Ex ac ion and cha ac e isa ion o gela ine om yellow in una skin
p e ea ed wi h a eu ec ic sol en
C is ina Gallego, E a Rodil, H´
ec o Rod íguez, Ana So o
*
CRETUS, Depa men o Chemical Enginee ing, Uni e sidade de San iago de Compos ela, E-15782, San iago de Compos ela, Spain
ARTICLE INFO
Keywo ds:
Eu ec ic mix u e
Gela ine
Fish by-p oduc s
Yellow in una
Thunnus albaca es
ABSTRACT
The accumula ion and managemen o was e gene a ed du ing he p ocessing o sea ood p oduc s is one o he
majo cu en challenges in he ishe y indus y. Among hese esidues, ish skin s ands ou o i s high collagen
con en , om which high-quali y gela ine can be ob ained. Howe e , con en ional me hods o gela ine
ex ac ion a e ime-consuming and complex p ocesses ha in ol e he use o s ong acidic and alkaline solu ions,
leading o se ious en i onmen al conce ns. This esea ch s udied he use o a eu ec ic sol en , o med by wo sa e
and en i onmen ally iendly compounds such as sodium ace a e ihyd a e and u ea, o p e ea he skin o
yellow in una be o e he ex ac ion o gela ine. Two di e en imes o p e ea men we e es ed, and he sub-
sequen ex ac ion was ca ied ou in wa m wa e , esul ing in much highe yields o gela ine (0.3 g/g we ish
skin) han p e ious s udies. Addi ionally, using FT-IR analysis and amino acid p o iling, i was obse ed ha he
p e ea men ime did no signi ican ly a ec he s uc u e o composi ion o he gela ine. Howe e , some
a ia ion was ound in i s molecula weigh dis ibu ion and he e o e in i s heological p ope ies: wi h an
inc ease in he p e ea men ime, bo h he molecula weigh and he s o age modulus o he gel also inc eased,
as well as he gela ion and mel ing empe a u es. This con ibu ion o e s a simpli ied p ocess ha educes
en i onmen al impac and p o ides he oppo uni y o ailo he inal p oduc .
1. In oduc ion
The ishe y indus y gene a es una oidably many esidues du ing
ish and shell ish p ocessing, accoun ing o as much as 50–70% o he
o al mass o he eeds ock, depending on he species (Ideia e al., 2020;
Muhammad e al., 2017). Toge he wi h an inc ease >60% in ishe ies
and aquacul u e p oduc ion in he las h ee decades (FAO, 2022), his
has led o he gene a ion o a as amoun o ish meal bu also was e.
Al hough he p ope managemen o hese by-p oduc s is essen ial o
ensu e he sus ainabili y o he indus y, hey ha e been adi ionally
conside ed as was e and disca ded ei he a he sea (p ope ly done, i
e u ns nu ien s o he en i onmen ) o in land ills. This s a egy ep-
esen s no only a h ea o he en i onmen bu also economic losses
and esou ce unde u ilisa ion i conside ing ha hese ma ine
by-p oduc s ha e been poin ed ou as, e.g., a sou ce o added alue
biomolecules (Cooney e al., 2023; F aga-Co al e al., 2022).
Fish skin is a good example o an unde u ilised esidue wi h po en ial
o alo isa ion, since i can cons i u e up o 30% o he o al esidues
a e ish ille ing and usually con ains la ge amoun s o p o ein
(G´
omez-Guill´
en e al., 2002), essen ially collagen.
Collagen is he majo s uc u al p o ein in an animal’s body, ac-
coun ing o ~30% o he o al p o ein composi ion (Chen e al., 2019;
Song e al., 2021). The chemical s uc u e o collagen consis s o h ee
α
chains, each con aining he epea ing Gly-X-Y amino acid sequence
(whe e “Gly” is glycine and he mos common X and Y amino acids a e
p oline and hyd oxyp oline, espec i ely), wis ed a ound each o he o
o m a igh -handed iple helix. This helical s uc u e is s abilised p i-
ma ily by hyd ogen bonds be ween he pep ide amine o glycine and
pep ide ca bonyl g oups in adjacen chains, wi h hyd ophobic in-
e ac ions playing a mino ole. Fu he mo e, in mammalian species,
collagen molecules a e c oss-linked o one ano he h ough co alen
bonds, which con ibu es o he s eng h and s abili y o endons, skins
o bones (Bhowmick & Fields, 2013; Tang e al., 2022; Va e, Undeland,
& Abdollahi, 2022). This highly o ganised and igh ly packed s uc u e,
essen ial o he unc ionali y and pe o mance o hese issues, esis s
luid pene a ion, ende ing na i e collagen insoluble in wa e . Pa ial
hyd olysis o collagen leads o he un olding o he iple helical s uc-
u e along wi h he b eakage o some polypep ide bonds, hus esul ing
* Co esponding au ho .
E-mail add ess: [email p o ec ed] (A. So o).
Con en s lis s a ailable a ScienceDi ec
Food Hyd ocolloids
jou nal homepage: www.else ie .com/loca e/ oodhyd
h ps://doi.o g/10.1016/j. oodhyd.2024.110652
Recei ed 23 Ap il 2024; Recei ed in e ised o m 30 Augus 2024; Accep ed 16 Sep embe 2024
Food Hyd ocolloids 159 (2025) 110652
A ailable online 19 Sep embe 2024
0268-005X/© 2024 The Au ho s. Published by Else ie L d. This is an open access a icle unde he CC BY-NC license ( h p://c ea i ecommons.o g/licenses/by-
nc/4.0/ ).
in gela ine, a mo e soluble ma e ial wi h wide applicabili y
(Duconseille, As uc, Quin ana, Mee sman, & San e-Lhou ellie , 2015;
Liu, Nikoo, Bo an, Zhou, & Regens ein, 2015). Due o i s physical and
mechanical p ope ies, especially i s capaci y o o m he mo- e e sible
gels in wa e , gela ine is la gely used in ood, cosmeceu ical, pha ma-
ceu ical, and pho og aphic indus ies (Cao, Nguyen, Nguyen, Le, &
Razumo skaya, 2017; Regens ein & Zhou, 2006). Cu en ly, skin and
bones om bo ine o po cine species se e as he main sou ces o
gela ine o human consump ion. Ne e heless, collagen and gela ine
om ish and sea ood a e o in e es , in pa because o eligious and
cul u al cons ain s combined wi h isks associa ed wi h li es ock, e.g.,
bo ine spongi o m encephalopa hy ou b eaks (Al es e al., 2022;
Regens ein & Zhou, 2006), al hough gela ine has no been ound o
con ain he i us.
T adi ional me hods o ob ain gela ine om ish skin include acid
and alkaline p e ea men s as p e ious s ages o he ex ac ion in ho
wa e , o enzyma ic hyd olysis (G´
omez-Guill´
en e al., 2002; G ossman &
Gan, 1992; Gudmundsson & Ha s einsson, 1997). The i s me hod in-
ol es mul iple neu alisa ion s eps, a la ge amoun o wa e o impu-
i y emo al and issue washing, and he gene a ion o high amoun s o
e luen s ha mus be adequa ely ea ed o comply wi h he p inciples
o g een chemis y and he ci cula economy. Such complex p ocesses
o en lead o low quali y p oduc s and ha e la ge chemical, ene gy, and
ime equi emen s.
This s udy aims o use eu ec ic sol en s as al e na i es o ha sh
chemicals, alkalis and acids, he eby imp o ing on he adi ional
me hods o ex ac ion. Eu ec ic sol en s esul om he lique ying e ec
associa ed wi h a eu ec ic beha iou in he solid-liquid equilib ium o
mix u es o wo o mo e compounds ha , indi idually, would no be
liquid (and he e o e no useable as sol en s) a he ope a ing empe -
a u e. A pa icula cha ac e is ic o eu ec ic sol en s is hei unabili y,
as i is possible o selec pa en compounds based on he speci ic
applica ion equi emen s (Ma ins, Pinho, & Cou inho, 2019; Smi h,
Abbo , & Ryde , 2014). Fo example, in he ex ac ion o gela ine o
po en ial u ilisa ion in cosmeceu ical o ood indus ies, pa en com-
pounds om he ood addi i es o he GRAS (“gene ally ega ded as
sa e”) lis would be a ac i e. Such is he case o he eu ec ic con igu ed
by u ea and sodium ace a e ihyd a e, which has p e iously been
s udied (Gallego, Rod íguez, & So o, 2023). A he eu ec ic composi ion
(u ea mole ac ion o 0.60) i s mel ing empe a u e is 31 ᵒC, hus
enabling i s u ilisa ion as a sol en a empe a u es abo e ha alue.
Mo eo e , he low/negligible apou p essu e o he eu ec ic, oge he
wi h i s good he mal s abili y, enables i s u ilisa ion as a sol en in a
b oad empe a u e ange wi h li le apo isa ion p oblems.
Abou he use o eu ec ic sol en s o he ex ac ion o biomolecules
om ish by-p oduc s, p e ious s udies ha e p oposed a me hod ha
consis s o solubilising he ish esidue in he eu ec ic sol en , leading o
he need o subsequen ly eco e he alue-added compound om he
dissolu ion medium (Bai, Wei, & Ren, 2017; Ba is a, Fe n´
andez, Gaspa ,
B onze, & Dua e, 2022; Bish , Ma ins, Dias, Ven u a, & Cou inho,
2021; Liu e al., 2020). On he o he hand, a mace a ion s age wi h he
eu ec ic sol en , ollowed by ex ac ion wi h wa e , is p oposed in his
wo k.
2. Expe imen al
2.1. Ma e ials
Skin o cu s o yellow in una (Thunnus albaca es), ob ained as by-
p oduc s du ing he ish ille ing p ocess, we e kindly supplied by
Jealsa Foods (Boi o, Galicia, Spain). The una was caugh in he A lan ic
Ocean (FAO a ea 34) using pu se seine and longline ishing me hods.
The ishing season s a s on Ap il 1s and is closed on Oc obe 31s . On
he boa , he una is s o ed by eezing i in b ine a −18 ◦C. Samples
we e b ough o he labo a o y ozen and s o ed a −20 ◦C o a
maximum o 4 wk. The composi ion o he skin, p o ided by he
supplie , is shown in Table 1.
U ea (Bioul a g ade, >99.5 w %) and sodium ace a e ihyd a e
(>99 w %) we e pu chased om Sigma Ald ich (S einheim, Ge many)
and Scha lau (Sen mena , Ba celona, Spain), espec i ely, and hey we e
used as ecei ed. The eu ec ic sol en was p epa ed by mixing bo h
chemicals (u ea mole ac ion o 0.60) a 60 ◦C and s i ing. Because he
mel ing empe a u e o he eu ec ic mix u e is 31 ◦C (Gallego, Rod í-
guez, & So o, 2023), a homogeneous liquid was o med om he wo
solids. A comme cial gela ine (Sigma-Ald ich) wi h an a e age molec-
ula weigh (Mw) o 60 kDa, ob ained om cold wa e ish skin, was
used as a con ol sample o compa ison.
2.2. Ex ac ion
A e de os ing a oom empe a u e (22 ±2 ◦C), skin was manually
sepa a ed om mea and scales. Then he cleaned skin was cu in o
pieces o ~0.5 ×~0.5 cm
2
, and 4.0 g o hese pieces we e p ocessed pe
ba ch. In he i s s ep, he skin pieces we e p e ea ed by mace a ion.
Tha is, he solid skin was soaked in he liquid eu ec ic sol en a a solid-
o-liquid a io o 1:3 (w/ ) a 35 ᵒC wi h mechanical s i ing. Two p e-
ea men imes we e es ed: 30 min (0.5 h) and 3.0 h. A e p e ea -
men , he samples we e d ained and insed wice wi h dis illed wa e a
oom empe a u e o emo e any aces o he eu ec ic solu ion (as
e i ied by he measu emen o a neu al pH in he washing wa e s,
gi en ha bo h componen s o he eu ec ic lead o basic pH). The
ex ac ion s age wi h wa m wa e was ca ied ou o e nigh wi h a solid-
o-liquid a io o 1:3 (w/ ) a 45 ᵒC. Cen i uga ion (O oal esa -Mad id,
Spain- Digicen 21R cen i uge) a 18,000×g was applied o 15 min o
elimina e small agmen s o skin. The clea supe na an was d ied using
a JP Selec a Con e m 2000209 o en (Selec a, Ab e a, Ba celona) a 40
ᵒC (a empe a u e selec ed o p e en p o ein he mal deg ada ion and
o p ese e he s uc u al, unc ional, and heological p ope ies o he
gela ine) o 48 h. Each ex ac ion es was done in iplica e. As a
con ol sample, o compa ison, an ex ac ion wi hou p e ea men was
also done.
2.3. Cha ac e isa ion
2.3.1. Ex ac ion yield
The ex ac ion yield was de e mined conside ing he weigh o we
skin be o e ex ac ion and he weigh o d ied ex ac ed p oduc (la e
p o en o be gela ine), acco ding o he ollowing equa ion (La ime ,
2023):
Ex ac ion yield (%) = Weigh o d ied gela ine (g)
Weigh o we skin (g)×100 [1]
An equi alen exp ession was also used wi h he weigh o d y skin in
he denomina o , o calcula e a yield no sensi i e o luc ua ions in he
mois u e con en o he skin. All analyses we e done in iplica e.
2.3.2. P oxima e composi ion
The mois u e and ash o ex ac ed gela ines we e de e mined ac-
co ding o AOAC s anda d me hods (950.46 and 900.2A, espec i ely)
using g a ime y (La ime , 2023). O ganic ma e was calcula ed by
di e ence. To al p o ein con en in he sample was de e mined using he
Table 1
Composi ion o yellow in una skin, as p o ided by he supplie .
Composi ion Con en (g/100 g o we skin)
Mois u e 47.4 ±0.2
P o ein
a
34 ±2
Lipid 8.5 ±0.7
Ash 9.8 ±0.9
a
De e mined by he Kjeldahl me hod. The p o ein con en was
calcula ed as he p oduc o o al ni ogen imes he ac o 5.6.
C. Gallego e al.
Food Hyd ocolloids 159 (2025) 110652
2
Kjeldahl me hod (In e na ional O ganiza ion o S anda diza ion, 2023)
wi h a Kjelda he m Diges o and Vapodes 50s dis illa ion sys em (C.
Ge ha d & Co., K¨
onigswin e , Ge many). The p o ein con en was
calcula ed as o al ni ogen imes 5.6 (Ma io i, Tom´
e, & Mi and, 2008).
All analyses we e done in duplica e.
2.3.3. Fou ie - ans o m in a ed (FT-IR) spec oscopy
The g oups and he in e ac ions be ween he bonds o he gela ines
we e analysed using FT-IR spec oscopy. Measu emen s we e done using
a Va ian (Palo Al o, Cali o nia) 670 IR spec ome e , scanning om
4000 o 400 cm
−1
a a esolu ion o 4 cm
−1
. All spec a we e ob ained as
32 scans (110 scans/s) a 25 ᵒC. Analysis o he spec al da a was ca ied
ou using he B uke Opus 7.8 da a collec ion so wa e (B uke Op ik,
2015).
2.3.4. Amino acid p o ile
The amino acid composi ion o he gela ines was de e mined by
quan i a i e analysis a Cen o Tecnol´
oxico da Ca ne (San Cib ao das
Vi˜
nas, Spain). The samples we e hyd olysed wi h aqueous HCl (6 N) o
24 h a 110 ᵒC. Then, he ex ac s we e de i a ised using he AccQ-Tag
Ul a De i a iza ion Ki (Wa e s, Mil o d, MA, USA) and subsequen ly
analysed using HPLC-FL wi h a Wa e s 2695 Sepa a ions module
equipped wi h a Wa e s 2475 Mul i Fluo escence de ec o and a Wa e s
AccQ-Tag Amino Acids C18 analysis column. Quan i ica ion was ca ied
ou using he ex e nal s anda d me hod wi h he Amino Acid S anda d H
(The mo Scien i ic, Rock o d, IL, USA). As a esul o he hyd olysis,
aspa agine and glu amine a e deamida ed esul ing in aspa ic acid and
glu amic acid, espec i ely. The e o e, he composi ion de ec ed co e-
sponds o he sum o bo h componen s. Two independen measu emen s
we e done o each sample.
2.3.5. Sodium dodecyl sulpha e polyac ylamide gel elec opho esis (SDS-
PAGE)
The Mw pa e n was de e mined acco ding o a modi ied e sion o
he me hod by Laemmli (1970). Gela ines we e dissol ed in dis illed
wa e a 5
μ
g powde /
μ
L and subsequen ly mixed wi h NuPAGE LDS
sample bu e 4X (Li e Technologies, Ca lsbad, CA, USA) in he p esence
o NuPAGE sample educing agen 10X (Li e Technologies) o ge a inal
concen a ion o 2
μ
g powde /
μ
L. The mix u es we e hea ed o 10 min
a 70 ᵒC ( ollowing he manu ac u e ’s ins uc ions) o o al p o ein
dena u a ion. An aliquo o 20
μ
L o each sample was loaded on o a
p ecas 4% s acking and 8% esol ing polyac ylamide gel (Li e Tech-
nologies). Also, 5
μ
L o PageRule Plus P es ained P o ein Ladde
(The mo Fishe Scien i ic Bal ics UAB, Vilnius, Li huania) wi h Mw
anging om 10 o 250 kDa we e loaded along wi h he samples. The gel
was imme sed in MES SDS Running Bu e (Li e Technologies), and
elec opho esis was un a a cons an ol age o 60 V o he s acking gel
and a 90 V o he esol ing gel using a Powe Ease 300W powe supply
(Li e Technologies). P o ein bands we e s ained wi h Coomassie b illian
blue R250 (The mo Fishe , Rock o d, IL, USA) o 1 h shaking using a JP
Selec a Ro abi Shake 3000974 (Selec a, Ab e a, Ba celona). Finally,
he gel was des ained, using dis illed wa e , shaking o e nigh . Each
sample was p epa ed and un in duplica e.
2.3.6. Gel pe mea ion ch oma og aphy – size exclusion ch oma og aphy
(GPC-SEC)
The Mw dis ibu ion o he gela ines was analysed using gel
pe mea ion ch oma og aphy a he Ins i u o de In es igaci´
ons Ma i˜
nas
(IIM-CSIC, Vigo, Spain) wi h an Agilen 1260 LC sys em (Agilen Tech-
nologies, San a Cla a, CA, USA) consis ing o a qua e na y pump
(G1311B), injec o (G1329B), column o en (G1316A), diode a ay
(G1315C), e ac i e index (G1362A), and dual angle s a ic ligh sca -
e ing (G7800A) de ec o s. S anda d and samples we e dissol ed a 2 g/
L in a 0.15 M ammonium ace a e/0.2 M ace ic acid bu e (pH =4.5) and
pumped h ough ou columns: P o eema p ecolumn (5
μ
m, 8 ×500
mm), P o eema 100 Å (5
μ
m, 8 ×300 mm), P o eema 300 Å (5
μ
m, 8 ×
300 mm) and P o eema 1000 Å (5
μ
m, 8 ×300 mm) (PSS GmbH, Mainz,
Ge many). The column o en and he ligh sca e ing de ec o we e kep
a 30 ᵒC, and he e ac i e index de ec o was main ained a 40 ᵒC.
De ec o s we e calib a ed wi h a polye hylene oxide s anda d (PSS,
Mainz, Ge many) o 106 kDa (Mw) and polydispe si y index 1.05. Mw
alues we e es ima ed wi h e ac i e index inc emen s o 0.19 (Meye
& Mo gens e n, 2003).
The numbe a e age molecula weigh (M
n
), he weigh a e age
molecula weigh (M
w
) and he polydispe si y index (PDI) we e calcu-
la ed wi h he ollowing equa ions, espec i ely:
Mn=∑MwiNi
∑Ni[2]
Mw=∑M2
wiNi
∑MwiNi[3]
PDI =Mw
Mn[4]
whe e N
i
is he numbe o p o ein molecules and M
wi
is hei molecula
weigh .
2.3.7. The mal cha ac e isa ion
The mal s abili y and decomposi ion beha iou o gela ines was
s udied using he mog a ime ic analysis (TGA). D ied gela ine (~5–20
mg) was placed in an open pla inum pan and loaded in o he measu ing
chambe o a TA Ins umen s Q500 he mog a ime ic analyse (TA
Ins umen s, New Cas le, DE, USA). Samples we e hea ed om oom
empe a u e o 800 ᵒC a 5 ᵒC/min in an ine ni ogen gas (99.999%,
Nippon Gases Ibe ia, Mad id, Spain) a mosphe e. To a oid con amina-
ion, ni ogen gas was also used as he balance pu ge gas and as he
sample pu ge gas ( low a es o 40 and 60 mL/min, espec i ely). The
eco ded he mog ams we e analysed using he Uni e sal Analysis 2000
so wa e o TA Ins umen s.
2.3.8. Rheological beha iou
Rheological p ope ies o he ex ac ed gela ines we e s udied in an
An on Paa MCR 102 (An on Paa , G az, Aus ia) modula compac
heome e , using a cone-pla e geome y (1ᵒ cone angle, 50 mm cone
diame e , 0.098 mm gap) con o ming o he small ampli ude oscilla o y
shea (SAOS) me hodology (S e e, 1996). The expe imen s we e done
ollowing he p o ocol by Al es e al. (2022) wi h sligh modi ica ions.
Solu ions a 6.67 w % we e p epa ed a 45 ᵒC o acili a e dissolu ion,
and hen hey we e allowed o cool o oom empe a u e be o e s a ing
he measu emen s. The dynamic heological p ope ies we e s udied a a
ixed equency and s ain o 1 Hz and 10%, espec i ely, which we e
wi hin he linea iscoelas ic egion p e iously assessed using s ain
sweep es s. The p o ocol consis ed o a hea ing amp om 2 o 30 ᵒC
ollowed by a cooling amp om 30 o 2 ᵒC, bo h a a a e o 1 ᵒC/min.
The s o age modulus (G
′
) and he loss modulus (G") we e measu ed as a
unc ion o empe a u e. Addi ionally, he mechanical p ope ies o he
gels we e e alua ed using equency sweeps om 10
−1
o 10
2
Hz a 2 ᵒC
(lowes wo king empe a u e).
3. Resul s and discussion
3.1. Ex ac ion yield
The p e ea men applied o he aw ma e ial p io o he ex ac ion
s ep has a majo e ec on he yield, and also on he p ope ies o he inal
p oduc (Al es e al., 2022; G´
omez-Guill´
en e al., 2002; G´
omez-Guill´
en,
Gim´
enez, L´
opez-Caballe o, & Mon e o, 2011; Milo ano ic & Hayes,
2018). The e ec o he con ac ime be ween he skin and he eu ec ic
sol en on bo h he yield and he p ope ies o he ex ac ed p oduc was
s udied. Al hough he ex ac ion yield is o en based on he o al mass o
C. Gallego e al.
Food Hyd ocolloids 159 (2025) 110652
3
we skin, he esul s ob ained in his way canno be compa ed since a
a ia ion in he mois u e o aw ma e ial will dis o he esul s.
The e o e, he ex ac ion yield was also calcula ed on a d y basis o
yellow in una skin. As shown in Table 2, he eco e y in he case o he
p e ea ed samples was 31–33 g gela ine/100 g we skin, which ep e-
sen s 60–62 g gela ine/100 g d y skin; and compa es a ou ably o he
yield ob ained wi h he non-p e ea ed sample ( ow labelled as p e-
ea men ime o 0 h in Table 2). These alues we e highe han he
alues o 6–19 g gela ine/100 g we skin ha ha e been epo ed (Ka im
& Bha , 2009). No signi ican di e ence was obse ed be ween he yield
a 0.5 h p e ea men and ha a 3 h p e ea men .
3.2. P oxima e composi ion
P oxima e composi ion is shown in Table 3. The ideal mois u e
con en o powde ed gela ine is in he ange 8–12% since le els o
mois u e >16% ca y he isk o lumping and mic obiological de e io-
a ion, while alues <6–8% may lead o hyg oscopic gela ines (Is¸ık
e al., 2024). These gela ines showed a sui able mois u e con en . The
aw ma e ial and i s p ese a ion me hod a ec he ash con en , anging
om 1.1 o 3.7% in he case o gela ines om yellow in una skin
(P ano o, Ma seno, & Rahmawa i, 2011; Sousa, V´
azquez, P´
e ez-Ma ín,
Ca alho, & Gomes, 2017). The ash alues o he samples ex ac ed wi h
p e ea men in Table 3 a e on he highe end o ha ange. Fu he
s udies will be equi ed o asce ain whe he i is due o a a ou ed
solubilisa ion o mine als om he skin in he eu ec ic sol en o o a
minimum p esence o sodium coming om he eu ec ic pa en com-
pounds. The sample wi h he sho es p e ea men ime had 88.8%
o ganic ma e , compa ed o 87.2% a e 3 h o p e ea men . Howe e ,
he p o ein con en was s a is ically equi alen in bo h cases wi hin he
unce ain y: 83 ±1 % and 84 ±1%, espec i ely. All he alues,
including hose o he sample ex ac ed wi hou p e ea men and o
he comme cial gela ine, we e wi hin he ange o alues ob ained when
applying adi ional ex ac ion sys ems.
3.3. FT-IR spec a
Fo he ish gela ines ob ained a e p e ea men wi h he eu ec ic
sol en and subsequen wa m wa e ex ac ion, he mos cha ac e is ic
peaks o gela ine s uc u e could be iden i ied (Fig. 1), namely hose
peaks iden i ied as amide A, amide B, amide I, amide II, and amide III.
S a ing a he highes wa enumbe , he i s in ense and b oad band
is seen a 3286 cm
−1
, co esponding o he amide-A signal (Kong & Yu,
2007) associa ed wi h he O-H s e ching and he N-H s e ching coupled
wi h hyd ogen bonding (Me ina, Suguna, Ka pu am, Vijaylakshmi, &
Renuka, 2017; Shah alizadeh e al., 2021). This band is somewha
o e lapping he amide-B band, obse ed a 2937 cm
−1
and associa ed
wi h C-H s e ching ib a ions. P e ious s udies (Muyonga, Cole, &
Duodu, 2004; Sil a, Bandei a, & Pin o, 2014) epo ed ha he wide
ampli ude o his band may be associa ed wi h he inc eased p esence o
pep ide chains, sugges ing mo e deg ada ion o he p o ein du ing he
p ocess o ex ac ion. The exis ence o hese low Mw agmen s implies
he p esence o mo e pep ide bonds, hus acili a ing he possibili y o a
ne wo k in e ac ion ha may lead o he o ma ion o gels.
Rep esen a i e bands o amide-I and amide-II we e ound, espec-
i ely, a 1635 and 1529 cm
−1
, in ag eemen wi h p e ious esul s (Kong
& Yu, 2007; Me ina e al., 2017; Sil a e al., 2014; Valca cel,
He mida-Me ino, Pi˜
nei o, He mida-Me ino, & V´
azquez, 2021). The
abso p ion zone o he amide-I co esponds o he s e ching ib a ions
o he C=O bond wi hin he pep ide backbone o he p o ein. On he
o he hand, he peak o he amide-II is associa ed mainly wi h he C-N
s e ching and he N-H bending (Abdollahi & Undeland, 2018; Kong &
Yu, 2007). Amide III is associa ed wi h he s e ching ib a ion o C-N
and N-H de o ma ion om amide bonds, and in his case is seen a 1225
cm
−1
. I has been associa ed wi h he p esence o a molecula diso de ,
p obably ela ed o he loss o he iple helix s uc u e (Kuma , Chan-
d a, Ela a asan, & Shamasunda , 2018; Sin husam an, Benjakul &
Kishimu a, 2014).
As shown in Fig. 1, no signi ican di e ences we e ound be ween he
spec a o gela ines ob ained a e he wo di e en p e ea men imes.
This sugges s ha , be ween he wo imes s udied, he con ac ime does
no ha e an impo an in luence on he obse able con o ma ion o he
ex ac ed gela ine. Mo eo e , hese key cha ac e is ic bands o gela ines
a e also obse ed in he FT-IR spec a o he sample ex ac ed wi h no
p e ea men and o he comme cial gela ine sample (Figs. S1 and S2 in
he Suppo ing In o ma ion).
3.4. Amino acid p o ile
The amino acid composi ion o gela ines has an impo an ole in
de e mining hei quali y and cha ac e is ics. The amino acid p o iles
a e shown in Table 4.
Table 2
Gela ine ex ac ion yields ( alues a e a e age o h ee eplica es ±s anda d
de ia ion).
Yield (g gela ine/100 g skin)
P e ea men ime (h) Basis: we skin Basis: d y skin
0 27 ±1 51 ±1
0.5 33 ±1 62 ±2
3 31 ±1 60 ±2
Table 3
Composi ion o gela ine samples ( alues a e he a e age o wo eplica es ±
s anda d de ia ion).
Con en (g/100 g sample)
P e ea men ime (h) Mois u e Ash O ganic
ma e
C ude
p o ein
0 8.3 ±0.3 2.1 ±
0.2
90 ±1 82 ±1
0.5 8.1 ±0.2 3.1 ±
0.1
88.8 ±0.5 83 ±1
3 9.2 ±0.2 3.7 ±
0.1
87.2 ±0.5 84 ±1
Comme cial sample 9.6 ±0.6 0.2 ±
0.0
90 ±1 88 ±1
Fig. 1. FT-IR spec a o ish gela ines ob ained using wa m wa e ex ac ion
om he p e ea ed aw ma e ial. F om op o bo om: 3 h p e ea men (blue
line), 0.5 h p e ea men ( ed line). Ve ical dashed lines co espond o he
wa enumbe o he cha ac e is ic peaks o p o eins and polypep ides.
C. Gallego e al.
Food Hyd ocolloids 159 (2025) 110652
4
The mos p ominen amino acids o conside o ish gela ine a e
glycine, p oline and hyd oxyp oline. The composi ion o hese h ee
amino acids in he samples ob ained a e p e ea men was, espec-
i ely, a ound 25, 12.5 and 7.5%, wi h no signi ican in luence o he
p e ea men ime. The esul s we e consis en wi h p e ious esea ch
whe e he ange was 22–35% o glycine, 9–15% o p oline and 5–10%
o hyd oxyp oline, and simila gela ine con en s we e ob ained (Al es
e al., 2022; De kach, Vo on’ko, Kuchina, & Kolo o a, 2020;
G´
omez-Guill´
en e al., 2002; Gudmundsson & Ha s einsson, 1997; Nu -
ilmala, Su yama e i a, Husein Hizbullah, Jacoeb, & Ochiai, 2022).
Gela ine con en can be es ima ed h ough hyd oxyp oline con en by
he me hod o Sa o, Ohashi, Oh suki, and Kawaba a (1991). Using a
con e sion ac o o 11.42 (acco ding o he p opo ion o hyd oxy-
p oline in he o al amino acid con en in collagen), he gela ine o he
samples ex ac ed wi h p e ea men was ~85%, lowe han he alue o
~97% ob ained o he sample wi h no p e ea men o o he com-
me cial gela ine, and he e o e meaning ha non-collagenous p o eins
we e also ex ac ed. P oline and hyd oxyp oline ha e an impo an ole
in he s abilisa ion o he iple helix, especially hyd oxyp oline because
o i s capaci y o o m hyd ogen bonds using he hyd oxy g oup (Piez &
G oss, 1960). When he empe a u e d ops below he coil- o-helix
ansi ion empe a u e, he collagen s uc u e unde goes pa ial egen-
e a ion. Segmen s o polypep ide chains ich in py olidine amino acids
adop a helical con o ma ion and o m junc ion zones capable o
e aining wa e , he eby acili a ing gel o ma ion (Haug, D age , &
Smids ød, 2004; Kasankala, Xue, Weilong, Hong, & He, 2007). The e-
o e, he combined p oline and hyd oxyp oline con en o gela ine a -
ec s i s physical p ope ies, bo h in e ms o heological beha iou and
he mal s abili y (G´
omez-Guill´
en e al., 2002; Lin, Regens ein, L , Lu, &
Jiang, 2017). The combined con en ep esen s ~20% in all he gela-
ines included in Table 4, which is consis en wi h p e ious alues o
he skin o wa m wa e ish such as yellow in una (highe han alues
ob ained o cold wa e species bu lowe han he 30% ound in
mammalian gela ines) (Fa is, Schaich, Liu, Pie gio anni, & Yam,
2009).
3.5. SDS-PAGE
Mw dis ibu ion o gela ines a ec s hei physical, chemical, and
biological p ope ies. Fig. 2 shows he esul o SDS-PAGE, whe e A and
B ep esen he wo eplica es o he same sample, and 1 and 2 deno e
he gela ines ex ac ed a e 3 and 0.5 h o p e ea men , espec i ely. I
can be obse ed ha bo h samples show e y simila pa e ns: a he -
e ogeneous dis ibu ion o bands anging om 15 o 130 kDa, pa icu-
la ly concen a ed a ound 55–70 kDa. Some bands appea in he
100–130 kDa ange, which could be indica i e o he p esence o he
esidual
α
1
and
α
2
chains o collagen. Howe e , he e is also e idence o
he o al absence o any β-dime (composed o wo
α
chains linked by
co alen bonds) o γ-chain ( ime composed o h ee c oss-linked
α
chains) (Al es e al., 2022). This in e p e a ion is somewha consis en
wi h he FT-IR spec a: he iple helix s uc u e o collagen has been
comple ely un olded du ing he ex ac ion p ocedu e. Fu he mo e, he
p esence o lowe Mw polypep ide chains con i ms he pa ial deg a-
da ion o he gela ine. These esul s a e consis en wi h hose o Yu e al.
(2023), who cha ac e ised gela ines ex ac ed om he skin o o he
wa m wa e ish species a e hei p e ea men wi h sodium hyd oxide
and ace ic acid, and subsequen ex ac ion wi h ho wa e a 50 ᵒC o 6
h. A simila pa e n was also obse ed o he sample ob ained wi hou
p e ea men ; whe eas o he comme cial gela ine, wi h an a e age Mw
o 60 kDa, e y blu ed bands we e obse ed (Fig. S3 in he Suppo ing
In o ma ion).
3.6. GPC-SEC
A quan i a i e analysis o he Mw dis ibu ion o he gela ines ob-
ained wi h p e ea men was done using GPC-SEC. The esul s a e
shown in Table 5, ob ained om he co esponding eluog ams shown in
Table 4
Amino acid con en (exp essed in g o amino acid/100 g o o al amino acids) o
he gela ines ob ained om he ish skin, using di e en imes o he p e-
ea men wi h he eu ec ic sol en . Values a e ep esen ed as he a e age o wo
eplica es ±s anda d de ia ion.
a
Concen a ion (g/100 g o al
amino acids)
Amino acid 0 h 0.5 h
p e ea men
3 h
p e ea men
Comme cial
Alanine 9.0 ±0.7 7.0 ±0.8 7.0 ±0.1 9.1 ±0.3
A ginine 9.0 ±0.2 7.6 ±0.7 7.9 ±0.1 8.9 ±0.2
Aspa ic acid
b
5.0 ±0.7 4.8 ±0.5 4.7 ±0.1 5.4 ±0.9
Cys eine n. d. n. d. n. d. n.d.
Glu amic acid
c
9.0 ±0.3 9 ±1 8.8 ±0.2 9.4 ±0.7
Glycine 23 ±1 24.6 ±0.3 24.9 ±0.4 23 ±1
His idine 2.00 ±0.03 5.0 ±0.5 4.79 ±0.03 2.0 ±0.1
Isoleucine 1.27 ±0.06 3.4 ±0.3 3.4 ±0.1 1.3 ±0.1
Leucine 2.6 ±0.1 0.9 ±0.1 0.92 ±0.03 2.1 ±0.1
Lysine 3.29 ±0.04 2.7 ±0.3 2.5 ±0.1 3.4 ±0.2
Me hionine 2.1 ±0.1 2.2 ±0.2 2.3 ±0.1 2.1 ±0.1
Hyd oxyp oline 9 ±1 7.5 ±0.6 7.4 ±0.1 8 ±1
Phenylalanine 2.17 ±0.01 2.0 ±0.1 2.2 ±0.1 2.09 ±0.01
P oline 11.3 ±0.8 12.4 ±0.1 12.5 ±0.2 11.2 ±0.6
Se ine 5 ±2 3.9 ±0.4 3.9 ±0.1 5 ±2
Th eonine 3.06 ±0.06 4.3 ±0.4 4.3 ±0.1 3.05 ±0.04
Ty osine 0.40 ±0.09 0.32 ±0.04 0.36 ±0.04 0.38 ±0.07
Valine 2.4 ±0.3 2.5 ±0.3 2.46 ±0.02 2.29 ±0.05
a
n.d.: no de ec ed.
b
Including aspa agine, which is deamida ed du ing he hyd olysis p ocess o
aspa ic acid.
c
Including glu amine, which is deamina ed du ing he hyd olysis p ocess o
glu amic acid.
Fig. 2. SDS-PAGE pa e ns o gela ines. The code M e e s o a p es ained
p o ein ladde ma ke (10–250 kDa); he codes 1A and 1B co espond o ep-
lica es o he sample ex ac ed a e 3 h o p e ea men ; and he codes 2A and
2B co espond o eplica es o he sample ex ac ed a e 0.5 h o p e ea men .
C. Gallego e al.
Food Hyd ocolloids 159 (2025) 110652
5

Fig. 3.
Simila o SDS-PAGE, GPC esul s showed a he e ogeneous dis i-
bu ion o he Mw o gela ines ega dless o he p e ea men ime. In
bo h cases, h ee di e en egions could be iden i ied: he i s egion
co esponds o chains wi h M
w
o 171–193 kDa o he gela ines ob-
ained om bo h ea men s. This egion, which is associa ed wi h he
p esence o β-dime s (Rigue o e al., 2023), ep esen s a small pa o he
peak in bo h cases (2.4 and 7.8%). The second egion co esponds o
molecules wi h M
w
o 83–88 kDa. I is compa ible wi h he M
w
o
α
chains (Rigue o e al., 2023). In his case, he peak a ea inc eases up o
16 and 29%, espec i ely. The la ges egion o bo h gela ines co e-
sponds o a M
w
o 26–30 kDa and ep esen s 82 and 64% o he o al peak
a ea. The polydispe si y index (PDI) o his egion was 30–50% highe
han in he o he egions. Since PDI is a measu e o he b oadness o he
M
w
dis ibu ion, high alues a e o en ela ed o in amolecula clea age
and some deg ee o selec i e hyd olysis (Eys u ska d, Haug, Elha aoui,
Djabou o , & D age , 2009; Rbii, Su el, B amba i, Buche , & Violleau,
2011), which is consis en wi h he low alues o M
w
in his egion.
By compa ing he esul s wi h bo h gela ines, he M
w
o all egions
and he peak a eas o he i s and he second egions (high M
w
) a e
highe in he gela ine ob ained a e he longe p e ea men ime. This
is consis en wi h Cui e al. (2021), who in es iga ed he solubili y and
in e ac ions be ween he same eu ec ic sol en and comme cial po cine
gela ine. They ound ha he M
w
o he gela ine inc eased a e being
dissol ed in he sodium ace a e ihyd a e-u ea eu ec ic sol en and
subsequen ly egene a ed, p obably due o a andom e- o ming o
la ge molecules using hyd ogen bonding.
3.7. The mal cha ac e isa ion
The mal s abili y and decomposi ion beha iou o gela ines a e
a ec ed by hei in e nal s uc u e, Mw dis ibu ion and amino acid
composi ion (Lin e al., 2017). The TGA cu e ( a ia ion o sample
weigh wi h empe a u e, exp essed as a pe cen age o he ini ial sample
mass) is shown in Fig. 4 oge he wi h i s de i a i e, o he gela ines
ob ained wi h p e ea men . Two main decomposi ion s eps we e
obse ed: he i s one, wi h a maximum a e o decomposi ion a ~140
ᵒC and ep esen ing a mass loss o ~20%, was p obably associa ed wi h
he e apo a ion o adso bed and bound wa e (Co eia e al., 2013); and
he second one was ela ed o he decomposi ion o p o ein and he
subsequen loss o amino acids (Ma ins e al., 2018), wi h a maximum
Table 5
Molecula weigh dis ibu ion o gela ines ob ained a e wo di e en p e-
ea men imes. Values a e ep esen ed as he a e age o wo eplica es ±
s anda d de ia ion.
P e ea men ime
(h)
Region
a
M
nb
(kDa)
M
wc
(kDa)
PDI
d
Peak a ea
(%)
0.5 1 160 ±
10
171 ±7 1.077 2.4 ±0.6
2 80 ±6 83 ±4 1.040 16 ±2
3 17 ±1 26 ±3 1.526 82 ±5
3 1 183 ±
19
193 ±14 1.056 7.8 ±0.9
2 83 ±9 88 ±6 1.053 29 ±3
3 21 ±2 30 ±2 1.396 64 ±7
a
See Fig. 3 o iden i ica ion o he di e en egions on he basis o hei
elu ion ime.
b
M
n
: numbe a e age molecula weigh .
c
M
w
: weigh a e age molecula weigh .
d
PDI: polydispe si y index.
Fig. 3. GPC eluog ams o yellow in una gela ines ob ained a e (a) 0.5 h o (b) 3 h o p e ea men wi h he eu ec ic sol en . Signals o he di e en de ec o s ( om
op o bo om): low angle ligh sca e ing (g een line); igh angle ligh sca e ing (blue line); ul a iole (232 nm) (black line); and e ac i e index ( ed line). Ve ical
dashed lines de ine h ee anges (labelled wi h he ca dinals 1, 2, and 3) o elu ion ime o easie in e p e a ion o he esul s – see Table 5 and associa ed discussion
in he main ex .
Fig. 4. TGA cu es (solid lines), and hei de i a i es wi h empe a u e (dashed
lines), o he gela ines ex ac ed om yellow in una skin wi h wo di e en
p e ea men imes.
C. Gallego e al.
Food Hyd ocolloids 159 (2025) 110652
6
decomposi ion a e a ound 300 ᵒC and inc easing he decomposed
pe cen age up o ~75%. Onse decomposi ion empe a u e (T
onse
),
empe a u e o he maximum decomposi ion a e (T
max
), and mass loss
associa ed wi h each o hese decomposi ion s eps a e shown in Table 6.
Minima in he de i a i e cu es a ~600 ᵒC sugges he beginning o a
hi d decomposi ion s ep. Since he TGA uns we e s opped a 800 ◦C,
he empe a u e o he maximum decomposi ion a e o his addi ional
decomposi ion s ep was no asce ained. In any case, he esidue
emaining a he inal poin o each un is also included in Table 6.
P e ious s udies epo ed a simila pa e n o he mal decomposi-
ion o ish gela ines, al hough some di e ences in speci ic empe a u e
alues a e wo h men ioning. Ma ins e al. (2018) did he TGA o ilapia
scale gela ine, while Valca cel e al. (2021) in es iga ed he he mal
decomposi ion o gela ines om he skin o seab eam, seabass, and
ainbow ou . In bo h cases i was obse ed ha , as in he p esen s udy,
he mal decomposi ion occu ed in wo dis inc s ages. The T
max
o he
i s s age in he case o ilapia scale gela ine was ound a 65.4 ᵒC,
subs an ially lowe han he cu en alues (116 ᵒC and 111 ᵒC o he
gela ines ob ained wi h he wo p e ea men s, espec i ely). As shown
by Valca cel e al. (2021), his pa ame e a ies depending on he spe-
cies om which he gela ine is ob ained and on he ex ac ion p ocess.
The con ol sample ex ac ed wi h no p e ea men showed a p e ious
decomposi ion s ep (see Fig. S4 in he Suppo ing In o ma ion and
Table 6) a low empe a u es (<50 ᵒC), associa ed wi h he e apo a ion
o adso bed wa e . Rega ding he second decomposi ion s ep, expe i-
men al and li e a u e alues a e mo e homogeneous: he cu en T
max
alues a e close o hose p e iously ob ained: ~325 ◦C ( ilapia) by
Ma ins e al. (2018) and 301–316 ᵒC (o he species) by Valca cel e al.
(2021). Despi e he di e ences in T
max
alues, he o e all mass losses
a e each o he s eps epo ed by Ma ins e al. (2018) we e consis en
wi h his s udy: 17–21 and 74–78%, espec i ely. The esidue a 800 ᵒC
o 15–17% o he ini ial mass was close o he 19% ob ained by Ma ins
e al. (2018), and sligh ly lowe han he 27–31% ob ained by Valca cel
e al. (2021). In he case o he comme cial gela ine sample, he absence
o decomposi ion s eps a ound 100–150 ◦C sugges s he absence o wa e
associa ion wi hin he gela ine s uc u e (Fig. S5 in he Suppo ing In-
o ma ion), which could a ec he gelling p ope ies. Conside ing he
b oadness o he anges and he s ong in luence o he o igin o he
gela ine in i s he mal decomposi ion beha iou , i can be assumed ha
he cu en esul s a e consis en wi h p e ious epo s.
3.8. Rheological beha iou
The iscoelas ic p ope ies o gela ine a e use ul o assess he quali y
o he p oduc and de e mine i s po en ial applica ions. Upon cooling an
aqueous solu ion o gela ine below i s sol-gel ansi ion empe a u e,
gela ines unde go con o ma ional changes, o ming iple chain helices
(junc ion poin s) o c ea e he h ee-dimensional ne wo k gel s uc u e.
Wa e emains apped in his ma ix, gi ing he gel i s cha ac e is ic
ex u e and consis ency. The o ces go e ning his ansi ion a e phys-
ical (speci ically hyd ogen bonds and an de Waals bonds), which
makes he gela ion p ocess he mo e e sible (Ahmed, 2017). The e o e,
one o he mos impo an ou comes o heological s udy o gela ines is
he de e mina ion o he gelling empe a u e (T
gel
) and he mel ing
empe a u e (T
mel
), which a e de e mined om he in e sec ion o he
s o age modulus (G
′
) and he loss modulus (G”) cu es du ing cooling
and hea ing o he sample, espec i ely. Fig. 5 shows he changes o G
′
and G
″
wi h empe a u e in a hea ing amp and in a cooling amp, o
aqueous solu ions o he gela ines p e ea ed wi h he eu ec ic sol en a
6.67 w %. Bo h gela ines showed a quali a i ely simila beha iou , wi h
some nume ical di e ences. A he beginning o he hea ing s ep, G
′
is
signi ican ly highe han G
″
, indica ing ha he solu ion shows a p e-
dominan ly solid-like beha iou a low empe a u es. When empe a-
u e inc eases, he h ee-dimensional ne wo k s a s weakening and he
consequence is a dec ease in bo h G
′
and G”. Once abo e he mel ing
poin , G
″
becomes highe han G
′
, ma king he ansi ion om gel o
liquid s a e. A highe empe a u es, G
″
emains highe han G
′
, sug-
ges ing he single s and a angemen o polypep ide chains (Kokol,
Po a ha a, Mihelˇ
ciˇ
c, & Pe ˇ
se, 2021). Du ing he cooling s ep, G
′
and G
″
s ongly inc ease as a esul o he o ma ion o junc ion zones and he
ein o cemen o he gel ne wo k h ough hyd ogen bonding, an de
Waals o ces, sel -assembly, and hyd ophobic associa ions (Da Sil a,
Bode, G illo, & D eiss, 2015). A a gi en empe a u e du ing he cooling
amp, G’ again becomes g ea e han G”. The c osso e o bo h cu es
de e mines he gelling poin , a which gela ine makes he ansi ion
om liquid-domina ed phase o solid-domina ed phase (Ahmed, 2017;
Huang e al., 2017).
Table 7 shows he alues o T
mel
and T
gel
o he gela ine solu ions,
including he ones o he sample ex ac ed wi h no p e ea men and o
he comme cial gela ine. The mel ing and gelling empe a u es o
gela ine a e no mally di e en e lec ing i s he mal hys e esis (Ra e &
Raza i, 2017). The gela ine ob ained a 3 h p e ea men showed highe
mel ing and gelling empe a u es han he gela ine ex ac ed in he
p ocedu e wi h a 0.5 h p e ea men , sugges ing ha a longe con ac
be ween he ish skin and he eu ec ic sol en esul ed in a mo e s uc-
u ed gel ne wo k wi h highe esis ance o empe a u e, which is
consis en wi h he p esence o highe Mw chains as shown using he
GPC-SEC. This may be ela ed o he exis ence o some pa icula in-
e ac ions be ween he eu ec ic sol en and he gela ine, since some
s udies ha e shown ha he componen s o eu ec ic sys ems can in e ac
wi h polypep ide chains and become pa o he gela ine s uc u e (Cui
e al., 2021; Sanchez-Fe nandez e al., 2022; Wan, Zhu, & Sun, 2024).
This is consis en wi h he gela ine ob ained wi hou p e ea men (see
Fig. S6 and Table 7) showing lowe mel ing and gelling empe a u es.
P e ious s udies epo ed gelling and mel ing empe a u es o ish
gela ines in he anges 8–25 and 11–28 ᵒC, espec i ely (Huang e al.,
2019). Howe e , o yellow in una skin, he esul s we e signi ican ly
lowe han he gelling empe a u e o 18.7 ᵒC and he mel ing empe -
a u e o 24.3 ᵒC epo ed by Cho, Gu, and Kim (2005). This may be due
o mo e pa ial deg ada ion o gela ine. Howe e , he aw ma e ials
canno be conside ed ully equi alen : in he s udy by Cho e al. (2005),
he skins we e esh; bu he cu en skins we e b ined. As shown by
Al es e al. (2022), gela ines de i ed om sal ed skins showed lowe gel
s eng h, which is seen as lowe alues o G’, T
mel
, and T
gel
. The lowe
Mw dis ibu ion esul ing om his mo e e ec i e deg ada ion allows
he molecules o en angle and o m a ne wo k a lowe ene gy le els,
di ec ly a ec ing he iscoelas ic beha iou (En ione e al., 2020). The
lowe gelling empe a u es migh ep esen a echnical ad an age o e
mammalian-de i ed gela ines in applica ions ha equi e be e elease
o a oma and s onge la ou (Choi & Regens ein, 2000). A simila
alue was ound by Sousa e al. (2017), who epo ed a T
mel
o 21.9 ᵒC.
On he o he hand, as shown in Fig. S7, and al hough he comme cial
gela ine o igina es om a cold-wa e ish and is soluble in wa e , i is
Table 6
Onse decomposi ion empe a u e (T
onse
), empe a u e o maximum decompo-
si ion a e (T
max
), and mass loss (wi h espec o he ini ial sample mass) asso-
cia ed wi h each o he wo main decomposi ion s eps iden i ied using TGA in he
he mal cha ac e isa ion o he gela ines. The inal esidue a 800 ◦C, as a pe -
cen age o he ini ial sample weigh , is also included.
P e ea men
ime (h)
Decomposi ion
s ep
T
onse
(ᵒC)
T
max
(ᵒC)
Mass
loss
(%)
Residue a
800 ◦C (%)
0 0
h
34 41 9.7 17.8
1s 154 161 13.7
2nd 263 306 78.9
0.5 1s 116 144 20.1 15.5
2nd 261 299 78.4
3 1s 111 136 18.2 16.2
2nd 253 289 74.4
Comme cial 1s 41 55 12.1 25.3
2nd 256 302 71.0
C. Gallego e al.
Food Hyd ocolloids 159 (2025) 110652
7
unable o gel.
F equency sweep es s we e done o analyse he mechanical p op-
e ies o he bes -quali y gels. Fig. 6 shows bo h G
′
and G
″
as a unc ion o
equency o he gela ines ob ained wi h p e ea men . As expec ed, he
highe alues o G
′
indica ed a g ea e con ibu ion om elas ici y han
om iscosi y, and hence a solid-like beha iou . Mo eo e , he alues o
G’ a e a leas one o de o magni ude g ea e han hose o G” o e-
quencies <10 Hz, indica i e o a comple ely de eloped and s able gel
s uc u e (Mo is, Nishina i, & Rinaudo, 2012). Bo h empe a u e and
equency sweeps show ha he elas ic modulus o he gela ine ob ained
a e he longe p e ea men ime was highe and, consequen ly, so is
he gel s eng h.
4. Conclusions
The use o a eu ec ic sol en as p e ea men agen o he ex ac ion
o gela ine om yellow in una skin was s udied. This me hod used, as a
p e ea men sol en , a mix u e o compounds ha in he pu e s a e a e
solid a he ope a ing empe a u e. I was es ed wi h wo di e en
p e ea men imes o 0.5 and 3 h, a e which ex ac ion was ca ied ou
wi h wa m wa e as in he adi ional p ocedu e. I was con i med ha
he eu ec ic sol en can in e ac wi h he skin o yellow in una, such
ha he subsequen ex ac ion o gela ine is possible.
Compa ing he gela ines ob ained wi h he p oposed p ocess o a
gela ine ex ac ed wi h wa e , i can be con i med ha he p e ea men
inc eases he ex ac ion yield. Addi ionally, he gela ines showed be e
heological p ope ies han he one jus ex ac ed wi h wa e (no p e-
ea men ) and a comme cial gela ine ob ained om cold-wa e ish
skin.
Rega ding he samples ex ac ed wi h p e ea men , he o e all
p ocess yields, which exceed li e a u e alues by up o 70%, we e ound
o be independen o he p e ea men ime. Besides he yield, o he
pa ame e s ela ed o gela ine cha ac e isa ion ha we e no a ec ed by
he p e ea men ime we e: chemical composi ions, FT-IR spec a, and
amino acid p o iles. These we e consis en wi h hose o gela ines ob-
ained using he adi ional me hod in p e ious s udies. Howe e , sig-
ni ican di e ences we e ound in he Mw dis ibu ion: he gela ine
ob ained a e a longe p e ea men ime showed a highe Mw, which
di ec ly a ec s i s unc ionali y. This inc ease in Mw is e lec ed in he
heology s udies, whe e i was obse ed ha his gela ine showed highe
alues o he s o age modulus, as well as highe gelling and mel ing
empe a u es. Thus, he p e ea men ime could be selec ed based on
he desi ed applica ion o he gela ine.
In summa y, he use o eu ec ic sol en s as an al e na i e o adi-
ional me hods o ex ac ing gela ine om ish was e may esul in
imp o emen s o a ious unc ional p ope ies: i enables he u ilisa ion
as p e ea men sol en s o compounds ha , in he pu e s a e, a e solid
a he ope a ing empe a u e; i simpli ies he p ocess in compa ison
wi h he adi ional me hods used, educing he en i onmen al impac ;
and i allows he uning o in e ac ions be ween he ish by-p oduc and
he sol en ( h ough judicious selec ion o he componen s o he
eu ec ic sys em) o design he inal p oduc wi h he desi ed p ope ies
o a speci ic applica ion. This op ion should be also conside ed o mo e
challenging a ge s such as collagen ex ac ion.
Funding
G an PID2021-123622OB-I00 unded by MCIN/AEI/10.13039/
501100011033 and by “ERDF A way o making Eu ope” by he Eu o-
pean Union.
CRediT au ho ship con ibu ion s a emen
C is ina Gallego: W i ing – o iginal d a , Visualiza ion, Valida ion,
Fig. 5. Va ia ion o s o age modulus (G
′
) and loss modulus (G”) o aqueous gela ine solu ions wi h empe a u e: hea ing amp (a) and cooling amp (b).
Table 7
Mel ing and gelling empe a u es o aqueous solu ions o he gela ines.
P e ea men ime (h) T
mel
(ᵒC) T
gel
(ᵒC)
0 14.6 7.3
0.5 17.0 8.5
3 19.4 11.0
Comme cial 5.1 –
Fig. 6. F equency sweep es s o he gels a a cons an empe a u e o 2 ᵒC.
C. Gallego e al.
Food Hyd ocolloids 159 (2025) 110652
8
Me hodology, In es iga ion, Fo mal analysis. E a Rodil: W i ing – e-
iew & edi ing, Me hodology, Concep ualiza ion. H´
ec o Rod íguez:
W i ing – e iew & edi ing, Supe ision. Ana So o: W i ing – e iew &
edi ing, Supe ision, P ojec adminis a ion, Me hodology, Funding
acquisi ion, Concep ualiza ion.
Decla a ion o compe ing in e es
The au ho s decla e ha hey ha e no known compe ing inancial
in e es s o pe sonal ela ionships ha could ha e appea ed o in luence
he wo k epo ed in his pape .
Da a a ailabili y
No da a was used o he esea ch desc ibed in he a icle.
Acknowledgemen s
The au ho s a e g a e ul o Jos´
e An onio V´
azquez om he REVAL
G oup o he Ins i u e o Ma ine Resea ch (IIM-CSIC) in Vigo, o his help
wi h he GPC-SEC s udies. The au ho s also hank Jealsa Foods S.A.U.
(Boi o, Galicia, Spain) o he kind supply o ish skin o he
expe imen s.
Appendix A. Supplemen a y da a
Supplemen a y da a o his a icle can be ound online a h ps://doi.
o g/10.1016/j. oodhyd.2024.110652.
Re e ences
Abdollahi, M., & Undeland, I. (2018). S uc u al, unc ional, and senso ial p ope ies o
p o ein isola e p oduced om salmon, cod, and he ing by-p oduc s. Food and
Biop ocess Technology, 11(9), 1733–1749. h ps://doi.o g/10.1007/s11947-018-
2138-x
Ahmed, J. (2017). Rheological p ope ies o gela in and ad ances in measu emen . In
J. Ahmed, P. P aszek, & S. Basu (Eds.), Ad ances in ood heology and i s applica ions
(pp. 377–404). Camb ige, UK: Woodhead Publishing. h ps://doi.o g/10.1016/
B978-0-08-100431-9.00015-2.
Al es, A. L., F aguas, F. J., Ca alho, A. C., Valc´
a cel, J., P´
e ez-Ma ín, R. I., Reis, R. L.,
e al. (2022). Cha ac e iza ion o cod ish gela in: A compa a i e s udy o esh and
sal ed skins and di e en ex ac ion me hods. Food Hyd ocolloids, 124, A icle
107238. h ps://doi.o g/10.1016/j. oodhyd.2021.107238
Bai, C., Wei, Q., & Ren, X. (2017). Selec i e ex ac ion o collagen pep ides wi h high
pu i y om cod skins by deep eu ec ic sol en s. ACS Sus ainable Chemis y &
Enginee ing, 5(8), 7220–7227. h ps://doi.o g/10.1021/acssuschemeng.7b01439
Ba is a, M. P., Fe n´
andez, N., Gaspa , F. B., B onze, M. R., & Dua e, A. R. C. (2022).
Ex ac ion o biocompa ible collagen om blue sha k skins h ough he con en ional
ex ac ion p ocess in ensi ica ion using na u al deep eu ec ic sol en s. F on ie s in
Chemis y, 10, 1–10. h ps://doi.o g/10.3389/ chem.2022.937036
Bhowmick, M., & Fields, G. B. (2013). S abiliza ion o collagen-model, iple-helical
pep ides o in i o and in i o applica ions. Me hods in Molecula Biology, 1081,
167–194. h ps://doi.o g/10.1007/978-1-62703-652-8_11
Bish , M., Ma ins, M., Dias, A. C. R. V., Ven u a, S. P. M., & Cou inho, J. A. P. (2021).
Unco e ing he po en ial o aqueous solu ions o deep eu ec ic sol en s on he
ex ac ion and pu i ica ion o collagen ype I om A lan ic cod ish (Gadus mo hua).
G een Chemis y, 23(22), 8940–8948. h ps://doi.o g/10.1039/d1gc01432c
B uke Op ik. (2015). OPUS 7.8 da a collec ion. Ge many: E lingen. e sion 7.8.
Cao, T. H., Nguyen, T. T. O., Nguyen, T. M. H., Le, N. T., & Razumo skaya, R. G. (2017).
Cha ac e is ics and physicochemical p ope ies o gela in ex ac ed om scales o
seabass (La es calca i e ) and g ey mulle (Mugil cephalus) in Vie nam. Jou nal o
Aqua ic Food P oduc Technology, 26(10), 1293–1302. h ps://doi.o g/10.1080/
10498850.2017.1390026
Chen, Y., Jin, H., Yang, F., Jin, S., Liu, C., Zhang, L., e al. (2019). Physicochemical,
an ioxidan p ope ies o gian c oake (Nibea japonica) swim bladde s collagen and
wound healing e alua ion. In e na ional Jou nal o Biological Mac omolecules, 138,
483–491. h ps://doi.o g/10.1016/j.ijbiomac.2019.07.111
Cho, S. M., Gu, Y. S., & Kim, S. B. (2005). Ex ac ing op imiza ion and physical p ope ies
o yellow in una (Thunnus albaca es) skin gela in compa ed o mammalian gela ins.
Food Hyd ocolloids, 19(2), 221–229. h ps://doi.o g/10.1016/j.
oodhyd.2004.05.005
Choi, S. S., & Regens ein, J. M. (2000). Physicochemical and senso y cha ac e is ics o
ish gela in. Jou nal o Food Science, 65, 194–199. h ps://doi.o g/10.1111/j.1365-
2621.2000. b15978.x
Cooney, R., de Sousa, D. B., Fe n´
andez-Ríos, A., Melle , S., Rowan, N., Mo se, A. P., e al.
(2023). A ci cula economy amewo k o sea ood was e alo isa ion o mee
challenges and oppo uni ies o in ensi e p oduc ion and sus ainabili y. Jou nal o
Cleane P oduc ion, 392, A icle 136283. h ps://doi.o g/10.1016/j.
jclep o.2023.136283
Co eia, D. M., Pad ˜
ao, J., Rod igues, L. R., Dou ado, F., Lance os-M´
endez, S., &
Sencadas, V. (2013). The mal and hyd oly ic deg ada ion o elec ospun ish gela in
memb anes. Polyme Tes ing, 32(5), 995–1000. h ps://doi.o g/10.1016/j.
polyme es ing.2013.05.004
Cui, Y., Zhu, Y., Dai, R., Shan, Z., Yi, J., & Chen, H. (2021). The solubili y and
in e ac ions o gela in in “wa e -in-sodium ace a e ihyd a e/u ea-DES” sys em.
Colloids and Su aces A: Physicochemical and Enginee ing Aspec s, 625, A icle 126916.
h ps://doi.o g/10.1016/j.colsu a.2021.126916
Da Sil a, M. A., Bode, F., G illo, I., & D eiss, C. A. (2015). Explo ing he kine ics o
gela ion and inal a chi ec u e o enzyma ically c oss-linked chi osan/gela in gels.
Biomac omolecules, 16(4), 1401–1409. h ps://doi.o g/10.1021/acs.
biomac.5b00205
De kach, S. R., Vo on’ko, N. G., Kuchina, Y. A., & Kolo o a, D. S. (2020). Modi ied ish
gela in as an al e na i e o mammalian gela in in mode n ood echnologies.
Polyme s, 12(12), 1–10. h ps://doi.o g/10.3390/polym12123051
Duconseille, A., As uc, T., Quin ana, N., Mee sman, F., & San e-Lhou ellie , V. (2015).
Gela in s uc u e and composi ion linked o ha d capsule dissolu ion: A e iew. Food
Hyd ocolloids, 43, 360–376. h ps://doi.o g/10.1016/j. oodhyd.2014.06.006
En ione, J., Cha , C., Pepczynska, M., Padilla, C., Gonz´
alez-Mu˜
noz, A., Olguín, Y., e al.
(2020). Rheological and s uc u al s udy o salmon gela in wi h con olled molecula
weigh . Polyme s, 12(7), 1–17. h ps://doi.o g/10.3390/polym12071587
Eys u ska d, J., Haug, I. J., Elha aoui, N., Djabou o , M., & D age , K. I. (2009).
S uc u al and mechanical p ope ies o ish gela in as a unc ion o ex ac ion
condi ions. Food Hyd ocolloids, 23(7), 1702–1711. h ps://doi.o g/10.1016/j.
oodhyd.2009.01.008
FAO. (2022). The s a e o wo ld ishe ies and aquacul u e 2022. Towa ds blue
ans o ma ion. Rome, I aly: Food and Ag icul u e O ganiza ion. h ps://doi.o g/
10.4060/cc0461en
Fa is, S., Schaich, K. M., Liu, L. S., Pie gio anni, L., & Yam, K. L. (2009). De elopmen o
polyion-complex hyd ogels as an al e na i e app oach o he p oduc ion o bio-
based polyme s o ood packaging applica ions: A e iew. T ends in Food Science and
Technology, 20(8), 316–332. h ps://doi.o g/10.1016/j. i s.2009.04.003
F aga-Co al, M., Ronza, P., Ga cia-Oli ei a, P., Pe ei a, A. G., Losada, A. P.,
P ie o, M. A., e al. (2022). Aquacul u e as a ci cula bio-economy model wi h
Galicia as a s udy case: How o ans o m was e in o e alo ized by-p oduc s. T ends
in Food Science and Technology, 119, 23–35. h ps://doi.o g/10.1016/j.
i s.2021.11.026
Gallego, C., Rod íguez, H., & So o, A. (2023). Solubili y o amino acids in he eu ec ic
sol en cons i u ed by sodium ace a e ihyd a e and u ea and in i s mix u e wi h
wa e . In e na ional Jou nal o Molecula Sciences, 24(2), A icle 1550. h ps://doi.
o g/10.3390/ijms24021550
G´
omez-Guill´
en, M. C., Gim´
enez, B., L´
opez-Caballe o, M. E., & Mon e o, M. P. (2011).
Func ional and bioac i e p ope ies o collagen and gela in om al e na i e sou ces:
A e iew. Food Hyd ocolloids, 25(8), 1813–1827. h ps://doi.o g/10.1016/j.
oodhyd.2011.02.007
G´
omez-Guill´
en, M. C., Tu nay, J., Fe n´
andez-Díaz, M. D., Ulmo, N., Liza be, M. A., &
Mon e o, P. (2002). S uc u al and physical p ope ies o gela in ex ac ed om
di e en ma ine species: A compa a i e s udy. Food Hyd ocolloids, 16(1), 25–34.
h ps://doi.o g/10.1016/S0268-005X(01)00035-2
G ossman, I. S., & Gan, R. (1992). P ocess o he p oduc ion o gela in om ish skins. U.
S. Pa en No. 5093474.
Gudmundsson, M., & Ha s einsson, H. (1997). Gela in om cod skins as a ec ed by
chemical ea men s. Jou nal o Food Science, 62(1), 37–39. h ps://doi.o g/
10.1111/j.1365-2621.1997. b04363.x
Haug, I. J., D age , K. I., & Smids ød, O. (2004). Physical and heological p ope ies o
ish gela in compa ed o mammalian gela in. Food Hyd ocolloids, 18(2), 203–213.
h ps://doi.o g/10.1016/S0268-005X(03)00065-1
Huang, T., Tu, Z., Shangguan, X., Sha, X., Wang, H., Zhang, L., e al. (2019). Fish gela in
modi ica ions: A comp ehensi e e iew. T ends in Food Science and Technology, 86,
260–269. h ps://doi.o g/10.1016/j. i s.2019.02.048
Huang, T., Tu, Z., Xinchen-Shangguan, Wang, H., Zhang, L., & Sha, X. (2017).
Rheological and s uc u al p ope ies o ish scales gela in: E ec s o con en ional
and ul asound-assis ed ex ac ion. In e na ional Jou nal o Food P ope ies, 20(2),
1210–1220. h ps://doi.o g/10.1080/10942912.2017.1295388
Ideia, P., Pin o, J., Fe ei a, R., Figuei edo, L., Spínola, V., & Cas ilho, P. C. (2020). Fish
p ocessing indus y esidues: A e iew o aluable p oduc s ex ac ion and
cha ac e iza ion me hods. Was e and Biomass Valo iza ion, 11(7), 3223–3246.
h ps://doi.o g/10.1007/s12649-019-00739-1
In e na ional O ganiza ion o S anda iza ion. (2023). Mea and mea p oduc s -
de e mina ion o ni ogen con en . ISO S anda d No. 937:2023.
Is
¸ık, Ç., Pa lak, M. E., Kı aç Demi el, F. T., Odabas
¸, H.˙
I., Da˘
gdelen, A. F., Yilmaz, M. T.,
e al. (2024). Gela in ex ac ion om chicken skin by con en ional and Ohmic
hea ing me hods and compa ison wi h comme cial halal gela ins. Food Hyd ocolloids,
150, A icle 109694. h ps://doi.o g/10.1016/j. oodhyd.2023.109694
Ka im, A. A., & Bha , R. (2009). Fish gela in: P ope ies, challenges, and p ospec s as an
al e na i e o mammalian gela ins. Food Hyd ocolloids, 23(3), 563–576. h ps://doi.
o g/10.1016/j. oodhyd.2008.07.002
Kasankala, L. M., Xue, Y., Weilong, Y., Hong, S. D., & He, Q. (2007). Op imiza ion o
gela ine ex ac ion om g ass ca p (Ca enopha yngodon idella) ish skin by esponse
su ace me hodology. Bio esou ce Technology, 98(17), 3338–3343. h ps://doi.o g/
10.1016/j.bio ech.2006.03.019
Kokol, V., Po a ha a, Y. B., Mihelˇ
ciˇ
c, M., & Pe ˇ
se, L. S. (2021). Rheological p ope ies o
gela ine hyd ogels a ec ed by low- and ho izon ally-induced cooling a es du ing
C. Gallego e al.
Food Hyd ocolloids 159 (2025) 110652
9