Academic Edi o : Wei Cui
Recei ed: 31 Janua y 2025
Re ised: 18 Feb ua y 2025
Accep ed: 20 Feb ua y 2025
Published: 22 Feb ua y 2025
Ci a ion: Mon ei o, M.A.F.; Fa ia, B.;
Mo aes, I.C.F.; Hilliou, L. Hyb id
Ca ageenans Ve sus Kappa–Io a-
Ca ageenan Blends: A Compa a i e
S udy o Hyd ogel Elas ic P ope ies.
Gels 2025,11, 157. h ps://doi.o g/
10.3390/gels11030157
Copy igh : © 2025 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
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A ibu ion (CC BY) license
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licenses/by/4.0/).
A icle
Hyb id Ca ageenans Ve sus Kappa–Io a-Ca ageenan Blends:
A Compa a i e S udy o Hyd ogel Elas ic P ope ies
Ma ia Alice F ei as Mon ei o 1, B uno Fa ia 1, Izabel C is ina F ei as Mo aes 2and Loic Hilliou 1,*
1Ins i u e o Polyme s and Composi es, Uni e si y o Minho, 5800-048 Guima ães, Po ugal;
[email p o ec ed] (M.A.F.M.); [email p o ec ed] (B.F.)
2Depa men o Food Enginee ing, Facul y o Animal Science and Food Enginee ing (FZEA),
Uni e si y o São Paulo (USP), Pos g adua e P og amme in Ma e ials Science and Enginee ing,
Pi assununga 13635-900, SP, B azil; [email p o ec ed]
*Co espondence: [email p o ec ed]
Abs ac : A compa ison be ween he gel p ope ies o blends o kappa- and io a-ca ageenans
(K+Is) and hyb id ca ageenans (KIs) wi h equi alen chemical composi ions is he e p e-
sen ed. The objec i e is o assess unde which condi ions hyb id ca ageenans a e aluable
al e na i e o blends o kappa- and io a-ca ageenans o gelling applica ions and o con-
ibu e o he iden i ica ion o phase-sepa a ed s uc u es o co-agg ega ed helices. Phase
s a es cons uc ed in sodium chlo ide and in po assium chlo ide con i m ha KIs build gels
unde a much na owe ange o ionic s eng h and polysaccha ide concen a ion. Hyb id
ca ageenans displayed sal speci ici y, o ming gels in KCl bu no in NaCl, highligh ing
hei limi ed gelling po en ial in Na
+
en i onmen s. A wo-s ep gela ion mechanism was
ound in bo h sys ems a lowe ionic s eng hs and when io a ca ageenan is he majo
componen . The shea elas ic moduli o KI gels a e o e all smalle han hose o blends,
bu he opposi e is obse ed a lowe ionic s eng hs in KCl and in sys ems iche in io a-
ca ageenans. The nonlinea elas ic p ope ies o gels do no ela e o he use o blends o
hyb id ca ageenans o hei o mula ion. Ins ead, la ge con en s in io a-ca ageenans
lead o gels able o sus ain la ge s ains be o e yielding o a luid s a e. Howe e , hese gels
a e mo e p one o s ain so ening, whe eas s ain ha dening is measu ed in gels con aining
mo e kappa-ca ageenan, i espec i e o hei blend o hyb id s uc u e.
Keywo ds: ca ageenan; hyd ogel; molecula mass; shea s o age modulus
1. In oduc ion
Ca ageenans a e a amily o sul a ed polysaccha ides, ex ac ed om ed seaweeds,
which ha e long been in eg al o he ood indus y due o hei unique gelling, hickening,
and s abilizing p ope ies [
1
]. They play a c ucial ole in he o mula ion o a wide ange
o p oduc s, including dai y goods, mea p oduc s, and plan -based al e na i es [
1
,
2
].
Ca ageenan is cu en ly he leading seaweed-de i ed ood hyd ocolloid and accoun s o
he six h-la ges sha e o he global hyd ocolloid ma ke (in e ms o alue), a e gua gum,
gela in, xan han gum, cellulose gum, and a abic gum [
3
]. The global ca ageenan ma ke
was wo h USD 850 million in 2022 and is expec ed o expand a an a e age yea ly g ow h
a e o 6.2%, eaching USD 1.55 billion by 2032 [4].
The ocus o mos comme cial applica ions has been on he use o kappa-ca ageenans
and io a-ca ageenans, ei he in pu e o ms o as blends, o achie e desi ed ex u al
p ope ies [5–7]
. Ex ensi e esea ch has been conduc ed on hese ca ageenans, pa ic-
ula ly in e ms o hei chemical s uc u e, gelling mechanisms, and in e ac ions wi h o he
Gels 2025,11, 157 h ps://doi.o g/10.3390/gels11030157
Gels 2025,11, 157 2 o 18
ood ing edien s [
8
]. Howe e , he inc easing demand o kappa- and io a-ca ageenans
in bo h ood and non- ood indus ies [
9
] has ecen ly exe ed signi ican p essu e on ca -
ageenan p oduc ion and seaweed a ming [
1
], making i u gen o look o unde exploi ed
and sus ainable esou ces.
In his con ex , kappa/io a-hyb id ca ageenans (KIs) [
10
], which consis o a andom
block copolyme o sequences o kappa-ca ageenan (
κ
) and io a-ca ageenan (
ι
) diads
(disaccha ides o D-galac ose and 3,6-anhyd ogalac ose uni s linked by speci ic glycosidic
bonds, see Figu e 1), ha e eme ged as p omising al e na i es o adi ional comme cial
blends. Indus ially known as weak kappa o Kappa-2 [
11
,
12
], hese copolyme s o m
he mo- e e sible hyd ogels wi h in e media e elas ici y in compa ison o hose o kappa-
and io a-ca ageenan, o e ing unique s uc u al and pe o mance ad an ages in ce ain
applica ions [
11
,
12
]. Mo eo e , hey a e p edominan ly sou ced om wild seaweeds in cold
wa e s, unlike kappa- and io a-ca ageenans, which a e mainly ob ained om he in ense
a ming o wa m-wa e species, wi h associa ed p oduc ion issues [
13
]. The a io be ween
kappa-ca ageenan and io a-ca ageenan diads in a KI is se by biology, ha is, he amily
and genus o seaweeds used o ex ac he KI. So, ins ead o mixing a di e en a ios o une
he p ope ies o he blend, he p ope ies o a KI a e uned by choosing he co ec seaweed
and isola ing he co esponding copolyme [
10
,
14
]. This makes hyb id ca ageenans no
only an a ac i e op ion o al e na i e sou cing bu also a po en ially mo e cos -e ec i e
solu ion by elimina ing he need o blending di e en ypes o ca ageenans.
Gels 2025, 11, x FOR PEER REVIEW 2 o 19
p ope ies [5–7]. Ex ensi e esea ch has been conduc ed on hese ca ageenans, pa icu-
la ly in e ms o hei chemical s uc u e, gelling mechanisms, and in e ac ions wi h o he
ood ing edien s [8]. Howe e , he inc easing demand o kappa- and io a-ca ageenans
in bo h ood and non- ood indus ies [9] has ecen ly exe ed signi ican p essu e on ca -
ageenan p oduc ion and seaweed a ming [1], making i u gen o look o unde ex-
ploi ed and sus ainable esou ces.
In his con ex , kappa/io a-hyb id ca ageenans (KIs) [10], which consis o a andom
block copolyme o sequences o kappa-ca ageenan (κ) and io a-ca ageenan (ι) diads
(disaccha ides o D-galac ose and 3,6-anhyd ogalac ose uni s linked by speci ic glycosidic
bonds, see Figu e 1), ha e eme ged as p omising al e na i es o adi ional comme cial
blends. Indus ially known as weak kappa o Kappa-2 [11,12], hese copolyme s o m
he mo- e e sible hyd ogels wi h in e media e elas ici y in compa ison o hose o kappa-
and io a-ca ageenan, offe ing unique s uc u al and pe o mance ad an ages in ce ain
applica ions [11,12]. Mo eo e , hey a e p edominan ly sou ced om wild seaweeds in
cold wa e s, unlike kappa- and io a-ca ageenans, which a e mainly ob ained om he
in ense a ming o wa m-wa e species, wi h associa ed p oduc ion issues [13]. The a io
be ween kappa-ca ageenan and io a-ca ageenan diads in a KI is se by biology, ha is,
he amily and genus o seaweeds used o ex ac he KI. So, ins ead o mixing a diffe en
a ios o une he p ope ies o he blend, he p ope ies o a KI a e uned by choosing he
co ec seaweed and isola ing he co esponding copolyme [10,14]. This makes hyb id
ca ageenans no only an a ac i e op ion o al e na i e sou cing bu also a po en ially
mo e cos -effec i e solu ion by elimina ing he need o blending diffe en ypes o ca a-
geenans.
Figu e 1. Chemical s uc u es o he kappa-ca ageenan diad (κ) and he io a-ca ageenan diad (ι)
a anged in sequences in he KI block copolyme s o making up he whole polysaccha ide chain in
he co esponding homopolyme s.
Despi e hei p omise, hese copolyme s p esen se e al ongoing esea ch challenges.
Thei chemical s uc u e is in luenced by a ious ac o s, such as he species o algae, he
ime and place o ha es , and he pa ame e s and me hods used du ing ex ac ion [14].
These ac o s do impac KI unc ionali y, as much as hey con ol he gelling p ope ies o
comme cial kappa- and io a-ca ageenans [15]. While hese a iables affec he mechani-
cal and s uc u al cha ac e is ics o he esul ing hyd ogels, he ela ionship be ween he
gela ion mechanism, he gel s uc u e, and he chemical s uc u e o hyb id ca ageenans
emains complex and poo ly unde s ood [10]. Addi ionally, while he gelling p ope ies
Figu e 1. Chemical s uc u es o he kappa-ca ageenan diad (
κ
) and he io a-ca ageenan diad (
ι
)
a anged in sequences in he KI block copolyme s o making up he whole polysaccha ide chain in
he co esponding homopolyme s.
Despi e hei p omise, hese copolyme s p esen se e al ongoing esea ch challenges.
Thei chemical s uc u e is in luenced by a ious ac o s, such as he species o algae, he
ime and place o ha es , and he pa ame e s and me hods used du ing ex ac ion [
14
].
These ac o s do impac KI unc ionali y, as much as hey con ol he gelling p ope ies o
comme cial kappa- and io a-ca ageenans [
15
]. While hese a iables a ec he mechanical
and s uc u al cha ac e is ics o he esul ing hyd ogels, he ela ionship be ween he
gela ion mechanism, he gel s uc u e, and he chemical s uc u e o hyb id ca ageenans
emains complex and poo ly unde s ood [
10
]. Addi ionally, while he gelling p ope ies
o comme cial kappa- and io a- ca ageenans a e ela i ely well-documen ed, hei gel
s uc u e–elas ic p ope ies ela ionships a e a om being ully cla i ied [16].
Gels 2025,11, 157 3 o 18
Na u ally, his is also he case o blends o kappa- and io a-ca ageenans (he e labeled
as “K+I”) o which con o e sial models a e p oposed (see, e.g., [
17
] and e e ences
he ein). By compa ing he expe imen al esul s o K+I gel elas ici y wi h blending ules,
phase-sepa a ed ne wo ks [
18
] signaled by a wo-s ep gela ion [
19
–
21
], in e pene a ed
ne wo ks [
22
], and co-agg ega ed ne wo ks whe e helices o kappa- and io a-ca ageenans
a e a leas pa ially sel -assembling [
23
] o o m an in e ac i e and associa i e ne wo k [
24
]
ha e been p oposed. The block copolyme na u e o hyb id ca ageenans impedes he
phase sepa a ion o kappa- and io a-ca ageenan blocks in he helical o coil con o ma ion.
The e o e, a sys ema ic compa ison o he solu ion and gel phases o KI and K+I appea s as
an elegan ou e o he elucida ion o he blend mic os uc u e in he gel phase. In e es ingly,
his ou e has no been much explo ed in he li e a u e, hough such a compa ison u ned
ou o be a s ong a gumen in a o o he copolyme na u e o KI by aking ad an age o
he ion speci ici y o kappa-ca ageenan in he p esence o po assium ca ions [
25
]. Indeed,
kappa-ca ageenan can be sepa a ed om io a-ca ageenan in K+I sys ems p epa ed in
po assium chlo ide, whe eas KIs do no show any phase sepa a ion [
25
]. KI and K+I
compa a i e phase s a es in bo h po assium chlo ide and sodium chlo ide we e p oduced
o a ange o composi ions a ying be ween 47 mol.% and 78 mol.% o
κ
[
26
]. These
s udies we e a he applica ion-o ien ed as he main objec i e was o sc een o egimes o
polysaccha ide concen a ion and ionic s eng h whe e KIs could be aluable al e na i es
o K+I gels.
He e, he ange o blends and copolyme composi ions is ex ended beyond he bal-
anced 50% usually ocused on in he li e a u e [
18
–
25
], o nea ly pu e kappa- and io a-
ca ageenans. Following a ecen s udy whe e a se o hyb id ca ageenans we e ex ac ed
om comme cial seaweeds [
27
], hese copolyme s a e he e alkali- ea ed o p oduce KI wi h
i ually no diads o he mu-ca ageenan (
µ
) o nu-ca ageenan (
ν
) ypes. These diads a e
mo e sulpha ed han
κ
and
ν
diads and a e known o limi o impede gel o ma ion [
10
]. The
la ge ampli ude oscilla o y shea (LAOS) beha io o p oduced KI and K+I gels is epo ed
as i has ecei ed less a en ion in he ca ageenan li e a u e [
10
,
16
,
28
]. Al hough LAOS is
less equen ly used nowadays due o he lack o a uni ied in e p e a ion o he esul s [
29
],
i has gi en in e es ing s uc u al cha ac e iza ion when he esul s a e i ed wi h ilamen
ne wo k heo ies o colloidal gel heo y o s ain-ha dening gels
(see, e.g., [16,30–32]).
LAOS cha ac e is ics a e expec ed o complemen he s uc u al in o ma ion in e ed om
he gels’ mechanical spec a measu ed o a ious polysaccha ide and sal concen a ions.
O e all, he app oach se he e is expec ed o con ibu e o he iden i ica ion o di e en gel
s uc u es in KI and K+I bu also o de ine he condi ions unde which hyb id ca ageenans
can be mos e ec i ely used o eplace io a- and kappa-ca ageenan blends, in pa icula
du ing indus ial p ocessing whe e la ge de o ma ions a e a play.
2. Resul s and Discussion
2.1. Cha ac e iza ion o P oduced Ca ageenans
The chemical s uc u es o all ex ac ed and alkali- ea ed ca ageenans a e displayed
in Table 1. The p o on NMR spec a used o he quan i a i e es ima ion o he ca ageenan
diads can be ound in Figu es S1 and S2 in he Supplemen a y Ma e ials. All ex ac ed
polysaccha ides a e copolyme s, whe eas he comme cial kappa-ca ageenan is a homopoly-
me since only
κ
could be de ec ed in he espec i e NMR spec um. All samples ha e no
o negligible amoun s o
µ
and
ν
(gi en he limi o esolu ion o p o on NMR), wi h he
excep ion o samples F and B
′
. While he compa ison be ween samples E and F allows
o assessing he impac o ew
ν
diads on he phase s a e, sample D
′
was ex ac ed om
ano he seaweed o eplace sample B
′
, since D
′
has he same
ι
con en bu wi h i ually
no
ν
. A ce ain amoun o Flo idean s a ch (ci ca 10 mol.% o o al polysaccha ides) was
Gels 2025,11, 157 4 o 18
ound in samples F, E′, and F′. The e o e, a new KI, sample H′, was p oduced and ea ed
wi h NaOH o compa e wi h sample E′and hus assess he impac o Flo idean s a ch.
Table 1. Chemical composi ions (ca ageenan diads om he kappa-ca ageenan amily in mol.%),
molecula mass Mw (in 10
5
g/mol), and polydispe si y index PDI o he hyb id ca ageenans
ex ac ed om comme cial seaweeds and alkali modi ied wi h KOH o NaOH. The chemical compo-
si ions o he comme cial kappa-ca ageenan (KAPPA) and he comme cial io a-ca ageenan (IOTA)
a e also gi en.
Sample
KOH
Sample
NaOH
ν µ ι κ Mw PDI ν µ ι κ Mw PDI
B 0 0 96 ±5 4 ±2 5.4 ±0.1 4.1 B′1±1 4 ±4 90 ±2 5 ±1 6.1 ±1.5 4.7
C
3
±
1
0 91 ±1 6 ±2 5.8 ±0.1 1.6 C′0±5 0 95 ±2 5 ±4 8.9 ±1.2 6.2
E 0 0 44 ±6 56 ±4 3.1 ±0.1 2.1 E′1±5 0 42 ±6 57 ±3 4.4 ±0.1 2.8
F
5
±
5
0 47 ±1 48 ±1 2.5 ±0.1 4.6 F′1±5 0 45 ±5 54 ±3 2.9 ±0.1 2.4
I 0 0 10 ±5 90 ±4 9.6 ±0.3 3.4 I′0 0 10 ±5 90 ±5 7.9 ±0.3 4.5
J 0 0 13 ±3 87 ±5 8.4 ±0.1 2.5 J′0 0 12 ±6 88 ±4 10.6 ±0.1 2.6
K 0 0 5 ±5 95 ±5 5.8 ±0.1 2.9 K′0 0 7 ±5 93 ±4 9.9 ±0.1 2.7
M 0 0 31 ±2 69 ±1 6.5 ±0.1 2.8 M′0 4 ±5 29 ±1 67 ±3 5.3 ±0.1 2.9
KAPPA
0 0 0 100 ±1 11.3 ±0.5 2.7 D′2±2 0 88 ±1 10 ±6 14.7 ±0.2 3.9
IOTA 0 0 92 ±1 8 ±5 9.0 ±0.2 4.7 H′0±5 1 ±5 37 ±6 62 ±1 3.1 ±0.1 2.6
The molecula mass dis ibu ions o all ca ageenans a e shown in Table 1. In e -
es ingly, he ype o alkali modi ica ion has a s ong e ec on bo h he weigh -a e aged
molecula mass Mw and he wid h o he dis ibu ion (PDI) o samples C and C
′
and
samples K and K
′
. Fo hese wo pai s o samples, he modi ica ion by NaOH (samples
C
′
and K
′
) gi es hyb id ca ageenans wi h a la ge Mw and e y di e en PDI when
compa ed wi h he polysaccha ides ea ed wi h KOH (samples C and K). S ong alkali
ea men s a e known o educe he Mw o ca ageenans [
33
]. Also, he endogenous sal s
b ough by he d ied seaweeds [
34
] may add o he alkali modi ica ion o he molecula
mass dis ibu ion. We hus suspec ha he seaweeds used o p oduce samples C and K a e
ich in po assium sal , he eby inc easing he ionic s eng h o KOH and p omo ing a la ge
chain scission han wi h NaOH. In con as o his, he emaining hyb id ca ageenans show
ei he simila molecula mass dis ibu ions (see samples B-B
′
, E-E
′
, and F-F
′
) o sligh ly
di e en Mw (see samples I-I
′
, J-J
′
, and K-K
′
). Mw is known o be a c i ical pa ame e o
gel o ma ion in ca ageenans. In pa icula , i has been ecen ly shown ha he c i ical
mass Mc below which no gel can be o med in KCl depends on he chemical s uc u e o
he ca ageenans [
35
]. Longe io a-ca ageenan chains a e needed o ob ain a gel in 0.1 M
KCl a a polysaccha ide concen a ion o 1 w .% when compa ed wi h KI chains. No e
he e ha all Mw alues epo ed in Figu e 1a e abo e he Mc ound o a ious hyb id
ca ageenans and non-comme cial kappa- and io a-ca ageenans [35].
2.2. Phase S a es in KCl and NaCl
The gel o solu ion s a e o all hyb id ca ageenans and blends o kappa- and io a-
ca ageenan p epa ed unde a ious sal condi ions and polysaccha ide concen a ions a e
p esen ed in Figu es 2and 3 o KCl and NaCl, espec i ely. Elemen al analysis showed
ha , due o he alkali ea men , all p oduced ca ageenans b ough Na
+
and K
+
in amoun s
a ying be ween 11 and 40 w .%. Thus, a he la ges ca ageenan concen a ions es ed
in Figu es 2and 3(2 w .%), he ac ual ionic s eng hs a e modi ied by 0.02 M o K
+
and
0.03 M
o Na
+
, a mos . Rep esen a i e images o he samples’ phases so ed in o solu ions,
suspensions (s able o se led), and gels (wi h o wi hou wa e syne esis) a e shown in
Figu e S3 in he Supplemen a y Ma e ials. O e all, he compa ison o he phases in he
igh and le columns in Figu es 2and 3con i med no able di e ences be ween KI and
blends [
12
,
26
]. No e he e ha he phase s a es ound o pu e io a-ca ageenan esemble
Gels 2025,11, 157 5 o 18
he phases epo ed he e o he blend wi h composi ion 10K+90I, wi h he excep ion o
low sal condi ions (0.01 M), whe e Michel e al. [
36
] epo ed he absence o gels. As o a
compa ison o pu e kappa-ca ageenan wi h he 90K+10I blend, he same au ho s ound
simila phases, excep a 0.01 M KCl whe e pu e kappa-ca ageenan did no o m gels a
0.5 and 1 w .%, and also in NaCl whe e pu e kappa-ca ageenan only o med gels a 2 w .%
o ionic s eng hs la ge han 0.1 M [36].
Gels 2025, 11, x FOR PEER REVIEW 5 o 19
o K
+
and 0.03 M o Na
+
, a mos . Rep esen a i e images o he samples’ phases so ed
in o solu ions, suspensions (s able o se led), and gels (wi h o wi hou wa e syne esis)
a e shown in Figu e S3 in he Supplemen a y Ma e ials. O e all, he compa ison o he
phases in he igh and le columns in Figu es 2 and 3 con i med no able diffe ences be-
ween KI and blends [12,26]. No e he e ha he phase s a es ound o pu e io a-ca agee-
nan esemble he phases epo ed he e o he blend wi h composi ion 10K+90I, wi h he
excep ion o low sal condi ions (0.01 M), whe e Michel e al. [36] epo ed he absence o
gels. As o a compa ison o pu e kappa-ca ageenan wi h he 90K+10I blend, he same
au ho s ound simila phases, excep a 0.01 M KCl whe e pu e kappa-ca ageenan did
no o m gels a 0.5 and 1 w .%, and also in NaCl whe e pu e kappa-ca ageenan only
o med gels a 2 w .% o ionic s eng hs la ge han 0.1 M [36].
Figu e 2. Phases o med in KCl by hyb id ca ageenans (KI, le columns) and by ca ageenan blends
(K+I, igh columns) wi h equi alen chemical composi ion in κ and ι, as a unc ion o 2 ac o s:
polysaccha ide concen a ion (KI o K+I, in w .%) and ionic s eng h o K
+
(in mol/L).
Gels (labeled in g een in Figu es 2 and 3) in he p esence o K
+
occu ed mo e eadily
in blends han in KI. Fo ι- ich samples (B and C), gela ion was mo e dependen on he
polysaccha ide concen a ion, occu ing only a copolyme concen a ions ≥ 1 w .%, indi-
ca ing a h eshold o o ming a s able ne wo k. Below his concen a ion, KI samples
o med s able suspensions o agg ega es, sugges ing ha he la e a e insufficien in num-
be and size o o m a ully connec ed ne wo k. Gi en he high cha ge densi y o ι, his
Figu e 2. Phases o med in KCl by hyb id ca ageenans (KI, le columns) and by ca ageenan blends
(K+I, igh columns) wi h equi alen chemical composi ion in
κ
and
ι
, as a unc ion o 2 ac o s:
polysaccha ide concen a ion (KI o K+I, in w .%) and ionic s eng h o K+(in mol/L).
Gels (labeled in g een in Figu es 2and 3) in he p esence o K
+
occu ed mo e eadily
in blends han in KI. Fo
ι
- ich samples (B and C), gela ion was mo e dependen on
he polysaccha ide concen a ion, occu ing only a copolyme concen a ions
≥
1 w .%,
indica ing a h eshold o o ming a s able ne wo k. Below his concen a ion, KI samples
o med s able suspensions o agg ega es, sugges ing ha he la e a e insu icien in
numbe and size o o m a ully connec ed ne wo k. Gi en he high cha ge densi y o
ι
,
his beha io is expec ed unde low K
+
concen a ions, whe e mono alen sal s like KCl
p o ide enough ionic shielding bu no enough o s able gel ne wo k o ma ion [
8
]. In
con as , all blends o med gels ac oss simila condi ions.
Gels 2025,11, 157 6 o 18
Gels 2025, 11, x FOR PEER REVIEW 7 o 19
Figu e 3. Phases o med in NaCl by hyb id ca ageenans (KI, le columns) and by ca ageenan
blends (K+I, igh columns) wi h equi alen chemical composi ion in κ and ι, as a unc ion o 2 ac-
o s: polysaccha ide concen a ion (KI o K+I, in w .%) and ionic s eng h o Na
+
(in mol/L). Phase
labeling is as in Figu e 2.
No ably, o samples F′, E′, H′, and M′ wi h 50–70 mol.% κ, no gels o med unde any
condi ion, con as ing s a kly wi h he co esponding blends. The diffe ences wi h sam-
ples E′ and F′ in Figu e 3 sugges ha he dis ibu ions o κ and ι blocks as well as hei
leng hs a e chemical a iables in luencing he o e all gelling pe o mance o KI in NaCl.
The effec o Flo idean s a ch on he gelling abili y o KI in NaCl could no be assessed as
samples H′ and E′ did no o m gels.
O e all, as much as he gelling unc ionali ies o ca ageenans a e a play, he esul s
displayed in Figu es 2 and 3 show ha hyb id ca ageenans can be a aluable al e na i e
o blends o kappa- and io a-ca ageenan bu only a speci ic sal and polysaccha ide con-
cen a ions. None heless, heological es ing is essen ial o a compa a i e es ima ion o
gel elas ici y and esis ance o de o ma ion.
Figu e 3. Phases o med in NaCl by hyb id ca ageenans (KI, le columns) and by ca ageenan
blends (K+I, igh columns) wi h equi alen chemical composi ion in
κ
and
ι
, as a unc ion o
2 ac o s
:
polysaccha ide concen a ion (KI o K+I, in w .%) and ionic s eng h o Na
+
(in mol/L). Phase labeling
is as in Figu e 2.
Fo
κ
- ich KI samples, ionic s eng h played a mo e signi ican ole, wi h gel o ma ion
pa icula ly challenged a ele a ed K
+
concen a ions (
≥
0.5 mol/L o samples J, and
>1 mol/L
o he co esponding blend). While po assium ions mo e e ec i ely induce he
helical and agg ega ed s a es o kappa-ca ageenan, sal -ou e ec s a la ge ionic s eng h
may lead o p ecipi a ion, comp omising gel o ma ion. A simila high sal sensi i i y was
obse ed in samples E and M a a K
+
concen a ion o 1 mol/L, whe e s able suspensions o
solu ions a e obse ed, whe eas low ionic s eng hs and low polysaccha ide concen a ions
hinde ed he gela ion.
Sample F showed di icul y in o ming gels, which only occu ed a high polysaccha-
ide concen a ions (2 w % KI) and ionic s eng hs o 0.5–1 mol/L, in e iden con as o he
blend wi h equi alen composi ion in kappa- and io a-ca ageenan diads. The con en o
p ecu so uni s in sample F (
≈
5 mol.%, see Table 1) may explain such a disc epancy as he
p esence o
ν
and
µ
diads in he KI chains is known o hei loss in gel abili y [
10
,
37
], which
is con i med by he gelling abili y o he p ecu so - ee sample E showing simila Mw.
Gels 2025,11, 157 7 o 18
Al e na i ely, he p esence o Flo idean s a ch in F could hinde he gel phase seen in he
co esponding blend. The exis ence o a cu -o con en in
κ
(ci ca 50 mol.%) needed o he
gel o ma ion o hyb id ca ageenan unde speci ic KCl and polysaccha ide concen a ions
eme ges om he di e ences be ween sample F and samples E and M, whose phases in KCl
compa e well wi h hose o he blends and he phase s a e ound o a hyb id ca ageenan
con aining 53 mol.%
κ
[
26
]. The la e s udy epo ed ha KI p oduced gels wi h no wa e
syne esis, in con as o gels made om he blends. The da a in Figu e 2con i m he
syne esis in blends, bu samples E, F, and M a e also p one o wa e expulsion om he
ne wo k. This disc epancy sugges s ha possible di e ences in he Mw as well as in he
dis ibu ions o
κ
and
ι
sequences along he KI chains, which a e inhe en o he di e en
algal sou ces used he e, gi e di e en gel syne esis. No e also ha sligh di e ences in he
expe imen al p o ocols employed (in pa icula he he mal his o y du ing cooling) o in
he assessmen o wa e syne esis (subjec ed o expe imen alis s’ c i e ions) can also lead o
such disc epancies.
In NaCl, he di e ences be ween KI and blends a e e en mo e s iking, which poin s o-
wa d he Na
+
speci ici y o hese copolyme s when hey con ain be ween 50 and
90 mol% κ.
The gel o ma ion o blends con aining mo e kappa-ca ageenan was mos ly condi ioned
by low polysaccha ide concen a ions, no showing much dependence on he ionic s eng h
(con a ily o wha is seen in KCl). As expec ed, due o he non-speci ic ion esponse o io a-
ca ageenan [
8
],
ι
- ich KI samples (C
′
and D
′
) demons a ed consis en gelling beha io s in
bo h Na
+
and K
+
. In con as ,
κ
- ich samples (I
′
, J
′
, and K
′
) equi ed highe polysaccha ide
concen a ions o o m gels in NaCl han in KCl.
No ably, o samples F
′
, E
′
, H
′
, and M
′
wi h 50–70 mol.%
κ
, no gels o med unde any
condi ion, con as ing s a kly wi h he co esponding blends. The di e ences wi h samples
E
′
and F
′
in Figu e 3sugges ha he dis ibu ions o
κ
and
ι
blocks as well as hei leng hs
a e chemical a iables in luencing he o e all gelling pe o mance o KI in NaCl. The e ec
o Flo idean s a ch on he gelling abili y o KI in NaCl could no be assessed as samples H
′
and E′did no o m gels.
O e all, as much as he gelling unc ionali ies o ca ageenans a e a play, he esul s
displayed in Figu es 2and 3show ha hyb id ca ageenans can be a aluable al e na i e
o blends o kappa- and io a-ca ageenan bu only a speci ic sal and polysaccha ide
concen a ions. None heless, heological es ing is essen ial o a compa a i e es ima ion o
gel elas ici y and esis ance o de o ma ion.
2.3. Gel Fo ma ion Du ing Cooling
Samples C, E, K, C
′
, and K
′
(sample E
′
did no o m gels) we e chosen o heome y,
as hese di e ed no ably in
κ
- and
ι
-ca ageenan con en s, enabling he in es iga ion o
heological esponses ied o he
κ
-con en . This selec ion also acili a ed he e alua ion
o how polysaccha ide concen a ion and ionic s eng h in luence heology since hese
samples o med gels unde a b oade ange o condi ions, acili a ing di ec compa isons
wi hin each sample. Gi en he NMR- ela ed unce ain y in he
κ
-ca ageenan con en s in
samples C and C
′
, and because 10 w .% kappa-ca ageenan is expec ed o impac mo e
han 5 w .% on he gel heology, a K+I sample wi h a composi ion 10K+90I was chosen
o compa ison wi h samples C and C
′
. KI om M. s ella us, which esembles samples
M and M
′
, has been ex ensi ely s udied unde simila condi ions [
38
–
40
]. Thus, he
heological cha ac e iza ion o sample E was p e e ed he e. I gi es a unique oppo uni y
o explo e less-documen ed ca ageenan ypes, adding dep h o he heological indings, in
pa icula in a composi ion o
κ
and
ι
close o 50 mol.%, which has been much s udied in
blends o kappa- and io a-ca ageenan [
17
–
24
]. Simila concen a ions and ionic s eng hs
we e chosen o all selec ed KI and K+I whene e possible. Howe e , sample K and he
Gels 2025,11, 157 8 o 18
co esponding blend did no gel o o med gels wi h signi ican wa e syne esis, p ecluding
any heological compa ison. Thus, a di e en concen a ion o ionic s eng h was chosen.
Figu e 4gi es illus a i e he mo heological cu es measu ed du ing he cooling o
ho KI and K+I solu ions in o de o assess he sol–gel ansi ions. All cooling cu es a e
a ailable in Figu es S4 and S5 in he Supplemen a y Ma e ials, o gels o med in KCl and
NaCl, espec i ely. Th ee ypes o cooling we e obse ed. A clea sol- o-gel ansi ion
cha ac e ized by he c osso e be ween he shea s o age modulus G
′
and he shea loss
modulus G
′′
, occu ing a he gel ansi ion empe a u e Tg, can be in e ed om he da a
such as hose plo ed in Figu e 4a,c.
Gels 2025, 11, x FOR PEER REVIEW 8 o 19
2.3. Gel Fo ma ion Du ing Cooling
Samples C, E, K, C′, and K′ (sample E′ did no o m gels) we e chosen o heome y,
as hese diffe ed no ably in κ- and ι-ca ageenan con en s, enabling he in es iga ion o
heological esponses ied o he κ-con en . This selec ion also acili a ed he e alua ion o
how polysaccha ide concen a ion and ionic s eng h in luence heology since hese sam-
ples o med gels unde a b oade ange o condi ions, acili a ing di ec compa isons
wi hin each sample. Gi en he NMR- ela ed unce ain y in he κ-ca ageenan con en s in
samples C and C′, and because 10 w .% kappa-ca ageenan is expec ed o impac mo e
han 5 w .% on he gel heology, a K+I sample wi h a composi ion 10K+90I was chosen o
compa ison wi h samples C and C′. KI om M. s ella us, which esembles samples M and
M′, has been ex ensi ely s udied unde simila condi ions [38–40]. Thus, he heological
cha ac e iza ion o sample E was p e e ed he e. I gi es a unique oppo uni y o explo e
less-documen ed ca ageenan ypes, adding dep h o he heological indings, in pa icu-
la in a composi ion o κ and ι close o 50 mol.%, which has been much s udied in blends
o kappa- and io a-ca ageenan [17–24]. Simila concen a ions and ionic s eng hs we e
chosen o all selec ed KI and K+I whene e possible. Howe e , sample K and he co e-
sponding blend did no gel o o med gels wi h signi ican wa e syne esis, p ecluding
any heological compa ison. Thus, a diffe en concen a ion o ionic s eng h was chosen.
Figu e 4 gi es illus a i e he mo heological cu es measu ed du ing he cooling o
ho KI and K+I solu ions in o de o assess he sol–gel ansi ions. All cooling cu es a e
a ailable in Figu es S4 and S5 in he Supplemen a y Ma e ials, o gels o med in KCl and
NaCl, espec i ely. Th ee ypes o cooling we e obse ed. A clea sol- o-gel ansi ion
cha ac e ized by he c osso e be ween he shea s o age modulus G’ and he shea loss
modulus G″, occu ing a he gel ansi ion empe a u e Tg, can be in e ed om he da a
such as hose plo ed in Figu e 4a,c.
(a) (b) (c)
Figu e 4. Tempe a u e dependence o shea s o age modulus G’ and loss modulus G″ du ing he
cooling o (a) a blend 10% + 90% K+I a 1 w .% in 1 M KCl, (b) sample E a 2 w .% in 0.5 M KCl, and
(c) sample C a 2 w .% in 0.01 M KCl. The a ow in (c) indica es he empe a u e T2 whe e a second
s ep s a s du ing he gel o ma ion.
A mono onic inc ease o bo h moduli wi h he dec easing empe a u e, whe e no
c osso e is de ec ed and hus no Tg, is displayed in Figu e 4b. This ype o cooling sug-
ges s ha gela ion occu ed a empe a u es abo e 85 °C as G′ is la ge han G″ in he
whole empe a u e ange es ed. No e ha o sample C′ a 2 w .% in 1 M NaCl, G′ is
always smalle han G″ du ing he whole cooling down o 25 °C (see Figu e S5C). This
indica es ha sample C′ emained in he liquid s a e du ing cooling. The c osso e be-
ween G′ and G″ occu ed du ing he ime spen a 25 °C o he eco d o he emaining
gel p ope ies (see Figu e S9C showing G′ > G″ a la ge s ains). Finally, he cooling illus-
a ed in Figu e 4c shows wo s eps in he he mal e olu ion o G′ (o G″). A empe a u e
Figu e 4. Tempe a u e dependence o shea s o age modulus G
′
and loss modulus G
′′
du ing he
cooling o (a) a blend 10% + 90% K+I a 1 w .% in 1 M KCl, (b) sample E a 2 w .% in 0.5 M KCl, and
(c) sample C a 2 w .% in 0.01 M KCl. The a ow in (c) indica es he empe a u e T2 whe e a second
s ep s a s du ing he gel o ma ion.
A mono onic inc ease o bo h moduli wi h he dec easing empe a u e, whe e no
c osso e is de ec ed and hus no Tg, is displayed in Figu e 4b. This ype o cooling
sugges s ha gela ion occu ed a empe a u es abo e 85
◦
C as G
′
is la ge han G
′′
in he
whole empe a u e ange es ed. No e ha o sample C
′
a 2 w .% in 1 M NaCl, G
′
is
always smalle han G
′′
du ing he whole cooling down o 25
◦
C (see Figu e S5C). This
indica es ha sample C
′
emained in he liquid s a e du ing cooling. The c osso e be ween
G
′
and G
′′
occu ed du ing he ime spen a 25
◦
C o he eco d o he emaining gel
p ope ies (see Figu e S9C showing G
′
> G
′′
a la ge s ains). Finally, he cooling illus a ed
in Figu e 4c shows wo s eps in he he mal e olu ion o G
′
(o G
′′
). A empe a u e
T2 (indica ed by an a ow) lowe han Tg signals he onse o a second gelling p ocess
co esponding o he elas ic ein o cemen (inc ease in G
′
) o he gel ne wo k es ablished a
Tg. Two-s ep gela ion has been epo ed in K+I (see o ins ance [
19
,
20
,
22
,
24
,
41
]) and in
hyb id ca ageenan samples [
42
]. Tg and T2 a e lis ed in Table 2 o all samples es ed in
KCl and in Table 3 o all samples es ed in NaCl.
In KCl, bo h
ι
- ich hyb id ca ageenan samples (sample C) and hei co esponding
comme cial blends displayed wo-s ep gelling mechanisms. Despi e hei high
ι
-con en
(
≈
90 mol.%), hei gelling beha io de ia ed signi ican ly om he mono onic inc ease
in G
′
and G
′′
ypically obse ed in pu e io a-ca ageenan [
23
]. Because io a-ca ageenan
ypically gels a a highe Tg han kappa-ca ageenan, hough he di e ence in Tg depends
on he sal ype and concen a ion [
8
], he p esence o Tg and T2 sugges s ha e en small
κ
ac ions (
≈
10 mol.%) can o m mic odomains o seconda y agg ega es ha ein o ce he
io a-ca ageenan gel ne wo k and con ibu e o a second gela ion s ep. He e ogeneous mi-
c odomains ela ed o phase-sepa a ed o in e pene a ed kappa- and io a-ca ageenan as-
semblies ha e also been e idenced in K+I using sca e ing echniques [
18
,
23
],
NMR [18,43],
op ical mic oscopy [23], and pa icle acking [44].
Gels 2025,11, 157 9 o 18
Table 2. The mo heological p ope ies o gels o med in KCl. Gel ansi ion empe a u es (Tg and
T2), gel elas ic moduli G0 measu ed a equilib ium a 25
◦
C, and la ge de o ma ion p ope ies (LAOS)
quan i ied by he s ain
γF
o onse on gel- o- luid shea -induced ansi ion and quali ied by he
s ain-ha dening (HARD) o s ain-so ening (SOFT) beha io occu ing a a s ain smalle han γF.
Gelling Condi ions Samples Tg (◦C) T2 (◦C) G0 (Pa) γF(%) LAOS
2 w .%–0.01 M KCl C 43.6 ±0.2 31.7 ±0.1 2180 ±17 377 ±30 SOFT
10K + 90I 47.6 ±0.3 34 ±2 1022 ±20 118 ±13 SOFT/HARD
1 w .%–1 M KCl C >85 60 ±2 48.7 ±0.3 331 ±14 SOFT
10K + 90I 79.4 ±0.8 46 ±3 237 ±2 556 ±34 SOFT/HARD
2 w .%–1 M KCl C 85.4 ±4.3 74.3 ±0.5 985 ±6 404 ±30 SOFT/HARD
10K + 90I >85 - 11,281 ±238 388 ±30 SOFT
2 w .%–0.01 M KCl E 66.1±0.2 54 ±1 7831 ±23 37 ±5 SOFT
60K + 40I 47.5 ±0.4 39.5 ±0.5 15,351 ±46 60 ±7 SOFT/HARD
1 w .%–0.5 M KCl E >85 - 2391 ±14 35 ±4 SOFT
60K + 40I >85 - 157 ±1 104 ±10 SOFT
2 w .%–0.5 M KCl E >85 - 14,860 ±134 11 ±1 SOFT
60K + 40I >85 - 357 ±2 92 ±4 HARD
0.5 w .%–0.01 M KCl K 43.5 ±1.5 * - 490 ±4 123 ±6 HARD
90K + 10I 32 ±1 * 27 ±1 535 ±5 136 ±7 HARD
0.5 w .%–0.5 M KCl K >85 - 257 ±2 100 ±9 SOFT
90K + 10I >85 - 1650 ±61 25 ±2 SOFT
2 w .%–0.5 M KCl K >85 - 20,476 ±71 18 ±2 SOFT
90K + 10I >85 - 81,921 ±4012 3.7 ±0.6 SOFT
*: no c osso e be ween G′and G′′, a he he empe a u e whe e a s ep ise in G′occu s.
Table 3. The mo heological p ope ies o gels o med in NaCl. Gel ansi ion empe a u es (Tg and
T2), gel elas ic moduli G0 measu ed a equilib ium a 25
◦
C, and la ge de o ma ion p ope ies (LAOS)
quan i ied by he s ain
γF
o onse on gel- o- luid shea -induced ansi ion and quali ied by he
s ain-ha dening (HARD) o s ain-so ening (SOFT) beha io occu ing a a s ain smalle han γF.
Gelling Condi ions Samples Tg (◦C) T2 (◦C) G0 (Pa) γF(%) LAOS
2 w .%–0.01 M NaCl C′30.76 ±0.5 29.3 ±0.1 738 ±3 181 ±5 SOFT
10K + 90I 52.0 ±1.5 30 ±1 1400 ±128 155 ±13 SOFT/HARD
1 w .%–1 M NaCl C′83.9 ±3.4 - 100 ±60 391 ±47 SOFT
10K + 90I 55.6 ±0.3 - 800 ±400 25 ±3 SOFT
2 w .%–1 M NaCl C′<25 - 80 ±20 355 ±30 SOFT
10K + 90I >85 62 ±3 410 ±50 315 ±38 SOFT/HARD
2 w .%–0.01 M NaCl K′39.4 ±0.4 40.5 ±0.5 18,226 ±111 36 ±5 SOFT
90K + 10I 44.5 ±0.5 * - 3832 ±140 100 ±5 HARD
1 w .%–0.5 M NaCl K′53 ±1 * - 3384 ±82 41 ±2 HARD
90K + 10I 46.0 ±0.3 * - 6797 ±45 23 ±1 SOFT
2 w .%–0.5 M NaCl K′50 ±1 * - 3118 ±96 11 ±4 HARD
90K + 10I 54.7 ±0.3 * - 25,535 ±311 49 ±7 HARD
*: no c osso e be ween G′and G′′, a he he empe a u e whe e a s ep ise in G′occu s.
P e ious s udies [
20
,
22
] ha e epo ed he o ma ion o phase-sepa a ed and/o in e -
pene a ing ne wo ks in blends, highligh ing ha
κ
ac ions as low as 2.5% can signi ican ly
in luence hei heological beha io . Addi ionally, esidual
κ
-con en in he comme cial
io a-ca ageenan (see Table 1) likely ampli ies i s con ibu ion o ne wo k o ma ion in he
10K + 90I blend, aising he ac ual
κ
-con en o
≈
17.2 mol.%. This highe e ec i e
κ
ac ion
may explain he o ma ion o a seconda y gela ion phase in hese
ι
- ich sys ems. Howe e ,
since Tg and T2 also show up in hyb id ca ageenans, a wo-s ep gela ion canno be sys-
ema ically associa ed wi h phase sepa a ion. A la ge composi ions in kappa-ca ageenan
in he blend and
κ
-con en in he hyb id ca ageenan, gelling empe a u es we e only
eco ded a low sal and ca ageenan concen a ions, and a single he mal p ocess (Tg) was
mainly eco ded. Ei he o que limi a ion a highe empe a u es impedes a clea p obe o
he ne wo king o he smalle
ι
ac ion o he elas ici y o he
κ
ne wo k, building up a
empe a u es close o he ιne wo k [8], masks he much so e ιone.
Gels 2025,11, 157 16 o 18
and each column shows esul s a di e en concen a ions and ionic s eng hs. Top ow—sample
C s. K+I 10K+90I a (A) 2 w .% KI o K+I in 0.01 M KCl; (B) 1 w .% KI o K+I in 1 M KCl; middle
ow—sample E s. K+I 60K+40I a (C) 2 w .% KI o K+I in 0.01 M KCl; (D) 1 w .% KI o K+I in
0.5 M KCl; and bo om ow—sample K s. K+I 90K+10I a (E) 0.5 w .% KI o K+I in 0.01 M KCl;
(F) 0.5 w .%
KI o K+I in 0.5 M KCl; Figu e S7: Mechanical spec a (s o age modulus G
′
, ull squa es;
loss modulus G
′′
, open squa es) o hyb id ca ageenan (KI) samples (in blue) compa ed o hei
espec i e comme cial K+I ca ageenan blends (in ed) a a ious polyme concen a ions and ionic
s eng hs, in NaCl. Each ow ep esen s a di e en hyb id ca ageenan and blend pai : ( op) sample
C
′
s. K+I 10K+90I, a (A) 2 w .% in 0.01 M NaCl; (B) 1 w .% in 1 M NaCl; (C) 2 w .% in 1 M NaCl;
and (bo om) sample K
′
s. K+I 90K+10I, a (D) 2 w .% in 0.01 M NaCl; (E) 1 w .% in 0.5 M NaCl; (F)
2 w .% in 0.5 M NaCl; Figu e S8: La ge ampli ude oscilla o y shea es s (s o age modulus G
′
, ull
squa es; loss modulus G
′′
, open squa es, as a unc ion o he applied s ain) o hyb id ca ageenan
(KI) samples (in blue) compa ed o hei espec i e comme cial K+I ca ageenan blends (in ed) a
a ious polyme concen a ions and ionic s eng hs in KCl solu ions. Each ow ep esen s a di e en
hyb id ca ageenan and blend pai , while each column shows esul s a di e en concen a ions and
ionic s eng hs. Top ow—sample C s. K+I 10K+90I a (A) 2 w .% KI o K+I in 0.01 M KCl; (B) 1 w .%
KI o K+I in 1 M KCl; (C) 2 w .% KI o K+I in 1 M KCl; middle ow—sample E s. K+I 60K+40I a (D)
2 w .% KI o K+I in 0.01 M KCl; (E) 1 w .% KI o K+I in 0.5 M KCl; (F) 2 w .% KI o K+I in 0.5 M KCl;
and bo om ow—sample K s. K+I 90K+10I a (G) 0.5 w .% KI o K+I in 0.01 M KCl; (H) 0.5 w .% KI
o K+I in 0.5 M KCl; (I) 2 w .% KI o K+I in 0.5 M KCl; Figu e S9: La ge ampli ude oscilla o y shea
es s (s o age modulus G
′
, ull squa es; loss modulus G
′′
, open squa es, as a unc ion o he applied
s ain) o hyb id ca ageenan (KI) samples (in blue) compa ed o hei espec i e comme cial K+I
ca ageenan blends (in ed) a a ious polyme concen a ions and ionic s eng hs in NaCl solu ions.
Each ow ep esen s a di e en hyb id ca ageenan and blend pai , while each column shows esul s
a di e en concen a ions and ionic s eng hs. Top ow—sample C
′
s. K+I 10K+90I a (A) 2 w .%
KI o K+I in 0.01 M NaCl; (B) 1 w .% KI o K+I in 1 M NaCl; (C) 2 w .% KI o K+I in 1 M NaCl; and
bo om ow—sample K
′
s. K+I 90K+10I a (D) 2 w .% KI o K+I in 0.01 M NaCl; (E) 1 w .% KI o
K+I in 0.5 M NaCl; (F) 2 w .% KI o K+I in 0.5 M NaCl.
Au ho Con ibu ions: Concep ualiza ion, L.H.; me hodology, L.H.; in es iga ion, M.A.F.M. and
I.C.F.M.; esou ces, L.H.; da a cu a ion, M.A.F.M. and I.C.F.M.; w i ing—o iginal d a p epa a ion,
M.A.F.M., B.F. and L.H.; w i ing— e iew and edi ing, M.A.F.M., B.F., I.C.F.M. and L.H.; isualiza-
ion, M.A.F.M. and L.H.; supe ision, I.C.F.M., B.F. and L.H.; p ojec adminis a ion, L.H.; unding
acquisi ion, L.H. All au ho s ha e ead and ag eed o he published e sion o he manusc ip .
Funding: This wo k was suppo ed by he Fundação pa a a Ciência e Tecnologia (FCT), h ough he
E2B2-PHACAR p ojec , g an numbe : PTDC/BII-BIO/5626/2020 (h p://doi.o g/10.54499/PTDC/
BII-BIO/5626/2020). Addi ional inancial suppo by he FCT unde he amewo k o S a egic
Funding g an : UID/CTM/50025/2020 and g an : CEECINST/00156/2018 is also acknowledged.
Ins i u ional Re iew Boa d S a emen : No applicable.
In o med Consen S a emen : No applicable.
Da a A ailabili y S a emen : The aw da a suppo ing he conclusions o his a icle will be made
a ailable by he au ho s on eques .
Acknowledgmen s: The s ong suppo o Ca gill o he E2B2-PHACAR p ojec is acknowledged
and also CAPES o he g an awa ded o I.C.F.M.
Con lic s o In e es : The au ho s decla e no con lic s o in e es .
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Disclaime /Publishe ’s No e: The s a emen s, opinions and da a con ained in all publica ions a e solely hose o he indi idual
au ho (s) and con ibu o (s) and no o MDPI and/o he edi o (s). MDPI and/o he edi o (s) disclaim esponsibili y o any inju y o
people o p ope y esul ing om any ideas, me hods, ins uc ions o p oduc s e e ed o in he con en .
