Hybrid carrageenans as alternatives to commercial blends: rheological properties and performance in a food application

Uni e si y o Minho
School o Enginee ing
Ma ia Alice F ei as Mon ei o
Hyb id Ca ageenans as Al e na i es
o Comme cial Blends: Rheological
P ope ies and Pe o mance in a
Food Applica ion
Janua y 2025
Hyb id Ca ageenans as Al e na i es o Comme cial Blends:
Rheological P ope ies and Pe o mance in a Food Applica ion
Ma ia Alice F ei as Mon ei o
UMinho | 2025
Uni e si y o Minho
School o Enginee ing
Ma ia Alice F ei as Mon ei o
Hyb id Ca ageenans as Al e na i es o
Comme cial Blends: Rheological P ope ies
and Pe o mance in a Food applica ion
Janua y 2025
Mas e ’s Disse a ion
Mas e ’s in Chemical and Biological Enginee ing
Disse a ion supe ised by
P o . D . B uno Fa ia and
P o . D . Loïc Hilliou
ii
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ACKNOWLEDGMENTS
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To P o esso Izabel Mo aes, whom along he way I go he pleasu e o conside my iend Bel, my mos

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amewo k o S a egic Funding g an : UID/CTM/50025/2020 and g an : CEECINST/00156/2018 a e
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esea ch opics and p o iding he E2B2-PHACAR p ojec wi h comme cial seaweeds.
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Ma ia Alice F ei as Mon ei o
i
RESUMO
Ca ageninas Híb idas como Al e na i as a Mis u as Come ciais: P op iedades
Reológicas e Desempenho numa Aplicação Alimen a
O p incipal obje i o des e p oje o oi compa a as p op iedades eológicas e os mecanismos de geli icação
de ca ageninas híb idas (KI) e mis u as come ciais (K+I), com composição equi alen e de κ, sob
di e en es concen ações de sal e de polissaca ídeo. A ca ac e ização das ca ageninas ex aídas incluiu
¹H-RMN, FTIR e análise da dis ibuição de peso molecula . Os pe is de geli icação o am a aliados com
diag amas de ase, enquan o os mecanismos de geli icação e o compo amen o iscoelás ico o am
es udados eologicamen e. O desempenho das ca ageninas híb idas numa o mulação de gomas egan
ambém oi in es igado, compa ando-as com mis u as come ciais e a aliando a in luência de p ecu so es
biológicos, bem como a inco po ação de um adi i o na u al.
A geli icação das KI oi mais dependen e da concen ação de polissaca ídeo e da o ça iónica,
enquan o as K+I geli ica am sob a maio ia das condições. Em si uações especí icas, como al as
concen ações de polissaca ídeo em amos as icas em ι ou equilíb io κ/ι, e o ças iónicas baixas em
amos as icas em κ, obse ou-se uma con e gência nos pe is de geli icação. As K+I p oduzi am géis
mais ígidos e o es, mas as KI ap esen a am elas icidade supe io em algumas condições, des acando
o seu po encial em aplicações especí icas. Con udo, maio elas icidade implicou meno esis ência dos
géis à de o mação, esul ando em maio agilidade. Na o mulação de gomas, as KI mos a am po encial
como subs i u os das K+I em e mos de desempenho é mico, mas as mis u as p oduzi am géis mais
o es. O a amen o alcalino das KI aumen ou a igidez e a esis ência à de o mação dos géis, enquan o
a inco po ação do ma e ial biohíb ido melho ou a igidez, mas eduziu a esis ência, indicando a
necessidade de o imização pa a equilib a o desempenho écnico e a pe ceção do consumido .
Es es esul ados e idenciam o po encial das ca ageninas híb idas como al e na i as a mis u as
come ciais, com aplicações p omisso as em alimen os uncionais. Em úl ima análise, a iabilidade das
ca ageninas híb idas como subs i u os de mis u as adicionais depende do equilíb io desejado en e
o ça, elas icidade e esis ência à de o mação do gel.
Pala as-cha e: Ca agenina híb ida, Mis u as de ca ageninas, Reologia, Ca agenina K-2,
Indús ia alimen a
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ABSTRACT
Hyb id Ca ageenans as Al e na i es o Comme cial Blends: Rheological P ope ies and
Pe o mance in a Food Applica ion
This s udy p o ides a comp ehensi e compa ison be ween he he mal- heological p ope ies o hyb id
ca ageenans (KI) and comme cial K/I ca ageenan blends (K+I) wi h equi alen κ con en , ocusing on
hyd ogels o med unde a ying sal and ca ageenan concen a ions. 1H-NMR, FTIR, and molecula
weigh dis ibu ion analyses we e employed o cha ac e ize hyb id ca ageenans, while phase diag ams
and heological es s ( empe a u e, equency, and s ain sweeps) e alua ed gel o ma ion p o iles, gelling
mechanisms, and iscoelas ic p ope ies. Addi ionally, he s udy in es iga ed he pe o mance o hyb id
ca ageenans in egan gummy candy o mula ions, compa ing hem o comme cial blends and assessing
he in luence o biosyn he ic p ecu so s and he inco po a ion o a na u al addi i e.
KI showed g ea e dependence on polysaccha ide concen a ion and ionic s eng h o gel o ma ion
compa ed o K+I, which gelled mo e eadily unde mos condi ions. KI demons a ed compa able
pe o mance unde speci ic condi ions, such as highe polysaccha ide concen a ions o ι- ich and
balanced κ/ι samples o lowe ionic s eng hs o κ- ich samples. While K+I gene ally p oduced s onge
and s i e gels, KI su passed hem in elas ici y unde ce ain ionic condi ions, highligh ing i s po en ial
o a ge ed applica ions despi e ade-o s such as inc eased b i leness.
In gummy candy o mula ions, KI success ully p oduced gel-like ex u es and demons a ed po en ial
as a eplacemen o K+I in e ms o he mal pe o mance. While K+I p oduced s i e and s onge gels,
he alkaline ea men o hyb ids gene ally enhanced hei s i ness and s ain esis ance. The
inco po a ion o he biohyb id ma e ial imp o ed s i ness bu educed s ain esis ance, unde sco ing he
need o o mula ion-speci ic op imiza ion o balance echnical pe o mance and consume appeal.
These indings highligh he po en ial o hyb id ca ageenans as clean-label, sus ainable al e na i es
o comme cial blends, wi h p omising applica ions in unc ional oods. Ul ima ely, he iabili y o hyb id
ca ageenans as subs i u es o comme cial blends depends on he desi ed balance be ween gel s eng h,
elas ici y, and de o ma ion esis ance.
Keywo ds: Hyb id ca ageenan, Ca ageenan blends, Rheology, K-2 ca ageenan, Food indus y
xi
Blend 50κ50ι, wi h a balanced κ/ι a io; and C) Sample J and Blend 90κ10ι, ich in κ-ca ageenan.
........................................................................................................................................................ 86
Figu e 19: Mic oscopic images o gummy candy samples using un ea ed ca ageenan C (UT-C) (A, D),
alkali- ea ed ca ageenan C (T-C) (B, E), and comme cial blend 10κ90ι (C, F), wi hou (A-C) and wi h
(D-F) he addi ion o a biohyb id ma e ial (BH). The biohyb id, composed o an hocyanins adso bed on o
ben oni e, appea s as ed o g een pa icles (D-F). ............................................................................ 87
Figu e 20: FTIR-ATR spec a o gummy candies p epa ed, using un ea ed ca ageenan (UT-J), alkali
ea ed ca ageenan (T-J) and he chemical equi alen comme cial blend (90κ10ι), wi h and wi hou he
biohyb id ma e ial (BH), o sample J. ............................................................................................... 96

x
LIST OF TABLES
Table 1: FTIR band assignmen o each ca ageenan disaccha ide a ............................................... 31
Table 2: 1H-NMR chemical shi s (ppm) o he α-anome ic p o ons o ca ageenan disaccha ides b ... 32
Table 3: Fo mula o he gummy samples, exp essed as weigh pe cen age (% w/w) ......................... 47
Table 4: Syn hesized desc ip ion o he compa a i e sys ems s udied, and labelling o egan gummy
candies p oduced ............................................................................................................................. 48
Table 5: Phase diag ams o KI (le ) and o K+I blends ( igh ), in KCl ............................................... 64
Table 6: Phase diag ams o KI (le ) and o K+I blends ( igh ), in NaCl ............................................. 67
Table 7: Values o Tg, Ts a 1 and Ts a 2, along wi h hei associa ed e o s o each sample, o bo h KCl and
NaCl, as well as a cha ac e iza ion o he gela ion mechanism (single-s ep o wo-s ep p ocess) ......... 71
Table 8: Rheological p ope ies o hyb id ca ageenans and comme cial blends gelled in KCl and NaCl
unde a ying polysaccha ide and ionic condi ions. The able includes gel elas ici y in he LVE ange (G0),
limi ing s ain (γL), and low s ain (γF), as well as s ain ha dening and so ening beha io s unde la ge
ampli ude oscilla o y shea (LAOS). Cases whe e s ain so ening is ollowed by s ain ha dening a e
indica ed as “No hen Yes” ............................................................................................................... 80
Table 9: Elas ic and heological p ope ies o gummy candy samples, p epa ed using comme cial
ca ageenan blends (mol.% κ; mol.% ι), KOH- ea ed (T-) o un ea ed (UT-) hyb id-ca ageenans, wi h (+
BH) o wi hou he biohyb id ma e ial. The able p esen s he gel elas ici y in he LVE ange (G0), limi ing
s ain (γL), and low poin (γF), as well as s ain ha dening and so ening beha io s unde la ge ampli ude
oscilla o y shea (LAOS). Cases whe e s ain so ening is ollowed by s ain ha dening a e indica ed as
“No hen Yes” .................................................................................................................................. 92
x i
LIST OF ABBREVIATIONS
ACN
AFM
AG
ATR
BH
CG
CGS
CS
DRIFT
DS
DSC
EFSA
FDA
FTIR
GC
GRAS
IR
KI
K+I
LAOS
LVE
NMR
PDI
PSS
PSD
SAOS
SAXS
SEC
SEM
SS
An hocyanin
A omic Fo ce Mic oscopy
Anhyd ogalac ose
A enua ed To al Re lec ance
Biohyb id Ma e ial
Clea Gel
Clea Gel wi h Syne esis
Clea Solu ion
Di use Re lec ance In a ed Fou ie T ans o m Spec oscopy
Deg ee o Subs i u ion
Di e en ial Scanning Calo ime y
Eu opean Food Sa e y Au ho i y
Food and D ug Adminis a ion
Fou ie T ans o m In a ed Spec oscopy
Gummy Candy
Gene ally Rega ded as Sa e
In a ed
Hyb id Ca ageenan
Comme cial Ca ageenan Blends
La ge Ampli ude Oscilla o y Shea
Linea Viscoelas ic ( egion)
Nuclea Magne ic Resonance
Polydispe si y Index
Phase-Sepa a ed (se led) Suspension
Pa icle Size Dis ibu ion
Small Ampli ude Oscilla o y Shea
Small-Angle X- ay Sca e ing
Size Exclusion Ch oma og aphy
Scanning Elec on Mic oscopy
S able Suspension
x ii
TG
TGS
TPA
TS
T-KI
UT-KI
XAN
ZP
Tu bid Gel
Tu bid Gel wi h Syne esis
Tex u e P o ile Analysis
Tu bid Solu ion
T ea ed Hyb id Ca ageenans
Un ea ed Hyb id Ca ageenans
Xan han Gum
Ze a Po en ial
xix
LIST OF SYMBOLS

ι
λ
µ
ν
K
I
L
K-2
G’
G’’
G0
Mw
γL
γF
Tg
Ts a
Kappa-ca ageenan disaccha ide moie y
Io a-ca ageenan disaccha ide moie y
Lambda-ca ageenan disaccha ide moie y
Mu-ca ageenan disaccha ide moie y
Nu-ca ageenan disaccha ide moie y
Kappa-ca ageenan polyme
Io a-ca ageenan polyme
Lambda-ca ageenan polyme
Kappa-2 ca ageenan, kappa/io a hyb id o weak-gelling kappa-ca ageenan polyme
S o age o elas ic modulus (Pa)
Loss o iscous modulus (Pa)
Elas ic modulus in he linea iscoelas ic egion (Pa)
Weigh -a e age molecula mass (g/mol)
Limi ing s ain (%)
Flow s ain (%)
Gela ion empe a u e (ºC)
Gela ion onse empe a u e (ºC)

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1. INTRODUCTION
1.1 Con ex and Mo i a 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
(Blakemo e, 2016; Souza e al., 2023). 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 (Blakemo e, 2016;
Ho chkiss e al., 2016). The e sa ili y and unc ionali y o ca ageenans make hem indispensable in ood
echnology, con ibu ing o ex u e, mou h eel, and p oduc s abili y.
The ocus o mos comme cial applica ions has been on he use o K (kappa) and I (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. Ex ensi e esea ch
has been conduc ed on hese ca ageenans, pa icula 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 (de V ies, 2002; Souza e al., 2023). Howe e ,
he inc easing demand o K and I ca ageenans in bo h ood and non- ood indus ies has ecen ly exe ed
signi ican p essu e on ca ageenan p oduc ion and seaweed a ming (Aze edo e al., 2014; Bixle &
Po se, 2011), making i u gen o look o unde exploi ed and sus ainable esou ces.
In his con ex , K/I-hyb id ca ageenans, which consis o a andom block copolyme o κ-ca ageenan
and ι-ca ageenan sequences, ha e eme ged as p omising al e na i es o adi ional comme cial blends
(Bixle e al., 2001; Villanue a e al., 2004). Indus ially known as weak kappa o K-2, hese hyb ids o m
he mo e e sible hyd ogels wi h in e media e elas ici y in compa ison o hose o K and I, o e ing unique
s uc u al and pe o mance ad an ages in ce ain applica ions (Bixle e al., 2001; Hilliou, 2014;
Ho chkiss e al., 2016; Villanue a e al., 2004). Mo eo e , hey a e p edominan ly sou ced om wild
seaweeds in cold wa e s, unlike K and I ca ageenans, which a e mainly ob ained om in ense a ming
o wa m-wa e species, wi h associa ed p oduc ion issues (Wa d e al., 2020). This makes hyb id
ca ageenans no only a mo e sus ainable op ion bu also po en ially mo e cos -e ec i e, by elimina ing
he need o blending di e en ypes o ca ageenans (Souza e al., 2023).
Despi e hei p omise, hyb id ca ageenans 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 (Ficko-Blean e al., 2015). While hese
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a iables a ec he mechanical and s uc u al cha ac e is ics o he esul ing hyd ogel, he ela ionship
be ween he gela ion mechanism and he s uc u e o hyb id ca ageenans emains complex and poo ly
unde s ood (Souza e al., 2023; an de Velde, 2008). Addi ionally, while he p ope ies o comme cial K
and I ca ageenans a e ela i ely well-documen ed, his is no he case o hei blends o which
con o e sial models a e p oposed (Bha acha yya e al., 2024; Geonzon e al., 2023).
Fu he mo e, comp ehensi e s udies compa ing he gelling mechanisms, phase diag ams, and
heological p ope ies o hyb ids wi h adi ional blends a e s ill lacking (Aze edo e al., 2014; Souza e
al., 2023). Unde s anding he iscoelas ic p ope ies o hyb id ca ageenan gels unde la ge de o ma ion
egimes is also c ucial, as hese p ope ies di ec ly impac hei unc ionali y in indus ial p ocesses.
Con inued esea ch in his a ea will help o de ine he condi ions unde which hyb id ca ageenans can
be mos e ec i ely used, po en ially leading o new applica ions and imp o ed p oduc o mula ions.
This esea ch is d i en by bo h he scien i ic need o ill hese gaps and he p ac ical need o add ess
challenges wi hin he ood indus y. By explo ing hyb id ca ageenans and sys ema ically compa ing hem
wi h adi ional comme cial blends, his s udy aims o de e mine whe he hey can o e dis inc s uc u al
and unc ional ad an ages, such as enhanced s abili y, imp o ed ex u e, o g ea e p ocessing e iciency.
Mo eo e , hese indings po en ially con ibu e o a deepe unde s anding o he ela ionships be ween
chemical s uc u e, gelling beha io and iscoelas ic p ope ies o hyb id ca ageenans, unde small and
la ge de o ma ion egimes. Addi ionally, by e alua ing hei pe o mance in a speci ic ood applica ion,
his esea ch seeks o highligh whe he hyb id ca ageenans can p o ide conc e e bene i s o e
con en ional blends. These insigh s could help shape u u e esea ch di ec ions, guide indus y p ac ices,
and con ibu e o he de elopmen o inno a i e ood p oduc s ha mee e ol ing consume demands.
1.2 Resea ch Objec i es
The p ima y goal o his esea ch is o pe o m a comp ehensi e compa ison be ween he he mal-
heological p ope ies o hyb id ca ageenans and comme cial K and I ca ageenan blends—ensu ing
equi alen κ con en –, ocusing on he hyd ogels p oduced. By e alua ing hese wo ca ageenan
sys ems, he s udy seeks o de e mine he simila i ies and di e ences in how hey beha e unde di e en
sal and ca ageenan composi ions. This will p o ide insigh s in o how hese sys ems o m gels, hei
elas ici y, iscosi y, and o e all pe o mance in condi ions ha mimic eal-wo ld applica ions.
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The s udy also aims o compa e he pe o mance o hyb id ca ageenans in a speci ic ood applica ion
– egan gummy candies – wi h adi ional comme cial blends, c i ically assessing po en ial bene i s and
limi a ions o using hyb id ca ageenans as a iable al e na i e. Addi ionally, hyb id ca ageenans wi hou
alkaline ea men we e also es ed in he gummy o mula ions, o in es iga e he e ec s o biosyn he ic
p ecu so s on i s o e all pe o mance. Fu he mo e, he e ec s o inco po a ing a na u al addi i e in o he
o mula ion was also e alua ed ac oss all ca ageenan sys ems s udied.
1.3 Documen Ou line
This disse a ion is s uc u ed as ollows:
Chap e 1: In oduc ion p o ides an o e iew o he esea ch con ex , mo i a ion, and main
objec i es, emphasizing i s ele ance o bo h he ield and ma ke demands.
Chap e 2: S a e o he A e iews he cu en s a e o esea ch ela ed o his s udy, co e ing key
concep s and majo con ibu ions o he ield, while also add essing ongoing challenges and knowledge
gaps. Mo e speci ically, i p o ides insigh s in o dis inc ypes o ca ageenans, hei indus ial
applica ions, common ex ac ion and cha ac e iza ion me hods, gelling mechanisms, and heological
p ope ies. Addi ionally, his chap e also p o ides an o e iew o hei use in unc ional ood o mula ions.
Chap e 3: Me hodology ou lines he esea ch design and me hods used in his s udy. I co e s
ma e ial selec ion, hyb id ca ageenan ex ac ion and cha ac e iza ion, p epa a ion o comme cial blends,
and he me hods used o e alua e hei gelling mechanisms and heological p ope ies. The chap e also
de ails he o mula ion o ca ageenan-based gummies, and hei assessmen p o ocol.
Chap e 4: Resul s and Discussions p esen he s udy’s indings and a ho ough analysis o hei
signi icance. In he i s pa , he cha ac e iza ion o he hyb id ca ageenans ex ac ed is discussed,
ollowed by a sys ema ic compa ison o hei heological p ope ies, and phase diag ams, wi h hose o
he co esponding comme cial blends. In he second pa , he o e all pe o mance o hyb id ca ageenans
and adi ional comme cial blends in he o mula ion o egan gummy candies is assessed, highligh ing
po en ial ad an ages o disad an ages o using hyb id ca ageenans in ood indus y applica ions.
Addi ionally, he e ec s o biological ca ageenan p ecu so s in he hyb id chemical s uc u e, and he
impac o inco po a ing a na u al addi i e in he o mula ion a e also e alua ed.
Chap e 5: Conclusions and Fu u e Wo k summa izes key insigh s, s udy limi a ions, and
ecommenda ions o u u e esea ch.
23
2. STATE OF THE ART
This chap e p o ides a comp ehensi e e iew o he exis ing li e a u e and cu en ad ancemen s
ele an o his s udy.
2.1 Ca ageenans
Ca ageenans a e a ype o ma ine biopolyme s syn hesized by speci ic ed algae om he class
Rhodophy a (Ho chkiss e al., 2016; Souza e al., 2023), comp ising up o 75% o he d y weigh o he
algae (Pacheco-Qui o e al., 2020; Pe ei a, C i chley, e al., 2009). Thei main unc ion, wi hin he cell
wall o he ma ine algae, includes p o iding esis i e ac ion o oceanic s esses, such as osmose p essu e
and desicca ion du ing low ides (Ficko-Blean e al., 2015; Souza e al., 2023).
These hyd ophilic polysaccha ides ha e been well ecognized o he abili y o hei solu ions o o m
he mo e e sible hyd ogels, he eby making hem use ul o a ious indus ies as gelling, hickening, and
s abilizing agen s (Aze edo, To es, e al., 2015; Necas & Ba osiko a, 2013). Besides hei unc ional
p ope ies, hei bioac i e a ibu es, a o dabili y and ela i e abundance, make ca ageenans sui able o
ood, cosme ic and pha maceu ical applica ions (Ahmed e al., 2014; De Jesus Raposo e al., 2015;
Pacheco-Qui o e al., 2020; Udo e al., 2023), and mo e ecen ly bio emedia ion applica ions (Alshah ani
e al., 2021; Hao e al., 2023; Hossain e al., 2024; G. Sha ma e al., 2022).
2.1.1 Chemical S uc u e
Ca ageenans a e na u al complex polysaccha ides classi ied as sul a ed galac ans (Ficko-Blean e al.,
2015; Pacheco-Qui o e al., 2020), due o he p esence o 15% o 40% o es e -sulpha e wi hin hei
molecula s uc u e (Ciancia e al., 2020; Necas & Ba osiko a, 2013; Pacheco-Qui o e al., 2020; Véliz
e al., 2017). This sul u con en subs an ia es hei acidic and anionic na u e. The s uc u e o hese
polysaccha ides is cha ac e ized by linea backbones o al e na ing D-galac ose and 3,6-anhyd ogalac ose
(3,6-AG) uni s, linked by speci ic glycosidic bonds (α-1,3 and β-1,4). This o ms a epea ing suga uni
called ca abiose moie y (Ciancia e al., 2020; Ficko-Blean e al., 2015; Necas & Ba osiko a, 2013; an
de Velde, 2008). Ca ageenophy es di e om aga -p oducing algae (aga ophy es) because o he speci ic
s e eochemical a angemen o he bicyclic α-3,6-AG uni in he galac an chain, which is in he L-
con igu a ion in aga and in he D-con igu a ion in ca ageenans (Ciancia e al., 2020; Ficko-Blean e al.,
2015). While he chemis y o sul a ed galac ans om ed seaweeds is well-documen ed, he biosyn he ic
30
A e ex ac ion, solid algal esidues a e sepa a ed om he K-2- ich solu ion h ough il a ion o
cen i uga ion. The esul ing hyb id ca ageenan is ypically concen a ed by p ecipi a ion wi h alcohol o
KCl, ollowed by p essing o emo e excess liquid (Ho chkiss e al., 2016; Souza e al., 2023).
P ecipi a ion wi h KCl is o en p e e ed o eco e ing K-2 wi h enhanced gelling p ope ies (a highe κ
ac ion) o o pa ially emo ing non-ca ageenan componen s om ce ain ca ageenophy es (Ba ahona
e al., 2021; A. K a chenko e al., 2023; A. O. K a chenko e al., 2020; Pe ez Recalde e al., 2016).
Al e na i ely, hyb id ca ageenan can be eco e ed as a ilm o powde h ough wa e e apo a ion o
eeze-d ying o he K-2- ich solu ion (Souza e al., 2023). The pu i y o he inal ca ageenan depends on
he sepa a ion and eco e y me hods employed.
Based on ea lie ex ac ion s udies on a se o hyb id ca ageenophy es (Aze edo e al., 2013; Aze edo,
To es, e al., 2015; Hilliou, La o onda, e al., 2006), ex ac ion pa ame e s, namely, algal con en in
ex ac ion sol en , ex ac ion du a ion and empe a u e, alga-ca ageenan sol sepa a ion, and alkaline
modi ica ion p ocedu e we e employed as desc ibed in Sec ion 3.
2.3 Ca ageenans Cha ac e iza ion Techniques
The chemical s uc u e o ca ageenans can be assessed h ough se e al analy ical echniques, wi h NMR
spec oscopy and FTIR (Fou ie T ans o m In a ed Spec oscopy) being pa icula ly common (Pe ei a,
Amado, e al., 2009; Souza e al., 2023; an de Velde, 2008).
Vib a ional spec oscopy me hods like FTIR s and ou as p ac ical ools o quali a i ely assessing he
molecula s uc u e and composi ion o ma e ials (Pe ei a e al., 2003). FTIR is a as and non-des uc i e
echnique, which measu es he abso p ion o in a ed adia ion by a sample, equi ing only a small
amoun o ma e ial o accu a e measu emen s. The esul ing spec um gi es de ailed in o ma ion abou
he molecula bonds and chemical g oups p esen , as hese co espond o speci ic wa eleng hs (Gómez-
O dóñez & Rupé ez, 2011; Pe ei a, Amado, e al., 2009; Pe ei a e al., 2003). Recen esea ch combining
Fou ie ans o m algo i hms wi h a enua ed o al e lec ance (ATR) echniques has u he enhanced
adi ional in a ed spec oscopy, by inc easing he esolu ion o spec al da a, simpli ying sample
p epa a ion, and b oadening he ange o ma e ials ha can be analyzed (Gómez-O dóñez & Rupé ez,
2011; Pe ei a, Amado, e al., 2009).
When analyzing he s uc u e o ca bohyd a es, i e key equency egions a e ypically iden i ied in IR
spec a (4000–650 cm-1), as desc ibed by Ma hlou hi & Koenig (1987): (1) 3600–2800 cm-1: OH and CH

31
s e ching ib a ions; (2) 1500–1200 cm-1: local symme y egion; (3) 1200–950 cm-1: CO s e ching
ib a ions; (4) 950–700 cm-1: he anome ic egion; and (5) below 700 cm-1: he skele al egion. Fo
ca ageenans, ele an FTIR abso p ion bands a e obse ed a ound 2920, 1370, 1240, 930, 845, 820
and 805 cm-1, as egis e ed in Table 1. These bands p o ide insigh s in o hei s uc u e, on majo
ca ageenan ypes bu also mino componen s, such as μ and ν (Chopin e al., 1999; Gómez-O dóñez &
Rupé ez, 2011; Pe ei a, Amado, e al., 2009). Howe e , while FTIR is a powe ul ool, i does ha e
limi a ions. I o e s only a semi-quan i a i e analysis and may s uggle o clea ly iden i y he composi ion
o complex o he e ogeneous ca ageenan s uc u es. This is because mul iple chemical g oups can
con ibu e o he same abso p ion band, complica ing in e p e a ion (Chopin & Whalen, 1993; Rochas e
al., 1986).
Table 1: FTIR band assignmen o each ca ageenan disaccha ide a
Wa eleng h (cm-1)
Bond(s)/g oup(s) Assignmen
Found in Disaccha ides
2920
C-H o o al suga con en
κ, ι, μ, ν
1370, 1240–1260
S=O o sulpha e es e s
κ, ι, μ, ν
930, 1070 (shoulde )
C–O o 3,6-anhyd ogalac ose
κ, ι
840–850
C–O–SO3 on C4 o galac ose
κ, ι, μ, ν
820, 825 (shoulde )
C–O–SO3 on C2 o galac ose
ν
815–820, 867 (shoulde )
C–O–SO3 on C6 o galac ose
μ, ν
800–805, 905 (shoulde )
C–O–SO3 on C2 o 3,6-anhyd ogalac ose
ι
a Adap ed om Gómez-O dóñez & Rupé ez (2011), Aze edo e al. (2013) and Pe ei a, Amado, e al. (2009)
NMR spec oscopy, which elies on he esul ing signals om he in e ac ion be ween a omic nuclei
and an ex e nal magne ic ield, p o ides insigh s in o he chemical en i onmen , s uc u e, and
composi ion o samples (Dais & Spy os, 2012). Speci ically, 13C-NMR and 1H-NMR a ge ca bon-13 and
p o on a oms (hyd ogen), espec i ely (Souza e al., 2023). Al hough solid-s a e ¹³C-NMR spec oscopy
has shown po en ial o quali a i e e alua ion o ca ageenans in seaweed, i emains unde u ilized in his
con ex (Pe ei a, Amado, e al., 2009; Pe ei a e al., 2003; Souza e al., 2023).
1H-NMR spec oscopy allows o he quan i ica ion o a ious ca ageenan ypes by analyzing he
posi ion and in ensi y o he α-anome ic hyd ogen esonances om he epea ing disaccha ide uni s,
ypically ound be ween 5.1 and 5.7 ppm (Campo e al., 2009). The 1H-NMR chemical shi s (ppm) o he
α-anome ic p o ons o ca ageenan disaccha ides κ, ι, μ, and ν a e egis e ed in Table 2. Mo e
32
impo an ly, p o on NMR enables a quan i a i e assessmen o he ca ageenan chemical s uc u es, as
he peaks in ensi ies ela e o he mola con en o he co esponding nuclei.
Table 2: 1H-NMR chemical shi s (ppm) o he α-anome ic p o ons o ca ageenan disaccha ides b
Ca ageenan disaccha ide
Chemical shi s (ppm)
κ
5.09
ι
5.29
μ
5.24
ν
5.50
b Adap ed om Mo aes & Hilliou (2024) and Van De Velde e al. (2004)
2.4 Gelling Mechanism – Coil- o-Helix Ion-Speci ic T ansi ion
The gelling mechanism o ca ageenans is widely ecognized in li e a u e as a wo-s ep p ocess: a coil- o-
helix ansi ion upon cooling in he p esence o sal , ollowed by he sel -assembly o helices, in o a h ee-
dimensional ne wo k (Campo e al., 2009; Hilliou, 2014, 2021; an de Velde, 2008). The exac na u e
o he helices and supe s uc u es, and hei ela ion o gel elas ici y has long been deba ed, and se e al
models ha e been p oposed (Beaumon e al., 2021; Geonzon e al., 2020; Hilliou, 2021; Sche e e al.,
2015). Howe e , u he s udies a e equi ed o ully unde s and s uc u e-elas ici y co ela ions.
A omic o ce mic oscopy (AFM) has ecen ly p o ided de ailed images o p ima y coils, seconda y
helices, and la ge agg ega ed ne wo k s uc u es o K- and I-ca ageenans in he p esence o sodium,
calcium and po assium sal s (Diene e al., 2019; Sche e e al., 2015). As sal concen a ion inc eases,
elec os a ic epulsion be ween nega i ely cha ged sul a e g oups is educed, p omo ing he o ma ion o
in amolecula helical s uc u es. Expec edly, K-ca ageenan unde goes his ansi ion a lowe sal
concen a ions compa ed o I-ca ageenan. In NaCl and CaCl2 en i onmen s, a p og essi e ansi ion o
polyme chains om andom coils o helical con o ma ions is obse ed, wi h K-ca ageenan o ming od-
like, s i e helices and I-ca ageenan o ming cu lie , mo e lexible helices. In con as , KCl igge s u he
sel -assembly o hese helical polyme s ands in o sup amolecula s uc u es o highe complexi y a high
sal concen a ions.
In he p esence o po assium, Sche e e al. (2015) sugges ed ha K-ca ageenan o ms igid, wis ed
dime s ha agg ega e in o s i supe s ands, con ibu ing o b i le gel ne wo ks. Meanwhile, I-
33
ca ageenan o ms lexible, supe coiled mul i- ilamen s ands, con ibu ing o mo e elas ic gels. Diene
e al. (2019) imaged qua e na y s uc u es o s acked supe -s anded helices connec ed by helical
b anching in K-ca ageenan wi h K+, while I-ca ageenan appea ed as loosely in e wined single helices,
simila o i s beha io in NaCl. Thus, a c i ical eading o hese esul s sugges s ha bo h s and igidi y
and connec i i y in he ne wo k a e esponsible o he di e en elas ic p ope ies o K- and I-ca ageenan
gels.
The sc eening powe o he di alen ca ion Ca2+ is highe , when compa ed o he mono alen Na+, o
bo h K- and I-ca ageenan, esul ing in con o ma ional ansi ion a ela i e lowe sal concen a ions. In
gene al, he o de o e ec i eness o inducing coil- o-helix ansi ion o K-ca ageenan is K+ ≈ Ca2+ >
Na+, whe eas o I-ca ageenan Ca2+ > K+ > Na+ is ollowed (Sche e e al., 2015).
AFM images o K-2 ca ageenans helical assembly a e mos ly like K-ca ageenan, hough displaying
mo e b anching and a less od-like mo phology (Sokolo a e al., 2013; Souza e al., 2023). The coil- o-
helix ansi ions in K-2 a e dis inc o i s K and I blocks, simila o he beha io seen in K+I mix u es,
whe e bo h componen s unde go independen coil- o-helix ansi ions, ollowed by sel -assembly in o
helical s uc u es. These obse a ions we e con i med in di e en ial scanning calo ime y (DSC) s udies
pe o med on K-2 solu ions in he p esence o di e en sal s ( an de Velde e al., 2005) and in NaCl wi h
di e en ionic s eng hs (Souza e al., 2011). Rheological da a on K-2 solu ions cooled in NaCl also
showed a empe a u e dependence o wo s eps in bo h he shea s o age (G’) and loss modulus (G”),
sugges ing he agg ega ion o wo ypes o helical assemblies (Hilliou e al., 2014; Hilliou & Gonçal es,
2007; Souza e al., 2011; To es e al., 2017). Howe e , ca ageenan concen a ion, coun e ion ype
and ionic s eng h highly in luence he wo s eps gelling mechanism o K-2 and i s he mal hys e esis
(g ea e in he p esence o K+ (Chan ie e al., 2004)).
In gene al, Na+ p omo es wo-s ep gela ion in K-2 solu ions and a o s he independen coil- o-helix
ansi ions o I and K blocks, ollowed by he sel -assembly o he helical s uc u es. Con e sely, in KCl
o CaCl2 en i onmen s, a mo e s aigh o wa d single-s ep gela ion is usually seen in he heological
p o iles (Baha i e al., 2022; Cosenza e al., 2014; Hilliou, La on onda, e al., 2006; Hilliou & Gonçal es,
2007; Hughes e al., 2023; To es e al., 2018).
In K+I mix u es, elas ici y may a ise ei he om mic ophase sepa a ion o in e pene a ing ne wo ks
o ι- and κ- ich domains (B enne e al., 2014; Du e al., 2016; Geonzon e al., 2019; Hu e al., 2016;
Pa ke e al., 1993) o e en om co-agg ega ion o K and I helices in o complex supe s uc u es, which
could explain he la ge gel elas ici y o K+I compa ed o he sum o indi idual gel elas ici ies (Bui e al.,
34
2019). Al hough he gelling p ocess in K-2 esembles ha o K+I mix u es, he na u e o i s sel -assembly
and supe helical s uc u es, and he o igin o gel elas ici y, emain poo ly unde s ood.
Fu he compa a i e in es iga ions on K-2 and K+I blends unde a ying ca ageenan concen a ions,
sal ypes, and ionic s eng hs a e essen ial as s udies on blends nea ly exclusi ely ocused on equal
con en s o κ and ι. These s udies should include in-si u s uc u al cha ac e iza ion o be e unde s and
he hie a chical s uc u es and sou ces o elas ici y in hese gels (Souza e al., 2023). Al e na i ely, a
mo e comp ehensi e compa a i e s udy o phase diag ams and gel p ope ies o K-2 and K+I blends,
co e ing a wide ange o κ and ι composi ions han in Aze edo e al. (2014) and To es, Aze edo, e al.
(2016), could be pe o med, as p oposed he e.
2.5 Viscoelas ic P ope ies o Hyb id Ca ageenans
Despi e o K-2’s epo ed in e media e elas ic p ope ies be ween K and I ca ageenan gels, hei linea
iscoelas ic p ope ies a e a less s udied in li e a u e. In gene al, he mechanical spec a o K-2,
measu ed by Small Ampli ude Oscilla o y Shea (SAOS), show quali a i e simila i ies wi h K o I gels
(Souza e al., 2023). K-2 gels exhibi nea - equency-independen elas ic modulus (G') wi h no signi ican
wa e syne esis (Aze edo e al., 2013, 2014; Aze edo, To es, e al., 2015; Pon hie e al., 2020; Souza
e al., 2011; To es, Aze edo, e al., 2016; To es, Chenlo, e al., 2016; To es e al., 2017, 2018),
con as ing wi h K gels, which end o elease wa e du ing liquid- o-gel ansi ion, pa icula ly in he
p esence o K+ (Souza e al., 2023).
Howe e , he linea iscoelas ic p ope ies o K-2 gels a e also in luenced by a ious ac o s, such as
chemical composi ion, polysaccha ide concen a ion, sal ype and ionic s eng h.
2.5.1 E ec o K-2 Chemical Composi ion and Molecula Mass
The elas ic modulus o K-2 is signi ican ly in luenced by i s chemical composi ion, especially he mola
ac ion o κ. Highe κ con en co ela es wi h s onge gels, especially in KCl solu ions (Hughes e al.,
2023; an de Velde e al., 2005). Howe e , ew s udies ocus on he ela ionship be ween K-2’s
composi ion and i s iscoelas ic p ope ies (Souza e al., 2023). In con as , no clea co ela ion is
obse ed in NaCl, likely due o a ia ions in molecula mass (Mw). The size o he K-2 chain in luences
he elas ici y o gels o med in NaCl, wi h longe chains leading o gels wi h lowe G’ (Hilliou, La on onda,
e al., 2006; Souza e al., 2011; To es, Chenlo, e al., 2016), while no clea end is seen in KCl ( an de
35
Velde e al., 2005). The e o e, mo e s udies a e needed o unde s and he impac o K-2’s chemical
composi ion and molecula mass on he gel elas ic p ope ies, in NaCl and KCl.
2.5.2 E ec o Sal Type and Ionic S eng h
As al eady epo ed in Sec ion 2.4, he ype o sal and i s ionic s eng h signi ican ly impac he gela ion
beha io . The elas ic modulus inc eases wi h ionic s eng h, and gels o med in KCl and CaCl2 a e ypically
s onge han hose in NaCl, wi h K-ca ageenan e idencing g ea e ca ion speci ici y. O e all, K-2 exhibi s
ca ion-speci ic agg ega ion, especially o K-helices (Souza e al., 2023).
A s udy by To es e al. (2018) showed ha s onge K-2 gels we e ob ained in CaCl₂, while NaCl
p oduced he weakes . Howe e , he ange o polysaccha ide concen a ions and ionic s eng h o which
a gel is p oduced is e y di e en o each sal . This inding is e iden in s udies compa ing phase
diag ams o di e en hyb id ca ageenans concen a ions in di e en sal ypes and concen a ions, such
as Aze edo e al. (2014), To es, Aze edo, e al. (2016) and To es e al. (2018). These s udies ha e also
highligh ed how hose pa ame e s a ec he o ma ion o clea o u bid gels. Tu bid gels a e o en
associa ed wi h he o ma ion o pa icula e suspensions o K-2 ha ing mo e han 50 mol.% κ, along wi h
an inc ease in ionic s eng h.
2.5.3 E ec o Polysaccha ide Concen a ion
Simila ly o wha is epo ed in heological p ope ies o K and I gels, he elas ic modulus o K-2 inc eases
wi h polysaccha ide concen a ion, ollowing an exponen ial end (To es, Chenlo, e al., 2016), o a
powe law (Hilliou, 2021). Powe laws wi h di e se exponen alues ha e been sugges ed, due o he wide
ange o K-2 composi ions and sal condi ions used o p epa e he gels (Hilliou, 2021; Hilliou & Gonçal es,
2007; To es, Aze edo, e al., 2016). Howe e , i is possible o in e an inc ease o powe law exponen
alues (n) wi h highe ionic s eng h, sugges ing a shi om wo m-like o s i e od-like ilamen s uc u es
(Ca illo e al., 2013; Doi & Kuzuu, 1980; Jones & Ma ques, 1990; Meng & Te en je , 2016). Gel u bidi y
also co ela es wi h highe sal o κ con en , leading o coa se s uc u es in K-2 gels and la ge n
exponen alues (Souza e al., 2023).
2.5.4 Viscoelas ic P ope ies unde La ge De o ma ion
The s udy o he p ope ies o gels unde la ge de o ma ions a e o g ea indus ial ele ance and can
con ibu e aluable insigh s o unde s and he gel’s s uc u e. In he linea iscoelas ic egime, small

36
de o ma ions esul in a p opo ional esponse whe e p ope ies like he shea modulus (which quan i ies
s i ness) emain cons an . Howe e , unde la ge de o ma ions, ca ageenan gels de ia e om
p edic able, linea esponses, displaying ins ead complex nonlinea beha io s – i s shea modulus
changes, o en ha dening o so ening depending on he ilamen ous ne wo k s uc u e (Souza e al.,
2023). Ro a ional heome y applies simple shea (as opposed o comp essi e o inden ing o ces) o
s udy how he ma e ial beha es unde de o ma ion. Simple shea ensu es symme ic de o ma ion,
making i easie o measu e and in e p e s ess - s ain ela ionships (Souza e al., 2023).
Theo e ically, ilamen ous ne wo ks show s ain ha dening, which means he gel becomes s i e when
subjec ed o la ge s ain be o e i e en ually up u es, due o ini e ex ensibili y o he ilamen s –
i.e
. hey
a e s e ched be ween c osslinks in he ne wo k, and his s e ching causes hem o s i en as hey
app oach hei b eaking poin (Ca illo e al., 2013; Doi & Kuzuu, 1980; Hilliou, 2021; Meng & Te en je ,
2016). Such s e ching also supposes some deg ee o eedom be ween c osslinks, ha is, p e- ension
o ilamen s by he ne wo k is no ha big (Meng & Te en je , 2016).
Expe imen al da a om La ge Ampli ude Oscilla o y Shea (LAOS) shows ha K-2 gels, in KCl, unde go
s ain ha dening, showing a ela i ely so elas ici y, wi h shea elas ic modulus ypically below 1 kPa
(Aze edo e al., 2014; Baha i e al., 2022; Hilliou, 2021; Hilliou e al., 2009), when compa ed o K gels,
which ha e much s i e p ope ies (up o 10 kPa). S ain so ening, in which gels become less s i unde
inc eased s ain, was epo ed o ce ain K (Diene e al., 2019; Flo es e al., 2017; Hilliou, 2021), I
(Diene e al., 2019; Flo es e al., 2017), and K+I blends (Flo es e al., 2017), hough he mechanisms
behind his beha io emain unclea (Souza e al., 2023).
2.6 Hyb id Ca ageenans – Ma ke T ends and Applica ion in Func ional
Foods
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 ocolloids ma ke (in e ms o alue), a e gua gum, gela in, xan han gum,
cellulose gum, and a abic gum (Ho chkiss e al., 2016; Zhang e al., 2023). The global ca ageenan
ma ke was wo h $850 million in 2022 and is expec ed o g ow a an a e age yea ly g ow h a e o 6.2%,
eaching $1.55 billion by 2032 (Pulidindi & Ahuja, 2023).
Ca ageenans, wi h a molecula mass abo e 100 kDa (Necas & Ba osiko a, 2013), a e gene ally
ecognized as sa e (GRAS) o consump ion by bo h he FDA (Food and D ug Adminis a ion) and EFSA
37
(Eu opean Food Sa e y Au ho i y), whe e hey a e classi ied as ood addi i es E407 ( e ined) and E407a
(semi- e ined) (Rioux & Tu geon, 2015; Udo e al., 2023). Al hough oxicological assessmen s ha e
con i med ha ood-g ade ca ageenan has minimal physiological impac s, some s udies sugges ha
deg aded ca ageenans may cause gas oin es inal heal h p oblems (Go eland e al., 2020; Han e al.,
2019; O’Sulli an e al., 2010; Shang e al., 2017; Sun e al., 2019), aising conce ns abou i s sa e y
(Bixle , 2017; Necas & Ba osiko a, 2013). These con o e sies signi ican ly in luenced he ca ageenan
ma ke , d i ing consume demand owa ds pe cei ed sa e al e na i es like aga , xan han gum, and gua
gum (Pulidindi & Ahuja, 2023).
Beyond hei unc ional applica ions as ex u izing, s abilizing and gelling agen s, hyb id ca ageenans
also exhibi a ious bioac i e p ope ies and unique a ibu es, making hem aluable ac oss a b oad
ange o ields besides he ood indus y. These include applica ions in cosme ics and pe sonal ca e
p oduc s (Dubey & Dubey, 2020; Kim e al., 2018; Sha ie e al., 2022), he pha maceu ical and
biomedical indus ies (Cal o e al., 2019; De Jesus Raposo e al., 2015; Ha e al., 2022; Meh a i e al.,
2024; Pacheco-Qui o e al., 2020; Padhi e al., 2016; P adhan & Ki, 2023), and also bio emedia ion
echniques (Alshah ani e al., 2021; Hao e al., 2023; G. Sha ma e al., 2022).
2.6.1 Bioac i e, Nu i ional, and Heal h-P omo ing P ope ies
Rega ding unc ional ood applica ions, ca ageenans ha e been shown o p omo e heal h and p e en
diseases by exhibi ing an i i al, an ibac e ial, an ioxidan , an i umo al, an i-in lamma o y and immune-
boos ing e ec s (Bhuyan e al., 2023; Boulho e al., 2017; Dousip e al., 2014; P adhan e al., 2022;
P ema a hna e al., 2024; Valado e al., 2019; W. Wang e al., 2011). Ca ageenans also help imp o e
gu heal h by ac ing as p ebio ics, s imula ing he g ow h o bene icial gu mic obio a and inc easing he
p oduc ion o sho -chain a y acids, such as ace a e, p opiona e, and bu y a e (Che y e al., 2019;
Kuma i e al., 2023; Li e al., 2017; Liu e al., 2015). Mo eo e , he high le el o die a y ibe in hese
biopolyme s also adds o hei nu i ional alue, helping wi h weigh managemen and educing he isk
o ca dio ascula disease due o hei choles e ol-lowe ing e ec s (Dousip e al., 2014; Valado e al.,
2019).
In addi ion, ca ageenans ep esen impo an plan -based al e na i es o animal-de i ed ing edien s
like gela in, dai y, and p ocessed mea s, aligning wi h cu en ma ke ends (Blakemo e, 2016; Ho chkiss
e al., 2016; Ka im & Bha , 2008; Necas & Ba osiko a, 2013; Palanisamy e al., 2018; Pe ei a, C i chley,
e al., 2009; Rashmi & Mona, 2023; Russo Spena e al., 2024; Song e al., 2022; Udo e al., 2023).
38
In gene al, he la e desc ibed bioac i e and unc ional p ope ies o ca ageenans (syn he ized in
Figu e 3) make hem excellen candida es o inclusion in unc ional oods and nu aceu icals (Kuma i e
al., 2023; P ase yaning um e al., 2019), p o iding ex u e while also p omo ing heal h bene i s beyond
basic nu i ion.
2.6.2 In e ac ion wi h o he Hyd ocolloids and Ing edien s
Po en ial syne gis ic in e ac ions be ween ca ageenans and o he hyd ocolloids ha e been s udied o
modi y he ex u e, s abili y, and gela ion p ope ies o ood p oduc s (Ho chkiss e al., 2016; Song e al.,
2022; Tunie a e al., 2021). In pa icula , when combined wi h xan han gum, K-ca ageenans o m gels
wi h imp o ed iscoelas ic p ope ies, op imized ex u e, enhanced s abili y and educed wa e syne esis
(liquid sepa a ion om gels), allowing o he c ea ion o high-quali y egan o mula ions ha closely
eplica e he senso y p ope ies o adi ional gela in-based p oduc s (A allone e al., 2023;
Balasub amanian e al., 2018; B enne e al., 2015; Tunie a & Spi idono , 2020; Xu e al., 2022).
T adi ional gummy candies (GC) ha e gel-like s uc u es and a e made using a combina ion o suga s,
wa e , gelling agen s ( ypically gela in), and may also inco po a e ui s, acids, a omas, and ood colo an s
(Cappa e al., 2015; Ge e al., 2021; Mu lu e al., 2018; Teixei a-Lemos e al., 2021). Besides choosing
egan al e na i es o animal-based gela in, some o he ways o align GC o cu en ma ke ends and
consume demands, may comp ise u ilizing na u al colo an s, inco po a ing plan ex ac s, i amins, o
ui de i a i es, wi h bioac i e and unc ional p ope ies, and subs i u ing suga wi h o he swee ene s,
Figu e 3: Func ional and bioac i e p ope ies o hyb id ca ageenans. Adap ed om Pacheco-Qui o e al. (2020)
and P adhan & Ki (2023).
39
such as honey o s e ia (Mu lu e al., 2018; Palachum e al., 2023; Roudba i e al., 2024; Ta ahi e al.,
2023). These inno a ions aim o enhance he nu i ional p o ile o a adi ionally non-nu i ious p oduc ,
ans o ming i in o a heal hie op ion. This is pa icula ly signi ican gi en he widesp ead consump ion
o jellies and gummies, especially among child en and adolescen s unde 17 yea s old, which has been
associa ed wi h a high incidence o obesi y, oo h decay, and hype glycemia (G and View Resea ch, 2024;
Khawaja e al., 2019; Rippe & Angelopoulos, 2016; Ta ahi e al., 2023). Ca ageenan-based egan
gummy supplemen s ha e also been success ully used as a medium o deli e ing pha maceu icals
and/o nu i ional bioac i es, especially o child en (Cheng e al., 2022; Qiu e al., 2024; Rashmi &
Mona, 2023; Ta ahi e al., 2023b).
In ha con ex , bioac i e compounds such as an hocyanins (ACNs) ha e gained g ea a en ion as a
na u al ood colo an and unc ional ing edien (Lou enço e al., 2019; Rod iguez-Amaya, 2019). These
compounds a e esponsible o he ed, pu ple and blue hues obse ed in a ious ui s, ege ables,
lowe s and g ains and exhibi po en an ioxidan , an i-in lamma o y, an i-alle gic, an i-diabe ic, and an i-
ca cinogenic ac i i ies (Rod iguez-Amaya, 2019; Shipp & Abdel-Aal, 2010). Howe e , ACN ac i i y is highly
dependen on ex e nal ac o s such as ligh , pH, oxygen, empe a u e, and in e ac ions wi h o he
ing edien s, which limi s hei use in ce ain applica ions (Albuque que e al., 2021; Co ez e al., 2017;
Rod iguez-Amaya, 2019). To mi iga e hese limi a ions, s abiliza ion echniques a e commonly employed,
including copigmen a ion, exclusion o O2, encapsula ion/mic oencapsula ion wi hin biopolyme s and
adso p ion on o nanoclays (Ca alcan i e al., 2011; Coelho Leand o e al., 2021; Co ez e al., 2017;
Koop e al., 2024; Mahda i e al., 2014; Rod iguez-Amaya, 2019).
Thus, his wo k aimed o ex ac and cha ac e ize K/I hyb id ca ageenans sou ced om a di e se
ange o seaweed, ocusing on hei iscoelas ic p ope ies and hei gelling beha io , unde a ying sal
and ca ageenan concen a ions. These indings we e sys ema ically compa ed wi h comme cial
ca ageenan blends o equi alen κ and ι composi ion.
In esponse o cu en ma ke ends o plan -based unc ional p oduc s, a hyb id ca ageenan-based
egan gummy candy was de eloped and cha ac e ized. In his con ex , he hyb id ca ageenan’s
pe o mance in his speci ic ood applica ion was compa ed o ha o comme cial ca ageenan blends,
o iden ical composi ions. Mo eo e , he e ec s o including biosyn he ic ca ageenan p ecu so s and he
inco po a ion o an an hocyanin-based na u al addi i e we e de e mined by measu ing he iscoelas ic
p ope ies and o e all s abili y o he o mula ion.
46
samples we e chosen based on hei dis inc κ/ι a ios and gelling p o iles, as hey demons a ed gel
o ma ion ac oss a b oade ange o sal and polysaccha ide concen a ions. Fo each sample, h ee
di e en polysaccha ide concen a ions and ionic s eng hs we e selec ed o p o ide a comp ehensi e
unde s anding o hei iscoelas ic p ope ies.
3.5.2 Ro a ional Rheome y P ocedu e
The iscoelas ic p ope ies we e measu ed using a s ess-con olled o a ional AR-G2 heome e (TA
Ins umen s L d, New Cas le, DE, USA), equipped wi h a 25-mm pa allel-pla e geome y and a gap o 500
μm, a 85 °C. To p e en wa e e apo a ion du ing es ing, dodecane (Sigma-Ald ich, S . Louis, MO, USA)
was applied a ound he sample im. Da a acquisi ion was ca ied ou using Rheology Ad an age so wa e
(TA Ins umen s L d, New Cas le, DE, USA).
A ime sweep was pe o med, whe e ho solu ions we e cooled om 85 °C o 25 °C, o e 1.5 hou s
o assess he ca ageenan sol-gel ansi ion. The s ain ampli ude and equency we e se o 0.5% and
1.0 Hz, espec i ely, essen ially o a oid any s ain-induced change in he gel-se ing beha io . Du ing
cooling, he heome e ’s gap was adjus ed o accoun o he he mal expansion o he pla es, while he
no mal o ce was main ained a 0 ± 1 N o compensa e o po en ial olume changes du ing he sol- o-
gel ansi ion.
Subsequen ly, he mechanical spec um o he hyd ogels a 25 °C was measu ed using a equency
sweep om 100 o 0.01 Hz, wi h a s ain ampli ude o 0.5%, essen ially o keep he s ain wi hin he
linea egime o iscoelas ici y. Las ly, he linea and la ge de o ma ion iscoelas ic beha io o he
hyd ogels was analyzed a 25 °C by pe o ming a s ain ampli ude sweep be ween 0.1% and 1000%, wi h
a ixed equency o 1 Hz.
3.6 Vegan Gummy Candy Fo mula ion and P epa a ion
3.6.1 Raw ma e ials and Fo mula ion
Inspi ed by a s udy conduc ed by Song e al. (2022), he egan gummy candies (GC) o mula included a
mix u e o ca ageenans (KI o K+I), dis illed wa e , xan han gum (XAN, ood g ade), osema y honey, and
a na u al addi i e (Table 3). Xan han gum was used due o i s documen ed syne gis ic e ec s when mixed

47
wi h K-ca ageenan (A allone e al., 2023; Balasub amanian e al., 2018; B enne e al., 2015; Tunie a
& Spi idono , 2020; Xu e al., 2022), assuming simila beha io occu s wi h K/I hyb ids.
Rosema y honey se ed as a nu i ious swee ene and ex u ize , chosen o i s he apeu ic and
p e en i e e ec s agains in lamma ion-media ed ch onic diseases (Ranneh e al., 2021). Rich in
dis inc i e phenolic and la onoid compounds, honey is epo ed o ha e heal h bene i s, including
an ioxidan (Ranneh e al., 2018), an i-p oli e a i e (Jagana han e al., 2014), and an i-bac e ial ac i i y
(Khan e al., 2018).
The na u al addi i e was a biohyb id ma e ial (BH) composed o an hocyanins (ACNs) ex ac ed om
jambolan ui (
Syzygium cumini
), which we e s abilized by adso p ion on o ben oni e. The BH was kindly
p o ided by P o esso Ge mán Valencia, o he Depa men o Chemical and Food Enginee ing o he
Fede al Uni e si y o San a Ca a ina, Campus João Da id Fe ei a Lima (Flo ianópolis, B azil). I was used
due o i s an ioxidan and an imic obial ac i i ies, a ibu ed o an hocyanins, as well as i s inc eased
s abili y (Koop e al., 2024).
Fo he o mula ion o he egan gummy candies, h ee di e en alkali (KOH) ea ed hyb id
ca ageenans (T-KI) wi h signi ican ly a ying κ/ι a ios (samples C, F, and J) we e e alua ed, along wi h
hei espec i e comme cial blends o simila chemical composi ion. Addi ionally, o in es iga e he
po en ial e ec s o he ca ageenan p ecu so s μ and ν on he o e all pe o mance o he gummies,
un ea ed hyb id ca ageenans (UT-KI) ex ac ed om he same sou ces (ca ageenans C, F, and J) we e
also assessed.
In addi ion, each GC o mula ion was eplica ed wi hou he BH ma e ial o be e unde s and i s
impac on he s abili y, ex u e, and s uc u al p o ile o he inal p oduc .
Table 3: Fo mula o he gummy samples, exp essed as weigh pe cen age (% w/w)
Ing edien s
GC Samples wi h BH
(% w/w)
GC Samples wi hou BH
(% w/w)
Wa e
86,5
87
Honey
10
10
Ca ageenan (UT-KI, T-KI, o K+I)
2
2
Xan han gum
1
1
Biohyb id
0,5
--------
48
The e o e, a o al o 18 GC samples we e p oduced, conside ing h ee di e en κ/ι a ios es ed in
h ee di e en sys ems (UT-KI, T-KI, and K+I), each assessed wi h and wi hou he biohyb id ma e ial, as
shown in Table 4.
Table 4: Syn hesized desc ip ion o he compa a i e sys ems s udied, and labelling o egan gummy candies
p oduced
Un ea ed Hyb id
Ca ageenans (UT-KI)
Alkali- ea ed Hyb id
Ca ageenans (T-KI)
Comme cial Ca ageenan
Blends (K+I)
GC Samples
wi h BH
UT-C + BH
T-C + BH
10κ+90ι + BH
UT-F + BH
T-F + BH
50κ+50ι + BH
UT-J + BH
T-J + BH
90κ+10ι + BH
GC Samples
wi hou BH
UT-C
T-C
10κ+90ι
UT-F
T-F
50κ+50ι
UT-J
T-J
90κ+10ι
3.6.2 Gummy Candies P epa a ion P ocedu e
The p epa a ion p ocedu e o he gummies was inspi ed on Song e al. (2022). To p epa e 10 g o GC
samples, 0.2 g o ca ageenan and 0.1 g o xan han gum we e weighed and g adually dispe sed in o a
po ion o dis illed wa e , s i ing igo ously wi h a spa ula o p e en clumping. The solu ion was hen
hea ed o 80 °C wi h con inuous magne ic s i ing o 30 minu es.
Meanwhile, he emaining ing edien s ( he es o he wa e , honey, and he biohyb id, o jus wa e
and honey o he con ol g oups) we e weighed and mixed a oom empe a u e un il a homogenous
solu ion was ob ained. This mix u e was added o he ca ageenan and xan han gum solu ion a e he
ini ial 30-minu e hea ing pe iod o he la e , and he esul ing p epa a ion was hen hea ed and s i ed
o an addi ional 30 minu es.
Finally, he samples we e le o es a oom empe a u e o e nigh o p omo e gela ion. A e isually
inspec ing he phase s a e and colo a ion o he GC samples, hey we e s o ed in he idge o
p ese a ion and aken ou only o es ing pu poses.
49
3.6.3 Pa icle Size Dis ibu ion and Ze a Po en ial o he Biohyb id
The pa icle size dis ibu ion (PSD) and ze a po en ial (ZP) o he biohyb id ma e ial dispe sed in wa e
we e analyzed o e alua e he magni ude o he elec os a ic cha ge on he pa icle su aces and hei
size. These ac o s a e c ucial o unde s anding po en ial in e ac ions wi h o he ing edien s in he
o mula ion, as well as hei impac on he p oduc 's o e all s abili y and consis ency (Pa upudi e al.,
2022).
PSD was measu ed using a Mas e size 3000 (Mal e n Ins umen s L d., Wo ces e shi e, UK) based
on lase -ligh sca e ing p inciples. Due o equipmen da abase limi a ions, he sample was app oxima ed
as silica (SiO₂), wi h a e ac i e index o 1.457 and an abso p ion index o 0.010. Wa e was used as he
dispe san , wi h a e ac i e index o 1.330.
The ZP was measu ed using he Ze asize Nano ZS (Mal e n Ins umen s L d., Wo ces e shi e, UK),
which uses Lase Dopple Velocime y o de e mine elec opho e ic mobili y. Measu emen s we e
conduc ed in iplica e a 25 °C.
3.7 Gummy Candy Cha ac e iza ion
3.7.1 Op ical Mic oscopic Imaging
Op ical mic oscopic imaging was used o inspec he quali y o he dispe sion o biohyb id pa icles along
he o mula ion ma ix, using an op ical ansmission mic oscopy (Olympus BH2, 20x) coupled o a LEICA
DFC 280 digi al came a. Mel ed samples (≈ 85 °C) we e placed on mic oscope slabs and gen ly squeezed
wi h a glass co e .
3.7.2 pH Measu emen s
The pH o all samples was measu ed using a HI 2210 pH Me e (Hanna Ins umen s, Limena, I aly).
3.7.3 Rheological Cha ac e iza ion
All GC samples we e heological cha ac e ized ollowing he same me hodology as desc ibed in Sec ion
3.5.2.
50
3.7.4 FTIR-ATR Spec a Acquisi ion
The FTIR-ATR analysis was conduc ed o unde s and whe he he addi ion o he biohyb id ma e ial causes
chemical changes wi hin he ma ix. Fo his assessmen , a small sample o each gummy was collec ed,
hea ed unde e lux, a 80 °C, and inally d ied o e nigh , o o m a ilm. The FTIR-ATR spec a acquisi ion
p ocedu e used was he same as desc ibed in Sec ion 3.3.1.
51
4. RESULTS AND DISCUSSION
4.1 Chemical S uc u e o Hyb id Ca ageenans
Insigh s on he chemical cha ac e iza ion o he hyb id ca ageenans p oduced we e mainly ob ained by
FTIR-ATR, 1H-NMR and SEC, o iden i y he ma e ial's molecula bonds and chemical g oups, chemical
composi ion and molecula mass dis ibu ion, espec i ely.
4.1.1 FTIR-ATR Spec a
All ca ageenan samples, bo h KOH- and NaOH- ea ed, we e analyzed using FTIR-ATR. Howe e , since
hey exhibi ed simila spec al beha io s, only he spec a ob ained om he KOH- ea ed samples a e
p esen ed and discussed he e. Thus, Figu e 7 shows he FTIR-ATR spec a o he KOH- ea ed
ca ageenan samples wi hin he ange o 1400–600 cm−1. The FTIR-ATR spec a o he NaOH- ea ed
ca ageenan samples a e egis e ed in Figu e A.1, in Appendix A.1 o u he consul a ion.
The FTIR spec a o he sample ilms e ealed dis inc ca ageenan p o iles. In pa icula , samples A,
B, C and D displayed a mo e p onounced band a 805 cm-1 and a shoulde a ound 905 cm-1, bo h
associa ed o 3,6-anhyd o-D-galac ose-2-sulpha e, cha ac e is ic o ι-ca ageenan disaccha ide uni s.
Figu e 7: FTIR-ATR spec a o hyb id ca ageenan samples ea ed wi h KOH, highligh ing he mos cha ac e is ic
bands o ca ageenans a 1240, 930, 845, and 805 cm⁻¹.
No e: The FTIR-ATR spec a o samples F and N, in KOH,
a e no shown due o poo esolu ion.

52
Con e sely, samples I, J, K, L, M and O exhibi ed a p ominen band nea 930
cm-1, alongside a shoulde a 1070 cm-1, which a e assigned o 3,6-anyd o-D-galac ose moie ies ypically
ound in ι, bu also κ-ca ageenan uni s.
Samples E, G and H p esen ed a spec um wi h b oade and less well-de ined peaks a ound key
wa eleng hs (
e.g.
, 930 cm-1 and 805 cm-1), implying weake s uc u al con as s. This p o ile may co ela e
wi h a mo e balanced o homogeneous dis ibu ion o κ- and ι-ca ageenan ac ions, leading o an o e all
blending o ib a ional modes, in which he dis inc i e ea u es o pu e κ- o ι-ca ageenan would be less
p onounced. In addi ion, he FTIR-ATR spec a o samples F and N, in KOH, we e no p esen ed due o
poo esolu ion, likely caused by esidues o impu i ies in he ilm which may ha e hinde ed an accu a e
assessmen .
Common o all ca ageenan samples, one band appea ed a app oxima ely 2920 cm−1 on he FTIR
spec a (da a no shown) assigned o C–H s e ching ib a ions, co esponding o he o al suga con en .
Mo eo e , all samples showed bands a app oxima ely 845 and 1240 cm-1, co esponding o d-galac ose-
4-sulpha e and sulpha e es e g oups, espec i ely, which a e ound ac oss all ca abiose ypes (κ, ι, μ
and ν). Howe e , bands associa ed wi h μ- and ν-ca ageenan, ypically nea 820 cm-1, we e no iden i ied,
sugges ing ha hese ypes a e ei he p esen in lowe concen a ions o hei s uc u al ea u es a e less
de ec able unde he cu en condi ions.
Ra io o Abso bances a 1370/2920 cm- 1 – Sul a e- o-Suga Index
The peak abso bances associa ed wi h o al sul a e g oups a 1240 cm-1 and 1370 cm-1 can be
no malized using he peak co esponding o C-H s e ching ib a ions a 2920 cm-1, which ep esen s he
o al suga con en . The esul ing a ios, A1240/A2920 and A1370/A2920, se e as semi-quan i a i e
indica o s o he sul a e con en , exp essed as he deg ee o subs i u ion (DS)— he a e age numbe o
sul a e g oups (in moles) pe disaccha ide uni in ca ageenan (Aze edo e al., 2013; Rochas e al., 1986).
Acco ding o Rochas e al. (1986), he accu acy o sul a e con en es ima ion using hese a ios is eliable
o polyme s wi h a DS o less han 0.8 o A1240/A2920 and mo e han 0.4 o A1370/A2920, making
he la e app op ia e o de e mining sul a e con en ac oss a b oade ange o sul a ed galac ans. The
acui y o he DS de e mina ion wi h he A1370/A2920 a io was ecen ly e i ied wi h he FTIR analysis
o ilms made o 4 blends o κ and ι wi h di e en a ios, spanning heo e ical DS om 1 o 2 whe eas
he band a io anged om 1.3 o 1.9 (de Oli ei a e al., 2020).
53
Theo e ically, κ-ca ageenan con ains, on a e age, one sul a e g oup pe disaccha ide uni , while ι-
and μ-ca ageenans each con ain wo sul a e g oups, and ν-ca ageenan ca ies h ee sul a e g oups pe
disaccha ide (Rochas e al., 1986; an de Velde, 2008). Assuming ha he hyb id ca ageenans p oduced
consis only o hese ou componen s, each con ibu ing a ying sul a e g oups, i is expec ed ha he
DS sul a e alues would ideally be abo e 1. The e o e, he a io A1370/A2920 was used as a semi-
quan i a i e index ela ing o al sul a e o suga con en , as ep esen ed in Figu e 8, o samples ea ed
wi h KOH and NaOH. As all a ios in Figu e 8 a e abo e 2, a DS canno be ex ac ed om he da a using
he calib a ion ca ied ou elsewhe e (de Oli ei a e al., 2020). Thus, he p esen analysis o he sulpha e
con en is only quali a i e.
Acco ding o Figu e 8, he samples exhibi ed a ying deg ees o sul a ion, wi h NaOH- ea ed samples
gene ally showing highe sul a e- o-suga a ios compa ed o KOH- ea ed ones, especially o samples B,
E, G, H, M, N, and O. This sugges s ha NaOH- ea ed samples end o p ese e sul a ion be e han
KOH- ea ed samples, which could impac hei gela ion, iscosi y, and o e all pe o mance. Some
samples (A, I, J, K, and L) had simila a ios be ween ea men s, indica ing less di e ence in sul a e
e en ion. Samples I, J, K, and L showed consis en ly lowe A1370/A2920 a ios, possibly due o a highe
con en o less sul a ed moie ies, such as κ-ca ageenan.
Se e al ac o s beyond he ac ual p esence o sul a ed ca ageenan ac ions can con ibu e o highe
sul a e con en in hyb id ca ageenan ex ac s. Co-ex ac ed ma e ials such as lo idean s a ch, p o eins,
Figu e 8: Ra io o abso bance a 1370/2920 cm-1 (A1370/A2920), indica i e o he o al sul a e con en in
ca ageenan samples ea ed wi h KOH (ligh g ey) and NaOH (da k g ey).
No e: he s anda d de ia ion o each a io
alls wi hin he ange o 10
-3
o 10-4. While i has been included in he ba cha , i is no p ominen ly isible due o i s
ela i ely small magni ude.
54
o esidual sul a e sal s (due o he absence o p e-washing) may lead o misleading es ima ions o sul a e
le els (Moses e al., 2015). Addi ionally, ion pai o ma ion be ween sul a e g oups and coun e -ions (Na⁺
o K⁺) can enhance hyd a ion, al e he ib a ional ene gy, and inc ease abso bance a 1370 cm-1 (Rochas
e al., 1986). This in e ac ion could esul in a highe A1370/A2920 a io, e lec ing ionic in luences on
FTIR eadings.
Ra io o Abso bances a 805/845 cm-1 - Rela i e Amoun o ι-ca ageenan
Addi ionally, he abso bance o he 805 cm-1 peak (assigned o 3,6-anhyd o-D-galac ose-2-sul a e) was
no malized wi h espec o he 845 cm-1 peak (assigned o D-galac ose-4-sul a e), wi h he A805/A845
a io e lec ing he ela i e amoun o ι-ca ageenan (Aze edo e al., 2013). The A805/A845 a io is
p esen ed in Figu e 9, o bo h KOH and NaOH- ea ed samples.
The a io A805/A845 sugges s ha he NaOH and KOH ea men s yielded chemically dis inc
ca ageenans, wi h KOH- ea ed ca ageenans (ligh g ey) consis en ly showing a sligh ly highe ela i e
ι-ca ageenan con en , especially in samples F, G, H, J, M, N, and O. In bo h scena ios, sample N
p esen ed he highes A805/A845 a io.
A possible explana ion o he highe A805/A845 a ios obse ed in KOH- ea ed samples and,
pa icula ly, in sample N could be he p esence o highe concen a ions o p ecu so uni s in he
ca ageenan chain. The abso p ion bands be ween 815 and 825 cm-1, assigned o he sul a e g oups on
Figu e 9: Ra io o abso bance a 805/845 cm-1 (A805/A845), indica i e o he ela i e amoun o ι-ca ageenan
in samples ea ed wi h KOH (ligh g ey) and NaOH (da k g ey).
No e: he s anda d de ia ion o each a io alls wi hin
he ange o 10⁻³. While i has been included in he ba cha , i is no p ominen ly isible due o i s ela i ely small magni ude.
55
C2 and C6 o galac ose ound in ca ageenans μ and ν, could a ec he in ensi y o he band a 805 cm-1
a ibu ed o he sul a e g oup on C2 o 3,6-anhyd ogalac ose, leading o an o e es ima ion o he ela i e
ι-ca ageenan con en .
Acco ding o Figu e 9, in NaOH, samples A, B, C, D, and N exhibi ed a highe A805/A845 a io,
indica i e o g ea e ι-ca ageenan con en , ollowed by samples E, F, G, H, O, and M. Samples I, J, K,
and L showed he lowes ela i e ι-ca ageenan con en in bo h alkaline solu ions.
4.1.2 1H-NMR
FTIR spec oscopy da a we e con i med by NMR spec oscopy analysis, as he chemical composi ion o
all samples we e analyzed using 1H-NMR. Figu e 10 p esen s he ¹H-NMR spec a o h ee chemically
ep esen a i e samples, p ocessed wi h NMRium® so wa e, along wi h he chemical shi s co esponding
o he anome ic p o ons o he p ima y ca abiose uni s (κ, ι, μ, and ν). The mola ac ions (mol.%) o
each ca ageenan disaccha ide epea ing uni a e shown in Figu es 11A and 11B o KOH- and NaOH-
ea ed samples, espec i ely.
The 1H-NMR spec a e ealed sligh de ia ions om he p edic ed chemical shi s o each anome ic
p o on o he κ, ι, μ, and ν ca abiose uni s (Table 2), wi h κ-ca ageenan peaks appea ing be ween 5.07
and 5.11 ppm, ι-ca ageenan a ound 5.27 o 5.31 ppm, μ-ca ageenan be ween 5.19 and 5.21 ppm,
and ν-ca ageenan om 5.46 o 5.51 ppm.
Figu e 10: 1H-NMR spec a o h ee signi ican ly di e en KOH- ea ed samples, in e ms o chemical composi ion,
ob ained ia NMRium® so wa e: sample B, ich in ι-ca ageenan; sample F, wi h a mo e balanced κ/ι a io, and
sample I, ich in κ-ca ageenan. Ve ical do ed lines indica e he peaks o he assigned disaccha ide uni s showing
up a chemical shi s e e enced in Van De Velde e al. (2004).
62
in balanced κ/ι sys ems migh weaken in e -chain in e ac ions and loosen ne wo k s uc u es.
Addi ionally, o he po en ial ac o s in luencing molecula mass and chemical composi ion ela ionships
should be explo ed o p o ide a mo e comp ehensi e unde s anding o hese mechanisms.
Upon compa ing he molecula mass da a o he un ea ed samples wi h hose om Figu es 12A and
12B, i is e iden ha alkaline ea men gene ally led o a educ ion in molecula mass, wi h a ew
excep ions—speci ically, samples G and L in KOH, and C’, D’, G’, and L’ in NaOH. Alkaline ea men ,
pa icula ly a ele a ed empe a u es, can clea e he glycosidic bonds wi hin ca ageenan chains. This
p ocess, known as alkaline hyd olysis, b eaks he polyme in o smalle agmen s, esul ing in lowe
molecula mass alues (Fuchs e al., 2024). The ex en o his e ec depends hea ily on ea men
condi ions ( empe a u e, pH, and du a ion), wi h ha she condi ions inducing mo e signi ican chain
scission, especially in NaOH solu ions (Aze edo e al., 2013; Aze edo, To es, e al., 2015). Howe e , he
dec ease in molecula mass was mo e p ominen in mos KOH- ea ed samples, wi h excep ions such as
samples H and I, which exhibi ed lowe Mw alues in NaOH, and sample A, which showed a simila
dec ease in bo h alkaline solu ions.
4.2 Hyb id Ca ageenans
s
Comme cial Blends
Selec ed hyb id ca ageenans p oduced we e sys ema ically compa ed wi h comme cial ca ageenan
blends o equi alen chemical composi ion h ough phase diag ams, in NaCl and KCl, o samples ea ed
wi h NaOH and KOH, espec i ely. Subsequen ly, he heological p ope ies o selec ed gels we e
e alua ed using o a ional heome y unde small and la ge de o ma ion.
4.2.1 Phase Diag ams o KI
s
K+I in NaCl and KCl
The phase diag ams o he selec ed hyb id ca ageenans (KI), alongside he chemically equi alen
comme cial blends (K+I) a compa able sal and polysaccha ide concen a ions, a e p esen ed in Table
5 o KCl and in Table 6 o NaCl. These samples we e selec ed based on he c i e ia ou lined in Sec ion
3.4.1, ensu ing a b oad ange o chemical composi ions wi h minimal o no biological p ecu so con en
o in e e ing componen s, such as lo idean s a ch. As an excep ion, sample E' (NaOH- ea ed), was
in en ionally included o assess po en ial gelling beha io di e ences due o s a ch con en , as p e iously
men ioned in Sec ion 4.1.2.

63
The in luence o sal ype on he gelling beha io o K- and I-ca ageenans is well documen ed: K-
ca ageenan is mo e sal -speci ic, showing highe gelling e iciency in he p esence o K⁺ ions. In con as ,
I-ca ageenan can also gel wi h Na⁺ ions, hough he p ocess is slowe , p oducing so e , mo e elas ic
gels. O e all, K⁺ p omo es as e and i me gela ion in K-ca ageenan, while Na⁺ induces so e , mo e
lexible gels, especially o I-ca ageenan (Diene e al., 2019; Piculell, 2006; Sche e e al., 2015). In
con as , he sal dependency o K-2 ca ageenan is no as well unde s ood as pu e K- o I-ca ageenan.
O e all, K-2 exhibi s in e media e gelling beha io be ween he wo ypes o ca ageenans, showing ca ion-
speci ic agg ega ion, especially o K-helices (Souza e al., 2023). Howe e , he ange o polysaccha ide
concen a ions and ionic s eng h o which a gel is p oduced is e y di e en o each sal (Aze edo e
al., 2014; To es, Aze edo, e al., 2016; To es e al., 2018).
The e o e, he main pu pose o his sec ion is o di ec ly compa e he gelling beha io o hyb id
ca ageenans wi h hei comme cial blend coun e pa s, along a wide ange o composi ions, in o de o
de e mine simila i ies and di e ences in how hey beha e unde di e en sal and ca ageenan
composi ions. Seconda ily, he compa ison be ween NaOH and KOH sys ems mainly in ends o e alua e
how K-2 hyb id ca ageenans beha e in he p esence o di e en sal s: K+ and Na+, since he e is s ill a
lack in he li e a u e on his opic.
O e all, he compa ison o phase diag ams in he igh and le columns con i med no able di e ences
be ween K/I-hyb id ca ageenans and he comme cial K- and I-ca ageenan blends (Aze edo e al.,
2014; Villanue a e al., 2004). Gel o ma ion (in g een) in he p esence o K+ occu ed mo e eadily in
comme cial K+I blends han in KI ca ageenans, happening ac oss almos all chemical composi ions (in
e ms o κ and ι con en ), polysaccha ide concen a ions, and ionic s eng hs, whe eas KI ca ageenans
showed highe sensi i i y o hose pa ame e s.
Fo ι- ich samples (B and C), gela ion was mo e dependen on he polysaccha ide concen a ion,
occu ing only a KI ≥ 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, sugges ing ha while he polysaccha ide chains a e p esen , hey
a e insu icien o o m a ully connec ed gel. Ins ead, hey may c ea e mic odomains o pa ial ne wo ks
ha do no comple ely immobilize he sol en , esul ing in a suspension s a e a he han a solid gel.
Gi en he high cha ge densi y o ι-ca ageenan, 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 (Piculell, 2006). In con as , all K+I samples o med gels ac oss simila condi ions.
64
κ- ich KI samples, pa icula ly sample K (95 mol.% κ), beha ed compa ably o hei comme cial
coun e pa s. Gene ally, o samples wi h a high κ composi ion, 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 I,
J, and K, and 1 mol/L o he K+I blend). While po assium ions mo e e ec i ely induce he helical and
agg ega ed s a es o K-ca ageenan, abo e he op imal sal concen a ion sol a ion and/o sal -ou e ec s
may lead o p ecipi a ion, comp omising gel o ma ion.
Simila high sal sensi i i y was obse ed in samples E and M (60-70 mol.% κ) a K⁺ concen a ions
o 1 mol/L. These esul s align wi h o he s udies, which epo ed he o ma ion o s able pa icle
suspensions ac oss all polyme concen a ions a [K⁺] = 1 mol/L o KI samples wi h 75 mol.% κ (To es,
Aze edo, e al., 2016) and 70 mol.% κ (Aze edo e al., 2014). Howe e , samples E and M gela ion we e
also hinde ed by low ionic s eng hs and low polysaccha ide concen a ions. This obse a ion con as s
wi h he p e iously men ioned s udies, whe e samples o med clea o u bid gels unde simila condi ions
(Aze edo e al., 2014; To es, Aze edo, e al., 2016) in ha mony wi h he end ound o κ- ich KI
samples.
0.01 0.1 0.5 1 0.01 0.1 0.5 1
0.5 SS SS SS SS
1SS TG TG TG
2TG TG TG TG
0.5 SS SS SS SS
1SS TG SS TG
2TG TG TG TG
0.5 PSS SS SS SS 0.5 CG CGS CG TS
1 PSS SS PSS TS 1CG CGS TG TG
2TS TGS TGS TS 2CG CG TG TG
0.5 CS TG TGS TS 0.5 CG CGS TS TS
1 PSS TGS TGS TS 1CG CGS TG TG
2 TGS TGS TGS TS 2CG CGS TG TG
0.5 SS TG CG TS 0.5 CG CGS TS TS
1 TGS TGS TG TS 1 CGS CGS TG TG
2 TGS TG TGS SS 2CG CGS TGS TG
0.5 CGS CGS SS SS
1 TGS TGS TGS SS
2 TGS TGS SS PSS
0.5 CGS CGS TS PSS
1 TGS TGS SS PSS
2 TGS TG SS TS
0.5 CGS TGS TGS SS
1 TGS TGS TS PSS
2 TGS TGS TG PSS
CS Clea Solu ion SS S able Suspension CG Clea Gel CGS Clea Gel wi h Syne esis
TS Tu bid Solu ion PSS Phase Sepa a ed (se lled) Suspension TG Tu bid Gel TGS Tu bid Gel wi h Syne esis
Phase Diag am in KCl
CG
TG
TG
CGS
TGS
TS
J
(87 κ + 13 ι)
1
CGS
CGS
TG
TS
M
(69 κ + 31 ι)
70 κ + 30 ι
I
(90 κ + 10 ι)
90 κ + 10 ι
0.5
CG
K
(95 κ + 5 ι)
2
CGS
TG
TG
F
(48 κ + 47 ι)
50 κ + 50 ι
E
(56 κ + 44 ι)
60 κ + 40 ι
TG
1
CG
CG
TG
TG
B
(4 κ + 96 ι)
10 κ + 90 ι
0.5
CG
CG
CG
C
(6 κ + 91 ι)
2
CG
CG
KI Sample
(%.mol κ + %.mol ι)
KI (w %)
[K+] (mol/L)
K+I Sample
(%.mol κ + %.mol ι)
K+I (w %)
[K+] (mol/L)
Table 5: Phase diag ams o KI (le ) and o K+I blends ( igh ), in KCl
65
Sample F (K-2 ca ageenan wi h ≈50 mol.% κ and 50 mol.% ι) exhibi ed he mos 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 0.1-0.5
mol/L, in e iden con as o i s K+I coun e pa .
In e es ingly, while sample F (48 mol.% κ + 47 mol.% ι) di e s om sample E (56 mol.% κ + 44 mol.%
ι) in chemical composi ion, he ex en o his di e ence is compa able o ha be ween samples E and M
(69 mol.% κ + 31 mol.% ι), ye he la e wo exhibi simila gelling beha io s. This sugges s ha ac o s
beyond κ and ι composi ion a e in luencing gela ion. One signi ican ac o could be he high
polydispe si y index (PDI) o sample F, coupled wi h i s a e age molecula mass being close o he lowe
limi (Figu e 12A), po en ially esul ing in a subs an ial p opo ion o chains oo sho o o m gels. This
could also accoun o he di e ences obse ed wi h i s comme cial blend coun e pa , as comme cial K-
and I-ca ageenans ha e signi ican ly highe molecula masses (Figu e B.2 – Annex B.2). Addi ionally,
he ela i ely highe con en o p ecu so uni s (≈ 5 mol.%) in sample F (Figu e 11A) may hinde i s abili y
o es ablish he obus ne wo k s uc u es necessa y o gela ion unde b oade sal and polysaccha ide
condi ions.
To es, Aze edo, e al. (2016) epo ed a K-2 ca ageenan wi h a simila chemical composi ion o
sample F (53 mol.% κ and 47 mol.% ι), ex ac ed om
A. de oniensis
, ha o med gels wi hou syne esis
o all condi ions, excep o 0.5-1 KI w .%, a 0.01 mol/ [K+] which yielded u bid solu ions. These
di e ences sugges ha in insic species-speci ic ac o s, such as K-2 chain leng h dis ibu ions and block
leng h a iabili y, also in luence he o e all gelling pe o mance o ex ac ed KI ca ageenans, as he
molecula masses o sample F and ex ac om
A. de oniensis
a e bo h in he lowe limi .
This inhe en a iabili y, common o na u al p oduc s, complica es s aigh o wa d co ela ions
be ween K-2 hyb ids' gelling p ope ies and hei κ o ι ac ions. Explo ing di e se algae sou ces could
help cla i y his a iabili y and es ablish a clea e κ con en h eshold, abo e which KI hyb ids beha e
mo e like comme cial mix u es in e ms o gelling abili y. Howe e , i is wo h no ing ha bo h comme cial
ca ageenans exhibi highe molecula masses han sample F, u he complica ing di ec compa isons.
Syne esis (depic ed in ligh and da k luo escen g een) inc eased wi h highe κ-con en (unno iced o
κ < 50 mol.%), especially in KI samples, whe e wa e sepa a ion occu ed in a g ea pa o gels. In
comme cial blends, syne esis was mo e common a 0.1 mol/L K⁺ o samples wi h ≥ 50 mol.% κ. These
indings did no en i ely align wi h a s udy by Aze edo e al. (2014), who obse ed no syne esis in KI gels
wi h 70 mol.% κ in KCl, while K+I gels exhibi ed wa e elease a highe K⁺ concen a ions. In his s udy,
syne esis occu ed in bo h KI and K+I gels a [K⁺] < 1 mol/L. Bo h s udies ag ee on he di icul y o gel
66
o ma ion in κ- ich KI sys ems a 1 mol/L KCl. The disc epancies may esul om di e ences in seaweed
sampling (loca ion and ha es ime) and ea men me hodology, as Aze edo e al. (2014) applied alkali
ea men di ec ly o seaweed p e-ex ac ion, whe eas his s udy ea ed he ex ac ed ca ageenan. This
sugges s ha possible di e en Mw dis ibu ions, as well as κ and  dis ibu ions along he polysaccha ide
chain, gi e di e en gel syne esis, whe eas he chemical s uc u e is essen ially iden ical in e ms o κ
and  con en s. No e also ha di e ences in he phase diag ams o K+I gels also poin o possible
di e ences in he expe imen al p o ocols employed (in pa icula he he mal his o y du ing cooling), in
he assessmen o phases (subjec ed o expe imen alis s c i e ia) o he K and I used, as comme cial
samples we e sou ced om he same p o ide .
Clea gels (wi h o wi hou syne esis) we e mo e equen ly obse ed in he comme cial blend sys ems,
pa icula ly a low ionic s eng hs ([K+] ≤ 0.1 mol/L). This obse a ion aligns wi h indings by Aze edo e
al. (2014), who linked he ansi ion om clea o u bid gels o changes in gel s uc u e and a shi in he
concen a ion dependence o c i ical s ain o gel b eak up. In con as , nea ly all KI samples showed
some deg ee o u bidi y, wi h clea solu ions appea ing only occasionally a lowe polysaccha ide
concen a ions. The inhe en endency o KI o exhibi u bidi y likely esul s om se e al ac o s: KI
samples we e ex ac ed om unp ocessed and unwashed algae, allowing o possible co-ex ac ion o
soluble and insoluble impu i ies ha con ibu e o u bidi y, whe eas he comme cial ca ageenans in K+I
blends we e ob ained in a highly p ocessed, pu i ied s a e. Lea ing aside he op ical ole o impu i ies,
he u bidi y sugges s ha KI samples end o yield mo e he e ogeneous s uc u es, po en ially o ming
la ge , i egula mic odomains compa ed o comme cial blends. This s uc u al he e ogenei y inc eases
ligh sca e ing and u bidi y, especially a highe polyme concen a ions.
Simila ly o wha was seen in KCl, in he p esence o Na+ hyb id ca ageenans also demons a ed a
mo e complex and a iable gelling beha io compa ed o he comme cial blends (Table 6). In NaCl, gel
o ma ion o K+I samples 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). No ably, comme cial blends
in NaCl p oduced clea e gels and solu ions compa ed o KCl.
As expec ed, due o he non-speci ic ion esponse o ι-ca ageenan, ι- ich 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 K-2
ca ageenans (samples F’, E’, H’ and M’) wi h ≈50-70 mol.% κ, no gels o med unde any condi ion,
67
con as ing s a kly wi h K+I coun e pa s. The s udy by To es, Aze edo, e al. (2016), hough aligning
wi h he no able gelling di icul y o hyb id ca ageenans wi h 50-50 mol.% and 75-25 mol.% o κ-ι, s a ed
he o ma ion o gels a 1-2 w .% o polysaccha ide and 0.5-1 mol/L [Na+]. Simila ly, Aze edo e al. (2014)
documen ed hese esul s o 70-30 mol.% κ-ι hyb ids.
Fo samples E' (con aining s a ch) and H', bo h wi h simila κ and ι ac ions, he phase diag am
analysis e ealed no signi ican di e ence, sugges ing ha s a ch p esence did no ha e a no iceable
impac . Howe e , due o he inhe en gelling di icul y o K-2 hyb ids in NaCl, i is challenging o de ini i ely
conclude ha s a ch has no e ec on gelling p ope ies.
In e es ingly, some κ- ich gels egis e ed he occu ence o syne esis in NaCl, which is no commonly
epo ed in he li e a u e. This happened speci ically in he K+I blend wi h 70 %.mol κ, and o samples
I’, J’ and K’, a highe polyme and Na+ concen a ions. Al hough po assium ions a e known o encou age
syne esis in K-ca ageenan gels due o hei s ong binding a ini y, sodium ions gene ally ha e weake
in e ac ions, so syne esis is less likely. No ably, in he case o he K+I blend wi h 70 mol.% κ, he esul s
0.01 0.1 0.5 1 0.01 0.1 0.5 1
0.5 SS CS PSS SS
1 PSS TG TG TG
2TG TG TG TG
0.5 SS SS SS SS
1SS TG TG TG
2TG TG TG TG
0.5 SS SS SS SS 0.5 CS CS CS CS
1 PSS PSS SS SS 1CG CG CG CG
2 PSS PSS TS TS 2CG CG CG TG
0.5 PSS PSS PSS PSS
1 PSS PSS PSS TS
2 PSS PSS PSS TS
0.5 CS SS PSS TS
1SS SS PSS TS
2SS SS TS TS
0.5 CS SS SS SS 0.5 CS CGS CG CGS
1 PSS SS SS SS 1CG CGS CGS TGS
2 PSS TS SS SS 2CG CGS CG TGS
0.5 CS PSS TS TS
1SS TG TGS TS
2TG TG TGS TGS
0.5 PSS SS PSS SS
1 PSS SS PSS PSS
2TG TG TG TGS
0.5 PSS PSS PSS PSS
1 PSS TG TGS PSS
2TG TG TG TGS
CS Clea Solu ion SS S able Suspension CG Clea Gel CGS Clea Gel wi h Syne esis
TS Tu bid Solu ion PSS Phase Sepa a ed (se lled) Suspension TG Tu bid Gel TGS Tu bid Gel wi h Syne esis
[Na+] (mol/L)
KI Sample
(%.mol κ + %.mol ι)
KI (w %)
[Na+] (mol/L)
K+I Sample
(%.mol κ + %.mol ι)
K+I (w %)
C'
(5 κ + 95 ι)
10 κ + 90 ι
0.5
CG
CG
D'
(10 κ + 88 ι)
2
CG
CG
CG
1
CG
CG
CG
TG
CG
CG
CG
TG
M'
(67 κ + 29 ι)
70 κ + 30 ι
I'
(90 κ + 10 ι)
90 κ + 10 ι
0.5
CS
K'
(93 κ + 7 ι)
2
CG
Phase Diag am in NaCl
60 κ + 40 ι
0.5
2
CG
CG
CG
TG
CG
CG
F'
(54 κ + 45 ι)
50 κ + 50 ι
H'
(62 κ + 37 ι)
E'
(57 κ + 42 ι)
CG
1
CG
CG
CG
CS
CS
CG
CS
CG
CG
TG
CS
CS
CS
J'
(88 κ + 12 ι)
1
CG
Table 6: Phase diag ams o KI (le ) and o K+I blends ( igh ), in NaCl

68
a e e en mo e unusual, as o he s udies using he same expe imen al p o ocol and sou ce o comme cial
ca ageenans did no epo any syne esis (Aze edo e al., 2014; To es, Aze edo, e al., 2016). This
u he suppo s he possibili y o use - ela ed expe imen al e o s, al hough none could be speci ically
iden i ied. One po en ial explana ion could be ha , du ing cooling, wa e apo om he gel o solu ion
may condense on he coole walls o he lask, o ming a liquid ilm ha could be mis akenly iden i ied as
syne esis.
To summa ize his sec ion, phase diag ams highligh ed key di e ences be ween K/I hyb id
ca ageenans (KI) and comme cial blends (K+I). KI ca ageenans showed g ea e dependency on
polysaccha ide concen a ion and ionic s eng h o gel o ma ion compa ed o K+I. None heless, hyb id
ca ageenans exhibi ed gela ion beha io s simila o comme cial blends unde speci ic condi ions. These
included highe polysaccha ide concen a ions o ι- ich samples in bo h KCl and NaCl, balanced κ/ι
samples in KCl, and κ- ich samples in NaCl, as well as lowe ionic s eng hs o κ- ich samples in KCl.
Howe e , hyb id ca ageenans consis en ly o med mo e u bid gels. In compa ing KI samples, KCl
acili a ed mo e consis en gel o ma ion ac oss concen a ions, while NaCl esul ed in di e se gelling
beha io s, including mo e suspensions and phase sepa a ions.
Samples ich in κ-ca ageenan showed mo e speci ici y owa ds KCl, whe eas ι- ich samples
p esen ed simila phase diag ams in NaCl and KCl. K-2 ca ageenans displayed sal -speci ic esponses,
o ming gels in KCl bu no in NaCl, unde sco ing hei limi ed gelling po en ial in Na+ en i onmen s,
consis en wi h Aze edo e al. (2014).
In conclusion, KI ca ageenans may se e as iable al e na i es o K+I unde speci ic polysaccha ide
concen a ions and ionic s eng hs conduci e o gel o ma ion in bo h. Howe e , heological es ing is
essen ial o e alua e hei compa a i e elas ici y, modulus, and yield s ess o op imal applica ion.
Addi ionally, KI may ind unique alue as a s abilize in non-gelling applica ions, such as sauces,
d essings, o emulsions, whe e gel-like ne wo ks a en’ essen ial bu wa e -binding and s able suspension
p ope ies a e ad an ageous.
4.2.2 Rheological Cha ac e iza ion o Hyd ogels
In he heological assessmen , speci ic samples we e selec ed o p o ide insigh in o gel beha io ac oss
a ying condi ions, gi en limi ed ime o analysis. Gels om samples C, E, and K in KOH and C' and K'
in NaOH (sample E’ did no o m gels) we e chosen, as hese di e ed no ably in κ- and ι-ca ageenan
69
con en , allowing he in es iga ion o heological esponses ied o ι-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, enabling di ec compa isons wi hin each
sample. The e o e, h ee di e en polysaccha ide concen a ion-ionic s eng h condi ions we e chosen o
each sample, o allow o his compa ison.
Since sample M’s heology has been ex ensi ely s udied unde simila condi ions, as a ca ageenan
de i ed om
Mas oca pus
(Aze edo e al., 2014, 2022; Mo ei a e al., 2016; To es, Chenlo, e al.,
2016), 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 (Bui e al., 2019;
Ridou e al., 1996).
The mal- heological P ope ies o Samples in KCl and NaCl Solu ions
Each sample was subjec ed o a ime sweep as ho solu ions cooled om 85 °C o 25 °C o moni o
he sol-gel ansi ion and assess gela ion beha io . This app oach allows a de ailed examina ion o he
e ol ing iscoelas ic p ope ies (G' and G'') du ing cooling, highligh ing gela ion empe a u e (Tg) as he
poin whe e G' (s o age modulus) c osses G'' (loss modulus), ma king he shi om a iscous o a gel-
like s a e. The empe a u e-dependence g aphs o he iscoelas ic moduli (G' and G'') o selec ed samples
a e p esen ed in Figu e 13, whe eas he cooling cu es o all o he samples in KCl and NaCl a e p o ided
in Figu es A.2 and A.3 o Appendix A.2.
No ably, di e en beha io s we e obse ed among he samples. Fo ins ance, in he 1 w .% K+I
(10κ90ι) sample in 1M KCl (Figu e 13A), G’ and G’’ in e sec ed, allowing o he iden i ica ion o Tg. In
con as , o he samples, such as 2 w .% KI (sample E) in 0.5M KCl (Figu e 13B), main ained a gel s a e
(G’ > G’’) h oughou he cooling p ocess, sugges ing ha Tg occu s abo e 85 °C. In all cases, he s a
o gela ion (Ts a ), ma ked by a s eep inc ease in G’, was eco ded. This app oach enables compa ison
be ween samples, e en when he e is no clea c osso e poin .
A hi d beha io , in ol ing a wo-s ep gela ion mechanism was also obse ed in samples, such as he
2 w .% KI (sample C) in 1M KCl (Figu e 13C). The la e is desc ibed as a wo-s ep inc ease in he
iscoelas ic moduli, co esponding o independen coil- o-helix ansi ions o he K and I blocks a dis inc
empe a u es (Ts a 1 and Ts a 2). This ype o gelling beha io has been epo ed o K+I in he p esence o
a ious sal s and ionic s eng hs (B enne e al., 2014; Bui e al., 2019; Du e al., 2016; Geonzon e al.,
70
2019; Hu e al., 2016; Pa ke e al., 1993; Piculell e al., 1992), bu also o K-2 hyb id ca ageenans,
pa icula ly in he p esence o Na+ sal s (Hilliou e al., 2014; Hilliou & Gonçal es, 2007; Souza e al.,
2011; To es e al., 2017; an de Velde e al., 2005). Some s udies also epo ed ha he s ep-inc ease
empe a u es in moduli o K-2 hyb id ca ageenans (Souza e al., 2011, 2023), as well as he
empe a u es o he coil- o-helix ansi ions o each blocks (Souza e al., 2011; an de Velde e al., 2005)
ma ch hose o K+I blends wi h simila mola composi ions, as well as isola ed K o I solu ions a
equi alen concen a ions, sugges ing ha con o ma ional ansi ions and helical agg ega ion emain
una ec ed by he p esence o he o he componen in he blend (Bui e al., 2019; Du e al., 2016; Pa ke
e al., 1993; Souza e al., 2023; an de Velde e al., 2005).
Figu e 13: Tempe a u e-dependen iscoelas ic moduli (G' and G'') o selec ed ca ageenan samples illus a ing
di e en gela ion beha io s: (A) Gela ion empe a u e (Tg) in 1 w .% K+I (10κ90ι) in 1 M KCl, whe e G' and G''
in e sec ; (B) Absence o Tg o 2 w .% KI (sample E) in 0.5 M KCl, indica ing a gel s a e main ained h oughou
cooling; (C) Two-s ep gelling mechanism obse ed in 2 w .% KI (sample C) in 1 M KCl, associa ed wi h sequen ial
coil- o-helix ansi ions in K and I blocks.
A
B
C
71
The alues o Tg, Ts a 1 and Ts a 2, along wi h hei associa ed e o s o each sample, a e p esen ed in
Table 7 o bo h KCl and NaCl, as well as a cha ac e iza ion o he gela ion mechanism (single-s ep o
wo-s ep p ocess).
Table 7: Values o Tg, Ts a 1 and Ts a 2, along wi h hei associa ed e o s o each sample, o bo h KCl and NaCl, as
well as a cha ac e iza ion o he gela ion mechanism (single-s ep o wo-s ep p ocess)
*Fo hese samples, he c osso e poin Tg was no de ec able due o he equipmen 's limi ed sensi i i y a he de o ma ion le el
used in he measu emen . While his small de o ma ion se ing was insu icien o iden i y Tg, i was chosen o p e en sample
dis up ion and a oid in e e ence wi h he gela ion p ocess. As a esul , only he ini ial gela ion empe a u e Ts a is p esen ed o
hese samples.
Expe imen
Sample
Tg (ºC)
Ts a 1 (ºC)
Ts a 2 (ºC)
Gelling
Mechanism
Samples in KCl
2 w % in 0.01 M KCl
Sample C
43.6 ± 0.2
51.0 ± 1.0
31.7 ± 0.1
Two-s ep
10κ90ι
47.6 ± 0.3
61.0 ± 2.0
34.0 ± 0.5
Two-s ep
1 w % in 1 M KCl
Sample C
-*
74 ± 1.0
60.0 ± 2.0
Two-s ep
10κ90ι
79.4 ± 0.8
81.0 ± 1.0
46.0 ± 3.0
Two-s ep
2 w % in 1 M KCl
Sample C
85.4 ± 4.3
> 85
74.0 ± 0.5
Two-s ep
10κ90ι
> 85
> 85
-
Single-s ep
2 w % in 0.01 M KCl
Sample E
66.1 ± 0.2
67.0 ± 0.3
54.0 ± 1.0
Two-s ep
60κ40ι
47.5 ± 0.4
53.0 ± 1.0
39.5 ± 0.5
Two-s ep
1 w % in 0.5 M KCl
Sample E
> 85
> 85
-
Single-s ep
60κ40ι
> 85
> 85
-
Single-s ep
2 w % in 0.5 M KCl
Sample E
> 85
> 85
-
Single-s ep
60κ40ι
> 85
> 85
-
Single-s ep
0.5 w % in 0.01 M KCl
Sample K
-*
43.5 ± 1.5
-
Single-s ep
90κ10ι
-*
32.0 ± 1.0
-
Single-s ep
0.5 w % in 0.5 M KCl
Sample K
> 85
> 85
-
Single-s ep
90κ10ι
> 85
> 85
-
Single-s ep
2 w % in 0.5 M KCl
Sample K
> 85
> 85
-
Single-s ep
90κ10ι
> 85
> 85
-
Single-s ep
Samples in NaCl
2 w % in 0.01 M NaCl
Sample C'
30.7 ± 0.5
38.0 ± 0.5
29.3 ± 0.1
Two-s ep
10κ90ι
52.0 ± 1.5
55.5 ± 1.0
30.0 ± 1.0
Two-s ep
1 w % in 1 M NaCl
Sample C'
83.9 ± 3.4
> 85
-
Single-s ep
10κ90ι
55.6 ± 0.3
58.3 ± 0.3
-
Single-s ep
2 w % in 1 M NaCl
Sample C'
< 25
25.0 ± 1.0
-
Single-s ep
10κ90ι
> 85
> 85
62.0 ± 3.0
Two-s ep
2 w % in 0.01 M NaCl
Sample K'
39.4 ± 0.4
40.5 ± 0.5
-
Single-s ep
90κ10ι
-*
44.5 ± 0.5
-
Single-s ep
1 w % in 0.5 M NaCl
Sample K'
-*
53.0 ± 1.0
-
Single-s ep
90κ10ι
-*
46.0 ± 0.3
-
Single-s ep
2 w % in 0.5 M NaCl
Sample K'
-*
50.0 ± 1.0
-
Single-s ep
90κ10ι
-*
54.7 ± 0.3
-
Single-s ep
78
p edominan ly iscoelas ic, cha ac e ized by G’’>G’ (Figu e 15C). These obse a ions align wi h he
empe a u e sweep analysis p e iously conduc ed o he 2 w .% sample C’ in 1 M NaCl, which e ealed
ha no gel had o med by he end o he es (T=25 ºC).
In he p esence o NaCl, while inc easing polysaccha ide concen a ion led o an inc ease in elas ici y
(wide gap be ween G’ and G’’) o comme cial blends, i did no exe signi ican di e ences o hyb id
ca ageenans C’ and K’. On he o he hand, excep o comme cial blend 90κ10ι, g ea e sal
concen a ion ypically esul ed in he loss o elas ici y and ne wo k s abili y. Howe e , κ- ich samples
consis en ly esul ed in mo e s able and elas ic gels in compa ison o ι- ich samples.
Gel P ope ies unde La ge De o ma ion
In heological s udies o gels, se e al c i ical pa ame e s p o ide insigh in o hei mechanical beha io
and s uc u al in eg i y. The gel elas ici y in he linea iscoelas ic (LVE) ange, ep esen ed by G0 (elas ic
modulus), e lec s he gel's s i ness and abili y o esis de o ma ion unde small applied s ains, whe e
he s uc u e emains undamaged. The limi ing s ain (γL) ma ks he bounda y o his linea ange, beyond
which he gel s uc u e begins o de o m i e e sibly o ansi ion in o a nonlinea iscoelas ic esponse.
To calcula e γL, he ole a ed de ia ion om he gel elas ici y in he LVE ange was de ined as 5%,
i.e.
, all
he G’ alues below 95% (in s ain so ening beha io s) o abo e 105% (in s ain ha dening beha io s) o
he pla eau alue we e conside ed o be ou side o he LVE ange. Ano he c ucial poin is he low poin
(γF), de ined by he c osso e o s o age (G’) and loss moduli (G’’). A his poin , he gel ansi ions om
a p edominan ly solid-like beha io (G’>G’’) o a liquid-like beha io (G’’>G’), ep esen ing he onse o
low (Mezge , 2006). A s ain sweep es illus a ing hese pa ame e s is shown in Figu e 16A.
Addi ionally, unde la ge de o ma ion, gels can exhibi s ain ha dening o s ain so ening beha io ,
depending on he s and-like s uc u e o ca ageenan gels, modula ed by he bending igidi y o he
ilamen s and he opology o he ne wo k (Ca illo e al., 2013; Meng & Te en je , 2016; Piculell, 2006).
S ain ha dening (Figu e 16B) e e s o an inc ease in s i ness as s ain inc eases, be o e up u ing, o en
indica ing s ong s e ching o ilamen s. Con e sely, s ain so ening (Figu e 16A) e lec s a dec ease in
s i ness wi h inc easing s ain, ypically associa ed wi h s uc u al b eakdown o ea angemen (Hilliou,
2021; Mezge , 2006). P ac ically, hese beha io s in luence he gel's esilience and unc ionali y in eal-
wo ld applica ions, as s ain-ha dening gels p esen highe up u e esis ance unde s ess, while s ain-
so ening gels may ail mo e easily unde high s ain condi ions. A dis inc i e beha io whe e samples
p esen ed a s ain so ening ollowed by a s ain ha dening esponse was also obse ed in some cases

79
(Figu e 16C). Toge he , hese pa ame e s, egis e ed in Table 8, p o ide a comp ehensi e unde s anding
o he gel's mechanical p ope ies.
Hyb id ca ageenans and comme cial blends unde he same ionic en i onmen and polysaccha ide
concen a ion, yielded di e en mechanical p ope ies, such as s eng h and de o mabili y. In he LVE
egion, comme cial ca ageenans gene ally p oduced s onge and s i e gels (highe G0 alues).
Howe e , sample E, a K-2 hyb id ca ageenan, signi ican ly su passed he elas ici y o i s comme cial
coun e pa , unde high ionic s eng hs. The same was also seen occasionally in low ionic s eng hs (
e.g.
,
sample C s. 10κ90ι, in KCl, and sample K’ s. 90κ10ι, in NaCl). None heless, in mos cases, g ea e
elas ici y came a he cos o inc eased gel b i leness (
e.g.
, sample E and sample 90κ10ι, in KCl, and
sample 10κ90ι, in NaCl). In addi ion, inc easing κ-con en , especially in KCl, and inc easing
polysaccha ide concen a ion usually enhanced gel elas ici y.
Figu e 16: Rep esen a ion o A) s ain so ening, B) s ain ha dening, and C) s ain so ening ollowed by s ain
ha dening beha io s. The s ain sweep example in A ep esen s a gel-like sample, showing he elas ic modulus,
(G0), he limi ing s ain (γL) a he limi o he LVE ange, and he low poin (γF) whe e G’=G’’.
γL
γF
G0
LVE ange
A
B
C
80
Table 8: Rheological p ope ies o hyb id ca ageenans and comme cial blends gelled in KCl and NaCl unde
a ying polysaccha ide and ionic condi ions. The able includes gel elas ici y in he LVE ange (G0), limi ing s ain
(γL), and low s ain (γF), as well as s ain ha dening and so ening beha io s unde la ge ampli ude oscilla o y shea
(LAOS). Cases whe e s ain so ening is ollowed by s ain ha dening a e indica ed as “No hen Yes”
Expe imen
Sample
G0 (Pa)
γL (%)
γF (%)
S ain Ha dening?
Samples in KCl
2 w % in 0.01M KCl
Sample C
2180 ± 17
1.40 ± 0.15
377 ± 30
No
10κ90ι
1022 ± 20
0.69 ± 0.04
118 ± 13
No hen Yes
1 w % in 1M KCl
Sample C
48.7 ± 0.3
1.50 ± 0.10
331 ± 14
No
10κ90ι
237 ± 2
1.96 ± 0.13
556 ± 34
No hen Yes
2 w % in 1M KCl
Sample C
985 ± 6
3.08 ± 0.18
404 ± 30
No hen Yes
10κ90ι
11281 ± 238
0.61 ± 0.03
388 ± 30
No
2 w % in 0.01M KCl
Sample E
7831 ± 23
0.93 ± 0.08
37 ± 5
No
60κ40ι
15351 ± 46
2.04 ± 0.14
60 ± 7
No hen Yes
1 w % in 0.5M KCl
Sample E
2391 ± 14
0.94 ± 0.06
35 ± 4
No
60κ40ι
157 ± 1
0.95 ± 0.07
104 ± 10
No
2 w % in 0.5M KCl
Sample E
14860 ± 134
0.30 ± 0.02
11 ± 1
No
60κ40ι
357 ± 2
1.58 ± 0.05
92 ± 4
Yes
0.5 w % in 0.01M KCl
Sample K
490 ± 4
1.38 ± 0.11
123 ± 6
Yes
90κ10ι
535 ± 5
0.96 ± 0.04
136 ± 7
Yes
0.5 w % in 0.5M KCl
Sample K
257 ± 2
0.63 ± 0.05
100 ± 9
No
90κ10ι
1650 ± 61
0.35 ± 0.04
25 ± 2
No
2 w % in 0.5M KCl
Sample K
20476 ± 71
0.81 ± 0.06
18 ± 2
No
90κ10ι
81921 ± 4012
0.37 ± 0.04
3.7 ± 0.6
No
Samples in NaCl
2 w % in 0.01M NaCl
Sample C’
738 ± 3
0.81 ± 0.05
181 ± 5
No
10κ90ι
1400 ± 128 *
0.31 ± 0.02
155 ± 13
No hen yes
1 w % in 1M NaCl
Sample C’
100 ± 60 *
<100 *
391 ± 47
No
10κ90ι
800 ± 400 *
<1 *
25 ± 3
No
2 w % in 1M NaCl
Sample C’
80 ± 20 *
- *
355 ± 30
No
10κ90ι
410 ± 50 *
4.57 ± 0.23
315 ± 38
No hen yes
2 w % in 0.01M NaCl
Sample K’
18226 ± 111
1.17 ± 0.11
36 ± 5
No
90κ10ι
3832 ± 140 *
1.38 ± 0.02
100 ± 5
Yes
1 w % in 0.5M NaCl
Sample K’
3384 ± 82
0.64 ± 0.02
41 ± 2
Yes
90κ10ι
6797 ± 45
1.21 ± 0.17
23 ± 1
No
2 w % in 0.5M NaCl
Sample K’
3118 ± 96
0.35 ± 0.04
11 ± 4
Yes
90κ10ι
25535 ± 311
3.50 ± 0.16
49 ± 7
Yes
*The s ain sweep analysis o hese samples in NaCl e ealed ce ain inconsis encies o expe imen al a i ac s, as e idenced
by he absence o a well-de ined linea iscoelas ic (LVE) egion (see Figu e A.6, in Appendix A.4). This limi a ion hinde ed he
accu a e de e mina ion o key pa ame e s such as he linea elas ic modulus (G0) and he limi ing s ain (γL). The lack o a
clea LVE egion sugges s po en ial de ia ions in he gel s uc u e o ex e nal ac o s, such as ins umen sensi i i y, sample
he e ogenei y, o ionic in e ac ions speci ic o NaCl sys ems.
In ι- ich samples in KCl, bo h sal and polysaccha ide concen a ions signi ican ly in luenced he gels'
elas ic and heological p ope ies. A low ionic s eng h (0.01 M KCl), he hyb id ca ageenan (sample C)
exhibi ed highe elas ici y and g ea e esis ance o de o ma ion han he comme cial blend 10κ90ι.
81
Con e sely, a high ionic s eng h (1 M KCl), he comme cial blend consis en ly displayed g ea e
elas ici y. In e es ingly, in his ionic en i onmen , he comme cial blend a 1 w .% polysaccha ide and
sample C a 2 w .% polysaccha ide exhibi ed s ain-ha dening beha io s, which coincided wi h he
condi ions unde which each sample demons a ed hei highes esis ance o de o ma ion.
Despi e po en ial expe imen al a i ac s and inconsis encies in ι- ich samples in NaCl, he comme cial
blends gene ally o med s onge gels han he hyb id ca ageenans ac oss all condi ions s udied as
shown in Figu e A.6, Appendix A.4). A low ionic s eng h (0.01 M NaCl), he comme cial blend also
wi hs ood g ea e s ain be o e pe manen ly losing i s linea iscoelas ici y. Howe e , a highe ionic
s eng hs, de o ma ion esis ance was di icul o e alua e due o i egula and expe imen ally inaccu a e
s ain sweep g aphs. Excep o he 1 w .% polysaccha ide expe imen a 1 M NaCl, comme cial blends
and hyb id ca ageenans exhibi ed simila yield poin s unde compa able condi ions. To cla i y whe he
hese i egula i ies we e due o sample-speci ic ac o s o equipmen limi a ions, u he analysis unde
op imized and con olled condi ions, using di e en shea ing and/o gelling condi ions, is equi ed o
ensu e eliable and ep oducible esul s and check o possible slip o nonlinea s ess esponse.
In he case o sample E and comme cial blend 60κ40ι, a low ionic s eng h, he comme cial blend
exhibi ed supe io gel elas ici y and esis ance o mechanical s ess compa ed o he hyb id ca ageenan.
Howe e , a high ionic s eng h, sample E showed g ea e elas ici y bu endu ed less de o ma ion, losing
s uc u al in eg i y mo e easily.
No ably, while he gel elas ici y o hyb id ca ageenan E consis en ly inc eased wi h ising sal
concen a ion, he linea elas ici y o he comme cial blend 60κ40ι dec eased unde high ionic s eng h.
This obse a ion aligns wi h indings by Aze edo e al. (2014), who epo ed ha he linea elas ici y (G0)
o ca ageenan blends inc eases wi h sal concen a ion up o c i ical poin , beyond which he elas ici y
begins o decline, e lec ing a weakening o he gel ne wo k. In con as , he elas ici y o hyb id
ca ageenans ollowed a di e en end, inc easing s eadily wi h sal concen a ion un il eaching a
sa u a ion poin . Beyond his, G0 ei he became independen o u he sal addi ion o he c i ical sal
concen a ion laid ou side he s udied ange (0.01–1 M Na⁺ and K⁺).
In sys ems wi h highe κ-con en s, bo h in KCl and NaCl, he comme cial blends consis en ly exhibi ed
g ea e elas ici y han hyb id ca ageenans unde equi alen condi ions. In KCl, his highe elas ici y o en
came a he cos o inc eased b i leness. In con as , in NaCl, comme cial blends p oduced gels ha
we e no only s onge bu also so e , demons a ing a highe ole ance o de o ma ion be o e eaching
he i e e sible damage limi (γL) and he inal b eakdown poin (γF).
82
No ably, highe κ-con en s we e also co ela ed wi h enhanced gel elas ici y when compa ing ce ain
comme cial blends wi h lowe mol.% κ (
e.g
., 2 w .% sample 60κ40ι in 0.5 M KCl). These indings align
wi h p e ious s udies documen ing he inc ease in he elas ic modulus o mixed gels as κ-ca ageenan
con en ises (B enne e al., 2014; Rochas e al., 1989; an de Velde e al., 2005), which is u he
ampli ied by he p esence o po assium ions (Chan ie e al., 2004; Pa ke e al., 1993). Howe e , while
inc easing κ-con en signi ican ly inc eased gel b i leness in comme cial blends (consis en wi h B enne
e al. (2014)), his end was no obse ed in hyb id ca ageenans (
e.g.
, compa ing expe imen s 2 w .%
in 0.5 M KCl, o samples E and K, wi h 60 and 90 mol.% κ, espec i ely).
These obse a ions sugges undamen al s uc u al di e ences be ween he wo sys ems. While hyb id
ca ageenans displayed lowe elas ici y, hey exhibi ed an in insically highe capaci y o esis s ain.
Howe e , u he compa a i e s udies a e equi ed o con i m his, pa icula ly in ol ing hyb id
ca ageenans and comme cial blends wi h a ying chemical composi ions es ed unde iden ical
polysaccha ide and ionic condi ions. In e es ingly, bo h hyb id ca ageenans and comme cial blends ich
in κ exhibi ed a s ain-ha dening esponse a low ionic s eng hs and a s ain-so ening esponse a high
ionic s eng hs, highligh ing a sha ed mechanical beha io despi e s uc u al di e ences.
In conclusion, in e ms o iscoelas ic p ope ies hyb id ca ageenans and comme cial blends may
o e speci ic ad an ages depending on he applica ion. While s onge and s i e gels p oduced by K+I
make hem be e sui ed o applica ions equi ing obus s uc u al in eg i y and minimal de o ma ion,
he s udied K-2 hyb id ca ageenan, o example, showcased i s po en ial o applica ions whe e gelling
abili y esis ance o la ge amoun o sal is desi able. Howe e , since inc eased elas ici y o en came a
he expense o educed esis ance o de o ma ion, leading o g ea e gel b i leness, i is impo an o
align he choice o ca ageenan sys em wi h he speci ic unc ional equi emen s o he a ge applica ion.
4.3 Food Applica ion
Gi en ha he ood indus y is one o he la ges ma ke s o ca ageenan, and conside ing he sec o 's
eme ging ends, he o mula ion o unc ional and ege a ian gummies based on hyb id ca ageenans
was de eloped and es ed. The signi icance o simula ing a eal-wo ld applica ion wi h hyb id
ca ageenans lies in he ac ha hei applicabili y is no solely dependen on hei beha io in he
p esence o a ying ionic s eng hs, sal s, and polysaccha ide concen a ions, bu also on hei
in e ac ions and syne gis ic e ec s wi h o he ing edien s, pa icula ly o he biopolyme s such as xan han
gum.
83
This speci ic ood applica ion was chosen because I had he oppo uni y o collabo a e wi h P o esso
Ge mán Valencia om he Fede al Uni e si y o San a Ca a ina (Flo ianópolis, B azil) on an ongoing
p ojec led by him and his eam. In pa icula , his wo k aligns wi h one o hei p ojec ’s goals: s udying
he applica ion o a na u al biohyb id ing edien , based on an hocyanins ex ac ed om jambolan ui
and adso bed on o ben oni e, in ca ageenan hyd ogels. The o mula ion in hei wo k, based on a s udy
by (Song e al., 2022),was adap ed o mee he speci ic objec i es o my esea ch and he esou ces
a ailable. Addi ionally, I ecognized ha his applica ion aligns wi h cu en ma ke ends, se ing as a
ege a ian al e na i e o animal-based (gela in) gummies while apping in o he g owing nu aceu ical
segmen ocused on unc ional o o i ied gummies (Maha e al., 2020). Gi en he global con ec ione y
sec o 's posi ion as one o he as es -g owing indus ies (Rawa e al., 2024), I pe sonally saw signi ican
po en ial and in e es in explo ing his speci ic applica ion.
The p ima y goal o his sec ion was o compa a i ely e alua e he pe o mance o alkaline- ea ed
hyb id ca ageenans agains bo h comme cial ca ageenan blends and un ea ed hyb id ca ageenans.
On he one hand, compa ing alkali- ea ed hyb id ca ageenans (
i.e.
, wi h biological p ecu so s emo ed)
o comme cial blends aimed o assess whe he , in a p ac ical applica ion, hei pe o mance di e s
signi ican ly o i hey could se e as iable subs i u es. On he o he hand, we looked in o whe he he
p esence o biological p ecu so s in he composi ion o hyb id ca ageenans esul ed in a signi ican
di e ence in gummy pe o mance. This conside a ion holds indus ial in e es , as using un ea ed hyb id
ca ageenans could educe p ocessing cos s and ime. While i is known ha alkaline ea men
signi ican ly enhances he gela ion po en ial o hyb id ca ageenans, when e alua ed wi hin a o mula ion
con aining o he ing edien s and biopolyme s, hese di e ences may o may no be c i ical o he inal
p oduc ’s quali y.
The hyb id ca ageenan samples o his applica ion we e selec ed based on esul s om he phase
diag am in KCl (Table 5, Sec ion 4.2.1), as heological es s we e no ye comple ed a ha ime.
Addi ionally, ime and sample quan i y cons ain s u he in luenced he selec ion o samples C, F, and
J, limi ing he assessmen o o he hyb id ca ageenans. Ne e heless, hese h ee samples, wi h
signi ican ly di e en κ/ι a ios, p o ide a gene al idea o he in luence o ι-con en on gummy
pe o mance when inco po a ed in o a o mula ion wi h o he biopolyme s and unc ional ing edien s.
Only samples ea ed wi h KOH we e es ed, as his alkaline solu ion yielded a mo e comp ehensi e
gela ion p o ile compa ed o NaOH (Tables 5 and 6, Sec ion 4.2.1).

84
4.3.1 Pa icle Size Dis ibu ion and Ze a Po en ial o he Biohyb id
The pa icle size dis ibu ion (PSD) and ze a po en ial (ZP) o he biohyb id ma e ial (BH) we e assessed
o e alua e hei implica ions o s abili y and dispe sion quali y in he gummy candy ma ix. The pa icle
size dis ibu ion o he BH dispe sed in wa e is shown in Figu e 17.
The pa icle size dis ibu ion o he biohyb id ma e ial (ben oni e wi h adso bed an hocyanins) showed
a imodal dis ibu ion, e lec ing i s complex agg ega ion beha io . The i s peak, a ound 0.5 μm, likely
ep esen s small clus e s o ben oni e pa icles. The second dominan peak, a 16-17 μm, co esponds
o la ge agg ega es o med as an hocyanins adso b on o he clay su ace, likely p omo ing u he
clus e ing o pa icles. The hi d peak a 140 µm sugges s he p esence o la ge , mo e loosely associa ed
agg ega es, po en ially o med du ing he mixing o d ying p ocess.
This obse ed imodal dis ibu ion aligns wi h beha io s epo ed in he li e a u e, whe e ben oni e-
based ma e ials commonly exhibi a b oad pa icle size ange due o hyd a ion, swelling, and agg ega ion
p ocesses (Bun in e al., 2022; Magzoub e al., 2020).
Powde ed ben oni e, ich in mon mo illoni e, shows a ine and mo e uni o m dis ibu ion, wi h a
pa icle size dis ibu ion be ween 0.5 and 200 µm (And ade e al., 2021; V yzas e al., 2016). Howe e ,
when ben oni e is dispe sed in wa e , i s pa icles hyd a e and swell, causing signi ican changes in he
size dis ibu ion, especially since he clay pa icles can expand se e al imes hei d y size (Magzoub e
al., 2020). The la ge pa icle sizes in he biohyb id ma e ial could be a ibu ed o he agg ega ion o
mon mo illoni e laye s, especially since an hocyanins adso b on o he su ace, which migh b idge he
Figu e 17: Pa icle size dis ibu ion o he biohyb id ma e ial dispe sed in an aqueous solu ion.
85
clay pa icles and cause clus e ing. This could also be due o he d ying me hod o o he p epa a ion
s eps, which end o encou age agg ega ion.
The ze a po en ial (ZP) o any pa icle is de ined as he numbe o cha ges i ca ies, and, along wi h
pa icle size dis ibu ion, i s assessmen is a e y impo an pa ame e o in e ing he s abili y o colloidal
dispe sions (Pa upudi e al., 2022; M. Sha ma, 2019; Singh i e al., 2018). Gene ally, pa icles wi h ze a
po en ial alues anging wi hin − 10 o + 10 mV a e conside ed o ha e neu al su aces while hose wi h
la ge absolu e alues o 30 mV (+/−) a e conside ed o be s ongly ca ionic o anionic. The e o e, high
su ace cha ge on pa icle su ace leads o epulsion and hus p e en s agg ega ion (Mekhame , 2010;
Pa a ale e al., 2012; Singh i e al., 2018). The ze a po en ial o he biohyb id/wa e sys em was -19.4
mV, sugges ing a mode a e le el o colloidal s abili y, wi h pa icles likely ending o agg ega e o e ime.
This ZP alue allows o weak in e ac ions be ween pa icles, leading o he o ma ion o la ge clus e s,
pa icula ly in he mic on and submic on anges
Fo a mo e homogeneous dis ibu ion in u u e applica ions, imp o ing elec os a ic s abiliza ion (
e.g.
,
by adjus ing pH o su ace modi ica ion (Magzoub e al., 2020; Mekhame , 2010) could educe
agg ega ion and esul in a mo e uni o m pa icle size dis ibu ion, which is c ucial o enhancing he
ma e ial’s pe o mance in he ma ix.
4.3.2 Visual Cha ac e iza ion o Gummy Candy Samples
The isual appea ance o all GC samples p epa ed was egis e ed in Figu es 18 A-C, o all sys ems in
s udy, using samples C, F and J, espec i ely.
The ca ageenan ma ices used enabled gel o ma ion o all GC samples excep UT-F (Figu e 18B).
This ou come is consis en wi h he balanced composi ion in ι and κ o he KI, oge he wi h he highe
con en o p ecu so uni s (see Figu e B.1 in Annex B.1), nega i ely in luencing he gelling beha io .
Howe e , i could also be a ibu ed o less ca e ul handling du ing he physical phase assessmen o
sligh empe a u e a ia ions, as i s coun e pa (UT-F + BH) gelled wi hou issue. Al e na i ely, BH could
shi he gel o ma ion condi ions as less wa e is a ailable. In any case, he heological assessmen ,
conduc ed unde mo e con olled cooling condi ions, will p o ide u he insigh s in o he ue gelling
beha io o his UT-F sample.
86
Examining he images o he gummy candies, a no iceable colo a ia ion can be obse ed.
Speci ically, samples wi hou he biohyb id ma e ial (BH) exhibi ed a mo e yellowish hue, while i s addi ion
esul ed in a ed/pink one in he un ea ed KI and comme cial K+I samples, and a g een one in he
alkaline- ea ed KI samples. This colo a ia ion is ela ed o he BH’s esponse o di e en pH
en i onmen s, due o he inco po a ion o pH-sensi i e an hocyanins (ACNs) (Koop e al., 2024; Me z e
al., 2020). The de ailed da a on he pH alues o each gummy candy sample, as well as hose o he
osema y honey and biohyb id ma e ial used, a e ga he ed in Table A.1, in Appendix A.5.
In he p esence o he BH, un ea ed KI and comme cial K+I samples showed pH alues be ween 3.6
and 3.9, a which he an hocyanins exhibi a pink hue. In con as , alkali- ea ed samples p esen ed highe
pH alues (a ound 7.8), whe e ACNs a e oxidized, leading o a blue colo a ion (Koop e al., 2024). When
10κ90ι
10κ90ι + BH
T-C
T-C + BH
UT-C + BH
UT-C
T-F
UT-F
50κ50ι
UT-F + BH
T-F + BH
50κ50ι + BH
UT-J
T-J
90κ10ι
UT-J + BH
T-J + BH
90κ10ι + BH
A
B
C
Figu e 18: Images o egan gummy candies p epa ed wi h un ea ed KI samples (UT-), ea ed KI samples (T-),
and he espec i e comme cial ca ageenan blends (mol.% κ; mol.% ι), bo h wi h and wi hou he biohyb id ma e ial
(BH). A) Sample C and Blend 10κ90ι, ich in ι-ca ageenan; B) Sample F and Blend 50κ50ι, wi h a balanced κ/ι
a io; and C) Sample J and Blend 90κ10ι, ich in κ-ca ageenan.
87
combined wi h he yellow ma ix o he gummy candies, his blue colo a ion ga e ise o a g een colo in
he alkaline- ea ed KI samples. The inc eased pH in he alkali- ea ed KI samples can be a ibu ed o
esidual KOH om he ea men p ocess, lea ing behind hyd oxide ions ([OH⁻]), which aise he pH o
he medium, c ea ing a mo e basic en i onmen .
Mic oscopic images we e cap u ed o all GC samples o assess he dispe sion quali y o he biohyb id
ma e ial (BH). Since he dispe sion beha io was consis en ac oss he h ee sys ems (C, F, and J), a
de ailed discussion will ocus on he mic oscopic images o sample C (Figu e 19). These images illus a e
di e ences in he dis ibu ion and p esence o BH wi hin he ma ices.
In Figu es 19 D, E and F he biohyb id ma e ial, colo ed ed o g een, appea s as dis inc agg ega es
wi hin he ma ix, indica ing a non-homogeneous dispe sion. These agg ega es a y in size, wi h la ge
clumps and smalle pa icles dispe sed h oughou he ma ix, in line wi h he p e iously discussed
pa icle size dis ibu ion o he biohyb id and i s ze a po en ial o -19.4 mV, which e lec s mode a e
colloidal s abili y. The dispe sion, hough e iden , is incomple e, wi h pa icles ending o clus e a he
han dis ibu e e enly.
Wi h BH
Wi hou BH
UT-C
T-C
10κ90ι
A
D
B
C
E
F
Figu e 19: Mic oscopic images o gummy candy samples using un ea ed ca ageenan C (UT-C) (A, D), alkali-
ea ed ca ageenan C (T-C) (
B, E), and comme cial blend 10κ90ι (C, F), wi hou (A-C) and wi h (D-F
) he addi ion
o a biohyb id ma e ial (BH). The biohyb id, composed o an hocyanins adso bed on o ben oni e, appea s as ed o
g een pa icles (D-F).
94
b oade ange o s ains as κ-con en inc eased, whe eas un ea ed samples ended o lose s uc u al
in eg i y mo e quickly. In e es ingly, he BH e e sed hese ends: i educed γL in ea ed samples bu
inc eased i in un ea ed samples as κ-con en ose. These obse a ions highligh he nuanced in e play
be ween chemical composi ion, BH p esence, and gel ne wo k beha io .
An in iguing s ain-ha dening esponse o inc easing s ain was obse ed in some samples,
pa icula ly hose om G oup 1 (high ι-con en ), whe e samples consis en ly exhibi ed so ening beyond
hei LVE egion, ollowed by a ansien phase o s ain ha dening, o ming an appa en pla eau. This
esponse likely e lec s he ini ial dis up ion o weake physical in e ac ions wi hin he gel ne wo k, ollowed
by empo a y eo ganiza ion o alignmen o polysaccha ide chains in o a mo e s ess- esis an
con igu a ion, un il he e en ual collapse o he s uc u e’s in eg i y. These mechanisms highligh he
complexi y o ca ageenan gel ne wo ks, whe e nonlinea elas ic e ec s and mic os uc u al
ea angemen s can momen a ily coun e ac de o ma ion be o e he ne wo k ul ima ely b eaks down
(Hilliou, 2021; Mo aes & Hilliou, 2024).
Impac o Ca ageenan Sou ce
The sou ce o polysaccha ide—whe he a comme cial blend, un ea ed hyb id ca ageenan, o alkali-
ea ed hyb id ca ageenan—exe s a no able in luence on he heological p ope ies o GC samples,
pa icula ly hei elas ic modulus and esis ance o de o ma ion.
Comme cial blends consis en ly exhibi ed highe G0 alues compa ed o hyb id ca ageenan samples,
highligh ing hei abili y o o m s i e gels. Among he hyb id samples, alkali- ea ed ca ageenans
gene ally p omo ed s onge gels ela i e o un ea ed samples. Howe e , excep ions o his end we e
obse ed, pa icula ly in G oup 1 (high ι-con en ). In his g oup, un ea ed hyb id ca ageenan samples
(
e.g.,
UT-C, wi h o wi hou he BH) su passed he elas ic modulus o hei ea ed coun e pa s (e.g., T-
C). Con e sely, in G oup 2 (balanced κ/ι a ios), he addi ion o he BH in oduced addi ional complexi y,
as i al e ed he expec ed ends in G0.
The a iabili y in he esis ance o de o ma ion (γL) u he complica es conclusions ega ding he
impac o he polysaccha ide sou ce. The s ain esis ance o gels appea ed o be highly dependen on
he chemical composi ion, including κ/ι a ios, as well as he inco po a ion o addi i es such as he BH.
This a iabili y unde sco es he complexi y o hese sys ems and he challenge o gene alizing he e ec
o polysaccha ide sou ces on s ain esis ance.

95
O e all, he choice be ween un ea ed and ea ed hyb id ca ageenans should be guided by he
speci ic heological p ope ies desi ed o he inal o mula ion. Fo example, in o mula ions wi h highe
ι-con en , i may no be necessa y o use ea ed hyb id ca ageenans o achie e highe G0 alues, as
un ea ed samples can some imes yield compa able o e en supe io s i ness. Howe e , he decision
mus also conside he desi ed s ain esis ance and o he iscoelas ic p ope ies, as hese a e equally
in luenced by he chemical composi ion, he p esence o biosyn he ic p ecu so s, and he inclusion o
addi i es.
Gene al Conclusion
The in e play be ween polysaccha ide sou ce, chemical composi ion, and addi i es mus be ca e ully
conside ed o ailo gel p ope ies o speci ic applica ions. Whe he he goal is o maximize s i ness,
enhance s ain esis ance, o achie e a balanced combina ion, each ac o mus align wi h he in ended
unc ionali y o he inal p oduc . Fo gummy o mula ions, decisions ega ding polysaccha ide sou ce and
ea men a e highly con ex -dependen , equi ing a clea unde s anding o desi ed ex u al and
mechanical p ope ies.
Howe e , while heological measu emen s p o ide essen ial insigh s in o gel beha io unde s ess,
hey cap u e only pa o he s o y. Addi ional es s, such as ex u e p o ile analysis (TPA), shel -li e s udies,
mic os uc u al analysis (
e.g.
, scanning elec on mic oscopy, SEM) and echniques o assess he mal
s abili y (
e.g.
, di e en ial scanning calo ime y, DSC), a e needed o ully unde s and he complexi y o
ex u e and s abili y in ood applica ions (Maha e al., 2020; Peleg, 2019; V. Sha ma & Bha dwaj, 2019).
Senso y es ing wi h ained panels o consume su eys could also p o ide aluable quali a i e eedback
on he ex u e, la o , and o e all sa is ac ion wi h he p oduc . In eg a ing heological da a wi h hese
b oade es ing me hods would o e a mo e comp ehensi e assessmen o gummy pe o mance,
ensu ing he inal p oduc mee s bo h echnical equi emen s and consume expec a ions.
4.3.4 Chemical In e ac ions (FTIR-ATR)
The Fou ie ans o m-in a ed (FTIR) spec um is a well-known me hod o de ec ing simila i ies and
di e ences in chemical s uc u es. Unde s anding whe he he addi ion o a biohyb id ma e ial causes
s uc u al and chemical changes wi hin he ma ix is c ucial o indus ial applica ions, as i di ec ly
impac s he p oduc 's physical p ope ies, s abili y, and unc ionali y. I also ensu es consis ency in
p oduc quali y and aids in p edic ing scalabili y challenges. The e o e, he GC samples we e
cha ac e ized using FTIR-ATR o in e possible chemical in e ac ions be ween he ing edien s.
96
This analysis was conduc ed o all he condi ions s udied: un ea ed-KI, alkali ea ed-KI, and
espec i e comme cial K+I blend, wi h and wi hou he addi ion o he BH, using KI samples C, F and J.
Since he gene al FTIR spec a was simila o he h ee sys ems: C, F and J, only sample J is discussed
in de ail (Figu e 20). The spec a o sys ems using samples C and F a e shown in Figu es A.10 and A.11,
espec i ely, in Appendix A.9.
The abso bance peaks ound in he FTIR spec a (Figu e 20) we e in e p e ed conside ing he
ing edien s o he o mula ions: wa e , ca ageenans, xan han gum, honey and he biohyb id ma e ial,
composed o ben oni e and an hocyanins. Fi s ly, no di e ences we e epo ed be ween samples wi h
and wi hou he BH, con i ming ha i s addi ion did no cause signi ican chemical al e a ions wi hin he
ma ix. This was an expec ed ou come due o he low concen a ion o BH used (only 0.5 w .%), poin ing
owa ds a possible limi o sensi i i y o FTIR o de ec such chemical in e ac ions.
The b oad band a 3280 cm-1 is a ibu ed o O-H s e ching ib a ions, which e lec s he p esence o
wa e (Ilyas e al., 2016; Kędzie ska-Ma ysek e al., 2018) and hyd oxyl g oups om ca bohyd a es
(honey, xan han, and ca ageenans) (Dam o e al., 2023; Kędzie ska-Ma ysek e al., 2018; Said e al.,
2021). The smalle peak ound be ween 2880 and 2920 cm-1 a ises om C-H s e ching ib a ions,
indica ing he p esence o alipha ic chains om suga s in honey and polysaccha ides like xan han gum
Figu e 20: FTIR-ATR spec a o gummy candies p epa ed, using un ea ed ca ageenan (UT-J), alkali ea ed
ca ageenan (T-J) and he chemical equi alen comme cial blend (90κ10ι), wi h and wi hou he biohyb id ma e ial
(BH), o sample J.
97
and ca ageenans (Kędzie ska-Ma ysek e al., 2018; Pe ei a, Amado, e al., 2009; Rochas e al., 1986;
Said e al., 2021).
The p ominen peak a 1640 cm-1 co esponds o he O-H bending mode o wa e (Ilyas e al., 2016;
Kędzie ska-Ma ysek e al., 2018). The bands egis e ed be ween 1350 and 1450 cm-1 a e cha ac e is ic
o bending ib a ion o O-CH and C-C-H in he ca bohyd a e s uc u e o bending ib a ion coming om
OH in he C-OH g ouping, whe eas he band a 1250 cm-1 is, in u n, a ib a ion cha ac e is ic o he
s e ching ib a ion o he C-H o CO in ca bohyd a es (Kędzie ska-Ma ysek e al., 2018, Dam o e al.,
2023). Ano he possibili y is ha hese FTIR bands, speci ically a 1370 cm-1 and in he 1240–1260
cm-1, ange a e ela ed o he p esence o sul a e es e s in ca ageenans and o he sul a ed
polysaccha ides, a ising om ib a ional modes o he S=O bond (Pe ei a, Amado, e al., 2009; Rochas
e al., 1986).
The isible elbow a app oxima ely 1050 cm-1 (nex o he cha ac e is ic band a 1025 cm-1) may be
due o s e ching ib a ion o C-O-C es e s (p esen in honey and an hocyanins, while he band a 1025
cm-1 is associa ed wi h C-O s e ching ib a ions (Kędzie ska-Ma ysek e al., 2018; Me z e al., 2020; Swe
e al., 2018). In he egion be ween 950 and 750 cm-1, he peaks a e likely due o C-O-S and C-O s e ching
om he 3,6-anhyd ogalac ose uni s and sul a e g oups in ca ageenans, as well as saccha ide ing
ib a ions and C–H de o ma ion, cha ac e is ic o he anome ic egion o ca bohyd a es (Dam o e al.,
2023; Gómez-O dóñez & Rupé ez, 2011; Kędzie ska-Ma ysek e al., 2018; Pe ei a, Amado, e al., 2009).
Fo mula ions using alkali- ea ed ca ageenans (T-J and T-J + BH) exhibi ed sligh de ia ions compa ed
o he o he wo sys ems unde s udy. No ably, a mino shi was obse ed in he peak assigned o he
C–H s e ching ib a ion associa ed wi h he o al suga con en , wi h a mo e p onounced band appea ing
a ound 2880 cm-1. Addi ionally, a new peak eme ged app oxima ely a 1700 cm-1, and ins ead o a single
peak a 1250 cm-1, he alkali- ea ed samples displayed wo dis inc peaks (a a ound 1200 cm-1 and 1300
cm-1). These obse a ions a e consis en wi h he con e sion o hyd oxyl g oups in o ca bonyl o es e
g oups, likely induced by he alkaline en i onmen . The appea ance o he ca bonyl s e ching band
(≈1700 cm-1) and he spli ing o he C–O s e ching egion (≈1200–1300 cm-1) a e cha ac e is ic o
es e i ica ion o s uc u al ea angemen s in polysaccha ides (Moba aki & Hemma eenejad, 2011;
Smi h, 2018).
In conclusion, while he inco po a ion o he biohyb id ma e ial imposed some changes in he
heological beha io o GC o mula ions, he e we e no isible di e ences in he FTIR spec a, indica ing
ha no new chemical bonds we e o med.
98
5. CONCLUSIONS AND FUTURE WORK
Selec ed hyb id ca ageenans (KI), along wi h hei chemically equi alen comme cial blends (K+I), we e
e alua ed h ough phase diag ams unde compa able sal and polysaccha ide concen a ions. Gel
o ma ion occu ed mo e eadily in K+I han in KI ca ageenans, ac oss almos all chemical composi ions
(in e ms o κ and ι con en ), polysaccha ide concen a ions, and ionic s eng hs, whe eas KI
ca ageenans showed a highe dependency wi h hose pa ame e s. KI ca ageenans demons a ed
gela ion beha io compa able o K+I unde speci ic condi ions, such as highe polysaccha ide
concen a ions o ι- ich samples and κ/ι-balanced samples in KCl and NaCl, and lowe ionic s eng hs
o κ- ich samples in KCl. K-2 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.
KI and K+I exhibi ed compa able cooling heological p o iles unde iden ical condi ions, pa icula ly in
KCl a la ge ionic s eng hs. Highe ι-ca ageenan con en and lowe ionic s eng hs a o ed wo-s ep
gela ion mechanisms, while highe κ-con en and ionic s eng hs esul ed in single-s ep gela ion.
Expe imen al limi a ions, such as es ic ed empe a u e anges and s ain sensi i i y, likely obscu ed
sub le ansi ions in some sys ems. Fu u e esea ch should employ ad anced heological echniques wi h
imp o ed con ol o gela ion kine ics o di e se applica ions.
Unde La ge Ampli ude Oscilla o y Shea (LAOS) es ing KI and K+I yielded dis inc mechanical
p ope ies unde iden ical ionic en i onmen s and polysaccha ide concen a ions. K+I gene ally p oduced
s onge and s i e gels han KI. Howe e , unde high ionic s eng hs, he K-2 hyb id ca ageenan
signi ican ly ou pe o med i s comme cial coun e pa in gel elas ici y. Simila ly, a low ionic s eng hs,
ce ain hyb ids exhibi ed compa able o e en supe io elas ici y. Inc eased elas ici y o en came a he
expense o educed esis ance o de o ma ion, leading o g ea e gel b i leness. This ade-o
unde sco es he impo ance o aligning he choice o ca ageenan sys em wi h he speci ic unc ional
equi emen s o he a ge applica ion. Ul ima ely, he iabili y o hyb id ca ageenans as subs i u es o
comme cial blends depends on he desi ed balance be ween gel s eng h, elas ici y, and de o ma ion
esis ance.
In gummy candy o mula ions, bo h KI and K+I ca ageenans success ully p oduced gel-like ex u es
ac oss all sys ems. KI demons a ed po en ial as e ec i e eplacemen s o K+I, ega ding he mal
pe o mance, o e ing a Tg ange (65 °C–100 °C) ha balances p ocessing lexibili y and p oduc s abili y.
In e ms o mechanical p ope ies, K+ I p oduced s i e gels (G0) wi h supe io s eng h and elas ici y han
99
KI, consis en wi h p io indings. Alkaline ea men gene ally enhanced he s i ness and s ain esis ance
o hyb id ca ageenans, excep o high ι-con en samples, whe e un ea ed ca ageenans ou pe o med
hei ea ed coun e pa s. This sugges s ha while p ecu so emo al is o en bene icial, he addi ional
alkali ea men s ep may be unnecessa y o speci ic applica ions. The inco po a ion o he biohyb id
ma e ial o en inc eased s i ness bu educed s ain esis ance, pa icula ly in ea ed hyb ids and
comme cial samples wi h g ea e κ-con en .
These indings unde sco e he in ica e in e play be ween ca ageenan sou ce, chemical composi ion,
and addi i es in shaping gel pe o mance, emphasizing he need o o mula ion-speci ic op imiza ion.
Hyb id ca ageenans show signi ican po en ial as clean-label, egan, and unc ional ood ing edien s,
combining s uc u al and heal h-p omo ing bene i s. Re ining o mula ions and p ocessing condi ions,
alongside u he e alua ions o ex u e, he mal s abili y, and senso y p ope ies, will be key o ensu e
he inal p oduc mee s bo h echnical and consume equi emen s.

100
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A.3 Mechanical Spec a o Samples in KCl
Figu e A.4: 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 KCl solu ions. Each ow ep esen s a di e en hyb id
ca ageenan and blend pai , and each column shows esul s a di e en concen a ions and ionic s eng hs.
Top ow - sample C s. 10κ90ι 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. 60κ40ι 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. 90κ10ι 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.
Sample C s. Blend 10κ90ι
Sample E s. Blend 60κ40ι
Sample K s. Blend 90κ10ι
A
B
C
D
E
F

127
A.4 La ge Ampli ude Oscilla o y Shea Tes s
A.4.1 La ge Ampli ude Oscilla o y Shea Tes s o Samples in KCl
Figu e A.5: 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. 10κ90ι 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. 60κ40ι 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. 90κ10ι 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.
Sample C s. Blend 10κ90ι, in KCl
Sample E s. Blend 60κ40ι, in KCl
Sample K s. Blend 90κ10ι, in KCl
A
B
C
D
E
F
G
H
I
128
A.4.2 La ge Ampli ude Oscilla o y Shea Tes s o Samples in NaCl
Figu e A.6: 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. 10κ90ι 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; and bo om ow - sample K’ s. 90κ10ι 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.
Sample C’ s. Blend 10κ90ι, in NaCl
Sample K’ s. Blend 90κ10ι, in NaCl
A
B
C
D
E
F
129
A.5 pH Values o Gummy Candies
Sample
pH (T = 22 ºC)
C
Un ea ed
UT-C
4.02
UT-C + BH
3.48
Alkali-T ea ed
T-C
7.99
T-C + BH
7.90
Blend
10κ90ι
4.45
10κ90ι + BH
3.84
F
Un ea ed
UT-F
4.25
UT-F + BH
3.65
Alkali-T ea ed
T-F
7.58
T-F + BH
7.79
Blend
50κ50ι
4.38
50κ50ι + BH
3.81
J
Un ea ed
UT-J
4.32
UT-J + BH
3.71
Alkali-T ea ed
T-J
7.75
T-J + BH
7.64
Blend
90κ10ι
4.73
90κ10ι + BH
4.01
Rosema y Honey
3.54
BH + Dis illed Wa e
3.88
Table A.1: pH alues o gummy candies p epa ed wi h and wi hou he biohyb id ma e ial (BH), as well as he pH
o osema y honey and he biohyb id used in he p epa a ions
130
A.6 Tempe a u e Dependence o he S o age (G’) and Loss (G’’) Moduli in
Gummy Candies
Figu e A.7: Tempe a u e dependence o he s o age modulus (G’, ull squa es) and loss modulus (G’’, open
squa es) o gummy candy samples. Resul s a e p esen ed o comme cial blends (in ed), alkali- ea ed (T-) hyb id
ca ageenans (in blue) and un ea ed (UT-) hyb id ca ageenans (in g een). Each ow ep esen s a di e en se o
samples based on hei chemical composi ion: op ow (A, B) 10κ90ι, T-C and UT-C; middle ow (C, D) 50κ50ι,
T-F and UT-F; bo om ow (E, F) 90κ10ι, T-J and UT-J. G aphs in he igh column (B, C, D) show he e ec s o
inco po a ing he biohyb id ma e ial (+BH).
A
(
γ
L
)
B
(
γ
L
)
C
(
γ
L
)
D
(
γ
L
)
E
(
γ
L
)
F
(
γ
L
)
131
A.7 Mechanical Spec a o Gummy Candies
Figu e A.8: Mechanical spec a o gummy candy samples, showing he s o age modulus (G’, ull squa es) and
loss modulus (G’’, open squa es) as a unc ion o equency a ia ion. Resul s a e p esen ed o comme cial blends
(in ed), alkali- ea ed (T-) hyb id ca ageenans (in blue) and un ea ed (UT-) hyb id ca ageenans (in g een). Each
ow ep esen s a di e en se o samples based on hei chemical composi ion: op ow (A, B) 10κ90ι, T-C and
UT-C; middle ow (C, D) 50κ50ι, T-F and UT-F; bo om ow (E, F) 90κ10ι, T-J and UT-J. G aphs in he igh
column (B, C, D) show he e ec s o inco po a ing he biohyb id ma e ial (+BH).
A
B
C
D
E
F

132
A.8 La ge Ampli ude Oscilla o y Shea Tes s o Gummy Candies
Figu e A.9: La ge ampli ude oscilla o y shea (LAOS) es s showing he s o age modulus (G’, ull squa es) and
loss modulus (G’’, open squa es) as a unc ion o applied s ain o gummy candy samples. Resul s a e p esen ed
o comme cial blends (in ed), alkali- ea ed (T-) hyb id ca ageenans (in blue) and un ea ed (UT-) hyb id
ca ageenans (in g een). Each ow ep esen s a di e en se o samples based on hei chemical composi ion:
op ow (A, B) 10κ90ι, T-C and UT-C; middle ow (C, D) 50κ50ι, T-F and UT-F; bo om ow (E, F) 90κ10ι,
T-J and UT-J. G aphs in he igh column (B, C, D) show he e ec s o inco po a ing he biohyb id ma e ial (+BH).
A
B
C
D
E
F
133
A.9 FTIR-ATR Spec a o Gummy Candies
A.9.1 FTIR-ATR Spec a o Gummy Candies, o sys ems using Sample C and Comme cial Blend 10κ90ι
A.9.2 FTIR-ATR Spec a o Gummy Candies, o sys ems using Sample F and Comme cial Blend 50κ50ι
Figu e A.10: FTIR-ATR spec a o gummy candies p epa ed, using un ea ed ca ageenan (UT-C), alkali ea ed
ca ageenan (T-C) and he chemical equi alen comme cial blend (10κ90ι), wi h and wi hou he biohyb id ma e ial
(BH), o sample C.
Figu e A.11: FTIR-ATR spec a o gummy candies p epa ed, using un ea ed ca ageenan (UT-F), alkali ea ed
ca ageenan (T-F) and he chemical equi alen comme cial blend (50κ50ι), wi h and wi hou he biohyb id ma e ial
(BH), o sample F.
134
B. ANNEXES
B.1 Chemical Composi ion o Un ea ed Hyb id Ca ageenans
B.2 Molecula Mass Dis ibu ion o Un ea ed Hyb id Ca ageenans and
Comme cial Pu e K- and I-ca ageenans
Figu e B.1: Chemical composi ion o un ea ed hyb id ca ageenan samples.
Figu e B.2: Molecula mass dis ibu ion (Mw) and polydispe si y index (PDI) o un ea ed samples (A–L), as well
as comme cial pu e K- and I-ca ageenans.