Sequential extraction of natural products from carrageenophytes: focus on hybrid carrageenans and the impact of the molecular mass on their gel properties

Uni e si y o Minho
School o Enginee ing
Ma ia Gab iela A onso Gonçal es
Sequen ial ex ac ion o na u al p oduc s
om h ee ca ageenophy es: ocus on
hyb id ca ageenans and he impac o
molecula mass on hei gel p ope ies
Decembe 2024
Sequen ial ex ac ion o na u al p oduc s om h ee
ca ageenophy es: ocus on hyb id ca ageenans and he
impac o molecula mass on hei gel p ope ies
Ma ia Gab iela A onso Gonçal es
UMinho | 2024
Ma ia Gab iela A onso Gonçal es
Sequen ial ex ac ion o na u al p oduc s om
ca ageenophy es: ocus on hyb id ca ageenans
and he impac o he molecula mass on hei gel
p ope ies
Decembe 2024
Mas e ’s Disse a ion
Mas e s in Biological and Chemical Enginee ing
Disse a ion supe ised by
Loïc Hilliou, PhD
B uno Fa ia, PhD
ii
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iii
ACKNOWLEDGEMENTS
I am deeply g a e ul o e e yone who, in some way, con ibu ed o he ealiza ion o his hesis. This
wo k is no only he culmina ion o six mon hs o i e yea s bu a es amen o my con inuous g ow h as
a p o essional and as a pe son.
Fi s and o emos , my since e hanks o P o esso D . Loic Hilliou om he Ins i u e o Polyme s and
Composi es a he Uni e si y o Minho, o welcoming me in o his esea ch g oup and o his unwa e ing
belie in my capabili ies. I am g a e ul o his s aigh o wa d and scien i ic app oach du ing ou mee ings,
ne e allowing discou agemen o ake hold, e en when aced wi h se backs. His lexibili y in le ing me
shape he p ojec 's ocus o align wi h my in e es s was uly in aluable. I would also like o exp ess my
app ecia ion o P o esso D . B uno Fa ia, whose insigh ul con e sa ions pushed me o iew my wo k
om esh pe spec i es, always d i ing i s imp o emen . I am immensely hank ul o my labo a o y and
cou se colleague, Ma ia Alice Mon ei o, o he iendship, gene osi y and he suppo she o e ed wi h
such wa m h. Each con e sa ion wi h he was a sou ce o mo i a ion ha echa ged me. I also ex end
my hanks o Rui Ribei o, Izabel Mo aes, João Al es and Ma ga ida Gonçal es, whose assis ance was
indispensable o he p og ess o my labo a o y expe imen s.
To my dea iends, I am p o oundly g a e ul o you p esence and cons an encou agemen . Thank
you o belie ing in me, e en in momen s o doub , and o always being he e o li me up when I el
o e whelmed. You unwa e ing suppo , he momen s o laugh e , and he sha ed d eams we e wha
kep me going. I ca y a deep app ecia ion o each o you.
Finally, and mos impo an ly, I wish o exp ess my p o ound g a i ude o my pa en s, my sis e and
my g andpa en s. Mom and Dad, hank you o you uncondi ional lo e, o always being my sa e ha bou ,
and o ins illing in me he esilience o chase my d eams wi hou ea . Xana, hank you o being my
sou ce o endless inspi a ion. And g andpas, hank you e y much o e e y hing, you ai h in me is a gi
beyond wo ds.
This wo k was suppo ed by he Fundação pa a a Ciência e Tecnologia (FCT), h ough he E2B2-
PHACAR p ojec (h p://doi.o g/10.54499/PTDC/BII-BIO/5626/2020). Addi ional inancial suppo by
he FCT unde he amewo k o S a egic Funding g an : UID/CTM/50025/2020 and g an :
CEECINST/00156/2018 (h ps://doi.o g/10.54499/CEECINST/00156/2018/CP1642/CT0012) a e
also acknowledged.
To all o you, my deepes hanks.

i
STATEMENT OF INTEGRITY
I he eby decla e ha ing conduc ed his academic wo k wi h in eg i y. I con i m ha I ha e no used
plagia ism o any o m o undue use o in o ma ion o alsi ica ion o esul s along he p ocess leading o
i s elabo a ion.
I u he decla e ha I ha e ully acknowledged he Code o E hical Conduc o he Uni e si y o Minho.
Uni e si y o Minho, B aga, Decembe 2024
Ma ia Gab iela A onso Gonçal es
RESUMO
Ex ação sequencial de p odu os na u ais de ês ca agenó i as: oco em ca ageninas híb idas e o
impac o da massa molecula nas suas p op iedades de geli icação
As ca ageninas são polissaca ídeos sul a ados conhecidas pelas suas p op iedades de
geli icação e mo e e sí eis, amplamen e u ilizadas nas indús ias alimen a , a macêu ica e cosmé ica.
Recen emen e, as ca ageninas híb idas (K2) êm despe ado in e esse de ido às suas p op iedades de
geli icação in e mediá ias. Es e es udo em como obje i o desen ol e um mé odo de ex ação sequencial
(SE) pa a algas e melhas (
Chond us c ispus
,
Mas oca pus s ella us
,
Giga ina pis illa a
), alinhado ao
concei o de bio e ina ia, po ex usão ex a i a. Os p incipais obje i os des e abalho incluem a ex ação
e ca ac e ização de K2-ca ageninas, a aliando o impac o de ex ações p é ias e o empo de ex ação.
Adicionalmen e, p e ende-se es uda o e ei o da edução de massa molecula (Mw) e de e mina uma
massa molecula c í ica (Mc) pa a a geli icação da K2-ca agenina. Pa a isola icobilip o eínas, clo o ila-
a, ca o enoides e ca ageninas das algas, oi u ilizada a seguin e sequência: ex ação com água ia
(CWE), ex ação e anólica (EE), ex ação com água quen e (HWE) e ex ação alcalina quen e (HAE). O
endimen o oi de e minado pa a cada ação, e os ex a os de ca ageninas o am analisados quan o à
Mw, aos ní eis de sul a ação (FTIR, 1H-NMR) e às p op iedades eológicas dos seus géis e líquidos
esul an es. A edução da Mw oi alcançada a a és de ul assonicação, com os p odu os deg adados
sujei os a análises semelhan es às an e io es. Os esul ados indicam que a SE sepa a e icazmen e os
componen es das algas, acili ando a ecupe ação de ca ageninas com al a Mw, pa icula men e em
M.
s ella us
e
G. pis illa a
, com elas icidade de gel (G’) e iscosidade supe io es em compa ação com os
con olos. A HWE p oduz ca ageninas de al o Mw e com p op iedades eológicas melho adas, enquan o
os ex a os HAE ap esen am meno Mw e po encial de geli icação eduzido. Em ge al, ex ações
p olongadas melho am a Mw e a G', embo a
C. c ispus
enha mos ado uma endência dis in a. Os
esul ados da Mw des acam que o ul assom le a à diminuição e dessul a ação das ca ageninas ao
longo do empo de p ocessamen o. Ademais, a Mc depende da composição química, suge indo uma Mc
de ~1·10⁵ g/mol pa a híb idas icas em κ e ~3·10⁵ g/mol pa a híb idas icas em ι, além das quais a
deg adação adicional em impac o mínimo na geli icação. Em suma, es e es udo demons a o po encial
de adap ação de p o ocolos de ex ação pa a a ende às necessidades indus iais, con ibuindo pa a a
alo ização sus en á el das algas e melhas a a és de mé odos de bio e ina ia.
Pala as-cha es: Ca ageninas híb idas; ex ação sequencial; massa molecula ; eologia.
i
ABSTRACT
Sequen ial ex ac ion o na u al p oduc s om h ee ca ageenophy es: ocus on hyb id ca ageenans
and he impac o molecula mass on hei gel p ope ies
Ca ageenans a e sulpha ed polysaccha ides known o hei he mo- e e sible gelling p ope ies,
widely used in he ood, pha maceu ical, and cosme ic indus ies. Hyb id ca ageenans (K2) ha e ecen ly
gained in e es o hei in e media e gelling p ope ies. This s udy aims o de elop a sequen ial ex ac ion
(SE) me hod o ed seaweeds (
Chond us c ispus, Mas oca pus s ella us, Giga ina pis illa a
), aligning
wi h he concep o ex ac i e ex usion bio e ine y. The p ima y objec i es o his s udy include ex ac ing
and chemically cha ac e izing K2-ca ageenans, e alua ing he impac o p io ex ac ions and ime o
ex ac ion. Addi ionally, his wo k seeks o assess he in luence o molecula mass (Mw) educ ion on
gelling p ope ies and o de e mine a c i ical molecula mass (Mc) o gela ion. To isola e
phycobilip o eins, chlo ophyll-a, ca o enoids, and ca ageenans om he algae, he ollowing sequence
was used: cold-wa e ex ac ion (CWE), e hanolic ex ac ion (EE), ho wa e ex ac ion (HWE), and ho
alkaline ex ac ion (HAE). Each ac ion's yield was de e mined, wi h ca ageenan ex ac s analysed o
Mw, sulpha ion le els ( ough FTIR, 1H-NMR), and hei heological p ope ies in gels and liquids.
Molecula mass educ ion was achie ed h ough ul asonica ion, and he deg aded p oduc s we e
cha ac e ized in a simila manne as be o e. Resul s indica e ha SE e ec i ely sepa a es algal
componen s, acili a ing he eco e y o high-Mw ca ageenans, pa icula ly in
M. s ella us
and
G.
pis illa a
, wi h supe io gel elas ici y (G’) and iscosi y compa ed o he con ols. HWE yields high-Mw
ca ageenans wi h enhanced heological p ope ies, whe eas HAE ex ac s exhibi lowe Mw and educed
gela ion po en ial. P olonged ex ac ion gene ally imp o es Mw and G’, al hough
C. c ispus
showed a
dis inc end. The Mw esul s highligh ha ul asonica ion leads o he deg ada ion and desulpha ion o
ca ageenans o e p ocessing ime. Fu he mo e, he Mc depends on he chemical composi ion,
sugges ing an Mc o ~1·10⁵ g/mol o κ- ich hyb ids and ~3·10⁵ g /mol o ι- ich hyb ids, beyond which
u he deg ada ion has minimal impac on gela ion. In summa y, his s udy demons a es he po en ial
o adap ing ex ac ion p o ocols o mee indus ial demands, con ibu ing o he sus ainable alo isa ion
o ed algae h ough bio e ine y me hods.
Keywo ds: Hyb id ca ageenan; molecula mass; heology; sequen ial ex ac ion.
ii
CONTENTS
1. In oduc ion .............................................................................................................................. 17
1.1 Con ex .............................................................................................................................. 17
1.1.1 Ca ageenans ............................................................................................................. 17
1.1.2 P ojec E2B2 – PHACAR ............................................................................................. 17
1.2 Mo i a ion .......................................................................................................................... 18
1.2.1 Bio e ine y concep ..................................................................................................... 18
1.2.2 Impac o Mw in he heological p ope ies .................................................................. 19
1.3 Goals ................................................................................................................................. 19
1.4 S uc u e o he disse a ion ............................................................................................... 20
2. S a e o a ................................................................................................................................ 21
2.1 Red Algae .......................................................................................................................... 21
2.1.1 Composi ion ............................................................................................................... 22
2.1.2 Pigmen s o Red Algae ................................................................................................ 24
2.2 Ca ageenan ...................................................................................................................... 27
2.2.1 Rele ance .................................................................................................................. 27
2.2.2 Chemical s uc u e o ca ageenans ............................................................................ 28
2.2.3 Ex ac ion ................................................................................................................... 30
2.2.4 Gel P ope ies ............................................................................................................ 32
2.2.5 Molecula Mass, Mw ................................................................................................... 34
2.3 Sequen ial Ex ac ion ......................................................................................................... 37
2.3.1 Bio e ine y S a egies o he Valo isa ion o Red Seaweeds ......................................... 38
2.3.2 Ex ac i e – Ex usion ................................................................................................. 40
3. Ma e ials and Me hods .............................................................................................................. 42
xi
ACRONYMS
1H-NMR
P o on Nuclea Magne ic Resonance
AFM
A omic Fo ce Mic oscopy
ATR
A enua ed T ansmission Re lec ance
CAGR
Compound Annual G ow h Ra e
Ca o
Ca o enoid
Chlo o-a
Chlo ophyll-a
Con
Con ol
CWE
Cold Wa e Ex ac ion
D-uni
α-D-galac opy anose
d.w.
D y Weigh
DA-uni s
3,6-anhyd ogalac ose
E2B2-PHACAR
Ex ac i e Ex usion-Based Bio e ine y o polyhyd oxyalkanoa es and ca ageenans
EE
E hanolic Ex ac ion
FTIR
Fou ie T ans o m In a ed Spec oscopy
G-uni
β-D-galac opy anose
G’
Dynamic/elas ic moduli
G’’
Loss moduli
G0
G’ a 1 Hz a 25ºC o gels
G1
G’ a 1Hz and 25 ºC o liquids
ɣ
Shea s ain
HAE
Ho Alkaline Ex ac ion
HWE
Ho Wa e Ex ac ion
I
Io a (a ype o ca ageenan)
K
Kappa (a ype o ca ageenan)
K2
Hyb id K/I (a ype o ca ageenan)

x
L
Lambda
MAE
Mic owa e-Assis ed Ex ac ion
Mc
C i ical Molecula Mass
Mn
Numbe -a e age Molecula Mass
Mu
G4S-D6S
Mw
Molecula Mass
Mwgel
Mw equi ed o gel o ma ion
Nu
G4S-D2S,6S
PDI
Polydispe si y Index
PUFAs
Omega-3 and Omega-6 polyunsa u a ed a y acids
R-PE
R-phycoe y h in
RTD
Residence Time Dis ibu ion
SE
Sequen ial Ex ac ion
Time
T
Tempe a u e
Tg
Gela ion Tempe a u e
Tm
Mel ing Tempe a u e
TON
Onse Tempe a u e
UAE
Ul asound-Assis ed Ex ac ion
w .%
% To al Weigh
η
Appa en Shea Viscosi y
ι
G4S-DA2S
κ
G4S-DA
ω
F equency
x i
LIST DE SYMBOLS
h
Hou s
min
Minu es
ºC
Deg ees Celsius
17
1. INTRODUCTION
1.1 Con ex
1.1.1 Ca ageenans
Ca ageenans a e sulpha ed polysaccha ides, p esen in he cell walls o ed algae (
Rhodophy a
)
(Abdul e al., 2018; Campo e al., 2009; Shukla e al., 2016). They a e comme cially impo an due o
hei he mo- e e sible gelling p ope ies (Hilliou, 2021), and widely used as ood addi i es (E407) o
hickening, s abilizing, and gelling, in p oduc s like ice c eam, sauces, mea , and as a egan gela ine
al e na i e (Souza e al., 2023; S ephen e al., 2006). The unique cha ac e is ics o ca ageenans a ise
om a ia ions in hei molecula s uc u e, including di e en sulpha e g oups and molecula mass.
Unde s anding hese p ope ies is essen ial o ailo ing hei applica ion in a ious ields, especially gi en
he inc easing demand o na u al and sus ainable bio-based p oduc s.
1.1.2 P ojec E2B2 – PHACAR
This s udy is pa o he E2B2-PHACAR p ojec , which aims o de elop a no el bio e ine y concep
called he Ex ac i e Ex usion-Based Bio e ine y (E2B2). The E2B2 app oach ocuses on he con inuous
and sequen ial ex ac ion o a ange o aluable p oduc s, including polyhyd oxyalkanoa es (PHA), na u al
compounds, and gelling hyb id ca ageenans (CAR). So a , comme cial ex ude s ha e been employed
o p e- ea men o e y ew biomasses (Vauchel e al., 2008) o o he ex ac ion o single p oduc s
(Sugiono e al., 2019). The E2B2 p ojec seeks o expand hese applica ions by eeding biomass in o an
ex ude , o ex ac mul iple p oduc s in a cascade manne , as seen in Figu e 1. Wi hin his amewo k,
e men a ion b o hs o PHA om mixed mic obial cul u e and seaweeds con aining hyb id ca ageenans
a e being s udied. The esea ch eam has con i med he block copolyme s uc u e o K2-ca ageenans
(Souza e al., 2023) and iden i ied complex in e ac ions be ween hei chemical composi ion and he
mechanical and s uc u al p ope ies o he esul ing gels (Hilliou, 2021). Howe e , he exac mechanism
and s uc u al cha ac e is ics o K2-ca ageenan gels emain un esol ed. Add essing hese challenges is
he second key objec i e o he E2B2-PHACAR p ojec .
18
Figu e 1. Model o he ex ac i e-ex usion concep o he E2B2-PHACAR p ojec .
1.2 Mo i a ion
1.2.1 Bio e ine y concep
Seaweeds possess unique bioac i e componen s no ound in e es ial biomass, posi ioning hem
as an excep ional and sus ainable esou ce. Thei po en ial is pa icula ly e iden in seaweed-based
bio e ine ies, which align wi h he p inciples o ci cula economy and sus ainabili y by p omo ing he
e icien use o esou ces, educing en i onmen al impac , and con ibu ing o job c ea ion and economic
g ow h. This is also in line wi h key Sus ainable De elopmen Goals ou lined by he Uni ed Na ions, such
as clean ene gy, economic g ow h, esou ce-use e iciency, esponsible consump ion and p oduc ion, and
clima e ac ion. (Ál a ez-Viñas e al., 2019; Balina e al., 2017; To es, K aan, e al., 2019).
Sequen ial ex ac ion as a bio e ine y app oach holds p omise by allowing o he eco e y o
mul iple aluable p oduc s. Howe e , as said be o e, comme cial ex ude s ha e adi ionally been limi ed
o deli e ing a single ex ac and an ex uda e. Es ablishing a con inuous bio e ine y concep capable o
e ining biomass in o a cascade o p oduc s di ec ly along he ex ude is a goal ha emains unexplo ed
(see Figu e 1).
In he cu en s a e, K2-ca ageenans a e ba ch-ex ac ed h ough a esou ce-in ensi e p ocess,
in ol ing alkali p e- ea men ollowed by ho wa e ex ac ion (Bianchi e al., 2022; Souza e al., 2023).
Such me hods equi e subs an ial chemical inpu s and gene a e signi ican was e. The E2B2 p ojec
add esses hese limi a ions by in es iga ing he easibili y o sequen ial ex ac ion wi hin an ex ude ,
whe e seaweed is p ocessed wi h wa e and alkali, allowing o mul iple p oduc s o be eco e ed wi h
19
minimal en i onmen al oo p in compa ed o con en ional me hods. Al hough E2B2’s applica ion has so
a ocused on K2-ca ageenans, ed seaweeds also con ain o he aluable componen s, such as p o eins
and pigmen s, which could be explo ed in u u e de elopmen s.
1.2.2 Impac o Mw in he heological p ope ies
The molecula mass (Mw) o ca ageenans plays a pi o al ole in de e mining hei gelling
p ope ies, including elas ici y and mechanical s eng h (Souza e al, 2023). In he con ex o he E2B2-
PHACAR p ojec , unde s anding how Mw a ia ions a ec he gels mechanical p ope ies is c ucial, as
he p ojec aims o e ine and expand he sequen ial ex ac ion p ocess, and he ex ac ion condi ions
di ec ly in luence he Mw o ca ageenans. Howe e , despi e i s impo ance, he li e a u e lacks
comp ehensi e s udies speci ically on he Mw-dependen gelling beha iou o K2-ca ageenans. As pa
o he second key objec i e o he E2B2-PHACAR p ojec , his s udy aims o add ess hese knowledge
gaps by explo ing he ela ionship be ween Mw and gel o ma ion, ocusing on K2-ca ageenans ex ac ed
h ough his no el bio e ine y concep .
1.3 Goals
The p ima y goal o his hesis is o design a p elimina y p ocedu e o he sequen ial ex ac ion o
K2-ca ageenans, o be used la e wi h an ex ude unde he ex ac i e-ex usion echnique, in alignmen
wi h he objec i es o he E2B2-PHACAR p ojec . The aim is o ex ac a ious p oduc s om algal biomass
using di e en sol en s and ex ac ion condi ions, and o chemically and heological cha ac e ize he K2-
ca ageenans ob ained, assessing he impac o p io ex ac ions on sequen ial p oduc s. Fu he mo e,
his s udy seeks o es ablish ela ionships be ween ex ac ion du a ion and gel elas ici y.
The seconda y goal o his hesis is o e alua e he e ec o molecula mass educ ion on he gelling
p ope ies o K2-ca ageenans, using ul asonica ion. Addi ionally, i seeks o de e mine whe he a c i ical
molecula mass (Mc) o K2-ca ageenan gela ion exis s and, i so, o iden i y his h eshold.

20
1.4 S uc u e o he disse a ion
This documen is o ganized as ollows:
• 2. S a e o he A : This chap e p o ides a comp ehensi e o e iew o he key opics ele an o
his disse a ion. I begins by discussing he composi ion o ed algae and explo ing he di e en
ypes and chemical s uc u es o ca ageenans, wi h a pa icula ocus on K2-ca ageenans. I
also co e s cu en ex ac ion me hods and he exis ing knowledge on he in luence o molecula
mass on gel p ope ies. Addi ionally, i in oduces he concep o sequen ial ex ac ion and he
ex ac i e-ex usion echnique, si ua ing hese app oaches wi hin he b oade con ex o
sus ainable and e icien bio e ine y p ac ices.
• 3. Ma e ials and Me hods. This chap e de ails he ma e ials and me hodologies employed o
ca y ou he p oposed asks in his s udy. I includes a ho ough desc ip ion o he sequen ial
ex ac ion p ocedu e, and he cha ac e iza ion echniques used o analyse he ex ac ed and
ul asonica ed K2-ca ageenans.
• 4. Resul s and Discussion. This sec ion p esen s he esul s ob ained h oughou he s udy,
accompanied by an in-dep h analysis and discussion. I begins wi h he yield o ex ac ed
p oduc s, ollowed by he heological and chemical p ope ies o he a ious ca ageenan ex ac s.
Then, he nex pa add esses he impac o ul asonica ion on K2-ca ageenans, examining
molecula mass and heological beha iou o p o ide a holis ic unde s anding o he e ec s o
molecula b eakdown.
• 5. Conclusions and u u e wo k. This concluding chap e summa izes he key indings o he
s udy, emphasizing he main con ibu ions and insigh s gained. I also ou lines po en ial a enues
o u u e esea ch, ocusing on un esol ed challenges and oppo uni ies o ad ancing he
sequen ial ex ac ion p ocess and op imizing ca ageenan gel p ope ies.
21
2. STATE OF ART
Ma ine mac oalgae, commonly known as seaweed, a e complex pho osyn he ic o ganisms p esen
in a ious aqua ic en i onmen s. They ha e been common since ancien imes o alimen a y and
medicinal pu poses, especially in Asian coun ies (Ba bo e al., 2016). Ad ancemen s in scien i ic
esea ch and no el me hodologies ha e acili a ed he iden i ica ion o compounds de i ed om seaweed.
These compounds ha e been ex ensi ely s udied o hei po en ial he apeu ic e ec s on he human
body, including hei an ioxidan , an imic obial, and an i umo al p ope ies (Loma i e e al., 2021).
Mo eo e , seaweeds migh be also employed in se e al indus ial applica ions, such as he p oduc ion o
e ilize s (Balina e al., 2017; To es, K aan, e al., 2019), aquacul u e eed, bio uels, in was ewa e
ea men s o o p oduce inno a i e, and ecologic ma e ials o eplace plas ic equi alen s (Kim e al.,
2017; Raiko a e al., 2019), con ibu ing owa ds a sus ainable solu ion o p o ec he en i onmen om
he discha ge o non-biodeg adable plas ic. Also, ma ine mac oalgae p esen economic and
en i onmen al ad an ages o e e es ial biomass, including apid g ow h a e, no compe i ion o
ag icul u al land and he depolyme isa ion o seaweeds be o e he biological con e sion in o bio uels is
acili a ed due o he high ca bohyd a e and low lignin con en , among o he ad an ages (Ál a ez-Viñas e
al., 2019). So, seaweeds a e an in e es ing op ion o he bio e ine y concep .
Seaweeds a e classi ied in o h ee main g oups acco ding o hei colou , which is de e mined by
he p esence o ce ain pigmen s. B own seaweeds (phylum
Och ophy a
, class
Phaeophyceae
) ha e an
abundance o pigmen s ha a y om yellow o da k b own (Seely e al., 1972). Red seaweeds (phylum
Rhodophy a
) a e cha ac e ized by hei high amoun s o ca o enoids, chlo ophyll-a, phycoe y h in,
phycocyanin, and allophycocyanin (Denis e al., 2009). G een seaweeds (phylum
Chlo ophy a
) possess
mainly he pigmen chlo ophyll, which has a c ucial ole in pho osyn hesis and gi es he cha ac e is ic
g een colou o plan s, algae, and cyanobac e ia (A yee e al., 2018).
2.1 Red Algae
Red seaweeds a e o economic impo ance, hey ep esen 61 % o global seaweed p oduc ion
(Penuela e al., 2018), and a e p ima ily used o ex ac ing aga and ca ageenan. Mo eo e , hey also
con ain o he compounds o in e es , in amoun s a ying quali a i ely and quan i a i ely among di e en
22
species, especially since he ex ac ion me hod is no gene ally s anda dized and o he ac ha a ious
abio ic and bio ic ac o s, such as he g ow h s age, ha es loca ion and pe iod, dep h, nu ien quan i y
and quali y, empe a u e, salini y, ligh exposu e, may also di e (Gene alić Mekinić e al., 2023).
To p o ide a comp ehensi e unde s anding o he ed seaweed, he ollowing sec ion will i s explo e
he gene al composi ion o ed algae, be o e ocusing on speci ic compounds ele an o his s udy
(pigmen s and ca ageenans). These compounds will be s udied in h ee dis inc ed algal species:
Chond us c ispus
,
Giga ina pis illa a
, and
Mas oca pus s ella us.
P o iding a aluable oppo uni y o
explo e hei po en ial wi hin he amewo k o a bio e ine y app oach.
2.1.1 Composi ion
I is undamen al o ha e some knowledge abou he composi ion o he ed algae and hei mos
s udied biological p ope ies, o pa e he way o he u iliza ion o his esou ce. So, in he nex lines he
gene al composi ion o he ed algae is discussed.
Ca bohyd a es
Acco ding o hei p e alence in algal sou ces, aga and ca ageenan, bo h sulpha ed
polysaccha ides, known as phycocolloids, a e he mos ele an ca bohyd a es in ed seaweeds,
accoun ing o up o 40–50 % o hei d y weigh (d.w.) (To es, Fló ez-Fe nández, e al., 2019) ollowed
by o he polysaccha ides ound in signi ican ly lowe amoun s, such as xylans, sulpha ed galac ans and
po phy ans (Ø e land e al., 2019).
P o eins
Among he di e en algae classes ound in ma ine ecosys ems, ed seaweeds exhibi he highes
con en o p o eins (Belghi e al., 2017; Ø e land e al., 2019). Gene ally, he p o ein con en o algae
anges be ween 5 % and 20 %, al hough ed algae may achie e g ea e p opo ions, wi h maximum alues
eaching 47 % o o al d.w. (Cian e al., 2012; P a een e al., 2019; Rud ana ip e al., 2018). Ne e heless,
p o eins depic a species-dependen occu ence, conside ing ha some species, such as hose om
G acila ia
genus p esen a low p o ein con en (below 5 % d.w.) whe eas o he s like
Py opia
ene a
show
a p o ein con en o 37 % d.w. (Hold & K aan, 2011). Fu he mo e, in e ms o o al p o eins,
phycobilip o eins a e he p edominan p o eins in ed seaweeds, cons i u ing up o 50 % o he o al
23
p o ein con en and con ibu ing o he eddish colo a ion cha ac e is ic o hese algae (Niu e al., 2010).
Rega ding he amino acid composi ion o ed algae p o eins, a high concen a ion o essen ial amino
acids has been documen ed, wi h aspa ic acid and glu amic acid being he mos abundan esidues,
comp ising 22–44 % o he o al amino acids ound in ed algae p o eins (Cian e al., 2012).
Lipids
Despi e low lipid con en , ed algae a e ich in nu i ionally impo an PUFAs (Omega-3 and
Omega-6 polyunsa u a ed a y acids). Thei omega-3:omega-6 a io is highe han in e es ial sou ces
and could be used as nu aceu icals o hei an i-hype choles e olemic, an ioxidan , an icance ,
an idiabe ic, an ihype ensi e, and an i-in lamma o y ac i i ies (Chan & Ma anjun, 2017; Kuma i e al.,
2013).
Vi amins and Mine als
Rhodophyceae
species con ain impo an mac omine als, like po assium, sodium, calcium and
magnesium (6,1–21,9 g K/100 g d.w., 1,8–8,1 g Na/100 g, 0,2–0,9 g Ca/100 g d.w. and 0,2–0,5 g
Mg/100 g d.w.) (Baghel e al., 2014; Kuma e al., 2011; Ma anjun e al., 2009). In he g oup o ace
mine als, i on was ound o be he mos abundan , ollowed by zinc, coppe , and selenium. They could
be po en ial ing edien s o unc ional oods, p o iding a supplemen a ion in de ici a y elemen s in die s
(Nunes e al., 2018). Red seaweeds also con ain i amins, wi h 2,1–2,7 mg p o- i amin A/g, 0,05–1,54
mg i amin B2/g, 3,8–4,8 mg i amin B6/g, 0,4–1,0 mg i amin B9/g, 2,5–501 mg i amin C/ 100
d.w., 1,34 mg i amin E/g d.w. and 4,61 µg α- ocophe ol/g d.w.(Ál a ez-Viñas e al., 2019; Chan &
Ma anjun, 2017). Indeed, i amins om ma ine sou ces ha e been al eady used o he en ichmen o
unc ional oods (Šima e al., 2020).
Phenolic Compounds
Red seaweeds con ain a a ie y o phenolic compounds, including na u al phenolics like phenolic
acids and la onoids, as well as speci ic ma ine-de i ed compounds, such as phlo o annin and
b omophenol. These compounds a e known o hei powe ul an ioxidan p ope ies. Thus, phenolic acids
epo ed in ed algae a e couma ic acid, ca eic acid, salicylic acid, hypogallic acid, and chlo ogenic acid
(Ca pena e al., 2023).
30
ca ageenan disaccha ides in o less sulpha ed ca ageenans (Souza e al., 2023). Figu e e ie ed om: Souza e
al., (2023).
I-ca ageenan is essen ially a homopolyme o G4S-DA2S disaccha ide uni s, con aining a e y
low amoun , o he o de o 5 mol %, o G4S-DA disaccha ide uni s. K-ca ageenan is somewha mo e
he e ogeneous, al hough s ill p ac ically a homopolyme , wi h up o 10 mol % o G4S-DA2S disaccha ide
uni s, discon inuous wi h blocks o disaccha ide uni s, G4S-DA (Hilliou, 2021; Souza e al., 2023). Mo e
simply, hese ca ageenans consis o polysaccha ide chains con aining bo h κ and ι uni s, as ep esen ed
in Figu e 3, anging om nea ly pu e I-ca ageenan o nea ly pu e K-ca ageenan (Campo e al., 2009).
Wi hin he mo e he e ogeneous ca ageenans, he hyb ids, K2-ca ageenan has a polyme ic s uc u e
( an de Velde, 2008), wi h sequences o G4S-DA (which make up 45-80 mol % o he hyb id chain) and
G4S-DA2S (which ep esen 20-50 mol % o he chain) (Hilliou, 2021), hence i s de ini ion in he indus y
as kappa-2 o weak kappa ( an de Velde, 2008). So, hyb id K2-ca ageenan a e copolyme s made o
blocks o κ and ι wi h a ious leng hs and andom dis ibu ion in he chain (Guibe e al., 2008). The
block size along he K2 chain, a ies om chain o chain, and i s dis ibu ion in he chain is andom
(s a is ic copolyme ) and speci ic o he seaweed biology ha p oduces i , as well as he ex ac ion
p ocess. So, K2-ca ageenans a e s a is ic block copolyme s, wi h polydispe si y bo h in he chain and in
block size (Campo e al., 2009; Souza e al., 2023).
Figu e 3. Schema ic ep esen a ion o he s uc u e o ca ageenans (K, I and hyb ids). The κ -uni s a e ep esen ed
in blue, he ι-uni s in ed and he biological p ecu so s (nu/mu) in g een.
2.2.3 Ex ac ion

31
Ca ageenans a e adi ionally ex ac ed om
C. c ispus
wild popula ions in Canada, I eland,
Po ugal, Spain and F ance, and om
Giga ina
collec ed in Sou h Ame ica and Sou he n Eu ope.
Howe e , he g owing demands o ca ageenans mo i a ed he es ablishmen o mac oalgae a ming
sys ems, wi h
Eucheuma sp
. in Philippines (Ca pena e al., 2023; Hedbe g e al., 2018) becoming he
majo p oduce wo ldwide and sp eading mac oalgae cul i a ion along o he Asian coun ies, p omo ing
he p oduc ion o
Po phy a sp.
(no i),
Kappaphycus al a ezii
( ich in K) and
Eucheuma den icula um
( ich
in I) (Ca pena e al., 2023).
The cu en ex ac ion p ocesses a e slow and complica ed, in ol ing se e al s eps. Fu he mo e,
i ’s qui e di icul o op imize and con ol he p oduc ion o ca ageenan, due o i s unique in insic
p ope ies, which a e highly dependen on he o igin o he seaweed, he geog aphical loca ion and he
ha es ing season (Abdul e al., 2018). No mally, ca ageenan indus ial ex ac ion p ocesses a e di ided
in o d ying and milling, alkaline p e- ea men , ho wa e ex ac ion, p ecipi a ion and d ying (Souza e al,
2023).
Na i e ex ac s, wi hou alkaline ea men , gene ally exhibi poo o absen gelling p ope ies. In
con as , an alkaline ea men a highe concen a ions and ex ended du a ions esul s in ca ageenan
wi h signi ican ly enhanced gel quali y (Aze edo e al., 2015). So, an alkaline ea men is usually
pe o med p io o ex ac ion, bu can also be done a e , o educe he numbe o sulpha ed g oups (o
p ecu so s) in K2-ca ageenan. This alkaline p ocess can be adjus ed o each seaweed (Abdul e al.,
2018; Souza e al., 2023). Aze edo e al., (2015) when s udying he ex ac ion o K2-ca ageenan om
wo algae,
C. c ispus
and
Ahn el iopsis
de oniensis
, obse ed ha excessi e KOH concen a ion p omo es
he depolyme iza ion o K2 and, consequen ly, he elas ici y o he gel is educed (see sec ion 2.2.5). The
p e ious s udy also pe cei es ha a e h ee hou s o he alkaline p e- ea men and one hou o na u al
ex ac ion a 90 ºC, yields o
C. c ispus
achie ed wi h NaOH a e la ge han hose achie ed wi h KOH
(45-70 % agains 35-60 %), whe eas a e e se end is obse ed o
A. de oniensis
(10-20% agains 15-
30%) (Aze edo e al., 2015). Using he same p o ocol Aze edo e al. (2013) shown ha o
M. s ella us
yields ob ained wi h NaOH we e la ge han hose achie ed wi h KOH (40 - 50% agains 30 - 43%), in
ag eemen wi h esul s ob ained wi h
C. c ispus
. So, o his wo algae NaOH equi es lowe
concen a ions and sho e p e- ea men du a ions compa ed o KOH o isola e K2 wi h op imal gel
p ope ies, indica ing ha NaOH is mo e e ec i e in con e ing he p ecu so s in o κ and ι-ca ageenan
(Aze edo e al., 2015).
The K2 ex ac ion p ocess usually begins by dispe sing he algae in ho wa e (Abdul e al., 2018)
I has ecen ly been demons a ed ha he solubili y o hyb id ca ageenans in wa e is s ongly dependen
32
on he ype o ca ions p esen in he algae a e being ha es ed, he ime o con ac wi h he wa e and
he empe a u e (T) used du ing ex ac ion (Aze edo e al., 2015). In o de o achie e op imal yields,
indus ial ca ageenan ex ac ions a e known o be pe o med a 80 ºC o 6 h. Addi ionally, i has been
epo ed ha he composi ion and heological beha iou o ca ageenan a e di e en when ex ac ed a
di e en empe a u es, o example be ween 60 ºC and 90 ºC (Baha i, Moelan s, Wallecan, e al., 2021).
As a ule, a highe T p oduces a less sulpha ed K2, howe e , he e a e excep ions, e.g. o
Gymnogong us
enuis
, a less sulpha ed ca ageenan was ob ained in cold wa e han in wa e a 80 ºC (Pe ez Recalde
e al., 2016)
A e ex ac ion, he solid esidues (sal s o med om ca ions o he alkaline ea men and om
he seaweeds) a e sepa a ed om he K2- ich solu ion by il a ion o cen i uga ion. K2 is hen eco e ed
om he solu ion, usually by p ecipi a ion wi h alcohol o wi h KCl (Abdul e al., 2018). Al e na i ely, K2-
ca ageenan no subjec ed o alkaline ea men can be eco e ed in ilm o powde o m by e apo a ing
wa e om solu ions wi h high K2 concen a ions o by eeze-d ying. Howe e , his me hod is sui able
only o K2 wi h high κ con en and su icien sal s o o m a s ong gel. Finally, pu i ica ion o K2 can be
achie ed using enzyma ic ea men s, among o he me hods (Souza e al., 2023).
2.2.4 Gel P ope ies
The capaci y o o m gels is unique o ca ageenans ha ha e he abili y o o m o de ed helical
s uc u es, being gela ion one o he se e al possible consequences o helix o ma ion (S ephen e al.,
2006).
Gel o ma ion mechanism
The mechanism o gel o ma ion in ol es he coil- o-helix ansi ion, ob ained by cooling ho
ca ageenan aqueous solu ions, ollowed by he agg ega ion o ca ageenan helices, esul ing in a h ee-
dimensional ne wo k. This mechanism is widely ecognized in li e a u e, bu his was ecen ly challenged
by Wes be y e al., (2024) who p oposed ha he gel o ma ion p ocess is mo e in ica e, in ol ing
coope a i e in e ac ions and he p esence o p e-exis ing helical s uc u es in he diso de ed s a e. This
new unde s anding could p omp a econside a ion o he mechanisms behind ca ageenan gela ion and
ela ed polysaccha ides. As o he s uc u e o he ne wo k ha pe mea es he olume and p o ides
elas ici y is a opic on which he e is li le ag eemen among au ho s. The e a e s udies ha epo he
33
o ma ion o single helices and o he s ha suppo he o ma ion o he double helix (Hilliou, 2021). Thus,
se e al models ha e been p oposed ying o desc ibe he s uc u e o he ne wo k. Recen ly, i was
possible o isualize by a omic o ce mic oscopy (AFM) he p ima y (coils), seconda y (helices) and supe
helical s uc u es o solu ions and gels. Fo K gels, od-shaped helices we e isualized, while mo e cu ed
s uc u es we e epo ed o , I gels (Hilliou, 2021). Fu he mo e, in he p esence o K⁺, i was possible o
obse e qua e na y s uc u es o s acked helices linked by helical b anching o o he domains in K gels.
Howe e , o I gels, independen ly o he ype o sal p esen , he single helix mesh was always obse ed.
Also, he o ma ion o ne wo ks a he supe helical le el, o K gels esul s in ha d gels, in con as o I
gels, which a e so e (Hilliou, 2021; Souza e al., 2023; S ephen e al., 2006).
The s udy o K2 gels is s ill unde way. Howe e , he ew publica ions wi h AFM images sugges
ha he helices and s uc u e o K2 a e simila o hose o K, wi h sligh ly mo e b anching and a
mo phology less simila o a “ od”. Acco ding o Souza e al. (2023) he mechanism o o ma ion o K2
gel esembles ha o K+I mix u es, essen ially wi h coil-helix ansi ions, independen o each ype o
block and i s agg ega ion. Ne e heless, i ’s indica ed ha hese simila i ies do no help in he
iden i ica ion o he na u e o he agg ega ion/s uc u ing and e en less in unde s anding he o igin o he
elas ici y o K2 gels (Souza e al. 2023). As K2 gels a e di e en om K+I mix u es, e en wi h a simila
G4S-DA (κ) mola con en , unde simila gelling condi ions ( o al ca ageenan concen a ion and ionic
s eng h), in K+I mix u es, I can be sepa a ed om K unde speci ic saline condi ions, whe eas in K2 his
sepa a ion is no possible. K2 is capable o adop ing helical con o ma ions independen ly o i s κ mola
ac ion, bu he e is s ill a long way o go un il a comple e unde s anding o he in e ac ion be ween such
mac omolecula s uc u es and he p ope ies o he K2 gel (Souza e al., 2023; S ephen e al., 2006).
Addi ionally, he e is no wa e syne esis in K2 gels, a leas in he li e a u e e iewed so a . Thus,
he s udied K2 gels may be a aluable al e na i e o he epo ed mix u es o comme cial K and I gels,
whe e wa e syne esis occu s du ing gel o ma ion o sys ems wi h ionic s eng hs abo e 0,1 M. Namely,
in applica ions whe e an ionic s eng h as high as 1 M is equi ed, K2 will success ully eplace comme cial
K+I mix u es as syne esis- ee gels wi h simila elas ici y a e o med (To es, Aze edo, e al., 2016).
P esence o sal s
Since ca ageenans a e polyelec oly es, hey a e highly sensi i e o he composi ion o he sal s
p esen in he aqueous medium (S ephen e al., 2006). So, i is known ha i is possible o adjus some
gel p ope ies, such as elas ici y, yield s ess and he mal s abili y, by con olling he amoun and ype o
34
sal added (S ephen e al., 2006). K-ca ageenan is ca ion-speci ic, meaning i s gel s i ness and
b i leness depend on he p esence o speci ic ca ions, such as K⁺ o Ca²⁺, which s abilize i s helical
s uc u e (Hilliou e al., 2006). In solu ions wi h KCl o CaCl₂, K-ca ageenan o ms s ong, s i , and b i le
gels, whe eas I-ca ageenan, which lacks his speci ici y, p oduces much weake gels ega dless o he
ca ion. Thus, he con ibu ion om ι blocks o he gel s uc u e and elas ici y in K2 is ac ually masked by
he con ibu ions om κ blocks, when in he p esence o he p e ious ca ions (K⁺/Ca²⁺). Howe e , in he
p esence o NaCl, K o ms weake gels, and hus i is easie o access i s con ibu ion in K2 gels. As
expec ed, simila esul s we e ob ained o he K2 gel, in which he weakes (and mos s able) gels we e
ob ained wi h NaCl, and he s onges o CaCl2. When no sal is used, K2 can show a gel beha iou , bu
la ge concen a ions and low empe a u es a e necessa y (To es e al., 2018).
Rheology
S udies o heological beha iou s, which ocus on he dynamic s o age/elas ic and shea loss
moduli (G’ and G’’, espec i ely) and he yield s ess, a e ele an o he indus ial applica ions o
ca ageenans, bu also o be e unde s and he gela ion mechanism (Souza e al., 2023). Acco ding o
Van de Velde (2008), in a mixed K⁺/Ca²⁺ solu ion, he gel elas ic modulus (G’) g adually dec eased wi h
he dec easing ac ion o κ uni s, indica ing ha G’ o K2 gels does no each a maximum a any speci ic
K⁺ concen a ion. This e lec s he in e media e na u e o K2 hyb ids, cha ac e ized by a con inuous
ansi ion in gel s i ness a he han a dis inc peak. Howe e , he gela ion empe a u e (Tg) o K2 hyb ids
wi h mo e han 50 mol% κ uni s emains independen o composi ion in he p esence o KCl bu becomes
highly sensi i e in NaCl solu ions, inc easing wi h highe κ con en and dec easing wi h lowe κ con en
(Souza e al., 2023). Addi ionally, he e is also a s ong dependence o he gel’s mel ing empe a u e
(Tm) on bo h he concen a ions o K-ca ageenan in K2 and NaCl (To es, Aze edo, e al., 2016; To es,
Chenlo, e al., 2016).
2.2.5 Molecula Mass, Mw
The molecula mass (Mw) o K2 ca ageenans is a complex cha ac e is ic in luenced by ex ac ion
me hods, empe a u e and he speci ic chemical s uc u e o he ca ageenan i sel . The e ec o
molecula mass on he yield s ess and G’ o he gel was de e mined, o K-ca ageenan, wi h di e en
alues o polyme concen a ions and sal concen a ions by Rochas e al., (1990), ha concluded ha
35
he yield s ess is di ec ly p opo ional o he molecula mass, bu no e y sensi i e o he sal
concen a ion. In con as , he G’ inc eased s eadily wi h Mw, up o a c i ical alue (Mc) o 1,8·105 g/mol,
independen ly o he polyme concen a ion and ionic con en . Beyond his limi , he G’ emained cons an
and independen o he Mw, bu e y sensi i e o he sal concen a ion (Rochas e al., 1990), as seen in
Figu e 4. Fo K2-ca ageenan, he e is s ill insu icien in o ma ion o unde s and he e ec o a ying Mw
and whe he Mc depends on he chemical composi ion in κ and ι o K2.
Figu e 4. Illus a ion o he a ia ion o he elas ic modulus (G’) wi h molecula mass (Mw) o K-ca ageenan. I
s abilizes om Mc, ega dless o he 3 di e en polyme concen a ions (5, 10, 20 g/L). Adap ed om: Rochas e
al., (1990).
Impac o Ex ac ion Pa ame e s
The molecula mass o isola ed K2 samples is signi ican ly in luenced by he milling p ocess p io
o biopolyme ex ac ion. G inding seaweeds in o smalle pa icles inc eases he yield o K2-ca ageenans
du ing wa e ex ac ion, esul ing in compounds wi h highe molecula masses. Consequen ly, la ge
molecula masses a e ob ained om powde s wi h smalle pa icle sizes, while smalle molecula masses
a e associa ed wi h la ge pa icle sizes. Fu he mo e, he polydispe si y index (PDI), ep esen ed by he
Mw/Mn a io (whe e Mn is he numbe -a e age molecula weigh ), ends o inc ease wi h Mw (To es e
al., 2016). In he p e ious e e ence i was obse ed ha di e ences in K2 molecula mass we e likely
due o la ge K2 molecules expe iencing g ea e di usion esis ance h ough he seaweed pa icles du ing
ex ac ion. In con as , powde s wi h smalle pa icle sizes esul ed in a highe p opo ion o la ge K2
G’

36
molecules, likely due o inc eased su ace a ea and enhanced accessibili y o p ecu so s o he sol en .
These indings sugges ha pa icle size dis ibu ion and di usion cha ac e is ics signi ican ly impac he
yield and molecula composi ion o he ex ac ed ac ions (To es, Chenlo, e al., 2016).
Molecula mass is also highly in luenced by he ex ac ion pa ame e s used du ing he eco e y
o ca ageenan. I is well known ha s onge and longe alkaline p e- ea men s end o educe he chain
size o K2, esul ing in a lowe Mw (Souza e al., 2023; Van De Velde e al., 2005). On K2-ca ageenans
s udies showed ha NaOH is as e han KOH in con e ing he biological p ecu so s (mu e nu) in o he
disaccha ide uni s κ and ι (Aze edo e al., 2013, 2015). So, his e iciency is e lec ed in he Mw o he
esul ing K2, as sho e alkaline ea men imes a e equi ed o achie e he desi ed le els o sulpha ed
disaccha ide uni s (Velde e al., 2005; Souza e al., 2023).
The empe a u e a ia ions can also lead o changes in Mw, esul ing in di e en ypes o gels.
Du ing he d ying s ep, in he case o
M. s ella us
, highe d ying empe a u es led o a lowe Mw o K2-
ca ageenan (Mo ei a e al., 2016). Bu o K2 ex ac ed, om
Gymnogong us enuis,
using cold wa e ,
in he ex ac ion s ep, esul ed in a di e en Mw p o ile compa ed o a mo e sulpha ed hyb id ca ageenan
eco e ed a 80 °C, which does no quali y as K2-ca ageenan. Al hough, highe empe a u es gene ally
inc ease yield, excessi e hea can lead o hyd olysis, causing deg ada ion. The e o e, i is impo an o
ca e ully con ol he ex ac ion empe a u e o p e en excessi e b eakdown o he ca ageenan (Baha i,
Moelan s, Wallecan, e al., 2021; Mo ei a e al., 2016; Souza e al., 2023).
Ca ageenan hyd olysis can also occu in low pH condi ions (Gus aw & Mleko, 2003) o unde
high salini y condi ions, such as in he p esence o NaCl o KCl. High sal concen a ions can dis up he
in e molecula in e ac ions ha main ain ca ageenan s uc u e, making i mo e suscep ible o hyd olysis,
pa icula ly when combined wi h high empe a u es o acidic condi ions. Howe e , s udies on he e ec s
o sal s on ca ageenan ex ac ion emain su p isingly limi ed (Baha i, Moelan s, Wallecan, e al., 2021).
Toxici y
Unde s anding he Mw o K2-ca ageenans is essen ial o i s indus ial applica ions, pa icula ly
in ood and cosme ic indus ies whe e ex u al p ope ies a e c i ical. The abili y o manipula e Mw h ough
ex ac ion me hods can lead o ailo ed p oduc s ha mee speci ic consume needs, wi h he desi ed
ex u es and s abili y in p oduc s. Fo example, ood-g ade ca ageenan ypically has a molecula mass
g ea e han 100 kDa, wi h comme cial p oduc s anging om 200–800 kDa (To es, Fló ez-Fe nández,
e al., 2019). No ably, ca ageenan is nei he deg aded no abso bed in he gas oin es inal ac , bu i
37
should no con ain low molecula mass ac ions, as hese can ha e oxic e ec s, including gas oin es inal
i i a ion and po en ial ca cinogenic p ope ies a high doses (To es, K aan, e al., 2019; Weine , 2016).
Fu he s udies a e necessa y o explo e i s impac on diges i e p ocesses, he colon mic obiome, and
in lamma ion, especially in p edisposed popula ions (Wu e al., 2016). To despi e he p e iews claims in
a e iew a icle, Bixle , (2017) emphasize ha ca ageenan is app o ed o ood use by majo egula o y
agencies wo ldwide, including he example o i s ecen inclusion in liquid in an o mula, ha highligh s
i s sa e y in sensi i e applica ions, and ha he e is no
in i o
e idence o suppo he p e ious claims.
Thus, while egula o y bodies a i m i s sa e y, public scep icism uelled by misin o ma ion emains a
signi ican hu dle o he indus y.
In esume, he e is a ecognized need o mo e s udies ocusing on he op imiza ion o ex ac ion
pa ame e s o be e unde s and how hey a ec he Mw and chemical s uc u es o K2-ca ageenans. In
pa icula , he e is no published esul s (such as hose illus a ed in Figu e 4) on he e ec s o Mw on he
gel p ope ies o K2-ca ageenans, and his could lead o imp o ed applica ions and unc ionali ies o K2-
ca ageenans in a ious indus ial con ex s.
2.3 Sequen ial Ex ac ion
The cu en indus ial ex ac ion ou es in he ca ageenan sec o ha e se e al weaknesses,
p ima ily due o hei high consump ion o ime, ene gy and wa e . Addi ionally, la ge amoun s o chemical
eagen s and sol en s a e equi ed o achie e op imal yield and quali y. The pollu an s and e luen s
gene a ed om chemicals used du ing ex ac ion and alkaline ea men , such as KCl, KOH, and NaOCl,
pose signi ican isks o heal h and he en i onmen , necessi a ing s ic con ol measu es (Abdul e al.,
2018). In addi ion, he alkaline ea men s ep, in pa icula , is a ela i ely ha sh and des uc i e p ocess
o he algae, causing he b eakdown o he issue ma ix and he e icien solubiliza ion o nea ly all he
ca ageenan. The esidual algal biomass, s ill ich in o ganic componen s, is gene ally un eco e able and
was ed a e his s ep. So, sho ening he ime o he alkaline s ep, while s ill p oducing ca ageenans o
high heological quali y would undoub edly be o in e es o he indus y (Vauchel e al., 2008).
Consequen ly, g eene ex ac ion me hods ha use less ene gy, educe wa e consump ion, and employ
mo e en i onmen ally iendly sol en s a e eme ging. Mic owa e-assis ed ex ac ion (MAE) (Ál a ez-Viñas
e al., 2023) and Ul asound-Assis ed Ex ac ion (UAE) (Maia e al., 2023) ha e shown he bes esul s in
e ms o ex ac ion yields and K2 gel p ope ies, wi h a sho e ex ac ion ime and ecological impac (Kes
38
e al., 2017). Howe e , MAE and UAE a e di icul o apply in indus ies and expensi e (Sugiono e al.,
2019).
Ano he hypo hesis in ol es using a win-sc ew ex ude -assis ed ex ac ion, which o e s se e al
ad an ages: i equi es sho e p ocessing imes, educes he amoun o sol en and eac an s needed,
gene a es minimal was e, and is sa e and sui able o indus ial applica ions (Vauchel e al., 2008).
Addi ionally, he cu en ba ch indus ial p ocess o ca ageenan ex ac ion is limi ed o p oducing only
one polyme pe cycle, lea ing many aluable compounds un eco e ed and was ed, so he ull po en ial
o he algal biomass is no being u ilized. The global ca ageenan indus ies p ocess 202 500 d y ons o
ca agenophy es annually o p oduce 65 000 ons o ca ageenan (Penuela e al., 2018) and he
emainde is los as was e. Wi h he inc ease o ca ageenan p oduc ion expec ed in he coming yea s,
he alo isa ion o he was e ac ions becomes necessa y.
2.3.1 Bio e ine y S a egies o he Valo isa ion o Red Seaweeds
A succession o ex ac ion s eps can lead o he sequen ial sepa a ion o p oduc s o in e es ,
om he same biomass, maximizing i s alue and gene a ing ewe esidues, while main aining he ocus
on he p oduc ion o phycocolloid. Fo ca agenophy es, a bio e ine y app oach should p io i ize
ca ageenan ex ac ion as he p ima y objec i e, gi en i s well-es ablished and indus ialized p oduc ion
p ocess (Ál a ez-Viñas e al., 2019; Balina e al., 2017; To es, K aan, e al., 2019). E o s o alo ise
sides s eams and u ilize he en i e ed algae biomass h ough a bio e ine y app oach a e inc easingly
being discussed. Va ious models ha e been p oposed o seaweeds o p oduce a wide ange o p oduc s,
ypically concluding wi h he ex ac ion o phycocolloids, a e all o he aluable componen s ha e been
emo ed.
In ac , bio e ine y concep s ha e al eady been applied o
M. s ella us
indus ial was e om
con en ional ex ac ion and ha e demons a ed ha K2-ca ageenan and an ioxidan compounds can be
u he ex ac ed om he was e (Bianchi e al., 2022). Maia e al. (2023) de eloped a 3-s ep sequen ial
ex ac ion using UAE o eco e pigmen s, p o eins, and ca ageenans om
Chond us c ispus
. The s udy
epo s he p esence o chlo ophyll-a and ca o enoids wi h mean alues a ying om 289,2 and 432,2
µg/g ex ac d.w., o 39,9 and 59,9 µg/g d.w., espec i ely. O he ac ions ob ained h ough UAE
con ained p o eins and ca ageenans yields in he anges o 3,6–41 g/100 g and 29,7–36,1 g/100 g,
espec i ely. Mo eo e , Baha i e al. (2021) p oposed a me hod o ex ac bo h phycoe y h in and hyb id
39
ca ageenan om he same species, beginning wi h he ex ac ion o phycoe y h in unde mild
condi ions. Thei indings showed ha a an ex ac ion empe a u e o 22 °C, educing pa icle size
signi ican ly enhanced phycoe y h in concen a ion ( om 0,26 mg/g in >2000 µm pa icles o 2,30
mg/g in <50 µm ac ions). In con as , ca ageenan yield only sligh ly inc eased ( om no eco e y in
>2000 µm pa icles o 2,1 g/kg in <50 µm), emaining much lowe han he yields ob ained a highe
empe a u es o 45 ºC (5,4 g/kg) and 90 ºC (9,9 g/kg). These esul s sugges ha phycoe y h in
ex ac ion is su ace-a ea dependen , whe eas ca ageenan ex ac ion elies mo e hea ily on
empe a u e. A sequen ial ex ac ion app oach has also been es ed wi h
Solie ia ili o mis
o ob ain a
nu ien - ich ex ac , PUFA- ich lipids, and pu e I-ca ageenan, using MAE. This me hod demons a ed
he po en ial o maximizing yields while gene a ing only 6,3–10,4 % o esidues om he ini ial biomass,
a signi ican educ ion compa ed o he esidues gene a ed h ough di ec ex ac ion o each p oduc ,
which we e 15 imes highe (Penuela e al., 2018).
In a s udy by Baha i e al. (2021), he ex ac ion o K2-ca ageenan om
C. c ispus
was
e alua ed using a sequen ial app oach unde di e en imes and empe a u es, wi hou he use o
alkaline p e- ea men . This elimina ion aimed o explo e a cleane ex ac ion p ocess, hough i allowed
p ecu so uni s (mu and nu) o emain in he ca ageenan, educing i s gelling p ope ies. Besides, i has
been shown ha ca ageenan’s composi ion and heological beha iou can a y signi ican ly depending
on he ex ac ion empe a u e and du a ion. Knowing ha he con en ional indus ial p ocesses,
ca ageenan is ypically ex ac ed a 80ºC o 6 hou s, he p e ious s udy in es iga ed wo ypes o
ex ac ion p ocedu es: he i s was a wo-s ep p ocess, beginning wi h an ini ial wash (0,5 hou s) and
ex ac ion o he seaweed a oom empe a u e o 8 hou s, ollowed by a second (sequen ial) ex ac ion
a a speci ic empe a u e ( anging om 22 o 90ºC) and du a ion ( om 2 o 8 hou s); and he second
me hod in ol ed a di ec ex ac ion wi hou an ini ial wash, conduc ed a a speci ic empe a u e and
du a ion. Resul s indica ed a linea co ela ion be ween he yield o ca ageenan- ich p ecipi a e (CRP)
and he combina ion o empe a u e and ime, sugges ing ha ca ageenan solubili y and leaching
inc ease g adually wi h ising ene gy inpu . Addi ionally, he s udy ound ha emo ing endogenous sal s
imp o ed he yield o hyb id ca ageenan a lowe ex ac ion empe a u es.
The e ec s o sequen ial ex ac ion me hods on he unc ional p ope ies o aga s and
po phy ans de i ed om ed seaweeds, wi h he aim o imp o ing ex ac ion yields and alo ising by-
p oduc s, was in es iga ed by (Gomes-Dias e al., 2024). The s udy e alua ed h ee ex ac ion
echniques: cold-wa e ex ac ion (CWE), e hanolic ex ac ion (EE), and alkaline ex ac ion (HAE) — bo h
indi idually and in combina ion, o de e mine hei impac on he quali y and yield o he ex ac ed
46
was cen i uged a 8 000 g o 10 minu es, and he liquid ac ion, con aining ca ageenan and sal s, was
ans e ed o a plas ic con aine and d ied a 55 ºC o e nigh .
The nex day, he d ied sample (o he h ee eplicas) was weighed o de e mine he o al mass
a e HAE. Then, om his o al, a po ion (e.g., 5 g ams) (mass a e HAE used o he p ecipi a ion), was
dissol ed in wa e o o m a 2 % (w/ ) ca ageenan solu ion. This solu ion was hen subjec ed o e hanolic
p ecipi a ion o emo e excess sal and isola e he ca ageenan (Aze edo e al., 2013). The p ecipi a ed
ca ageenan was ans e ed o a con aine and d ied again in an o en a 55ºC o e nigh . The ollowing
day, he d ied p ecipi a e was weighed (mass p ecipi a ed) o ob ain he yield, YY, Equa ion (5).
(mass p ecipi a ed · o al mass a e HAE)
( o al algae mass · mass a e HAE used o he p ecipi a ion)
(5)
3.1.8 Cha ac e iza ion o hyb id ca ageenans
Gels p epa a ion and iscoelas ic cha ac e iza ion
K2 ex ac s 2% (w/ ) we e dissol ed in solu ion wi h 1 M NaCl and s i ed igo ously o 1 h a
80 ºC. The concen a ion and ionic s eng h we e speci ically selec ed o ob ain gels wi h su icien igidi y
a 25 ºC, wi hou wa e elease, and o main ain a single ca ion (Na⁺) o acili a e gel o ma ion, since he
alkaline ex ac ion was ca ied ou wi h NaOH (Aze edo e al., 2014). A e allowing he solu ions o cool
o 24 h, wo possible ou comes we e obse ed: a) gel o ma ion o b) no gel o ma ion.
a. Gel o ma ion) The samples ha gelled we e ehea ed o 85 ºC and he esul ing ho K2 solu ions
we e di ec ly ans e ed in o he p e-hea ed (80 ºC) Coue e accesso y (CC10) o a Paa Physica
MCR300 heome e (An on Paa , Aus ia). The geome y su ace was co e ed wi h dodecane oil o
p e en wa e e apo a ion. The samples we e hen cooled o 25 ºC wi hin 5 500 seconds (≈1,5 h).
Du ing he cooling o he solu ion a small ampli ude oscilla o y shea was applied (wi h 0,1 % s ain
a a ω=1 Hz, he ω being he equency) o p obe he empe a u e e olu ion o linea iscoelas ic
p ope ies such as an δ=G’’/G’. The empe a u e whe e an δ =1 was defined as he gel se ing
empe a u e, Tg, as seen in Figu e 6 a). Following he cooling and gel se ing, a a 25 ºC a equency
YY =

47
sweeps was pe o med wi h a 0,1 % s ain, which ensu ed he measu emen o equilib a ed gel’s
mechanical spec a in he linea egime and he de e mina ion o he G’ a 1 Hz, label as G0 (Figu e
6 b)). A shea s ain (ɣ) sweep om 0,01 o 1000 % wi h a equency o 1 Hz was hen made o e i y
ha he p oduced equency sweep and G0 we e acqui ed wi hin he linea egime o elas ici y (Souza
e al., 2011) and o cha ac e ize he gel beha iou unde la ge de o ma ion, as shown in Figu e 6 c).
Figu e 6. Example o he expe imen al p o ocol ollowed o he heological cha ac e iza ion o he gels, in he
Paa Physica MCR300 heome e (An on Paa , Aus ia).
b. No gel o ma ion) The solu ions we e placed in o a pla e-pla e geome y (40 mm diame e ) o a
s ess-con olled o a ional heome e (MCR 302, An on Paa ) a 25 ºC. Each sample was equilib a ed
du ing 300 seconds by applying an oscilla o y s ain o 10 % a 1 Hz and 25 ºC. This s ep was ollowed
by a ω sweep om 100 o 0,01 Hz, pe o med wi h a s ain o 50,3 %, in iew o eco d he
mechanical spec um o he solu ion and collec he G’ a 1 Hz, label as G1 (as seen in Figu e 7 a)).
Then, a shea a e (ɣ) sweep anging om 1000 s-1 o 1 s-1, immedia ely ollowed by sweep om 1 s-
1 o 1000 s-1, we e pe o med o measu e he shea a e dependence o he appa en shea iscosi y,
η, o measu e he low cu e o he solu ion and assess any possible hixo opic beha iou ela ed
wi h ca ageenan agg ega ion wi h ime, shown in Figu e 7 (b) and c)).
G’= G’’
48
Figu e 7. Example o he expe imen al p o ocol ollowed o he heological cha ac e iza ion o he liquids, in
s ess-con olled o a ional heome e (MCR 302, An on Paa ).
Spec oscopic analyses
The Fou ie T ans o m In a ed Spec oscopy (FTIR) me hod was used o he quali a i e chemical
cha ac e iza ion o he ex ac ed K2. The FTIR analyses we e pe o med on a Pe kin-Elme Spec um 100
FTIR spec ome e equipped wi h an A enua ed T ansmission Re lec ance (ATR) accesso y. The spec a
we e ob ained in a ange o 4000 o 600 cm−1 wi h 16 scans pe sample and a esolu ion o 1 cm-1. The
assignmen s o he FTIR spec a we e mos ly based on he p e ious wo k o (Van De Velde e al., 2004)
(see Table 5).
Nuclea Magne ic Resonance (NMR) spec oscopy was pe o med using a 400 MHz A ance III
spec ome e (B uke ) a 70 ºC o limi he line b oadening caused by he iscosi y o he 1 w .%
ca ageenan solu ions in D2O. Indeed, he sample solu ions we e ul a sonica ed du ing hou s (using a
DCG-300H ba h, MCR L d.) o lowe hei iscosi y p io o NMR cha ac e iza ion. Chemical shi s (ppm)
we e e e enced o he D2O lock signal (4,41 ppm) and u he adjus ed o ma ch he chemical shi o κ
(5,09 ppm), as p esc ibed by an De Velde e al., (2004). The mola ac ions (mol %) o he ca ageenan
epea ing uni s a e calcula ed as he in eg a ed in ensi y o he co esponding 1H-NMR peak (see Table 6)
o e he sum o he in eg a ed in ensi ies o all assigned ca ageenan anome ic p o ons (Van de Velde e
al.,2004). The py u ic acid ace als and lo idean s a ch a e also de ec ed in he 1H-NMR spec a by he
me hyl p o on esonances wi h chemical shi s, ela i e o DSS (an NMR s anda d), o 1,44 ppm o he
49
py u ic acid and 5,35 ppm and 4,98 ppm o lo idean s a ch (Van De Velde e al., 2004). The NMR
spec a we e p oduced by he chemical analyses se ice a he depa men o chemis y o he Uni e si y
o Minho.
Table 5. Iden i ica ion o ca ageenan ypes by in a ed spec oscopy (adap ed om (Chopin & Whalen, 1993)).
The in ensi y o he obse ed peaks is indica ed by symbols: ‘+’ (weak), ‘++’ (mode a e), ‘+++’ (s ong) and
‘s’ (shoulde peak)
Wa enumbe s
(cm-1)
Bond(s)/g oup(s)
Le e
Code
Type o ca ageenan
κ
mu
ι
nu
L
1240-1260
S=O o sulpha e es e s
-
+
++
++
+++
+++
970-975
Galac ose
D
+
s
+
s
-
930
C–O o 3,6-anhyd ogalac ose
DA
+
-
+
-
-
845
C–O–SO3 on C4 o galac ose
G4S
+
+
+
+
-
825-830
C–O–SO3 on C2 o galac ose
D2S
-
-
-
+
+
815-820
C–O–SO3 on C6 o galac ose
D6S
-
+
-
+
+
805
C–O–SO3 on C2 o 3,6-
anhyd ogalac ose
DA2S
-
-
+
-
-
Table 6. Chemical shi s (ppm) o he al a-anome ic p o ons o ca ageenans e e ed o DSS as in e nal
s anda d a 0 ppm (adap ed om: Van de Velde e al.,2004)
Types o ca ageenan
Monosaccha ide
Chemical shi (ppm)
κ
DA
5,093
Mu
D6S
5,238
ι
DA2S
5,292
Nu
D2S,6S
5,501
50
Molecula mass de e mina ion
The molecula mass dis ibu ion was ob ained by size exclusion ch oma og aphy and e ac i e
index de ec ion om a se ice pu chased o he Analise Labo a o y o he LAQV-REQUIMTE Associa e
Labo a o y, NOVA Uni e si y o Lisbon. A Wa e s 600 appa a us coupled o a Wa e s 2410 di e en ial
e ac i e index de ec o was used, whe eas he size exclusion ch oma og aph was a PolySep-GFC-P
Linea column (Phenomenex, Alcobendas, Spain). This column has been calib a ed wi h pullulan
s anda ds (Shodex, Munich, Ge many) anging om 6 300 o 642 000 g/mol. Duplica ed measu emen s
o Mw and PDI we e pe o med wi h 0,1 w % ca ageenan solu ions in 0,1 M NaCl a 40 ºC.
3.2 S udy o he Molecula Mass
3.2.1 Raw Ma e ial
The K2-ca ageenans u ilized in his s udy we e ho -wa e ex ac ed and hen subjec ed o a 3%
(w/ ) KOH alkaline ea men by ano he esea ch g oup, ollowing ea lie esul s epo ed by (Aze edo
e al., 2013). The ex ac ions we e conduc ed wi h i e comme cially sou ced algae, gene ously p o ided
by AlgaPlus® and Ca gill®. The esul ing hyb id ca ageenans we e assigned speci ic codes and had
a ying chemical composi ions o assess he impac o molecula s uc u e on he b eakdown o molecula
mass: B-87 (4 mol % κ and 96 mol % ι); Giga ina (43 mol % κ, 40 mol % ι, and 17 mol % nu); Mas oca pus
(70 mol % κ and 30 mol % ι); BAT 18-09 (56 mol % κ, 36 mol % ι, 4 mol % mu, and 4 mol % nu); and C-
25 (90 mol % κ and 10 mol % ι).
3.2.2 Reagen s
The eagen s used du ing his wo k a e desc ibed on Table 7, such as he companies ha
p o ided hose eagen s.
Table 7. Reagen s used du ing he ul asonica ion o K2-ca ageenans
Reagen s
Company
Po assium chlo ide
Sigma Ald ich
Dodecane
Sigma Ald ich
51
3.2.3 Ul asonica ion o ca ageenans and gel es
K2-ca ageenan samples we e dissol ed in dis illed wa e wi h igo ous s i ing a 80 ºC o 1 h
o ensu e comple e dissolu ion, a a concen a ion o 0,5% (w/ ), once is know ha a lowe
concen a ions he Mw b eakdown o he polyme is as e (Rochas e al, 1990). Following his, he
ca ageenan solu ions unde wen ul asonica ion o a ious du a ions in an ul asonic ba h (DCG-300H,
MCR L d.), ope a ed a 40 kHz and an ul asonic powe o 400 W in pulsed mode a 80 ºC o mechanically
educe hei molecula mass by ca i a ion, a he han by hyd olysis ou e which could a ec he hyb id
ca ageenan composi ion. Ul asonica ion was applied o a ious du a ions (1, 2, 3, 4, 12, 14, 16, and
20 h), gene a ing dis inc samples a each ime in e al. A gela ion es was pe o med on each sample
pos -ul asonica ion by p epa ing 1 % (w/ ) ca ageenan solu ions in 0,1 M KCl. The solu ions 0,5 % (w/ )
we e i s e apo a ed o each he p io concen a ion, ollowed by he addi ion o KCl in i s sal o m.
These we e hea ed o 80 ºC o 1 h and hen cooled o oom empe a u e o 24 h o induce gel o ma ion
(Aze edo e al., 2015). As a con ol, a non-sonica ed sample (0 h) was included, and all he ca ageenan
samples success ully o med gels a 0 h. The gelling p ope ies o he samples ollowing ul asonica ion
we e compa ed, e ealing ha some samples los hei abili y o gel a e 4 hou s o ea men , while
o he s emained gelled un il 20 hou s. The ul asonica ion du a ion o a ca ageenan solu ion s opped
when an aliquo sample emained liquid a e he gel es .
3.2.4 Rheology
A e he gela ion es , he K2-ca ageenan samples subjec ed o di e en ul asonica ion imes,
as well as he non-sonica ed con ol sample, we e cha ac e ized by o a ional heome y o compa e he
gelling beha iou ac oss he samples. The liquids we e cha ac e ized as desc ibed in sec ion 3.1.8 (Gels
p epa a ion and iscoelas ic cha ac e iza ion). As o he gel, a new geome y and heome e was used –
o accommoda e o he small numbe o a ailable samples. A di e en pla e-pla e geome y (40 mm
diame e ) o a s ess-con olled o a ional heome e (MCR 302, An on Paa ) was used, bu he
expe imen al p o ocol (Figu e 8) closely esembles he p e ious one (Figu e 6). I di e s in he con ol o
gel hickness, wi h he no mal o ce se o 0 N du ing cooling. A new cha ac e is ic is e alua ed: TON.
This empe a u e is measu ed wi h mo e p ecision han he in lec ion poin , Tg (see Figu e 8.a.1)). TON
can be iden i ied om he Gap (mm) educ ion, which indica es ha he sample is ansi ioning om a
liquid (whe e he empe a u e dependence o he gap is linea due o he he mal expansion o he
shea ing geome y) o a gel s a e (wi h he con ac ion o he sample’s olume), as illus a ed in Figu e

52
8.a.2). The mechanical spec a (Figu e 8 b)) and he shea s ain es (Figu e 8 c)) we e pe o med as
desc ibed be o e (in sec ion 3.1.8).
Figu e 8. Example o he expe imen al p o ocol ollowed o he heological cha ac e iza ion o he gels, in he
s ess-con olled o a ional heome e (MCR 302, An on Paa ).
3.2.5 Spec oscopic analyses
The FTIR me hod was employed o he quali a i e cha ac e iza ion o he chemical s uc u e o
K2-ca ageenan, compa ing he non-sonica ed sample (0 h) wi h he sample subjec ed o he longes
ul asonica ion ime. This analysis aimed o e i y whe he any modi ica ions occu ed in he chemical
s uc u e o he ca ageenan a e he ul asound. The expe imen al p o ocol ollowed is de ailed in sec ion
3.1.8 (Spec oscopic Analyses).
3.2.6 Molecula mass de e mina ion
To assess he molecula b eakdown, he molecula mass o he ca ageenans p io o and du ing
ul asonica ion was de e mined. The expe imen al p o ocol ollowed is de ailed in sec ion 3.1.8 (Molecula
mass de e mina ion).
53
3.3 S a is ical analysis
S a is ical analysis was pe o med o iden i y signi ican di e ences using one-way ANOVA in
O igin e sion 2024b (Lea ning Edi ion). Mul iple compa isons we e e alua ed h ough Tukey's es a a
95 % con idence in e al. Fo each seaweed species and ca ageenan sample, an analysis was conduc ed
o assess whe he he sequen ial ex ac ion and ul asound me hods p oduced signi ican di e ences (p
< 0,05) in he yield o ex ac ed ac ions, he Mw and he PDI. Resul s a e p esen ed as means ± s anda d
de ia ion, wi h a minimum o wo eplica es conduc ed unless o he wise speci ied.
54
4. RESULTS AND DISCUSSION
4.1 Sequen ial Ex ac ion
The sequen ial ex ac ion (SE) p ocess enables he ac iona ion o pigmen s and K2-
ca ageenans o e ime, isola ing ac ions wi h dis inc chemical and heological p ope ies. By analysing
yields, chemical composi ion, and heological beha iou ac oss ex ac ion s ages, his s udy assesses he
impac o SE and ex ac ion ime on he unc ional p ope ies o K2-ca ageenans. These indings a e
c ucial o op imizing ex ac ion ime o sc een o he po en ial o ex ac i e ex usion, and ailo ing
ca ageenans o speci ic indus ial applica ions.
Fi s , o alida e SE as a iable al e na i e o con en ional ba ch ex ac ion o K2-ca ageenans,
he yields o ac ions eco e ed a each s ep a e analysed in ela ion o he known composi ions o he
s udied seaweeds. Subsequen ly, he chemical s uc u es o he ca ageenans ob ained h ough SE a e
discussed, ollowed by an in-dep h assessmen o hei heological p ope ies.
4.1.1 Cold Wa e ex ac ion (CWE)
The i s ex ac ion consis s o an o e nigh cold wa e ex ac ion. The compound ex ac ed is a
known pigmen o he ed algae, bu also a p o ein, R-phycoe y h in (R-PE) (Baha i, Moelan s, Kloeck, e
al., 2021). This ex ac ion does no ha e a con ol, as i is he i s ex ac ion and he e o e no in luenced
by any p io ex ac ion. The abso bance alues a speci ic wa enumbe s o he a ious liquid ex ac s
( iplica ed samples) can be consul ed Appendix A.
Compa ing he R-PE yields shown in Figu e 9, speci ically, 0,343 ± 0,058 mg R-PE/g d.w. o
M.
s ella us
, and 0,214 ± 0,062 mg R-PE/g d.w. o
C. c ispus
, wi h li e a u e alues o 1,99 mg R-PE/g
d.w. o
M. s ella us
and 0,53 mg R-PE/g d.w. o
C. c ispus
(Nguyen e al., 2017; Pina e al., 2014), i
is e iden ha less han hal o he R-PE epo ed in he li e a u e is ex ac ed om he algae s udied he e.
As o
G. pis illa a
no alues a e a ailable in he li e a u e o compa e wi h he 0,307 ± 0,049 mg R-PE/g
d.w. shown in Figu e 9. Fu he mo e,
C. c ispus
shows compa a i ely lowe R-PE yields han he o he
wo algae in his s udy and in he li e a u e.
The low R-PE yield obse ed in his s udy may be a ibu ed o non-op imized ex ac ion
condi ions. Fo ins ance, Nguyen e al. (2017) achie ed signi ican ly highe yields o
M. s ella us
by using
a di e en me hod in ol ing phospha e bu e (20 mM, pH 7,1) and xylanase o deg ade he algal cell
55
wall, yielding up o 1,99 mg R-PE/g d.w. Fu he s udies should conside a ying he algae used wi hin
M. s ella us
and he o he species, o assess seasonal and geog aphical impac s on R-PE yield, as R-PE
con en may di e be ween win e and summe .
I is possible ha his ex ac does no only con ain R-PE, bu also sal s om he seaweed, as well
as sulpha ed ca ageenans (e.g. L-ca ageenans), as some s udies e eal ha i is possible o ex ac
ca ageenans a 25 ºC, a ex ended imes (Baha i e al, 2021). So, he liquid ex ac s we e analysed
u he and discussed in sec ion 4.1.5. In luence o he sequen ial s eps.
Figu e 9. Ex ac ion yield (mg/g d.w.) o he phycoe y h in, R-PE, wi h s anda d de ia ion e o ba s, o he h ee
di e en algae (
Mas oca pus s ella us, Chond us C ispus
and
Giga ina pis illa a
), om he o e nigh (16h) cold
ex ac ion, i s s ep o he sequen ial ex ac ion.
4.1.2 E hanolic Ex ac ion (EE)
The second ex ac ion, based on e hanol, showed a ied ou comes o he algae, he ex ac ion
yield o chlo ophyll-a (chlo o-a) and ca o enoids (ca o ), a e shown in Figu e 10 and Figu e 11,
espec i ely. De ails on he alues o each iplica ed sample and abso bance is gi en in Appendix B.
Fo
M. s ella us
,
C. c ispus
, and
G. pis illa a
, he chlo ophyll-a yield showed an inc ease o
s a is ical simila i y yield ac oss ex ac ion imes (10 min o 1 h), when analysing sepa a ely bo h
ex ac ion ypes (sequen ial and con ol). In
M. s ella us
, he 1 h SE ex ac p oduced a lowe yield han
he 1h con ol, bu o smalle imes (10–30 min) he yield was simila . Con e sely,
C. c ispus
exhibi ed
a sligh inc ease in chlo ophyll-a yield in SE ex ac ions, likely due o he ini ial CWE enhancing pigmen
di usion. Simila ly,
G. pis illa a
showed imp o ed eco e y wi h he 1 h SE ex ac ion compa ed o sho e
62
Table 9. Peaks associa ed wi h ca ageenan in he in a ed spec a o di e en alga ac oss speci ic wa eleng h
egions. The in ensi y o he obse ed peaks is indica ed by symbols: “-” (no peak), “+” (weak), “++” (mode a e),
and “+++” (s ong), wi h “no da a” ep esen ing samples ha we e no subjec ed o FTIR analysis. The yellow
columns a e he ones ela ed o κ o /and ι-ca ageenan, and he whi e columns ela e o mu and/o nu-
ca ageenan
4.1.5 In luence o he sequen ial s eps
The liquid ex ac s ob ained du ing he ini ial cold wa e ex ac ion (CWE) wi h
C. c ispus
and
G.
pis illa a
we e analysed o de e mine ca ageenan con en , and he p esence o non-gelling ca ageenan
in hese ex ac s was obse ed in he FTIR and NMR spec a. This is in ag eemen wi h Baha i e al.
1240-1260 970-975 930 845 825-830 815-820 805
CWE Sequen ial 16h - - - - - - -
1 h + + ++ ++ + + +
30 min + - ++ ++ + - +
10 min
1 h + + ++ ++ - - +
30 min + + ++ ++ - - +
10 min
1 h ++ + - + - - +
30 min - + + ++ - + +
10 min
1 h + + ++ +++ - - +
30 min + - - ++ - + +
10 min
CWE - 16 h + - ++ ++ - - +
1 h +++ ++++ +++ - - ++
30 min ++ ++++ +++ - - ++
10 min +++ ++++ +++ - - ++
1 h +++ ++++ +++ - - ++
30 min +++ ++++ +++ - - ++
10 min +++ ++++ +++ - - ++
1 h + + ++ ++ - - +
30 min + + ++ ++ - - +
10 min + + ++ ++ - - +
1 h + - - + - - +
30 min + - + +++ - - +
10 min - + + +++ - - +
CWE - 16 h + - ++ - + - +
1 h + - ++ + - - +
30 min + + ++ + - - +
10 min + + ++ + - - +
1 h - - ++ + + + -
30 min - + ++ + + + +
10 min - + ++ + + - -
1 h + + + ++ + - +
30 min + - - - - - +
10 min
1 h + + + - - - +
30 min + + + ++ - + +
10 min
Mas oca pus S ella us
ime
Ex ac ed K2-ca ageenans
Peaks a he wa eleng hs (cm¯¹)
Algae
Ex ac
Type
Con ol
Sequen ial
HWE
Con ol
Sequen ial
HWE
Con ol
Sequen ial
HAE
HWE
Con ol
Sequen ial
Giga ina pis illa a
no da a
no da a
no da a
no da a
no da a
no da a
Chond us C ispus
HAE
Con ol
Sequen ial
HAE
Con ol
Sequen ial

63
(2021), who epo ed app oxima ely 5 % yield o ca ageenan, om
C. c ispus,
ollowing an 8 h ex ac ion
a 22ºC, plus an addi ional 2 h ex ac ion a he same empe a u e. The p e ious s udy con i med ha a
small ac ion o ca ageenan, pa icula ly non-gelling, L-ca ageenans, as de e mined om FTIR, can be
eco e ed unde oom- empe a u e condi ions.
No de ec able K2-ca ageenan we e ound in he ex ac om
M. s ella us
, as he liquid ailed o
o m a ilm upon d ying. This esul s ongly sugges s ha he CWE in
M. s ella us
has no impac on he
eco e y o K2-ca ageenans. Thus, he o al ca ageenan yield o nea ly 50 % o he p e ious algae in
bo h SE p ocesses (HWE and HAE) is in ha mony wi h he ca ageenan yields epo ed elsewhe e o
hou s long alkali ex ac ion ou es (Aze edo e al., 2013), whe eas i is almos wice he yield epo ed
mo e ecen ly o a HWE pe o med a 80-90 ºC o a o al o 3 h (Sanz e al., 2023).
In he case o
G. pis illa a
and
C. c ispus
, bo h κ- and ι-ca ageenans we e iden i ied alongside
hei p ecu so s, mu- and nu-ca ageenans. The FTIR band a 825–830 cm⁻¹ o
G. pis illa a
is indica i e
o he subs an ial p esence o nu-ca ageenan, co obo a ed by he ¹H-NMR spec um. Mo eo e , he
cha ac e is ic peak a 5,54 ppm in he ¹H-NMR spec a con i med he p esence o L-ca ageenans in
hese ex ac s bu is no he majo componen as claimed elsewhe e om FTIR spec a (Baha i e al.,
2021). Essen ially, CWE p oduc s om
G. pis illa a
a e mo e sulpha ed han he sequen ial and con ol
HWE p oduc s.
The Mw o he ca ageenans ex ac ed h ough CWE was ound o be compa able o hose
ob ained om HAE o he same species, sugges ing ha he cold-wa e p ocess is capable o eco e ing
ca ageenan chains o simila leng hs, bu i needs a longe ime. Howe e , PDI o he CWE ex ac s om
C. c ispus
was no ably high (4 ± 0,038), e lec ing a b oad dis ibu ion o molecula masses, likely
in luenced by he mild ex ac ion condi ions and he p esence o p ecu so s and L-ca ageenans.
An impo an obse a ion is ha he emo al o ca ageenan du ing he CWE could be a
con ibu ing ac o o he educed sulpha ion le els obse ed in subsequen SE s eps. The p e e en ial
ex ac ion o mo e soluble and highly sulpha ed ca ageenans in he CWE may lea e behind a
ca ageenan ac ion iche in less sulpha ed hyb id ca ageenans. This hypo hesis aligns wi h he indings
o lowe sulpha e con en and enhanced heological p ope ies in SE ex ac s. So, he emo al o
p ecu so s du ing CWE no only shi s he composi ion o subsequen ex ac s bu also impac s hei
molecula cha ac e is ics, a ou ing he eco e y o less sulpha ed ca ageenans wi h imp o ed gel-
o ming abili ies.
64
Analysing he HWE esul s o
M.
s ella us
, FTIR spec a e ealed ha SE ca ageenans sha ed
simila peaks wi h he con ols. Howe e , in he NMR spec a he 1 h SE ex ac exhibi ed a highe
sulpha e con en han he con ol, a 30 min i was he opposi e. In he subsequen HAE, alkali ex ac s
(bo h SE and con ols) didn’ show p ecu so s, aligning wi h expec a ions since he alkaline medium
a ou s he ex ac ion less sulpha ed ca ageenans. An excep ion was obse ed o he 30 min con ol
alkaline ex ac , whe e ¹H-NMR iden i ied mu- and nu-ca ageenans, sugges ing ha p io s eps in he 30
min SE alkaline ex ac may ha e acili a ed he eco e y o less sulpha ed ca ageenan in his s ep.
Rega ding he impac o he SE on Mw and PDI, wa e -based
M. s ella us
SE ex ac s displayed inc eased
PDI, likely due o p io s eps b oadening he chain size dis ibu ion. The Mw o he 1 h SE wa e and
alkaline ex ac was highe han i s con ol, while o he 30 min, i was simila .
Fo
C. c ispus
, FTIR spec a showed minimal di e ences be ween wa e -based SE and con ol
ex ac s. Howe e , ¹H-NMR e ealed ha all he wa e ex ac s con ained p ecu so s (less han 16 mol%
in o al), wi h he SE ex ac s ha ing a sligh ly lowe mola con en o p ecu so s han con ols.
Acco dingly, gels a e ob ained wi h SE HWE ex ac s in con as o he liquids ob ained wi h ex ac s om
con ol expe imen s, as will be discussed in sec ion 4.1.8 Tex u izing beha iou . In he alkaline ex ac ion
s ep, di e ences we e obse ed in FTIR signals a 930 cm⁻¹ and 845 cm⁻¹ be ween SE and con ols.
Howe e , since hese signals a e common ac oss ca ageenans, i ’s challenging o a ibu e hem o
speci ic chemical changes caused by SE. No ably, ¹H-NMR showed s onge κ-ca ageenan signals in
con ols, which also con ained mu-ca ageenans absen in SE ex ac s. Toge he wi h he compu ed
yields, his again sugges s ha mo e sulpha ed ca ageenans we e ex ac ed du ing he HWE s ep,
whe eas pa s o such chains s ill esis he alkali ea men and a e hus ex ac ed du ing he con ol
expe imen s. Wi h espec o he molecula mass dis ibu ion, he sequen ial app oach in bo h HWE and
HAE has no impac , excep o sho e imes wi h HWE and longe imes o HAE, whe e sequen ial
ex ac ions ga e longe chains han he con ol in he HWE and ice- e sa in he HAE. The PDI alues
emained insensi i e o he ex ac ion p o ocols o
C. c ispus
.
Fo
Giga ina pis illa a
, SE p oduc s om HWE pe o med a sho e imes con ained ewe
p ecu so s han he con ols. In he alkaline s ep, SE and con ol ex ac s we e la gely simila . In his alga,
SE wa e ex ac s consis en ly displayed highe Mw han con ols, sugges ing ha he SE sequence
posi i ely impac ed Mw eco e y. In alkaline ex ac ions, he Mw o SE and con ol samples we e qui e
simila .
65
In summa y, Mw ends sugges ha SE gene ally con ibu es o a mo e consis en eco e y o
high Mw ca ageenans compa ed o con ols, pa icula ly du ing he HWE s ep. This e ec was mo e
p onounced in
M. s ella us
and
G. pis illa a
, while
C. c ispus
exhibi ed mo e s able Mw ac oss condi ions.
Combining hese obse a ions wi h FTIR and ¹H-NMR da a, i is e iden ha SE enhances he eco e y o
less sulpha ed ca ageenans, especially in sho e ex ac ion imes. This sugges s ha he CWE s ep likely
acili a ed he leaching o mo e sulpha ed polyme s which a e no ound in he subsequen ex ac ions.
Fu he mo e, SE ex ac s demons a ed supe io heological p ope ies, wi h highe G’ in gels and highe
(o simila ) iscosi y in liquid s a es compa ed o hei espec i e con ols. These indings highligh he
bene i s o inco po a ing sequen ial ex ac ion o imp o ed ca ageenan quali y, pa icula ly in op imizing
Mw, sulpha ion le els, and heological pe o mance.
4.1.6 E ec o ime
Fo his hesis, h ee ex ac ion imes we e s udy: 1 h, 30 min and 10 min. This pa ame e a ec s
he yield o he ex ac ion as discussed be o e, bu also he chemical composi ion o he ype o
ca ageenans ex ac ed.
Fo he HWE
M. s ella us
esul s, he 1H-NMR e eals ha he 30 min wa e SE K2-ca ageenan,
had no p ecu so s (mu and nu), whe eas 1 h had a 32,13% mola con en o p ecu so s. A simila end
is obse ed in he con ol samples, whe e he 1h ex ac ion esul s in he emo al o mo e p ecu so s
han he 30 min ex ac ion (13,64 % mola and 3,84 % mola , espec i ely). Finally,
M. s ella us
is no
sui ed o ex ac ion imes as sho as 10 minu es, once he yield was e y low and he ex ac s ha we e
e alua ed didn’ show K2-ca ageenan. As o he
C. c ispus
, 1H-NMR e eal ha a sho e imes (30 min
and 10 min) o ex ac ion he SE wa e ex ac s had less p ecu so s, which is eminiscen om he esul s
ound o
M. s ella us
. Howe e , he opposi e is ound o he con ols. In he alkaline s ep, no p ecu so s
we e de ec ed o he wo las algae. And i seems ha o
C. c ispus
he 10 min ex ac ion deli e s a κ-
iche K2-ca agenan han he 1 h, in he SE and con ols. Fo
G. pis illa a
, smalle SE HWE ex ac ion
imes led o lowe p ecu so con en s (see FTIR wi h less p onounced bands symp oma ic o sulpha ed
g oups and NMR esul s), in conco dance wi h he o he wo algae. In he alkaline s ep, he chemical
composi ion o K2-ca agenans does no seem o be a ec ed by he ime, wi h he excep ion o a small
mola pe cen age (4%) o nu in he 1h SE alkaline ex ac s.
So, he gene al end is ha wi h mo e ime, mo e ca ageenan (see Figu e 12) wi h mo e
sulpha e is leached ou om he seaweeds du ing he HWE. P ac ically, and in iew he op imized
66
ex u ing p ope ies o ca ageenans, mo e ex ac ion ime b ings bene i s when alkaline is used as mo e
ca ageenan (see Figu e 13) wi h less p ecu so s a e eco e ed.
As o he impac o ime in he molecula mass, o
M. s ella us
, longe ex ac ion imes
consis en ly leads o he isola ion o longe o simila hyb id ca ageenan chains wi h iden ical PDI. The
same end is ound wi h
C. c ispus,
excep o K2-ca ageenans ob ained om he sequen ial HAE whe e
smalle chains a e eco e ed a longe imes. No e ha his may explain he co esponding small
ca ageenan yield as smalle chains a e mo e di icul o p ecipi a e. In
G. pis illa a
, SE wa e ex ac ions
had a simila Mw ac oss he ime. As o he alkaline SE ex ac s he e is an inc ease Mw o e ime, while
he PDI main ains consis en . So, he o e all end is ha he lowe Mw polysaccha ides a e solubilized
i s a ea lie imes. These esul s also e eal ha he ime o ex ac ion, besides a ec ing he yield and
chemical s uc u e o he K2-ca ageenan, also signi ican ly in luence Mw. And consequen ly, he G’ and
he ɳ was highe o he ex ac s wi h longe ex ac ion imes, compa ing o he samples wi h simila
condi ions.
4.1.7 Na u al ca ageenans (HWE) s less sulpha ed ca ageenans (HAE)
I is well-es ablished in he li e a u e ha K2-ca ageenans ex ac ed wi h alkaline sol en s a e
less sulpha ed, while hose ob ained h ough wa e ex ac ion, e lec hei na u al, mo e sulpha ed s a e
(Souza e al., 2023). Compa ing na u al ( om he HWE) and less sulpha ed K2-ca ageenans ( om he
HAE) ac oss he h ee algae species e ealed ha he main dis inc ion in chemical composi ion is ha
HAE ex ac s a e consis en ly less sulpha ed han HWE ex ac s, as expec ed.
The Mw o ca ageenans wi hin he same species was signi ican ly highe (2–5 imes) in HWE
ex ac s compa ed o HAE ex ac s (see Table 8). This educ ion in Mw, indica es ha alkaline condi ions
p omo e ca ageenan deg ada ion, leading o lowe molecula masses (Van De Velde e al., 2005; Souza
e al., 2023). The b oade PDI obse ed in some HAE ex ac s, highligh s he deg ada ion caused by
alkaline condi ions, which likely agmen ca ageenan chains in o molecules o a ying leng hs. This
aligns wi h he expec a ion ha alkaline ea men s dis up he s uc u al in eg i y o na u al ca ageenans,
leading o less uni o m p oduc s (Van De Velde e al., 2005; Souza e al., 2023).
The compa ison be ween na u al and less sulpha ed K2-ca ageenans unde sco es he
complemen a y oles o wa e and alkaline ex ac ions in modula ing ca ageenan p ope ies. Wa e
ex ac ions e ain he na i e, mo e sulpha ed o m wi h highe Mw, while alkaline ex ac ions a ou
desulpha ion and lowe Mw. These indings sugges ha s a egic use o SE and ex ac ion condi ions can
op imize he ca ageenan eco e y and ailo i s p ope ies o speci ic applica ions, o e ing aluable
67
insigh s in o he bio e ine y po en ial o ed algae. Fo example, ood-g ade ca ageenan ypically has a
molecula mass g ea e han 100 kDa, wi h comme cial p oduc s anging om 200–800 kDa (To es,
Fló ez-Fe nández, e al., 2019). So, om he same algae a di e en ex ac ion imes and wi h di e en
sol en s, di e en ca ageenans can be ob ained o sa is y a ious ypes o p oduc s.
4.1.8 Tex u izing beha iou
The ex u izing capaci y o he ca ageenan is a key ac o in hei comme cial applica ion and
alue. Quan i a i e esul s ega ding he ex u izing beha iou (gel elas ici y, iscosi y and gelling
empe a u e) can be obse ed in Table 8.
Table 10 shows ha when 1 M NaCl was added o a 2% (w/ ) ca ageenan solu ion, all he
samples o 1 h and 30 min ho wa e SE gelled a e 24 h a 25 ºC, in all species, whe eas he 10 min
ho wa e SE ex ac ion samples emained liquid. In compa ison, he con ol samples did no o m gels,
excep o he 1 h ho wa e con ol om
C. c ispus
, which exhibi ed a simila elas ic modulus G0 o i s
espec i e sequen ial ex ac (as showed in Table 8). So, o e all, he “na u al” K2-ca ageenans ob ained
om he sequen ial HWE had be e elas ici y han he co esponding con ols, sugges ing ha he
sequen ial ex ac ion p ocess con ibu ed o imp o e he gel quali y. This is p obably due o he lowe
amoun in p ecu so s o he SE ex ac s, as many o he ca ageenan samples imaged in Table 10 show
simila Mw bu di e en gelling beha iou .
Resul s ecen ly epo ed o a se o hyb id ca ageenans ex ac ed om comme cial seaweeds
wi h ho wa e , showed ha when he hyb id ca ageenan composi ion is close o a balanced amoun in
I- and K-ca ageenans, no gel can be o med wi h 1 w .% in 1 M NaCl (Mo aes & Hilliou, 2024). This
obse a ion aligns wi h some o he non-gelling beha iou o he con ol samples, as sugges ed by he
chemical cha ac e is ics p esen ed in Table 8. I a h eshold o a leas 55 mol% o ei he I- o K-
ca ageenan is he e hypo hesized o gel o ma ion in NaCl, he lack o gela ion in he con ols can be
a ibu ed o hei composi ion, independen ly o he Mw e ec s. Bu i canno explain he o ma ion o
gels in HWE SE samples, when hey show a e y close ca ageenan composi ion and molecula masses
o he espec i e con ols.
In con as , all HAE samples (bo h SE and con ol) emained liquid. While he HWE samples, wi h
highe sulpha e con en (Table 8), we e expec ed o exhibi weake gels o lowe iscosi ies due o he
p esence o ca ageenan p ecu so s incapable o gelling, he la ge molecula mass o wa e -ex ac ed
K2-ca ageenans likely con ibu ed o gel o ma ion. This highligh s he signi icance o molecula mass in
gel se ing, as smalle polysaccha ides om he alkaline ex ac s may s uggle o agg ega e o o m

68
helices. Conside ing he abo e discussion o he HWE, he gel se ing o hyb id ca ageenans in NaCl
depends on a complex in e play be ween he chemical s uc u e, he leng h o κ and ι blocks and he
molecula mass. As such, he gelling p ope ies o hese ca ageenans ( om he HWE and HAE) a e
be e s udied in he p esence o KCl (Mo aes & Hilliou, 2024) o e en in mix u es o sal s ha a ou he
gelling o he K-ca ageenan blocks (Van de Velde e al., 2005). Ne e heless, gelling in he p esence o
a single ca ion (Na⁺, om NaCl and om he alkali chosen in ex ac ion) was p e e ed he e, in pa o
highligh he ole o ι blocks in he hyb id ca ageenan s uc u e.
Table 10. Compa ison o gels/liquids o solu ions o ca ageenan (2%(w/ ) ca + 1M NaCl), a e cooling o 24 h
a 25ºC, om
Mas oca pus s ella us, Chond us c ispus,
and
Giga ina pis illa a
, a e di e en ex ac ion imes (1h,
30min, 10min) using HWE, a 90ºC. The i s ow shows esul s om he sequen ial ex ac ion (SE), while he
second ow displays he con ols
Gels
The gelling empe a u e (Tg) is epo ed he e as he empe a u e o he gel poin , whe e he
o ma ion o helices and junc ion zones upon cooling leads o he de elopmen o a h ee-dimensional
elas ic ne wo k. Fo
C. C ispus
, o bo h he con ol and sequen ial 1 h ho wa e ex ac s, he g aphs did
no show a clea c osso e poin whe e G’ equals G’’, being he G’ always highe han he G’’, in he s udy
empe a u e ange (25 ºC – 85 ºC). In o he wo ds, he samples a e ne e uly liquid, making i
impossible o de e mine Tg accu a ely. So, i is assumed ha Tg is highe han 85 ºC (see Table 8), see
Appendix H o mo e de ailed in o ma ion abou he heological beha iou o he samples.
Algae
M. s ella us
C. c ispus
G. pis illa a
Time
1h
30min
1h
30min
10min
1h
30min
10min
SE
Con ol
69
When ex ac ion ime doubled om 30 min o 1 h, he Tg o
M. s ella us
dec eased, his
coincided wi h he p esence o p ecu so s (mo e han 30 mol %) in he 1 h K2-ca ageenan ex ac ,
whe eas no p ecu so s we e de ec ed in he 30 min ex ac . In con as , o
G. pis illa a
and
C. c ispus
,
Tg inc eased wi h ex ended ex ac ion ime, e en hough he 1 h K2-ca ageenan ex ac s also exhibi ed
highe p ecu so con en s, simila o
M. s ella us
. P io esea ch sugges s ha o K2-ca ageenans
con aining only κ- and ι-ca ageenan, Tg emains independen o ca ageenan composi ion ( an de Velde,
2005). Simila ly, s udies by Hilliou e al. (2006) and Aze edo e al. (2013, 2015) ound no co ela ion
be ween Tg and he chemical s uc u es o hyb id ca ageenans om
M. s ella us
,
C. c ispus
, and
Ahn el iopsis de oniensis
, which displayed composi ions simila o hose shown in Table 8. Also, Hilliou
e al., (2006) concluded ha he ime o ex ac ion (30 min – 6 h) didn’ seem o a ec he Tg. Thus, he
lack o a clea ela ionship be ween Tg and chemical s uc u e he e is consis en wi h p e ious indings.
E en so, he measu ed Tg lis ed in Table 8, anging om 26 - 39°C, a e simila o he Tg epo ed in he
li e a u e o simila gelling condi ions (Souza e al., 2011) and such alues a e sui ed o a wide ange o
ood and non- ood applica ions (To es e al., 2018). Also, K2-ca ageenans wi h Tg abo e 85 ºC can ind
applica ions in a ious indus ies due o hei s abili y and unc ional p ope ies unde hea (Chen e al.,
2022).
Among he samples ha o med gels, he 1 h ho wa e SE ex ac o
M. s ella us
p oduced he
mos elas ic K2-ca ageenan, wi h a G0 o 376,32 Pa, while he 30 min ho wa e SE ex ac o
G. pis illa a
yielded he leas elas ic gel, wi h a G0 o 30,28 Pa. These esul s highligh he impac o ex ac ion
condi ions and algae on he s uc u al and heological p ope ies o ca ageenan, pa icula ly i s gel
elas ici y. In
M. s ella us
and
G. pis illa a
, inc easing he ex ac ion ime om 30 min o 1 h signi ican ly
imp o ed gel elas ici y, wi h G0 inc easing app oxima ely 2,43- old. Howe e , in
C. c ispus
, he opposi e
end was obse ed: he 30 min SE ex ac exhibi ed highe G0 han he 1 h ex ac , wi h he la e showing
a educ ion o mo e han hal in gel elas ici y. This di e gence is consis en wi h p io s udies, such as
hose by Van de Velde e al. (2005), which demons a e a di ec co ela ion be ween κ-ca ageenan
con en and gel elas ici y. The 30 min SE ex ac o
C. c ispus
con ained ewe p ecu so s (2,73 mol %
mu-ca ageenan) and a highe κ con en (68,66 mol %) han he 1 h ex ac s (SE and con ol), which
showed highe p ecu so le els (7,66–10,78 mol %) and lowe κ con en (app oxima ely 60 mol %) (show
in Table 8). This chemical composi ion explains he supe io elas ici y o he sho e ex ac ion ime
sample, as he inc eased p ecu so con en in he 1 h ex ac likely hinde ed he o ma ion o obus
helices. The same G0 enhancemen wi h he la ge con en in κ-ca ageenan is ound wi hin gelling
70
ex ac s om
M. s ella us
and
G. pis illa a.
To no e ha al hough he 1h SE wa e ex ac om
G. pis illa a
has a highe κ con en and Mw compa ed o he same ex ac om
M. s ella us
, he PDI o he la e is
mo e han ou imes highe , sugges ing ha he highe G0 obse ed in
M. s ella us
could be a ibu ed o
i s b oade Mw dis ibu ion, indica ing he p esence o ca ageenan wi h longe chains.
As seen be o e he heological p ope ies o K2-ca ageenan gels a y signi ican ly be ween algae
and ex ac ion condi ions, hese di e ences di ec ly in luence hei po en ial applica ions. The highly
elas ic gel om
M. s ella us
(376,2 Pa) is ideal o i m ood p oduc s like jellies and puddings, as well as
du able non- ood uses such as wound d essings and cosme ic hyd ogels. In con as , he so e gel om
G. pis illa a
(30,28 Pa) sui s sp eads, d inkable gels, and lexible ilms. Also, he gel's yield s ain, when
G’=G’’ (see Table 8) e lec s i s esis ance o de o ma ion be o e luidiza ion and is a key pa ame e o
uning ex u e and pe o mance in a ious applica ions (F anck, n.d.). So, by op imizing ex ac ion
condi ions, hese gels can be ailo ed o mee speci ic indus ial needs (Mugdha Bha e al., 2020; Russo
Spena e al., 2024).
Liquids
To u he e alua e he heological beha iou o he liquid ca ageenan samples, iscosi y (ɳ) as
a unc ion o shea a e (ɣ) was analysed. This included bo h he amp-up (0 o 1000 s⁻¹) and amp-down
(1000 o 0 s⁻¹) in shea a es, allowing o e idencing any low hys e esis (see Table 11). The
C. c ispus
,
doesn’ ha e any da a o he alkaline samples, since he e wasn’ enough mass ex ac ed o do he es s,
as said be o e. As o he 10 minu es ho alkaline SE om he
G. pis illa a
he heome e was unable o
de ec he iscosi y, as i was close o he ins umen 's sensi i i y limi and simila o he iscosi y o wa e .
Fo compa a i e pu pose, he iscosi y alues a a shea a e o 500 s⁻¹ a e epo ed in Table 8,
since he solu ions show no low hys e esis when he shea a e is highe . Addi ionally, he elas ic moduli
(G') a 1 Hz and 25ºC, G1, was ex ac ed p o iding insigh in o hei s uc u al igidi y, and as expec ed
he G’ o he liquid samples a e e y low and close o he sensi i i y limi o he heome e , which may
no ully ep esen he ue elas ic moduli. The heological plo s a e gi en in Appendix H.
Analysing he iscosi y amps o ho wa e ex ac s, dis inc beha iou s we e obse ed among
he K2-ca ageenan solu ions om di e en species. Fo
M. s ella us
, bo h samples exhibi ed non-
New onian beha iou , and clea hys e esis was obse ed. This hys e esis indica es ha , unde low shea
a es, he iscosi y did no eco e i s ini ial alue wi hin he expe imen al ime ame, sugges ing
71
s uc u al changes unde shea s ess. These changes likely esul om he b eakdown o ea angemen
o ca ageenan agg ega es a highe shea a es, a phenomenon consis en wi h he polyme ’s high
molecula mass and agg ega ion p opensi y.
Con e sely, o he same ex ac ion (HWE),
C. c ispus
K2-ca ageenan solu ions, while also non-
New onian, exhibi ed limi ed hys e esis. Fo he 30 min con ol sample, he beha iou sugges s less
p onounced agg ega ion o smalle agg ega e sizes ha emain ela i ely una ec ed wi hin he applied
shea a e ange. This is likely due o he smalle molecula mass, o ewe agg ega e- o ming in e ac ions
compa ed o
M. s ella us
and
G. pis illa a.
Indeed, o he
G. pis illa a
, hys e esis was p ominen in all
HWE samples. The 30 min con ol sample exhibi ed a signi ican misma ch be ween low cu es nea
0,01 s⁻¹ shea a e, indica ing mo e agile ca ageenan agg ega es o a highe deg ee o b eakdown
unde shea s ess, limi ing iscosi y eco e y. In e es ingly, he 10 min con ol ex ac showed a iscosi y
app oaching ha o wa e (~1 mPa·s) (see Table 8), beha ing as a New onian luid a shea a es abo e
1 s⁻¹. A lowe shea a es, he heome e ’s o que sensi i i y limi ed he measu emen s, esul ing in
cu es wi h slopes app oaching -1, o he p e ious sample. This beha iou highligh s he in luence o
molecula s uc u e and agg ega ion s a e on he obse ed iscosi y p o iles. Addi ionally, he hys e esis
(Table 11) obse ed in he
M. s ella us
and
G. pis illa a
ex ac s sugges s ha hei ca ageenans had a
highe endency o ini ially agg ega e compa ed o
C. c ispus
, ha likely indica es ha he highe
molecula mass a ou s loose agg ega ion be ween chains, which a e b eaking down unde shea s ess,
and need mo e ime han he expe imen al ime o e-agg ega e a smalle shea a es.
Fo he HAE,
M. s ella us
sequen ial K2-ca ageenan ex ac s and con ol samples also exhibi ed
hys e esis. And, despi e ha ing simila molecula masses, di e ences in iscosi y could be a ibu ed o
composi ional a ia ions, such as p ecu so con en . In
G. pis illa a
, he 30 min and 10 min SE ex ac
beha ed essen ially as a New onian luid , whe eas da a sca e ing a lowe shea a es impedes a clea
conclusion abou low hys e esis o limi o o que sensi i i y.
O e all, when compa ing liquid samples (Table 8), sequen ial ex ac s gene ally exhibi ed highe
o compa able iscosi ies a 500 s⁻¹ compa ed o con ols. The e is an excep ion o
M. s ella us
, he 1
h con ol ex ac showed sligh ly highe iscosi y han i s sequen ial coun e pa , possibly due o a
ma ginally highe κ-ca ageenan mola con en (~ 4% highe ). Longe ex ac ion imes ac oss all samples
we e associa ed wi h highe iscosi ies, a ibu able o he eco e y o highe molecula mass
ca ageenans. Sho e ex ac ion imes yielded ca ageenans wi h sho e chain leng hs, which dissol ed
mo e eadily bu o med weake s uc u es. This aligns wi h s udies showing ha highe molecula mass
78
The esul s e ealed no able al e a ions in he chemical composi ion, mo e p onounced in
samples B-87 and
Giga ina
, whe e he appea ance o a p ominen abso p ion band a ound 1110 cm-1
was obse ed, a e ul asound. This band co esponds o O-H de o ma ion, C-O s e ching, and ing
ib a ions o polysaccha ides, such as ca ageenans (Masaki e al., 2017), ha may esul om he b eak-
up o he glycosidic bonds. Howe e , he modi ica ion in he 1110 cm-1 band was no obse ed in K2 wi h
highe κ-ca ageenan con en . Addi ionally, e idence o desulpha ion was de ec ed aligning wi h indings
om he e e enced s udy (Tecson e al., 2021). So, he s uc u al modi ica ions caused by he
mechanical b eakdown o he Mw encompasses he molecula s uc u e o ul asonica ed hyb id
ca ageenans and hus impac hei gelling p ope ies (Souza e al., 2011). The s abili y and gela ion
pe o mance o ul asonica ed hyb id ca ageenans, he e o e, appea o depend on bo h ene gy inpu
and he di e en s a ing mac omolecula chemical composi ion, as he e is a dis inc i e sulpha e
composi ion p o ided by he κ and ι uni s in he K2-ca ageenans, being ha he κ-ca ageenan has only
one sulpha e g oup and he ι-ca ageenan has wo sulpha e g oups (Souza e al., 2023). Al hough, he
dis inc ion in numbe s o sulpha e g oups alone does no p o ide conclusi e e idence o signi ican
di e ences in hei esis ance o mechanical b eakdown. Fu he chemical analyses a e ecommended
o con i m hese indings and p o ide deepe insigh in o he s uc u al changes a each ime poin .
4.2.3 The impac o he Mw on he Rheology
Some ca ageenans equi ed no mo e han 2 h o ul asonica ion o lose hei gelling capabili y
a 1 w .% in 0,1 M KCl, as shown in Table 13, while o he s needed o e 16 h o each ha s a e. This
was expec ed, gi en hei di e en ini ial chemical composi ions and Mw. The able lacks some da a due
o some easons: he B-87 samples we e he only ones measu ed in a heome e whe e he con ol o
he gel hickness was di e en and didn’ allow o ex ac Ton; o he
Giga ina
sample a 1 h, i was no
possible o de e mine Ton, as he solu ion con inued o inc ease in hickness h oughou he es ed
empe a u e ange (25 ºC–80 ºC). The co esponding heological g aphs om he di e en samples o
each algae can be ound in Appendix J

79
Table 13. Summa y o κ-con en (mol%), molecula mass (Mw), polydispe si y index (PDI), phase beha iou pos -
gel es o (0,1% (w/ ) ca ageenan + 0,1 M KCl), elas ic modulus G' (a 1 Hz, 25 ºC, unde 0,1% o 50,3% s ain
o gels (G0) and liquids (G1), espec i ely), gela ion empe a u e (Tg), onse empe a u e (Ton) o he gels, and
iscosi y (ɳ) a high shea s ain o he liquids samples o he di e en K2-ca ageenans ul asonica ed a a ious
imes. Wi h “nd” ep esen ing no da a and g ey lines ep esen ing he gel samples. Fo each ca ageenan and ime
poin , alues o Mw and PDI wi h di e en le e s indica e s a is ically signi ican di e ences (p < 0,05)
4.2.4 S o age Modulus, G’
The da a in Table 13 e eals a clea co ela ion be ween molecula mass and he s o age
modulus (G’), ep esen ing he elas ici y o each sample: as he Mw educes, he lowe he G’. The
molecula mass dependence o G’ is quali a i ely in conco dance wi h he single li e a u e which
concluded ha he elas ici y o samples o 1 w .% K-ca ageenan in 0,1 M KCl inc eases wi h la ge Mw
(Rochas e al., 1990). Such co ela ion is less e iden o sample B-87 since he ul asonica ion did no
p oduce ca ageenans wi h e y di e en molecula masses.
These esul s (in Table 13) con adic he conclusions o he s udy o Souza e al. (2011)
pe o med wi h hyb id ca ageenans dissol ed in a di e en sal , 0,1 M NaCl. Unde such gelling
condi ions, he highes elas ici y is ob ained wi h lowes molecula mass. Howe e , G’ is ound o dec ease
wi h he inc ease o he ι-con en in hyb id ca ageenans, ha ing simila Mw (Souza e al., 2011). He e,
Algae Time (h)
0Gel 20,76 >85 nd -
1 2,64 ± 0,46 a3,26 ± 0,09 aGel 25,05 >85 nd -
2 3,02 ± 0,10 a2,72 ± 0,06 aLiquid 0,0322 - - 10,09
3 2,52 ± 0,01 a2,51 ± 0,00 aLiquid 0,0466 - - 9,65
4 2,67 ± 0,10 a2,32 ± 0,06 aLiquid 0,856 - - 12,06
0 15,40 ± 0,81 a2,70 ± 0,16 aGel 638,19 60,03 53,27 -
1 5,57 ± 0,67 b2,12 ± 0,00 aGel 112,96 55 nd -
2 5,79 ± 0,04 b2,00 ± 0,05 aLiquid 1,64 - - 26,97
3 5,07 ± 0,03 b,c 1,96 ± 0,03 aLiquid 0,976 - - 24,37
4 3,29 ± 0,01 c1,97 ± 0,01 aLiquid 0,655 - - 17,03
06,49 ±0,06 a2,83 ± 0,03 aGel 3518,53 57,68 55 -
6 2,42 ± 0,01 c2,11 ± 0,07 aGel 4232,02 62,07 56,56 -
12 1,83 ± 0,01 d2,11 ± 0,04 aGel 3542,98 65,61 52,96 -
16 3,12 ± 0,03 b2,17 ± 0,06 aGel 2189,33 62,92 51,07 -
20 1,42 ± 0,01 e2,04 ± 0,02 aLiquid 4,85 - - 32,79
0 6,66 ± 0,17 a2,51 ± 0,01 aGel 4235,96 62,76 54,68 -
6 1,87 ± 0,06 c1,99 ± 0,01 aGel 809,77 58,36 43,7 -
12 1,53 ± 0,02 d1,97 ± 0,00 aGel 4460,8 68,96 56,56 -
14 2,37 ± 0,04 b2,00 ± 0,01 aGel 1188,12 66,78 50,91 -
16 1,08 ± 0,00 e2,01 ± 0,03 aLiquid 0,00466 - - 7,08
0 9,64 ± 0,26 a3,41 ± 0,00 aGel 5543,03 63,33 54,52 -
6 2,59 ± 0,03 b2,03 ± 0,01 aGel 3603,74 67,52 54,84 -
12 1,61 ± 0,02 c2,01 ± 0,01 aLiquid 1,38 - - 14,16
16 0,83 ± 0,01 d1,92 ± 0,03 aLiquid 0,401 - - 5,4
20 0,77 ± 0,01 d1,91 ± 0,03 aLiquid 0,55 - - 10,51
nd
nd
B-87
Hyb id Ca ageenans
κ (mol %)
4 ± 2,5
PDI
Tg (ºC)
TON (ºC)
ɳ (mPa/s) a
500 s¯¹
Phase ( 1%(w/ )
ca + 0,1 M KCl)
G0/G1(Pa)
Mas oca pus
Giga ina
BAT 18-09
C-25
Mw (g/mol) · 10⁵
42 ± 1,8
69 ± 0,4
56 ± 2,9
90 ± 4,5
80
only wo samples o e such a compa ison bu in KCl since
Mas oca pus
and BAT 18-09 show simila
ini ial Mw. Howe e , he hyb id ca ageenan chemical composi ion does no show su icien di e ence in
he ι-con en o d aw a clea conclusion.
The 6h BAT 18-09 sample appea s o be an ou lie , in e ms o he G0, as al hough he Mw
dec eased, as expec ed, i s G' alue is no ably lowe , compa ed o o he samples om he same alga
wi h simila Mw. This sugges s a possible expe imen al e o , du ing he heology phase, po en ially
ela ed o he addi ion o he sal (0,1 M KCl). Gi en he ac ha only a e y small amoun o KCl (0,0373
g) was equi ed, he e may ha e been inaccu acies in weighing, which could ha e a ec ed he sample's
heological p ope ies. Repea ing his sample measu emen would be necessa y o e i y i he esul was
an anomaly due o sample p epa a ion o an ac ual ma e ial p ope y change.
The di e ences in he beha iou o K2-ca ageenan, om di e en algae, when exposed o
ul asonica ion may also be a ibu ed o a ia ions in helix agg ega ion wi hin hei ca ageenan
s uc u es, whe e ι-helices migh exhibi g ea e esis ance o mechanical s ess compa ed o κ-helices.
This di e ence aligns wi h obse a ions o I gels displaying mo e cu ed helix s uc u es, in con as o
he od-like helices and mo e complex, b anched agg ega ion pa e ns seen in K gels (Hilliou, 2021). The
single-helix mesh in I gels esul s in so e ex u es, whe eas he supe helical ne wo k in K gels, especially
in he p esence o K⁺, p oduces i me gels (Souza e al., 2023; S ephen e al., 2006). Fo K2-
ca ageenans, ea ly s udies sugges a s uc u e esembling wi h K-ca ageenan, bu wi h addi ional
b anching and unique agg ega ion beha iou ha complica es unde s anding i s mechanical p ope ies
(Souza e al., 2023). Bu he e is s ill a long way o go un il a comple e unde s anding o he in e ac ion
be ween such mac omolecula s uc u es and he p ope ies o he K2 gel, so he discussion canno go
u he om he helix pe spec i e.
4.2.5 C i ical Molecula Mass, Mc
Acco ding o Rochas e al. 1990, he s o age modulus (G’) inc eases s eadily wi h Mw un il
eaching a c i ical alue, Mc, whe e i pla eaus o a gi en sal concen a ion. Fo pu e K-ca ageenan he
Mc gi en was 1,8·105 g/mol. Howe e , in his wo k i was no possible o ob ain clea cons an G’ alues
o gels, making i di icul o ind a Mc, as shown in Figu e 17. Howe e , Rochas e al. 1990 ex apola ed
om hei heological da a on gels ha o Mw below 0,4·105 g/mol, no K-ca ageenan gels can be o med
o 1 w .% ca ageenan in 0,1 M KCl. This c i ical mass is much below he ange o Mw (be ween 1,6·105
g/mol and 2,6·105 g/mol, see Table 13 and Figu e 17) ha sepa a es liquids om gels wi h sample C-
81
25 ( he κ- iche K2-ca ageenan). This sugges s ha he ex apola ion p oposed is no alid, o ha he
d op in he sulpha e con en in C-25 ha accompanies he dec ease in Mw impedes a ai conclusion on
such c i ical mass.
Figu e 17. S o age modulus, G’, as unc ion o he molecula mass, Mw, o he di e en p oduc s be o e and a e
ul asound (1 % (w/ ) K2-ca agenan + 0,1 M KCl) o he i e di e en algae. The hyb id ca ageenans wi h he
ini ial highe mola con en o κ-ca ageenan,
Mas oca pus
, BAT 18-09 and C-25, had simila ini ial Mw and kind
o simila beha iou , so i was possible o combine hem in a g aph, in he o ange squa e. The gel samples a e
ep esen ed by he ull symbols and he liquid sample as he emp y symbols.
The e is a possible seconda y h eshold Mc2, whe e (a e y low) Mw s abilizes wi h u he
ul asonica ion, showing li le addi ional change o e ime, as seen in Figu e 15, and discussed a he
beginning o sec ion 4.2.1, i seems ha κ- ich samples (like Mas oca pus and C-25) had a Mc2 close o
1·10⁵ g/mol, while mo e ι- ich hyb ids, such as Giga ina and B-87, may ha e Mc2 close o 3·105 g/mol.
The exis ence o an ul asound esis an Mc2 may be possible, since he bonds ha emain o be b oken
in ca ageenans a e e y s able (e.g., C-C). Fu he mo e, mo e in o ma ion is equi ed o unde s and he
heological meaning o he alue o Mc and i s possible ela ion o he mesh size o he gel.
Fo κ- ich K2-ca ageenans, he minimum Mw equi ed o gel o ma ion (he ea e e e ed o
as Mwgel) a he s udy condi ions, 1% w . o K2ca ageenan wi h 0,1 M KCl, appea s o be e y close o
he c i ical molecula mass, Mc2. In con as , B-87 K2-ca ageenan, which is p edominan ly ι-
Rich in κ mol.%
82
ca ageenan (96 mol%), displays weak gelling p ope ies om he ou se . Fo his ype, he liquid samples
exhibi G’ alues ha a e ela i ely insensi i e o Mw, likely because hei Mw alues (3,02–2,52·105
g/mol), which esul in minimal elas ici y, a e nea Mc2 (3·105 g/mol). Howe e , a sample o B-87 o ms
a gel a Mw 2,64·105 g/mol, and when examining he PDI i is e iden ha liquid samples (PDI ≈ 2) ha e
a na owe molecula size dis ibu ion compa ed o he gel- o ming B-87 sample (PDI = 3,3), e en hough
s a ically hey a e simila . This sugges s ha , e en when he a e age Mw is nea Mwgel, only a ac ion
o molecules wi h su icien ly high Mw may con ibu e o gela ion. Fo K2-ca ageenan de i ed om
Giga ina
, he ansi ion om gel o liquid phase occu s a a Mw b eakpoin nea 5,5·105 g/mol (Mwgel),
which is abo e Mc2 (3·105 g/mol). This disc epancy may s em om he p esence o p ecu so molecules,
speci ically 19 ± 5 mol% nu-ca ageenan. While nu-ca ageenan does no di ec ly a ec Mc2, i may
in luence he igidi y o he polyme chains, he eby educing he gela ion abili y o he sample.
4.2.6 The mal beha iou o he gels
The Tg o
Giga ina
ca ageenan samples dec eases wi h a educ ion in Mw, as seen in Table 13.
Howe e , o he mo e κ- ich K2-ca ageenans ( om
Mas oca pus
, BAT 18-09, and C-25), Tg ends o
inc ease as Mw dec eases, wi h BAT 18-09 6h s anding ou as an ou lie in his g oup, as discussed
be o e. A s udy by Van de Velde e al. (2005) on K2-ca ageenans om
Mas oca pus
, wi h a ious
chemical s uc u es, epo ed ha gelling empe a u e is independen o biopolyme chemis y in
polysaccha ides wi h mo e han 50 mol% κ-ca ageenan disaccha ide uni s. This sugges s ha Tg
dependency in hese ca ageenans may be mo e in luenced by Mw. Addi ionally, he g adual Tg a ia ion
o app oxima ely 2 - 10 ºC likely indica es shi s in he helical s uc u e.
Ul asonica ion ime is also expec ed o a ec TON by educing Mw o e ime, ye he ela ionship
be ween ul asonica ion du a ion and TON appea s inconsis en ac oss samples. This indica es ha he
he mal p ope ies o ca ageenans may be sensi i e o dis ibu ion and densi y o he polyme ne wo k
a he han me ely he a e age Mw alue.
4.2.7 Viscosi y o he liquids
The iscosi y o he liquid solu ions a e a ying du a ions o ul asonica ion is shown in Table
13. In gene al, as molecula mass declines, iscosi y also dec eases due o he smalle size and lowe
sulpha ion (con i med by he FTIR) o he K2-ca ageenan molecules, which educes esis ance o low,
as he p opo ion o sulpha e ac ions and he equilib ium o ca ions in he wa e solu ion de e mine he
83
iscosi y o solu ions o s eng h o gels o med by ca ageenans (Campos e al, 2009). Fo B-87, since
he Mw emains ela i ely cons an (close o 3·10⁵ g/mol), iscosi y also s ays app oxima ely he same.
The C-25 16h sample p esen s an unexpec ed esul : i s iscosi y is less han hal o he 20h
sample, despi e ha ing a simila Mw and compa able elas ici y (G1) (as seen in Table 13). This
disc epancy could be a ibu ed o mino inconsis encies du ing sample p epa a ion, such as sligh
a ia ions in sample hyd a ion, concen a ion, o solu ion he e ogenei y. Addi ionally, his es ing was
done in he summe season, and he highe empe a u e o he cooling sys em may ha e lowe ed he
iscosi y. Repea ing iscosi y measu emen s and doing a chemical s uc u e analysis o his sample
could help de e mine whe he his obse a ion is due o expe imen al a iabili y o a unique cha ac e is ic
o he K2-ca ageenan a his ul asonica ion s age.

84
5. CONCLUSIONS AND FUTURE WORKS
In conclusion, he sequen ial ex ac ion (SE) p ocedu e and subsequen ul asonica ion
expe imen s collec i ely ad ance he unde s anding o op imizing K2-ca ageenan ex ac ion and
molecula ailo ing om ca ageenophy es. In some algae, SE demons a ed yields o K2-ca ageenans
compa able o hose ob ained ia con en ional ex ac ion me hods wi h longe p ocessing imes, u he
suppo ing i s indus ial ele ance.
The SE p o ocol p o ed highly e ec i e in ac iona ing phycobilip o eins, chlo ophyll-a,
ca o enoids, and ca ageenans (na u al and less sulpha ed) in o dis inc ac ions, showcasing i s
po en ial o sus ainable indus ial applica ions. I s design aligns wi h bio e ine y p inciples, p omo ing
s eamlined and e icien p ocesses. While chlo ophyll-a and ca o enoids a e co-ex ac ed in he same
solu ion, implemen ing ad anced sepa a ion echniques like High-Pe o mance Liquid Ch oma og aphy
(HPLC) could p o ide deepe insigh s in o hei speci ic composi ion and concen a ions. When compa ing
he e iciency and quali y o SE o he con ol, o he ca ageenans ex ac ed, SE consis en ly acili a es
he eco e y o high molecula mass (Mw) K2-ca ageenans du ing ho wa e ex ac ion (HWE),
pa icula ly om
M. s ella us
and
G. pis illa a.
Mo eo e , o e all, SE ex ac s demons a e supe io
heological p ope ies unde he s udied condi ions, including enhanced gel elas ici y and compa able o
highe iscosi ies in liquid s a es. This is a ibu ed o he p e- ea men s, such as cold-wa e ex ac ion
(CWE), which yield less sulpha ed ca ageenans in subsequen s eps (in mos cases), he eby imp o ing
gelling p ope ies. Besides, K2-ca ageenans ex ac ed unde alkaline condi ions (HAE) a e less sulpha ed
bu exhibi signi ican ly lowe Mw and lowe heological p ope ies, compa ed o hose ob ained ia HWE.
Ul asonica ion e ec i ely educes molecula mass, a p ocess s ongly in luenced by he chemical
composi ion o K2-ca ageenans, u he elucida ing he ela ionship be ween Mw and he elas ic
p ope ies o gels. The educ ion in Mw co ela es wi h a dec ease in gel elas ici y, s abilizing nea Mc2
(~1·10⁵ g/mol o κ- ich and ~3·10⁵ g/mol o ι- ich hyb ids), whe e u he deg ada ion has minimal
impac . Al hough a c i ical molecula mass (Mc) o gela ion was no clea ly de ined, deg aded K2-
ca ageenans exhibi educed iscosi y and gel elas ici y, unde sco ing he essen ial ole o molecula
mass in de e mining unc ional p ope ies.
This in e play be ween chemical composi ion and Mw highligh s he complexi y o op imizing
ex ac ion pa ame e s o enhance bo h e iciency and p oduc unc ionali y. Ex ended ex ac ion imes
ypically esul in highe molecula mass ca ageenans wi h enhanced gel elas ici y, hough hey also lead
o highe sulpha ion le els, an appa en con adic ion. In e es ingly,
C. c ispus
displayed an in e se
85
beha iou in he HWE. Addi ionally, sho e ex ac ion imes occasionally p oduced compa able yields,
pa icula ly in pigmen ex ac ion, unde sco ing he necessi y o species-speci ic op imiza ion. Achie ing
a balance be ween ene gy e iciency and ex ac ion ou comes is essen ial.
The indings om he SE p ocess and he ul asonica ion s udy complemen each o he , o e ing
a comp ehensi e unde s anding o how o op imize K2-ca ageenan ex ac ion and ailo i s p ope ies.
This wo k unde sco es he c i ical in luence o ex ac ion sequence, du a ion, and sol en selec ion on
p oduc quali y. By add essing key gaps in he li e a u e, such as he impac o ex ac ion p e- ea men s
on ca ageenan sulpha ion and he ela ionship be ween Mw and gel unc ionali y, i lays he g oundwo k
o u u e inno a ions in ca ageenophy es alo isa ion. Addi ionally, i con ibu es o he de elopmen o
sus ainable and cos -e ec i e bio e ine y me hods, enhancing he alo isa ion o ed seaweed h ough
he ex ac i e-ex usion app oach.
Fu u e Resea ch
Fo u u e esea ch, e alua ing he gel s eng h o K2-ca ageenan ex ac s in he p esence o
di e en ca ions (e.g., KCl) is also sugges ed o enhance hei p ac ical applica ions in a ious indus ies.
In es iga ing he sequen ial ex ac ion p o ocol's o de , a ying ex ac ion imes, and empe a u e anges
is also ecommended o op imize yield and p oduc quali y. Addi ionally, comp ehensi e analyses o
phenolic compounds and soluble p o eins wi hin he ex ac s should be pe o med, accompanied by
bioac i i y scans Assessing esidual ac ions, pa icula ly he was e ac ion, h ough weighing and
composi ional analysis could e eal new oppo uni ies o alo isa ion, including he iden i ica ion o
bioac i e compounds ha suppo a holis ic u iliza ion o biomass esou ces. Fu he mo e, s udies on
alkaline ex ac s o
C. c ispus
should be comple ed, pa icula ly in a eas whe e cu en da a, limi ed by
insu icien mass, does no allow o clea conclusions.
In alignmen wi h he p ojec unde which his wo k is amed, u u e e o s a e o eseen o adap
he sequen ial ex ac ion p o ocol o use in an ex ude . This adap a ion could s eamline he p ocess,
enhancing scalabili y and e iciency while main aining eco- iendly p ac ices. To achie e his, i will be
necessa y o in es iga e he ope a ional pa ame e s o ex usion and hei impac on p oduc quali y and
yield, e ining he p o ocol o ensu e i s applicabili y o indus ial p ocesses.
Fo molecula mass s udies ocusing on he elas ic p ope ies o K2-ca ageenan, i is essen ial
o include pH moni o ing h oughou he p ocess and pe o m chemical analyses on all samples o
imp o e he accu acy o deg ada ion assessmen s. Explo ing addi ional condi ions, such as a ying he
86
ypes o sal s and ca ageenan concen a ions, may p o ide a mo e comp ehensi e unde s anding o
hei e ec s on gel elas ici y (G') and o he heological beha iou s. Fu he mo e, e alua ing addi ional
ime poin s du ing ul asonica ion is ecommended o be e cap u e he p og ession o molecula
deg ada ion and i s impac on unc ional p ope ies. This app oach would allow o a mo e de ailed
unde s anding o he deg ada ion p ocess. Finally, complemen a y s udies on pa ame e s like helix
o ma ion in K2-ca ageenans could p o ide aluable insigh s in o he complex in e ac ions and s uc u al
dynamics o hese polysaccha ides. Such in es iga ions would b oaden he scope o esea ch, o e ing a
deepe unde s anding and expanding he po en ial applica ions o ca ageenan-based ma e ials.
87
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94
APPENDIX
Appendix A – Abso bance o he liquid ex ac s om he CWE
Calcula ion example o yield o he R-PE o he di e en ex ac s:
Sample: Tes 1 o 60 min –
Mas oca pus s ella us
R-PE (mg/mL) = 0,1247· [(A564–A730) – 0,4583 · (A618– A730)] (1)
R-PE = 0,1247· [(0,0940–0,0170) – 0,4583 · (0,0460– 0,0170)] = 0,0079 mg/mL
(0,0079 mg/mL) / (2,004 g/60 mL) = 0,2365 mg/g o R-PE.
Table A.1. Abso bance, yield and S anda d De ia ion (SD) o he R-PE ex ac om he CWE o he sequen ial
ex ac ion (60, 30 and 10 minu es) o algae
M. s ella us
Table A.2. Abso bance, yield and S anda d De ia ion (SD) o he R-PE ex ac om he CWE o he sequen ial
ex ac ion (60, 30 and 10 minu es) o algae
C. C ispus
1 2 3 1 2 3 1 2 3
2,004 2,007 2,0015 2,0076 2,0083 2,001 2,0322 2,0168 2,0175
0,0940 0,1240 0,1340 0,2250 0,1840 0,1950 0,2020 0,2470 0,2100
0,0460 0,0720 0,0740 0,1530 0,1140 0,1250 0,1370 0,1710 0,1410
0,0170 0,0310 0,0320 0,0920 0,0610 0,0700 0,0830 0,1030 0,0840
0,0079 0,0093 0,0103 0,0131 0,0123 0,0124 0,0118 0,0141 0,0125
0,2379 0,2767 0,3093 0,3915 0,3677 0,3731 0,3470 0,4186 0,3704
Cold Wa e
Ex ac ion
(16h)
0,3436
Mas oca pus s ella us
60 min
30 min
10 min
R-PE (mg/mL)
R-PE (mg/g)
A e age R-PE (mg/g)
SD R-PE (mg/g)
Algae
Time
Tes
Ini ial d y algae mass (g)
Abs 564
Abs 618
Abs 730
0,0580
1 2 3 1 2 3 1 2 3
2,0101 2,018 2,0267 2,0076 2,0083 2,001 2,0052 2,0044 2,0166
0,0870 0,1540 0,0940 0,2290 0,2120 0,2210 0,1150 0,2170 0,1780
0,0670 0,1200 0,0720 0,1840 0,1700 0,1780 0,0880 0,1730 0,1410
0,0440 0,0800 0,0480 0,1270 0,1190 0,1270 0,0570 0,1200 0,0980
0,0040 0,0069 0,0044 0,0095 0,0087 0,0088 0,0055 0,0091 0,0075
0,1208 0,2064 0,1292 0,2828 0,2594 0,2641 0,1634 0,2714 0,2237
Cold Wa e
Ex ac ion
(16h)
0,2135
0,0624
Chond us c ispus
60 min
30 min
10 min
R-PE (mg/mL)
R-PE (mg/g)
A e age R-PE (mg/g)
SD R-PE (mg/g)
Algae
Time
Tes
Ini ial d y algae mass (g)
Abs 564
Abs 618
Abs 730

95
Table A.3. Abso bance, yield and S anda d De ia ion (SD) o he R-PE ex ac om he CWE o he sequen ial
ex ac ion (60, 30 and 10 minu es) o algae
G. pis illa a
Appendix B - Abso bance o he liquid ex ac s om he EE
Fo an example o he calcula ion see Appendix A, o mo e in o ma ion.
1.Sequen ial Ex ac ion
Table B.1.1. Abso bance, yield and S anda d De ia ion (SD) o he Chlo ophyll-a and Ca o enoid ex ac om he
EE o he sequen ial ex ac ion (60, 30 and 10 minu es) o algae
M. s ella us
1 2 3 1 2 3 1 2 3
2,0043 2,031 2,0618 2,0157 2,0045 2,0031 2,0019 2,0115 2,0123
0,1400 0,1420 0,1470 0,1680 0,1550 0,2000 0,1690 0,2050 0,2030
0,0900 0,0920 0,0940 0,1120 0,0990 0,1260 0,1030 0,1340 0,1360
0,0570 0,0590 0,0590 0,0710 0,0600 0,0770 0,0610 0,0820 0,0900
0,0085 0,0085 0,0090 0,0098 0,0096 0,0125 0,0111 0,0124 0,0115
0,2534 0,2500 0,2611 0,2903 0,2879 0,3756 0,3317 0,3689 0,3418
Cold Wa e
Ex ac ion
(16h)
0,3067
0,0490
60 min
30 min
10 min
Giga ina pis illa a
R-PE (mg/mL)
R-PE (mg/g)
A e age R-PE (mg/g)
SD R-PE (mg/g)
Algae
Time
Tes
Ini ial d y algae mass (g)
Abs 564
Abs 618
Abs 730
1 2 3 1 2 3 1 2 3
2,004 2,007 2,0015 2,0076 2,0083 2,001 2,0322 2,0168 2,0175
0,056 0,068 0,086 0,066 0,103 0,081 0,084 0,073 0,069
0,027 0,031 0,042 0,031 0,054 0,041 0,036 0,033 0,031
0,06 0,067 0,086 0,063 0,094 0,076 0,083 0,073 0,069
0,111 0,117 0,148 0,104 0,15 0,124 0,138 0,13 0,121
0,002 0,003 0,007 0,006 0,017 0,014 0,001 0,002 0,002
1,3383 1,3723 1,6971 1,1639 1,5720 1,3226 1,6121 1,5477 1,4320
40,0692 41,0245 50,8748 34,7860 46,9641 39,6582 47,5971 46,0452 42,5861
0,216 0,26 0,316 0,24 0,344 0,268 0,332 0,284 0,268
6,4671 7,7728 9,4729 7,1727 10,2773 8,0360 9,8022 8,4490 7,9703
6,1294
8,4954
1,6025
5,9820
7,9043
1,5072
Time
Tes
Ini ial d y algae mass (g)
43,9895
40,4694
Mas oca pus s ella us
60 min
30 min
10 min
Chlo o-a (μg /mL)
Algae
E hanolic
Ex ac ion
95% ( / )
Abs 480
Abs 632
Abs 652
Abs 665
Abs 750
2,5653
0,9501
45,4095
8,7405
Chlo o-a (μg /g)
A e age Chlo o-a (μg /g)
SD Chlo o-a (μg /g)
Ca o (μg/mL)
Ca o (μg /g)
A e age Ca o (μg /g)
SD Ca o (μg /g)
96
Table B.1.2. Abso bance, yield and S anda d De ia ion (SD) o he Chlo ophyll-a and Ca o enoid ex ac om he
EE o he sequen ial ex ac ion (60, 30 and 10 minu es) o algae
C. c ispus
Table B.1.3. Abso bance, yield and S anda d De ia ion (SD) o he Chlo ophyll-a and Ca o enoid ex ac om he
EE o he sequen ial ex ac ion (60, 30 and 10 minu es) o algae
G. pis illa a
1 2 3 1 2 3 1 2 3
2,0101 2,018 2,0267 2,0076 2,0083 2,001 2,0052 2,0044 2,0166
Chond us c ispus
60 min
30 min
10 min
Algae
Time
Tes
Ini ial d y algae mass (g)
0,029 0,028 0,025 0,047 0,027 0,029 0,035 0,032 0,032
0,021 0,021 0,016 0,03 0,018 0,02 0,026 0,025 0,024
0,043 0,045 0,035 0,057 0,038 0,042 0,048 0,046 0,043
0,084 0,094 0,072 0,111 0,075 0,082 0,091 0,087 0,08
0,003 0 0 0,001 0,002 0,002 0,007 0,007 0,008
1,0163 1,1881 0,9069 1,3546 0,9170 0,9999 1,0559 1,0063 0,9069
30,3355 35,3265 26,8496 40,4844 27,3949 29,9807 31,5960 30,1218 26,9840
0,104 0,112 0,1 0,184 0,1 0,108 0,112 0,1 0,096
3,1043 3,3300 2,9605 5,4991 2,9876 3,2384 3,3513 2,9934 2,8563
Chlo o-a (μg /g)
A e age Chlo o-a (μg /g)
SD Chlo o-a (μg /g)
Ca o (μg/mL)
Ca o (μg /g)
A e age Ca o (μg /g)
SD Ca o (μg /g)
E hanolic
Ex ac ion
95% ( / )
Abs 480
Abs 632
Abs 652
Abs 665
Abs 750
Chlo o-a (μg /mL)
29,5673
2,3555
3,0670
0,2556
30,8372
4,2607
3,1316
0,1863
32,6200
6,9324
3,9084
1,3833
1 2 3 1 2 3 1 2 3
2,0043 2,031 2,0618 2,0157 2,0045 2,0031 2,0019 2,0115 2,0123
60 min
30 min
10 min
Giga ina pis illa a
Algae
Time
Tes
Ini ial d y algae mass (g)
0,158 0,108 0,185 0,12 0,083 0,072 0,098 0,12 0,064
0,108 0,074 0,131 0,087 0,061 0,06 0,064 0,079 0,047
0,251 0,174 0,295 0,199 0,139 0,135 0,148 0,185 0,11
0,491 0,346 0,579 0,405 0,286 0,282 0,288 0,359 0,219
0,004 0,002 0,003 0,003 0,003 0,008 0,003 0,003 0,004
6,0822 4,3169 7,1785 5,0773 3,5907 3,5199 3,5561 4,4370 2,7142
182,0757 127,5297 208,8995 151,1313 107,4782 105,4347 106,5810 132,3486 80,9270
0,616 0,424 0,728 0,468 0,32 0,256 0,38 0,468 0,24
18,4404 12,5258 21,1854 13,9306 9,5784 7,6681 11,3892 13,9597 7,1560
Chlo o-a (μg /g)
A e age Chlo o-a (μg /g)
SD Chlo o-a (μg /g)
Ca o (μg/mL)
Ca o (μg /g)
A e age Ca o (μg /g)
SD Ca o (μg /g)
E hanolic
Ex ac ion
95% ( / )
Abs 480
Abs 632
Abs 652
Abs 665
Abs 750
Chlo o-a (μg /mL)
106,6189
25,7108
10,8350
3,4356
172,8350
41,4645
17,3839
4,4254
121,3481
25,8133
10,3924
3,2096
97
2. Con ols
Table B.2.1. Abso bance, yield and S anda d De ia ion (SD) o he Chlo ophyll-a and Ca o enoid ex ac om he
EE o he con ol ex ac ion (60, 30 and 10 minu es) o algae
M. s ella us
Table B.2.2. Abso bance, yield and S anda d De ia ion (SD) o he Chlo ophyll-a and Ca o enoid ex ac om he
EE o he con ol ex ac ion (60, 30 and 10 minu es) o algae
C. c ispus
1 2 3 1 2 3 1 2 3
2,0685 2,0323 2,0160 1,9999 2,0026 2,0100 2,0062 2,0011 2,0030
0,0950 0,1020 0,0940 0,1280 0,1940 0,1020 0,0570 0,2510 0,1520
0,0620 0,0640 0,0660 0,1120 0,1640 0,0830 0,0410 0,2330 0,1370
0,1220 0,1390 0,1220 0,1420 0,2220 0,1220 0,0710 0,2670 0,1720
0,2090 0,2460 0,2030 0,1880 0,3110 0,1820 0,1150 0,3150 0,2240
0,0160 0,0060 0,0220 0,0870 0,1200 0,0530 0,0190 0,2020 0,1060
2,3429 2,9032 2,1906 1,2261 2,3421 1,5802 1,1729 1,3397 1,4155
67,9582 85,7118 65,1968 36,7841 70,1728 47,1713 35,0773 40,1688 42,4001
0,316 0,384 0,288 0,164 0,296 0,196 0,152 0,196 0,184
9,16606 11,3369 8,57143 4,92025 8,86847 5,85075 4,54591 5,87677 5,51173
Mas oca pus s ella us
60 min
30 min
10 min
9,6915
51,3760
17,0869
6,5465
2,0640
72,9556
11,1332
Algae
Time
Tes
E hanolic
Ex ac ion
95% ( / )
Algae mass (g)
Abs 480
Abs 632
Abs 652
Abs 665
Chlo o-a (μg /mL)
Chlo o-a (μg /g)
A e age Chlo o-a (μg /g)
SD Chlo o-a (μg /g)
Ca o (μg/mL)
Ca o (μg /g)
A e age Ca o (μg /g)
SD Ca o (μg /g)
1,4557
39,2154
Abs 750
3,7533
5,3115
0,6877
1 2 3 1 2 3 1 2 3
2,0078 2,0014 2,0010 2,0060 2,0240 2,0277 2,0108 2,0131 2,0250
0,0400 0,0340 0,0440 0,0760 0,1040 0,0990 0,0470 0,0530 0,0940
0,0290 0,0280 0,0360 0,0720 0,1000 0,0950 0,0440 0,0480 0,0790
0,0470 0,0390 0,0480 0,0780 0,1060 0,1020 0,0510 0,0570 0,0990
0,0790 0,0580 0,0690 0,0900 0,1180 0,1160 0,0630 0,0720 0,1350
0,0120 0,0160 0,0240 0,0630 0,0910 0,0850 0,0350 0,0360 0,0550
0,8215 0,5038 0,5467 0,3183 0,3183 0,3680 0,3284 0,4180 0,9547
24,5497 15,1043 16,3933 9,5218 9,4372 10,8896 9,7981 12,4598 28,2887
0,112 0,072 0,08 0,052 0,052 0,056 0,048 0,068 0,156
3,34695 2,15849 2,3988 1,55533 1,5415 1,65705 1,43227 2,02672 4,62222
Abs 750
Algae
Time
Tes
E hanolic
Ex ac ion
95% ( / )
Algae mass (g)
Abs 480
Abs 632
Abs 652
Abs 665
Chlo o-a (μg /mL)
Chlo o-a (μg /g)
A e age Chlo o-a (μg /g)
SD Chlo o-a (μg /g)
Ca o (μg/mL)
Ca o (μg /g)
A e age Ca o (μg /g)
SD Ca o (μg /g)
Chond us c ispus
60 min
30 min
10 min
18,6824
9,9495
0,8152
1,5846
0,0631
5,1219
2,6347
0,6284
16,8489
9,9962
2,6937
1,6964
98
Table B.2.3. Abso bance, yield and S anda d De ia ion (SD) o he Chlo ophyll-a and Ca o enoid ex ac om he
EE o he con ol ex ac ion (60, 30 and 10 minu es) o algae
G. pis illa a
Appendix C - Yield o he “na u al” K2-ca ageenan om he HWE (SE and Con ol)
Calcula ion example o yield:
Sample: Tes 1 o 60 min –
Mas oca pus s ella us
1.Sequen ial ex ac ion
Table C.1.1. Yield and S anda d De ia ion (SD) o he K2-ca ageenan ex ac om he HWE o he sequen ial
ex ac ion (60, 30 and 10 minu es) o alga
M. s ella us
No e: Due o he insu icien mass, i was no possible u he chemical es ing and due he ac ha i does no
o m a ilm, i is no possible o con i m ha i is ca ageenan a e 10 minu es o ex ac ion.
1 2 3 1 2 3 1 2 3
2,0510 2,0268 2,0000 2,0356 2,0421 2,0051 2,0038 2,0015 2,0039
0,2280 0,0960 0,1360 0,1660 0,1740 0,3870 0,1000 0,0970 0,0980
0,1160 0,0520 0,0720 0,1100 0,1110 0,2930 0,0510 0,0460 0,0450
0,2550 0,1130 0,1580 0,1780 0,1870 0,3960 0,1100 0,1080 0,1080
0,4480 0,1970 0,2720 0,2590 0,2790 0,5230 0,1900 0,1890 0,1910
0,0100 0,0080 0,0090 0,0570 0,0540 0,2080 0,0070 0,0010 0,0000
5,2833 2,2900 3,1624 2,3719 2,6599 3,6955 2,2042 2,2575 2,3004
154,5567 67,7921 94,8708 69,9113 78,1524 110,5825 66,0017 67,6746 68,8777
0,872 0,352 0,508 0,436 0,48 0,716 0,372 0,384 0,392
25,509508 10,4204 15,24 12,8512 14,1031 21,425365 11,1388 11,5114 11,7371
Abs 750
Algae
Time
Tes
E hanolic
Ex ac ion
95% ( / )
Algae mass (g)
Abs 480
Abs 632
Abs 652
Abs 665
Chlo o-a (μg /mL)
Chlo o-a (μg /g)
A e age Chlo o-a (μg /g)
SD Chlo o-a (μg /g)
Ca o (μg/mL)
Ca o (μg /g)
A e age Ca o (μg /g)
SD Ca o (μg /g)
Giga ina pis illa a
60 min
30 min
10 min
86,2154
21,5010
16,1266
4,6314
67,5180
1,4444
11,4624
0,3021
105,7399
44,3917
17,0566
7,7069
1 2 3 1 2 3 1 2 3
2,004 2,007 2,0015 2,0076 2,0083 2,001 2,0322 2,0168 2,0175
Mas oca pus s ella us
60 min
30 min
10 min
Algae
Time
Tes
Ini ial d y algae mass (g)
0,1059 0,1076 0,1092 0,0370 0,0507 0,0488 0,0093 0,0025 0,0035
5,284% 5,361% 5,456% 1,843% 2,525% 2,439% 0,458% 0,124% 0,173%
0,0051
0,0037
0,252%
0,180%
Ca ageenan (g)
A e age Ca ageenan (g)
SD Ca ageenan (g)
Yield (% dw)
A e age Yield (% dw)
SD Yield (% dw)
0,371%
Ho Wa e
Ex ac ion
(90 ºC)
0,086%
0,0455
0,0074
2,269%
0,1076
0,0017
5,367%
Y=(sample mass)/(algae mass)
(4)
Y= 0,1049/2,004 · 100 = 5,284 % d.w.
99
Table C.1.2. Yield and S anda d De ia ion (SD) o he K2-ca ageenan ex ac om he HWE o he sequen ial
ex ac ion (60, 30 and 10 minu es) o alga
C. c ispus
Table C.1.3. Yield and S anda d De ia ion (SD) K2-ca ageenan ex ac om he HWE o he sequen ial ex ac ion
(60, 30 and 10 minu es) o alga
G. pis illa a
2.Con ols
Table C.2.1. Yield and S anda d De ia ion (SD) o he K2-ca ageenan ex ac om he HWE o he con ol
ex ac ion (60, 30 and 10 minu es) o alga
M. s ella us
Table C.2.2. Yield and S anda d De ia ion (SD) o he K2-ca ageenan ex ac om he HWE o he con ol
ex ac ion (60, 30 and 10 minu es) o alga
C. C ispus
1 2 3 1 2 3 1 2 3
2,0101 2,018 2,0267 2,0076 2,0083 2,001 2,0052 2,0044 2,0166
Chond us c ispus
60 min
30 min
10 min
Algae
Time
Tes
Ini ial d y algae mass (g)
0,6157 0,5230 0,7098 0,4674 0,5088 0,5903 0,2623 0,1927 0,2900
30,630% 25,917% 35,022% 23,282% 25,335% 29,500% 13,081% 9,614% 14,381%
26,039%
Ca ageenan (g)
A e age Ca ageenan (g)
SD Ca ageenan (g)
Yield (% dw)
A e age Yield (% dw)
SD Yield (% dw)
30,523%
Ho Wa e
Ex ac ion
(90 ºC)
3,169%
0,2483
0,0501
12,358%
2,464%
0,6162
0,0934
0,5222
0,0625
4,554%
1 2 3 1 2 3 1 2 3
2,0043 2,031 2,0618 2,0157 2,0045 2,0031 2,0019 2,0115 2,0123
60 min
30 min
10 min
Giga ina pis illa a
Algae
Time
Tes
Ini ial d y algae mass (g)
0,1253 0,2245 0,2400 0,1646 0,1389 0,1391 0,0854 0,0530 0,0732
6,252% 11,054% 11,640% 8,166% 6,929% 6,944% 4,266% 2,635% 3,638%
7,347%
3,513%
0,0622
0,0148
0,0164
9,649%
Ca ageenan (g)
A e age Ca ageenan (g)
SD Ca ageenan (g)
Yield (% dw)
A e age Yield (% dw)
SD Yield (% dw)
Ho Wa e
Ex ac ion
(90 ºC)
0,1966
0,1475
0,0705
2,956%
0,710%
0,823%
1 2 3 1 2 3 1 2 3
2,0173 2,0159 2,0042 2,0098 2,0161 2,0046 2,0082 2,0037 2,0157
0,1976 0,2493 0,2301 0,1868 0,1897 0,2333 0,0000 0,0000 0,0000
9,795% 12,367% 11,481% 9,294% 9,409% 11,638% 0,000% 0,000% 0,000%
Ca ageenan (g)
A e age Ca ageenan (g)
SD Ca ageenan (g)
Yield (% dw)
A e age Yield (% dw)
SD Yield (% dw)
Ho Wa e
Ex ac ion
(90 ºC)
Algae mass (g)
0,0000
0,0000
0,2257
0,0261
11,214%
1,306%
0,000%
0,000%
Algae
Time
Tes
0,2033
0,0261
10,114%
1,321%
Mas oca pus s ella us
60 min
30 min
10 min
1 2 3 1 2 3 1 2 3
2,0159 2,0134 2,0021 2,0148 2,0021 2,0039 2,0073 2,0023 2,0049
0,6950 0,7773 0,7618 0,8027 0,7612 0,6814 0,5078 0,4638 0,5135
34,476% 38,606% 38,050% 39,840% 38,020% 34,004% 25,298% 23,163% 25,612%
Ca ageenan (g)
A e age Ca ageenan (g)
SD Ca ageenan (g)
Yield (% dw)
A e age Yield (% dw)
SD Yield (% dw)
Ho Wa e
Ex ac ion
(90 ºC)
Algae mass (g)
Algae
Time
Tes
37,044%
Chond us c ispus
60 min
30 min
10 min
0,7484
0,0616
37,288%
2,986%
2,241%
0,7447
0,0437
1,332%
0,4950
0,0272
24,691%

100
Table C.2.3. Yield and S anda d De ia ion (SD) o he K2-ca ageenan ex ac om he HWE o he con ol
ex ac ion (60, 30 and 10 minu es) o alga
G. pis illa a
No e: Fo he sample es 2 a 10 minu es, he polysaccha ide clued o he ecipien , and i was no possible o
emo e in a way ha didn’ comp omise he weigh .
Appendix D - Yield o he “less sulpha ed” K2-ca ageenan om HAE (SE and Con ol)
Calcula ion example o yield:
Sample: Tes 1 o 60 min –
Mas oca pus s ella us
YY = (mass p ecipi a ed · o al mass a e HAE) / ( o al mass o algae · mass a e HAE used o he
p ecipi a ion) (5)
YY = (1,7953 · (2,4899 + 2,4895 + 2,3826))/((2,004 + 2,007 + 2,0015) · 5,0166)) · 100 = 43,82 %
d.w.
Using he o mula o he p opaga ion o e o :
SD YY=YY⋅((SD mass p ecipi a ed / mass p ecipi a ed)2+ (SD o al mass a e HAE / o al mass a e
HAE)2 + (SD o al mass o algae / o al mass o algae )2 + (SD mass a e HAE used o he p ecipi a ion
/ mass a e HAE used o he p ecipi a ion )2 )1/2
And, knowing ha he mass p ecipi a ed and he mass a e HAE used o p ecipi a ion didn’ ha e a
s anda d de ia ion e o , he ac ion equals ze o, he equa ion looks like his:
SD YY = 43,82 ⋅((0)2+ (0,06183 / (2,4899 + 2,4895 + 2,3826) )2 + (0,00275/ (2,004 + 2,007 +
2,0015))2 + (0)2 )1/2 = 0,369 % d.w.
1 2 3 1 2 3 1 2 3
2,0043 2,0012 2,0192 2,0265 2,0118 2,0046 2,0176 2,0293 2,0158
0,3265 0,4004 0,4081 0,3988 0,4329 0,3401 0,2619 - 0,2632
16,290% 20,008% 20,211% 19,679% 21,518% 16,966% 12,981% - 13,057%
Ca ageenan (g)
A e age Ca ageenan (g)
SD Ca ageenan (g)
Yield (% dw)
A e age Yield (% dw)
SD Yield (% dw)
Ho Wa e
Ex ac ion
(90 ºC)
Algae mass (g)
Algae
Time
Tes
Giga ina pis illa a
60 min
30 min
10 min
0,3906
0,0469
19,388%
2,290%
0,2626
0,0009
13,019%
0,054%
0,3783
0,0451
18,836%
2,208%
101
1.Sequen ial Ex ac ion
Table D.1.1. Yield and S anda d De ia ion (SD) o he K2-ca ageenan ex ac om he HAE o he sequen ial
ex ac ion (60, 30 and 10 minu es) o alga
M. s ella us
No e: The chemical es didn’ show any ca ageenans on he 10 min ex ac , so he yield doesn’ co espond o
ca ageenan.
Table D.1.2. Yield and S anda d De ia ion (SD) o he K2-ca ageenan ex ac om he HAE o he sequen ial
ex ac ion (60, 30 and 10 minu es) o alga
C. c ispus
Table D.1.3. Yield and S anda d De ia ion (SD) o he K2-ca ageenan ex ac om he HAE o he sequen ial
ex ac ion (60, 30 and 10 minu es) o alga
G. pis illa a
1 2 3 1 2 3 1 2 3
2,004 2,007 2,0015 2,0076 2,0083 2,001 2,0322 2,0168 2,0175
Mas oca pus s ella us
60 min
30 min
10 min
Algae
Time
Tes
Ini ial d y algae mass (g)
2,4899 2,4895 2,3826 2,8862 2,6986 2,5324 1,7948 1,6606 1,7625
Mass a e HAE (g)
Ho Alkaline
Ex ac ion
NaOH 3%
(w/ ) (90
ºC)
P ecipi a ion
To al mass (g)
Ca ageenan p ecipi a e (g)
Yield (% dw)
SD yield (% dw)
SD mass a e HAE (g)
SD alga mass (g)
5,0551
0,1216
2,069%
36,557%
0,369%
0,798%
0,0279%
5,0202
1,3604
5,0166
1,7953
43,820%
0,06183
0,00275
0,17701
0,00403
0,07004
0,00870
1 2 3 1 2 3 1 2 3
2,0101 2,018 2,0267 2,0076 2,0083 2,001 2,0052 2,0044 2,0166
Chond us c ispus
60 min
30 min
10 min
Algae
Time
Tes
Ini ial d y algae mass (g)
2,8926 2,6062 2,7683 3,0070 2,8799 2,4134 2,5872 2,0315 2,8086
Mass a e HAE (g)
Ho Alkaline
Ex ac ion
NaOH 3%
(w/ ) (90
ºC)
P ecipi a ion
To al mass (g)
Ca ageenan p ecipi a e (g)
Yield (% dw)
SD yield (% dw)
SD mass a e HAE (g)
SD alga mass (g)
0,166%
0,188%
0,2142
9,537%
4,987%
13,188%
0,1402
0,0723
2,0073
2,0001
2,0019
0,711%
0,14362
0,31255
0,40036
0,00830
0,00403
0,00682
1 2 3 1 2 3 1 2 3
2,0043 2,031 2,0618 2,0157 2,0045 2,0031 2,0019 2,0115 2,0123
60 min
30 min
10 min
Giga ina pis illa a
Algae
Time
Tes
Ini ial d y algae mass (g)
1,8314 1,7286 1,1391 1,8422 1,9988 2,1694 1,6494 1,7386 2,0993
Mass a e HAE (g)
Ho Alkaline
Ex ac ion
NaOH 3%
(w/ ) (90
ºC)
P ecipi a ion
To al mass (g)
Ca ageenan p ecipi a e (g)
Yield (% dw)
SD yield (% dw)
SD mass a e HAE (g)
SD alga mass (g)
2,5928
1,3496
4,20%
1,61%
1,06%
52,703%
59,221%
24,499%
4,5518
4,3688
5,0166
3,1126
0,37358
0,16365
0,23821
0,02877
0,00691
0,00579
102
2.Con ol
Table D.2.1. Yield and S anda d De ia ion (SD) o he K2-ca ageenan ex ac om he HAE o he con ol
ex ac ion (60, 30 and 10 minu es) o alga
M. s ella us
No e: The chemical es didn’ show any ca ageenans on he 10 min ex ac , so he yield doesn’ co espond o
ca ageenan.
Table D.2.2. Yield and S anda d De ia ion (SD) o he K2-ca ageenan ex ac om he HAE o he con ol
ex ac ion (60, 30 and 10 minu es) o alga
C. c ispus
1 2 3 1 2 3 1 2 3
Algae
Time
Tes
Mas oca pus s ella us
60 min
30 min
10 min
2,0111 2,0180 2,0008 2,0314 2,0234 2,0048 2,0069 2,0169 2,0055 2,0095 2,0086 2,0094 2,0167 2,0013 2,0280 2,0194 2,0194 2,0056 2,0120 2,0487 2,0204 1,9915 2,0097 2,0340 2,0016 2,0014 2,0011
1,7373 1,7409 1,6992 2,9664 2,4320 2,9625 1,9413 2,1652 1,0088 3,1357 2,9100 2,8512 2,7631 2,7377 2,6870 2,5981 3,0231 3,2390 1,6485 1,2648 1,7254 1,5231 1,6041 1,5887 1,9611 2,1966 2,1707
Ho Alkaline
Ex ac ion
NaOH 3%
(w/ ) (90 ºC)
Ini ial mass (g) (1)
0,6741
Algae mass (g)
Mass (g)
P ecipi a ion
Ca ageenan p ecipi a e (g) (1)
2,0019
0,0800
2
2,0034
0,6453
Yield (% dw)
SD Yield (% dw)
Ho Alkaline
Ex ac ion
NaOH 3%
(w/ ) (90 ºC)
0,136%
0,648%
3,28%
17,598%
0,8115
Ini ial mass (g) (2)
Ca ageenan p ecipi a e (g) (2)
TOTAL Ini ial mass (g)
TOTAL Ca ageenan p ecipi a e (g)
0,6741
4,3606
0,7315
6,3625
2
27,327%
2,0034
0,6453
28,940%
0,02311
0,30742
0,61332
0,00866
0,01365
0,00622
SD alga mass (g)
SD mass a e HAE (g)
1 2 3 1 2 3 1 2 3
Algae
Time
Tes
Chond us c ispus
60 min
30 min
10 min
2,0095 2,0086 2,0094 2,0167 2,0013 2,0280 2,0194 2,0194 2,0056 2,0120 2,0487 2,0204 1,9915 2,0097 2,0340 2,0016 2,0014 2,0011
3,1357 2,9100 2,8512 2,7631 2,7377 2,6870 2,5981 3,0231 3,2390 1,6485 1,2648 1,7254 1,5231 1,6041 1,5887 1,9611 2,1966 2,1707
Ho Alkaline
Ex ac ion
NaOH 3%
(w/ ) (90 ºC)
Ini ial mass (g) (1)
Algae mass (g)
Mass (g)
P ecipi a ion
Ca ageenan p ecipi a e (g) (1)
0,5205
2
2,0077
0,8764
2,0023
0,4210
Yield (% dw)
SD Yield (% dw)
Ho Alkaline
Ex ac ion
NaOH 3%
(w/ ) (90 ºC)
1,09%
0,334%
1,14%
2,7897
30,821%
7,0081
3,3102
63,967%
0,4210
Ini ial mass (g) (2)
Ca ageenan p ecipi a e (g) (2)
TOTAL Ini ial mass (g)
TOTAL Ca ageenan p ecipi a e (g)
5,0081
0,8764
64,433%
2,0077
2,0023
SD alga mass (g)
0,15019
0,03874
SD mass a e HAE (g)
0,32609
0,00049
0,01340
0,00797
103
Table D.2.3. Yield and S anda d De ia ion (SD) o he K2-ca ageenan ex ac om he HAE o he con ol
ex ac ion (60, 30 and 10 minu es) o alga
G. pis illa a
No e: The chemical es didn’ show any ca ageenans on he 10 min ex ac , so he yield doesn’ co espond o
ca ageenan.
Appendix E – FTIR spec a o he ex ac s om CWE, HWE and HAE
E.1.Ex ac s om he CWE
Figu e E.1.1. FTIR spec a om he CWE K2-ca ageenan ex ac s.
1 2 3 1 2 3 1 2 3
Algae
Time
Tes
Giga ina pis illa a
60 min
30 min
10 min
2,0120 2,0487 2,0204 1,9915 2,0097 2,0340 2,0016 2,0014 2,0011
1,6485 1,2648 1,7254 1,5231 1,6041 1,5887 1,9611 2,1966 2,1707
Ho Alkaline
Ex ac ion
NaOH 3%
(w/ ) (90 ºC)
Ini ial mass (g) (1)
Algae mass (g)
Mass (g)
P ecipi a ion
Ca ageenan p ecipi a e (g) (1)
2,0011
2
1,2345
0,5787
0,4189
2
Yield (% dw)
SD Yield (% dw)
Ho Alkaline
Ex ac ion
NaOH 3%
(w/ ) (90 ºC)
Ini ial mass (g) (2)
Ca ageenan p ecipi a e (g) (2)
TOTAL Ini ial mass (g)
TOTAL Ca ageenan p ecipi a e (g)
2,51%
0,244%
0,451%
47,084%
24,994%
22,076%
2,0500
0,7171
2
4,0511
2
1,2345
1,2958
0,4189
SD alga mass (g)
SD mass a e HAE (g)
0,24674
0,04301
0,12914
0,01923
0,02132
0,00025
110
Figu e H.1.1.3. S o age modulus (G′) (solid symbols) and loss modulus (G′′) (emp y symbols) as a unc ion o he
shea a e (ɣ), a a 25 ºC and 1 Hz, o K2-ca ageenan samples (1 hou and 30 min sequen ial wa e ex ac ion
om
Mas oca pus s ella us)
, in a 2 w .% + 1 M NaCl solu ion.
1.2. Liquids
Cold Wa e Ex ac ion
The ex ac didn´ ha e K2-ca ageenans.
Ho Wa e Ex ac ion
Figu e H.1.2.4. S o age modulus (G′) (solid symbols) and loss modulus (G′′) (emp y symbols) as a unc ion o he
oscilla ion equencies (ω), a a 25 ºC wi h a 0,1 % s ain, o K2-ca ageenan samples (1 hou and 30 min con ol
wa e ex ac ion om
Mas oca pus s ella us)
, in a 2 w .% + 1 M NaCl solu ion.

111
Alkaline Ex ac ion
Figu e H.1.2.5. S o age modulus (G′) (solid symbols) and loss modulus (G′′) (emp y symbols) as a unc ion o he
oscilla ion equencies (ω), a a 25 ºC wi h a 0,1 % s ain, o K2-ca ageenan samples (1 hou and 30 min con ol
alkaline ex ac ion ( op line); 1 hou and 30 min sequen ial alkaline ex ac ion (bo om line) om
Mas oca pus
s ella us)
, in a 2 w .% + 1 M NaCl solu ion.
112
H.2
.Chond us c ispus
2.1.Gels
Ho Wa e Ex ac ion
Figu e H.2.1.1. S o age modulus (G′) (solid symbols) and loss modulus (G′′) (emp y symbols) du ing cooling om
85 ºC o 25 ºC o K2-ca ageenan samples (1 hou and 30 min sequen ial and 30 min con ol wa e ex ac ion
om
Chond us c ispus
), in a 2 w .% + 1 M NaCl solu ion.
113
Figu e H.2.1.2. S o age modulus (G′) (solid symbols) and loss modulus (G′′) (emp y symbols) as a unc ion o he
oscilla ion equencies (ω), a a 25 ºC wi h a 0,1 % s ain, o K2-ca ageenan samples (1 hou and 30 min
sequen ial and 30 min con ol wa e ex ac ion om
Chond us c ispus
), in a 2 w .% + 1 M NaCl solu ion.
114
Figu e H.2.1.3. S o age modulus (G′) (solid symbols) and loss modulus (G′′) (emp y symbols) as a unc ion o he
shea a e (ɣ), a a 25 ºC and 1 Hz, o K2-ca ageenan (1 hou and 30 min sequen ial and 30 min con ol wa e
ex ac ion om
Chond us c ispus
), in a 2 w .% + 1 M NaCl solu ion.
2.2.Liquids
Cold Wa e Ex ac ion
Figu e H.2.2.1. S o age modulus (G′) (solid symbols) and loss modulus (G′′) (emp y symbols) as a unc ion o he
oscilla ion equencies (ω), a a 25 ºC wi h a 0,1 % s ain, o K2-ca ageenan samples (16h o e nigh cold wa e
ex ac ion om
Chond us c ispus
), in a 2 w .% + 1 M NaCl solu ion.
115
Ho Wa e Ex ac ion
Figu e H.2.2.2. S o age modulus (G′) (solid symbols) and loss modulus (G′′) (emp y symbols) as a unc ion o he
oscilla ion equencies (ω), a a 25 ºC wi h a 0,1 % s ain, o K2-ca ageenan samples ( 10 min and 30 min con ol
and 10 min sequen ial wa e ex ac ion om
Chond us c ispus)
, in a 2 w .% + 1 M NaCl solu ion.
Alkaline Ex ac ion
The e is no da a, once he mass ex ac ed wasn’ enough o hese es s.

116
H.3.
Giga ina pis illa a
3.1.Gel
Wa e Ex ac ion
Figu e H.3.1.1. S o age modulus (G′) (solid symbols) and loss modulus (G′′) (emp y symbols) du ing cooling om
80 ºC o 25 ºC o K2-ca ageenan samples (1 hou and 30 min sequen ial wa e ex ac ion om
Giga ina pis illa a
),
in a 2 w .% + 1 M NaCl solu ion.
Figu e H.3.1.2. S o age modulus (G′) (solid symbols) and loss modulus (G′′) (emp y symbols) as a unc ion o he
oscilla ion equencies (ω), a a 25 ºC wi h a 0,1 % s ain, o K2-ca ageenan samples (1 hou and 30 min
sequen ial and 30 min con ol wa e ex ac ion om
Giga ina pis illa a
), in a 2 w .% + 1 M NaCl solu ion.
117
Figu e H.3.1.3. S o age modulus (G′) (solid symbols) and loss modulus (G′′) (emp y symbols) as a unc ion o he
shea a e (ɣ), a a 25 ºC and 1 Hz, o K2-ca ageenan (1 hou and 30 min sequen ial and 30 min con ol wa e
ex ac ion om
Giga ina pis illa a
), in a 2 w % + 1 M NaCl solu ion.
3.2.Liquids
Cold Wa e Ex ac ion
Figu e H.3.2.1. S o age modulus (G′) (solid symbols) and loss modulus (G′′) (emp y symbols) as a unc ion o he
oscilla ion equencies (ω), a a 25 ºC wi h a 0,1 % s ain, o K2-ca ageenan samples (16h o e nigh cold wa e
ex ac ion om
Giga ina pis illa a)
, in a 2 w .% + 1 M NaCl solu ion.
118
Ho Wa e Ex ac ion
Figu e H.3.2.2. S o age modulus (G′) (solid symbols) and loss modulus (G′′) (emp y symbols) as a unc ion o he
oscilla ion equencies (ω), a a 25 ºC wi h a 0,1 % s ain, o K2-ca ageenan samples (1 hou , 30 min and 10 min
con ol and 10 min sequen ial wa e ex ac ion om
Giga ina pis illa a)
, in a 2 w .% + 1 M NaCl solu ion.
119
Ho Alkaline Ex ac ion
Figu e H.3.2.3. S o age modulus (G′) (solid symbols) and loss modulus (G′′) (emp y symbols) as a unc ion o he
oscilla ion equencies (ω), a a 25 ºC wi h a 0,1 % s ain, o K2-ca ageenan samples (1 hou and 30 min con ol
ex ac ion; 1 hou , 30 min and 10 min sequen ial alkaline ex ac ion) om
Giga ina pis illa a)
, in a 2 w .% + 1 M
NaCl solu ion.
SE
HAE
126
J.3. Alga
Mas oca pus
3.1.Gel
Figu e J.3.1.1. S o age modulus (G′) (solid symbols) and loss modulus (G′′) (emp y symbols) du ing cooling om
85 ºC o 25 ºC o K2-ca ageenan samples, om
Mas oca pus
, wi h 0h, 6h, 12h and 16h ul asound, in a 1 w .%
+ 0,1 M KCl solu ion.
Figu e J.3.1.2. Tempe a u e dependence o he hickness (mm) o K2-ca ageenan samples, om
Mas oca pus
,
wi h 0h, 6h,12h and 16h o ul asound, in a 1 w .% + 0,1 M KCl solu ion, du ing heological es ing.

127
Figu e J.3.1.3. S o age modulus (G′) (solid symbols) and loss modulus (G′′) (emp y symbols) as a unc ion o he
oscilla ion equencies (ω), a a 25 ºC wi h a 0,1 % s ain, o K2-ca ageenan samples, om
Mas oca pus
, wi h
0h, 6h, 12h and 16h ul asound, in a 1 w .% + 0,1 M KCl solu ion.
Figu e J.3.1.4. S o age modulus (G′) (solid symbols) and loss modulus (G′′) (emp y symbols) as a unc ion o he
shea s ain (%), a a 25 ºC and 1 Hz, o K2-ca ageenan om
Mas oca pus
, wi h 0h, 6h, 12h and 16h o
ul asound, in a 1 w .% + 0,1 M KCl solu ion.
128
3.2.Liquid
Figu e J.3.2.1. S o age modulus (G′) (solid symbols) and loss modulus (G′′) (emp y symbols) as a unc ion o he
oscilla ion equencies (ω), a a 25 ºC wi h a 50,3 % s ain, o K2-ca ageenan samples om
Mas o capus
wi h
20h o ul asound, in a 1 w % + 0,1 M KCl solu ion.
129
J.4. Alga BAT 18-09
4.1.Gel
Figu e J.4.1.1. S o age modulus (G′) (solid symbols) and loss modulus (G′′) (emp y symbols) du ing cooling om
85 ºC o 25 ºC o K2-ca ageenan samples, om BAT 18-09, wi h 0h, 6h, 12h and 14h ul asound, in a 1 w .% +
0,1 M KCl solu ion.
Figu e J.4.1.2. Tempe a u e dependence o he hickness (mm) o K2-ca ageenan samples, om BAT, wi h 0h,
6h,12h and 14h o ul asound, in a 1 w .% + 0,1 M KCl solu ion, du ing heological es ing.
130
Figu e J.4.1.3. S o age modulus (G′) (solid symbols) and loss modulus (G′′) (emp y symbols) as a unc ion o he
oscilla ion equencies (ω), a a 25 ºC wi h a 0,1 % s ain, o K2-ca ageenan samples, om BAT 18-09, wi h 0h,
6h, 12h and 14h ul asound, in a 1 w .% + 0,1 M KCl solu ion.
Figu e J.4.1.4. S o age modulus (G′) (solid symbols) and loss modulus (G′′) (emp y symbols) as a unc ion o he
shea s ain (%), a a 25 ºC and 1 Hz, o K2-ca ageenan om BAT 18-09, wi h 0h, 6h, 12h and 14h o ul asound,
in a 1 w .% + 0,1 M KCl solu ion.
131
4.2.Liquid
Figu e K.4.2.1. S o age modulus (G′) (solid symbols) and loss modulus (G′′) (emp y symbols) as a unc ion o he
oscilla ion equencies (ω), a a 25 ºC wi h a 50,3 % s ain, o K2-ca ageenan samples om BAT 18-09 wi h 16h
o ul asound, in a 1 w .% + 0,1 M KCl solu ion.
J.5. Alga C-25
5.1.Gel
Figu e J.5.1.1. S o age modulus (G′) (solid symbols) and loss modulus (G′′) (emp y symbols) du ing cooling om
85 ºC o 25 ºC o K2-ca ageenan samples, om C-25, wi h 0h and 6h ul asound, in a 1 w .% + 0,1 M KCl solu ion.

132
Figu e J.5.1.2. Tempe a u e dependence o he hickness (mm) o K2-ca ageenan samples, om C-25, wi h 0h
and 6h o ul asound, in a 1 w .% + 0,1 M KCl solu ion, du ing heological es ing.
Figu e J.5.1.3. S o age modulus (G′) (solid symbols) and loss modulus (G′′) (emp y symbols) as a unc ion o he
oscilla ion equencies (ω), a a 25 ºC wi h a 0,1 % s ain, o K2-ca ageenan samples, om C-25, wi h 0h and 6h
ul asound, in a 1 w .% + 0,1 M KCl solu ion.
Figu e J.5.1.4. S o age modulus (G′) (solid symbols) and loss modulus (G′′) (emp y symbols) as a unc ion o he
shea s ain (%), a a 25 ºC and 1 Hz, o K2-ca ageenan om C-25, wi h 0h and 6h o ul asound, in a 1 w .% +
0,1 M KCl solu ion.
133
5.2.Liquid
Figu e J.5.2.1. S o age modulus (G′) (solid symbols) and loss modulus (G′′) (emp y symbols) as a unc ion o he
oscilla ion equencies (ω), a a 25 ºC wi h a 50,3 % s ain, o K2-ca ageenan samples om C-25 wi h 12h, 16h
and 20h o ul asound, in a 1 w .% + 0,1 M KCl solu ion.