo aes &Hilliou, 2024). The da a also
show ha he empe a u e T
on
o he onse o gela ion ollows he same
end, o he whole ange o measu ed
ι
–con en in he seaweeds. This is
a a he no el esul since an ea lie s udy epo ed ha he gelling
empe a u e does no ela e wi h he chemical s uc u e o he hyb id
ca ageenans ( an de Velde e al., 2005). Howe e , bo h G
0
and T
on
measu ed wi h he gels made om ex ac s o seaweeds H a e o he
end shown by he emaining se o da a, as G
0
and T
on
a e oo small
when compa ed wi h hose om gels associa ed wi h equi alen low
ι
–con en . Looking a Fig. 4, i is clea ha such odd esul is ela ed o
he la ge con en in non-gelling
μ
disaccha ide uni s making up seaweed
H. Indeed, when compa ing bo h G
0
and T
on
wi h he
ι
–con en in he
ex ac ed hyb id ca ageenan, see Fig. 9b, hese ou lie poin s anish as
he ex ac ion p ocess signi ican ly educed he amoun in biological
p ecu so s, and he
ι
–con en is eadily shi ed o la ge alues.
E iden ly, mo e da a in he ange 40–70 mol.% o
ι
–con en in he
polysaccha ide chain is needed o explo e he chemical s uc u e-gel
elas ic ela ionships o his class o hyb id ca ageenan. Howe e , he
se o da a epo ed in Fig. 9b is o indus ial ele ance as hey show
chemical s uc u e-gelling empe a u e-elas ici y ela ionships o low
cos (no alkali modi ica ion) hyb id ca ageenans (Bixle , 1996;Villa-
nue a e al., 2004).
In e es ingly, he quali a i e analysis o he LAOS expe imen s pe -
o med on all ca ageenan gels sugges s ha he e is a cu -o in he
ι
–con en o hyb id ca ageenans, o he o de o 30 mol.% (compa e
Fig. 9a and b), below which he s ain ha dening anishes, and s onge
gels show a s ain so ening beha iou . Based on heo e ical a gumen s
(Ca illo e al., 2013;Meng &Te en je , 2017;S o m e al., 2005, Pas-
o e, MacKin osh, Lubensky, &Janney, 2005), one may conjec u e om
he LAOS beha iou ha hyb id ca ageenans con aining mo e han 30
mol.% o
ι
a e essen ially ib illa ne wo ks, whe eas hyb id
Fig. 8. S ain dependence o (a) he s o age (G
′
) and (b) he loss (G
″
) moduli o equilib a ed ca ageenan gels a 25 ◦C in 0.1 M KCl.
Fig. 9. Rela ionships be ween he gels linea elas ic modulus G
0
, he empe a u e T
on
o onse o gela ion, he s ain γ
F
o gel luidiza ion, and (a) he
ι
–con en in
ca ageenophy es, (b) he
ι
–con en in he ex ac ed hyb id ca ageenan. The ho izon al dashed line in (a) indica es he on ie be ween s ain ha dening and s ain
so ening hyb id ca ageenan gels. The ed ci cle in (a) highligh he da a om seaweed H which show up as an ou lie (see ex o de ail). The e ical line in (b)
indica es he cu -o in he
ι
–con en o hyb id ca ageenans ha delimi s he s ain ha dening ( o
ι
–con en la ge han he cu -o line) and s ain so ening
beha iou o co esponding gels.
L. Hilliou e al. Food Hyd ocolloids 162 (2025) 111007
7
ca ageenans wi h lowe
ι
–con en build gels wi h a dense o mo e
c osslinked s uc u e which does no allow o he s ain-induced
s e ching o s uc u al elemen s in he ne wo k. Al e na i ely, he
bending igidi y o he ilamen s, o he comp ession accumula ed on
ilamen s du ing gel o ma ion, would be esponsible o di e ences in
he LAOS beha iou whe eas he ne wo k s uc u e is essen ially he
same (Ca illo e al., 2013;Meng &Te en je , 2017). Howe e , s udies
wi h hyb id ca ageenans ee o
μ
and
ν
should be ca ied ou o ali-
da e his p oposed gel s uc u e-elas ici y ela ionship as well as he 30
mol.% cu -o con en in
ι
. No e he e ha a mo e quan i a i e analysis o
he la ge de o ma ion beha iou , o ins ance wi h he luidiza ion s ain
γ
F
signalling he s ain-induced gel o liquid ansi ion whe e G’=G
″
as
shown in Fig. 9b, does no pick up he e ec o
ι
–con en on he ca a-
geenan gel cha ac e is ics.
4. Conclusions
HR CP-MAS-HPD
13
C NMR spec oscopy di ec ly pe o med on d ied
and g inded seaweeds ga e access o he con en s in
ι
disaccha ide uni s
in he seaweeds. This solid-s a e NMR expe imen al ool and he quan-
i a i e analysis p esen ed he e a e o in e es o sc een o indus ial
ca ageenophy es. F om he s udy o 12 seaweeds and co esponding 12
hyb id ca ageenan ex ac s, a good co ela ion be ween he
ι
–con en s
in he seaweeds and in he ca ageenan was ound, which esul ed om
bo h a be e p ecision in he quan i ica ion o such disaccha ide uni s in
he seaweeds and a good span in he ange o
ι
–con en s in bo h ca a-
geenans and seaweeds. The hypo hesis ha he
ι
–con en in he sea-
weeds ules he hyd ogel elas ic p ope ies o ex ac ed ca ageenan
could hus be es ed, going beyond he classical ca ageenan s uc u e-
gel p ope ies ela ionships o en s udied based on he κ-con en , since
kappa-ca ageenans a e known o build s i e gels in wa e . Indeed,
seaweeds ich in
ι
–con en can be used o ex ac hyb id ca ageenans
wi h lowe gel se ing empe a u es, o he o de o 40 ◦C, wi h gel
elas ic moduli below 100 Pa o 1 w % in 0.1 M KCl, and wi h la ge
s ain esis ance cha ac e ized by s ain ha dening p ope ies. A mini-
mum amoun o 30 mol.% o
ι
disaccha ide uni s in he hyb id ca a-
geenan chemical s uc u e is needed o achie e such p ope ies. On he
basis o ib illa ne wo k heo ies, beyond 30 mol.% o
ι
con en , he
ne wo k is dense , o he bending igidi y o ilamen s is no mo e uled
by he
ι
disaccha ide uni s, esul ing in s i e and mo e b i le gels
o med a highe empe a u es. The heological cha ac e iza ion adop-
ed he e, based on gel se ing unde ze o no mal o ce and on he sys-
ema ic s udy o he gels LAOS beha iou , and he unde lying heo e ical
amewo k, a e p omising o e isi ing he chemical composi ion - gel
s uc u e - elas ic p ope ies ela ionships in kappa2-ca ageenan sys-
ems, which a e chemically simple han he hyb id ca ageenans
s udied he e, and in mix u es o kappa- and io a-ca ageenans. As o
u u e wo ks, i would be in e es ing o apply he solid-s a e NMR
app oach p esen ed he e o algal esidues as o assess he e iciency o
new ex ac ion ou es.
CRediT au ho ship con ibu ion s a emen
Loic Hilliou: W i ing – e iew &edi ing, W i ing –o iginal d a ,
Valida ion, Supe ision, Resou ces, P ojec adminis a ion, Me hodol-
ogy, In es iga ion, Funding acquisi ion, Fo mal analysis, Da a cu a ion,
Concep ualiza ion. Izabel C is ina F ei as Mo aes: W i ing – e iew &
edi ing, Valida ion, In es iga ion, Fo mal analysis, Da a cu a ion. Ped o
Lúcio Almeida: W i ing – e iew &edi ing, Valida ion, In es iga ion,
Fo mal analysis, Da a cu a ion.
Funding sou ces
This wo k was suppo ed by he Fundaç˜
ao pa a a Ciˆ
encia e Tecno-
logia (FCT), h ough he E2B2-PHACAR p ojec , g an numbe : PTDC/
BII-BIO/5626/2020. Addi iona suppo by he FCT unde he amewo k
o S a egic Funding g an UID/CTM/50025/2020 and g an CEE-
CINST/00156/2018 a e also acknowledged.
Decla a ion o compe ing in e es
The au ho s decla e ha hey ha e no known compe ing inancial
in e es s o pe sonal ela ionships ha could ha e appea ed o in luence
he wo k epo ed in his pape .
Acknowledgmen s
LH acknowledges he long-s anding suppo o Ca gill Tex u ing
Solu ions –Hyd ocolloids - F ance o he E2B2-PHACAR p ojec , om
he call applica ion, o he deli e y o samples and scien i ic discussions.
Appendix ASupplemen a y da a
Supplemen a y da a o his a icle can be ound online a h ps://doi.
o g/10.1016/j. oodhyd.2024.111007.
Da a a ailabili y
Da a will be made a ailable on eques .
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