In luence o di e en p ocessing echniques on mic oalgal
p o ein ex ac ion
Ca a ina Mo ei a
a
, Ped o Fe ei a-San os
a,b,c
, Ra aela Nunes
a
, Be na do Ca alho
d,e
,
Hugo Pe ei a
d,e
, Jos´
e A. Teixei a
a,
, C is ina M.R. Rocha
a, ,*
a
Cen e o Biological Enginee ing, Uni e si y o Minho, Campus de Gual a , 4710-057 B aga, Po ugal
b
Depa men o Chemical Enginee ing, Facul y o Science, Uni e si y o Vigo, As Lagoas, 32004 Ou ense, Spain
c
Ins i u o de Ag oecoloxía e Alimen aci´
on (IAA), Uni e si y o Vigo (Campus Auga), As Lagoas, 32004 Ou ense, Spain
d
G eenCoLab – Associaç˜
ao Oceano Ve de, Campus de Gambelas, Uni e si y o Alga e, 8005-139 Fa o, Po ugal
e
CCMAR - Cen e o Ma ine Sciences, Uni e si y o Alga e, Campus de Gambelas, 8005-139 Fa o, Po ugal
LABBELS - Associa e Labo a o y, Guima ˜
aes, B aga, Po ugal
ARTICLE INFO
Keywo ds:
Mic oalgae
P o ein
S uc u e
Cell dis up me hods
Ex ac ion
ABSTRACT
The nu ien - ich composi ion o mic oalgae biomass posi ions i as a highly p omising na u al ood ing edien .
This holds he po en ial o no only enhance he nu i ional alue o a ious ood p oduc s bu also simul a-
neously al e hei s uc u al a ibu es.
This wo k in es iga ed he e ec o i e p o ein ex ac ion echniques such as eeze- hawing, enzyma ic-
assis ed ex ac ion, high-p essu e homogeniza ion, ul asounds-assis ed ex ac ion, and pH adjus men (pH 7,
pH 10, and pH 13) in p o ein yield, and subsequen e cell and p o ein s uc u e o h ee mic oalgal suspensions,
namely, Chlo ella ulga is, Nannochlo opsis oceanica, and Te aselmis chui.
In Chlo ella ulga is, eeze- hawing and high-p essu e homogeniza ion exhibi ed a highe e ec in e ms o
p o ein yield (~26.60 g
p o ein
/100 g
p o ein mic oalgae
). The same occu ed o Nannochlo opsis oceanica wi h also
ul asounds-assis ed ex ac ions and pH 7 and 10 ha ing a p o ein yield abo e 30 %. Te aselmis chui was simila
o Chlo ella ulga is (>20.00 g
p o ein
/100 g
p o ein mic oalgae
) o eeze- hawing, high-p essu e homogeniza ion
and ul asound-assis ed ex ac ion. Enzyma ic-assis ed had a he lowe p o ein yield o all he h ee mic oalgae
(<10.00 g
p o ein
/100 g
p o ein mic oalgae
). The majo i y o p o eins ex ac ed om Chlo ella ulga is samples had
molecula weigh s exceeding 337 kDa, whe eas p o eins ex ac ed om Nannochlo opsis and Te aselmis had
molecula weigh s anging om 5 o 50 kDa.
α
-helices occu ed in p o eins ex ac ed om Chlo ella ulga is
h ough eeze- hawing and enzyma ic-assis ed ex ac ion, while Nannochlo opsis and Te aselmis only had
β-shee .
In conclusion, o op imal p o ein yield eco e y, me hodologies such as eeze- hawing and high-p essu e
homogeniza ion a e he mos e icien ac oss all s udied mic oalgae. The me hod selec ed o ex ac ion had a
g ea e impac on bo h he p o ein yield and s uc u e o sp ay-d ied cells.
1. In oduc ion
In esponse o he g owing “p o ein gap” ha has eme ged in ecen
decades, esea che s ha e been explo ing al e na i e sou ces o p o ein.
This gap is p ima ily a consequence o he apid inc ease in he global
popula ion, which has led o a heigh ened demand o ood. In addi ion,
he cu en ag icul u al p ac ices may no be sus ainable in he long
e m due o hei nega i e en i onmen al impac s, including he gen-
e a ion o g eenhouse gases, land clea ing, and nu ien un-o s, among
o he ac o s [1].
The apidly inc easing demand o p o ein has led o a signi ican
in e es in mic oalgae p o ein. This in e es is p ima ily d i en by he
a ibu es o mic oalgae, including hei p oduc i i y and e icien
esou ce u iliza ion. Mic oalgae display a high p o ein con en and an
amino acid p o ile, ich in essen ial amino acids, as se e al s ains
exhibi highe p o ein con en compa ed o con en ional sou ces like
chicken, eggs, and soybeans [2]. Consequen ly, mic oalgae p o ein has
eme ged as a highly p omising and inno a i e sou ce o p o ein.
* Co esponding au ho a : Cen e o Biological Enginee ing, Uni e si y o Minho, Campus de Gual a , 4710-057 B aga, Po ugal.
E-mail add ess: [email p o ec ed] (C.M.R. Rocha).
Con en s lis s a ailable a ScienceDi ec
Algal Resea ch
jou nal homepage: www.else ie .com/loca e/algal
h ps://doi.o g/10.1016/j.algal.2025.103958
Recei ed 11 Ma ch 2024; Recei ed in e ised o m 7 Feb ua y 2025; Accep ed 7 Feb ua y 2025
Algal Resea ch 86 (2025) 103958
A ailable online 13 Feb ua y 2025
2211-9264/© 2025 The Au ho s. Published by Else ie B.V. This is an open access a icle unde he CC BY license ( h p://c ea i ecommons.o g/licenses/by/4.0/ ).
The high p o ein con en o mic oalgae like Chlo ella ulga is
(C. ulga is), Nannochlo opsis oceanica (N. oceanica), and Te aselmis chui
(T. chui) makes hem sui able o use in ood p oduc s like mea and ish
subs i u es[3]. Howe e , ex ac ing speci ic componen s om mic o-
algae can be challenging due o he s uc u e and igidi y o hei cell
walls [4].
Fo example, C. ulga is is cha ac e ized by he de elopmen o a
p og essi ely hickening cell wall as i ma u es. This p ocess culmina es
in he o ma ion o a mic o ib illa laye consis ing o cellulose mic o-
ib ils [5]. N. oceanica has one dis inc i e ea u e: a s u dy bilaye ed
ilamina shea h ou e laye comp ising bo h cellulosic and hyd o-
phobic laye s. This unique s uc u al cha ac e is ic ende s i a unique
obs acle when a emp ing o dis up cell in eg i y, p ima ily because o
i s igid and obus na u e [6,7]. T. chui cell wall is o med by he usion
o cell body scales cha ac e is ic o he P asinophyceae, and p ima ily
comp ise mannose and glucose monosaccha ides in hei composi ion
[8].
An essen ial ini ial s ep in ol ing p o ein ex ac ion is o en equi ed
o o e come his na u al ba ie . This p ocess acili a es access o he
in acellula componen s and s eamlines subsequen ex ac ion p o-
cedu es. In an a emp o o e come he longs anding hu dle in he
ex ac ion o mic oalgae and plan -de i ed p o ein, nume ous me hods,
including eezing- hawing (F/T) [9], enzyma ic-assis ed ex ac ion
(ENZ) [10], pH adjus men [11], and o he echniques ha e been
employed in he pas [12,13]. Howe e , hese me hods ha e consis-
en ly encoun e ed he ba ie o low ex ac ion yields and/o low
scalabili y [4].
The concep o g een ex ac ion me hods has ga ne ed suppo om
many esea che s since i is a pa adigm ha emphasizes he need o
educe sol en usage, minimise ene gy consump ion, and mi iga e
en i onmen al pollu ion while concu en ly s i ing o maximize yield
[4,14]. Fueled by he p inciples o g een ex ac ion, no el p ocessing
echnologies, no ably ul asound-assis ed ex ac ion (UAE) and high-
p essu e homogeniza ion (HPH), ha e been inc easingly employed o
e ec i ely ex ac compounds om plan ma ices, signi ican ly
inc easing he yields. Typically, he e iciency o hese echniques is
assessed by examining he ex ac ion o a single componen be o e and
a e he applica ion o he ea men and/o he e alua ion o mea-
su emen o cellula in eg i y [15,16].
In addi ion o he chemical composi ion o mic oalgae, hei
mo phological and s uc u al cha ac e is ics also signi ican ly in luence
he e icacy o p o ein solubiliza ion. Ce ain p o eins p esen challenges
in solubiliza ion due o hei hyd ophobic na u e o he p esence o di-
sul ide bonds be ween p o ein molecules, esul ing in dec eased solu-
bili y [1,8–10,16,17]. P o ein solubili y can be enhanced in alkaline
media o o he dena u ing ea men s.
A comp ehensi e li e a u e e iew does no e eal a clea end bu
sugges s ha each mic oalgae esponds di e en ly o cell lysis, neces-
si a ing ailo ed p ocesses o each species. These in es iga ions p e-
dominan ly ocus on esh biomasses o hose subjec ed o p e-
ea men s, such as eeze-d ying o eezing. A s uc u ed app oach
and compa able da a a e impe a i e o in es iga e he po en ial o
p o ein ex ac ion in hese mic oalgae ho oughly.
The cen al ocus o he cu en s udy is o assess how di e en
p o ein ex ac ion echniques a ec he ex ac ion o p o eins om h ee
di e en mic oalgae species in an aqueous medium (C. ulga ia, N.
oceanica, T. chui). Di e en p ocessing echniques ( eeze- hawing, high-
p essu e homogeniza ion, ul asound-assis ed ex ac ion, enzyma ic
ex ac ion, and pH a ia ion) we e employed, and hei impac on
p o ein yield, and on he s uc u e o he eco e ed p o ein (molecula
weigh and p o ein con o ma ion) was e alua ed.
2. Ma e ials and me hods
2.1. Ma e ials
Chlo ella ulga is (C. ulga is, ba ch numbe L201950071) was g own
in he e o ophy, washed wi h decalci ied wa e (1:10 w/w), and p o-
cessed by mic o il a ion o ob ain a suspension wi h a concen a ion o
~60 g/L. Nannochlo ospis oceanica (N. oceanica, ba ch numbe
L202240131) and Te aselmis chui (T. chui, ba ch numbe L202130148)
we e g own au o ophically in econs i u ed saline wa e in ubula
pho obio eac o s in Allmic oalgae Na u al P oduc s S.A. (Pa aias,
Po ugal). The biomass was ha es ed by il a ion o ob ain a suspen-
sion o app oxima ely 60 g/L and he h ee mic oalgae pas es we e hen
pas eu ized and sp ay d ied.
Viscozyme® L was pu chased om Sigma-Ald ich Chemical Co. L d.
(S . Louis, MO, USA). All chemicals used we e analy ical g ade.
2.2. Mic oalgae cha ac e iza ion
Mic oalgae p o ein con en was es ima ed by quan i ica ion o o al
ni ogen a e sample acid diges ion using a Kjeldahl diges o (Foss
Analy ics, Hille oed, Denma k), applying he ni ogen con e sion ac o
(N ×4.78) [18]. Lipids we e de e mined using he Bligh & Dye me hod
wi h mino modi ica ions. Fo his, 1 mL mix u e chlo o o m/me hanol
(2:1, / ) was added o 50 mg mic oalgae biomass, and e-ex ac ion
cycles we e pe o med un il no pigmen a ion was de ec ed in he sol-
en . This s ep was ollowed by a e-ex ac ion in 2 mL chlo o o m and 1
mL me hanol, and 750
μ
L wa e we e added o p omo e phase sepa a-
ion and lipid ex ac ion. The o ganic phase was collec ed. The com-
bined phases we e d ied unde N
2
s eam and weighed. Each sample was
analyzed in iplica e, and he esul s we e p esen ed in pe cen age o
biomass d y weigh [19–21].
Fo he mois u e and ash con en de e mina ion 1 g algae biomass
was weighed in o ce amic c ucibles and d ied o e nigh a 105 ◦C. The
c ucibles we e allowed o cool o oom empe a u e in a desicca o and
weigh ed o calcula e he mois u e con en (%). Samples we e hen pu
in a mu le u nace (ECF 12/6, Len on, UK) a 575 ◦C o 16 h and
weighed (AOAC [22]).
Ca bohyd a e con en was assessed wi h he ollowing p ocedu e: 3
mg o each mic oalga was mixed wi h 3 mL 72 % H
2
SO
4
, and placed in o
a wa e ba h a 30 ±3 ◦C o 60 min. A e wa ds, each sample was
dilu ed o a 4 % H
2
SO
4
inal concen a ion and au ocla ed o 1 h a
121 ◦C. The samples we e analyzed by high-pe o med liquid ch oma-
og aphy wi h a e ac i e index de ec o using he ollowing pa ame-
e s: Aminex HPX-87H column a 60 ◦C; mobile phase o 0.05 M H
2
SO
4
a a 0.6 mL/min low a e. The quan i ica ion o monosaccha ides was
pe o med using a calib a ion cu e o each s anda d (glucose, galac-
ose, hamnose, a abinose, ucose, glucu onic acid and galac u onic
acid) a concen a ions be ween 0.0625 and 1.5 g/L [23]. A limi a ion o
HPX-87H column is he co-elu ion o xylose, mannose, and galac ose;
consequen ly, he esul s om composi ional analyses we e p esen ed as
he sum o hese h ee suga s (XMG) [24].
All expe imen s we e un in iplica e, and he esul s a e p esen ed
as pe cen ages.
2.3. Mic oalgae ea men s
2.3.1. F eeze/ hawing
A olume o 30 mL deionized wa e (dH
2
O) was in oduced o 3 g
mic oalgae. This mix u e unde wen a eezing phase (a −20 ◦C) las ing
90 min, ollowed by a hawing phase (a 20–25 ◦C) las ing 180 min. The
p elimina y assay encompassed a o al o 10 cycles, wi h he op imal
ou come eme ging a e 4 cycles (Table 8- supplemen a y ma e ial). All
samples we e hen cen i uged a 6000 ×g o 30 min, and he supe -
na an s we e eco e ed and lyophilized o u he use. All expe imen al
p ocedu es we e conduc ed in iplica e.
C. Mo ei a e al.
Algal Resea ch 86 (2025) 103958
2
2.3.2. High p essu e homogeniza ion
A quan i y o 200 g o powde ed o C. ulga is, T. chui o N. oceanica
a a a io o 1:10 (200 g / 2000 mL o dH
2
O) was pumped h ough he
HPH (Panda Plus 2000, GEA Ni o Soa i, Pa ma, I aly) a 1000 ±100
ba . The p ocess o homogeniza ion was pe o med in one cycle, and
only he i s s age o homogeniza ion was u ilized [25,26]. The em-
pe a u e was measu ed du ing all he ex ac ion (23 ±2 ◦C). A e
ea men , all solu ions we e cen i uged a 6000 ×g o 30 min and he
supe na an s we e eco e ed and lyophilized o u he use.
2.3.3. Enzyma ic ex ac ion
An enzyma ic me hod using Viscozyme® L (Sigma-Ald ich Chemical
Co. L d., S . Louis, MO, USA) was pe o med o e alua e i s impac on he
eco e y o p o eins om mic oalgae. Speci ically, 1.5 g algae we e
combined wi h 50 mL a 50 FPU enzyme solu ion in wa e a pH 4.5 and a
empe a u e o 50 ◦C, acco ding o he manu ac u e ’s op imum ec-
ommended condi ions o his enzyma ic cock ail. The ollowing incu-
ba ion imes we e es ed: 1, 2, 3, 4, 5, 6, 24, 48, 72 and 168 h, wi h he
op imal ou come eme ging a e 48 h. A e wa ds, all solu ions we e
cen i uged a 6000 ×g o 30 min and he supe na an s we e eco e ed
and lyophilized. Ex ac ions we e conduc ed in iplica e.
2.3.4. Ul asound-assis ed ex ac ion
An ul asound p obe (model c 334, Sonics, New own, USA) was
es ed o enhance p o ein ex ac ion. A olume o 120 mL dH
2
O was
added o 12 g algae and he ollowing exposu e imes we e es ed: 2, 5
and 10 min. The op imal ou come was ob ained a e 10 min. A e , all
solu ions we e cen i uged a 6000 ×g o 30 min and he supe na an s
we e eco e ed and lyophilized o u he use. Expe imen s we e con-
duc ed in iplica e.
2.3.5. pH adjus men
The mic oalgae s udied we e subjec ed o pH adjus men o s udy he
impac o pH on p o ein solubiliza ion o each species. Using he solid-
liquid a io o 1:10 ( inal olume o 50 mL), he p ocedu e was done
wi h dH
2
O, wi h concen a e solu ions o NaOH and HCl used o adjus
he pH o he a ge alue (7, 10 and 13). A e ha , he mic oalgae
suspensions we e s i ed o 1 h a 200 pm. A e , all solu ions we e
cen i uged a 6000 ×g o 30 min and he supe na an s we e eco e ed
and lyophilized o u he use. Expe imen al p ocedu es we e con-
duc ed in iplica e.
2.4. P o ein quan i ica ion
The p o ein con en was assessed h ough he quan i ica ion o o al
ni ogen a e sample acid diges ion using an au oma ic Kjeldahl
diges o uni (Foss Analy ics, Hille oed, Denma k), applying he ni o-
gen con e sion ac o o 4.78. All de e mina ions we e un in iplica e,
and esul s we e epo ed as pe cen age o p o ein [18,27].
B ad o d me hod was used o p elimina y in o ma ion ega ding he
F/T cycles. B ie ly, 180
μ
L ex ac and 20 mL B ad o d solu ion we e
mixed and incuba ed o 15 min. A s anda d cu e was made o BSA (0,
0.0625, 0.125, 0.25, 0.5 and 1 g/L) and abso bance was ead a 595 nm
[28].
2.5. Fou ie T ans o m In a ed Spec oscopy
The seconda y s uc u e in he lyophilized ex ac s was in es iga ed
wi h FTIR spec oscopy using an ALPHA II-B uke spec ome e
(E lingen, Ge many) wi h a diamond-composi e a enua ed o al
e lec ance (ATR) cell (FTIR-ATR). The FTIR spec a we e eco ded in
he 4000–400 cm
−1
ange by acqui ing 64 scan cycles pe sample wi h a
esolu ion o 4 cm
−1
. The maximum peaks we e assigned wi h peak
picking based on he second de i a i e as implemen ed in B uke OPUS
so wa e [29,30].
2.6. Molecula weigh
The p o ein’s molecula weigh (MW) dis ibu ion was assessed
h ough High-Pe o mance Liquid Ch oma og aphy (HPLC) gel pe me-
a ion ch oma og aphy (GPC), u ilizing a PolySep-GFC-P-4000 column
(300 ×7.8 mm, Phenomenex, USA). Lyophilized ex ac s we e dilu ed in
dH
2
O a a concen a ion o 1 mg/mL and hen subjec ed o elu ion wi h
ul apu e wa e , employing a low a e o 0.8 mL/min a a empe a u e
o 40 ◦C, wi h e ac i e index (RI) and ul a iole (UV) de ec ion
me hods. Linea eg ession calib a ion was execu ed using he s anda d
pullulan ki P-82 om ShodexTM, Japan, o e a ange spanning om
6.1 kDa o 337 kDa [31].
2.7. S a is ical analysis
Resul s we e p esen ed as mean ±s anda d de ia ion (SD) o a leas
h ee independen expe imen s. S a is ical analyses we e pe o med
using G aphPad P ism 9 (G aphPad So wa e, Inc., San Diego, CA).
Di e ences be ween samples we e es ed using - es , and he esul s
we e conside ed s a is ically di e en o alues o p <0.05.
3. Resul s
3.1. P oximal composi ion o mic oalgae biomass
The compa a i e p oximal (see Table 1) analysis demons a ed ha
T. chui exhibi ed a highe p o ein con en , whe eas C. ulga is demon-
s a ed a highe ca bohyd a e concen a ion, p ima ily a ibu ed o
glucan le els (see Table 2). Bo h N. oceanica and T. chui had compa able
lipid and ash le els.
3.2. P o ein ex ac ion me hods o p o ein eco e y
The p o ein ex ac ion e iciencies o h ee mic oalgae species we e
e alua ed using i e di e en p o ein ex ac ion echniques (Table 3). F/
T and HPH demons a ed he highes e icacy in p o ein ex ac ion,
while enzyma ic ea men wi h Viscozyme® esul ed in he lowes
p o ein yield. Applica ion o pH 7 du ing ex ac ion om C. ulga is and
N. oceanica did no yield s a is ically di e en esul s compa ed o UAE.
In N. oceanica, ex ac ion a pH 10 p oduced a p o ein yield equi alen
o ha o HPH. Ex ac ion unde alkaline pH led o he lowes yields in
T. chui, wi h pH 13 yielding he leas a o able esul s.
Ex ac s om C. ulga is ea ed by F/T, and pH a ia ion demon-
s a ed he highes p o ein con en , while hose ea ed wi h ENZ
exhibi ed he lowes . N. oceanica ex ac s wi h highe p o ein con en
we e F/T, pH 10, pH 13, and HPH. T. chui ex ac s wi h he highes
p o ein con en we e HPH, UAE, and pH 7, while ENZ, pH 10, and pH 13
ea men s esul ed in he lowes p o ein con en (Fig. 1) (See o e all
ex ac ion yield in Table 7- supplemen a y ma e ial).
3.3. Molecula weigh and seconda y s uc u e o ex ac ed p o ein
3.3.1. Chlo ella ulga is
The analysis o he MW o C. ulga is p o ein showed he p esence o
Table 1
Chemical composi ion o Chlo ella ulga is, Nannochlo opsis oceanica and Te a-
selmis chui biomass exp essed in g/100 g o biomass d y weigh . Values ep esen
he mean and co esponding s anda d de ia ion (n =3).
g/100 g d y weigh C. ulga is N. oceanica T. chui
P o ein 27.1 ±0.2 27.7 ±0.0 32.3 ±0.4
Ca bohyd a es 45.1 ±0.5 6.4 ±0.2 9.9 ±0.4
Lipids 12.2 ±0.1 21.9 ±1.1 22.8 ±0.9
Ashes 4.3 ±0.1 38.3 ±0.2 32.9 ±0.1
Mois u e 4.8 ±0.0 4.5 ±0.1 4.5 ±0.0
C. Mo ei a e al.
Algal Resea ch 86 (2025) 103958
3
agg ega es exceeding 337 kDa ac oss all p o ein ex ac ion echniques
employed. In F/T, HPH, ENZ, UAE, and pH 7 me hods, agg ega es
cons i u e o e 90 % o he p o ein composi ion. The p o ein ex ac ed a
pH 10 and pH 13 demons a ed ha ing he lowes MW (Tables 3–5).
The FTIR spec a o he mic oalgae ex ac s e ealed simila i ies
among he i e p o ein ex ac ion echniques wi h mino a ia ions in
he abso p ion in ensi y o speci ic cha ac e is ic peaks (Fig. 2A).
Pa icula ly o pH 7 and ENZ, highe in ensi ies we e obse ed be ween
1670 and 1600 cm
−1
(amine I) and 1400–1200 cm
−1
(amine III). The
second de i a i e spec a indica ed he p esence o he
α
-helix o m
solely in F/T and HPH ex ac s (Fig. 2B).
3.3.2. Nannochlo opsis oceanica
The analysis o he MW o N. oceanica p o ein showed a dis inc
singula peak o e e y echnique. F/T, and UAE exhibi ed MW lowe
han 6 kDa, while HPH, ENZ, pH 7, and pH 10 p esen ed a p o ein size o
15 kDa, and pH 13 showed MW o 50 kDa.
The FTIR spec a o he N. oceanica ex ac s displayed simila i ies
among he i e p o ein ex ac ion echniques, al hough in he amine I
Table 2
Ca bohyd a es composi ion o Chlo ella ulga is, Nannochlo opsis oceanica and
Te aselmis chui biomass exp essed in g/100 g o biomass d y weigh . Values
ep esen he mean and co esponding s anda d de ia ion (n =3).
g/100 g d y weigh C. ulga is N. oceanica T. chui
Glucose 36.1 ±0.7 1.9 ±0.0 5.1 ±0.2
A abinose 1.2 ±0.1 0.6 ±0.0 0.6 ±0.1
Rhamnose 1.2 ±0.1 0.6 ±0.0 0.6 ±0.1
XMG 3.1 ±0.1 1.2 ±0.0 2.4 ±0.1
Galac u onic acid 3.1 ±0.7 2.1 ±0.2 1.2 ±0.1
Table 3
P o ein yields (g
p o ein
/100 g
o al mic oalgae p o ein
) a e he p o ein ex ac ion echniques applica ion. Di e en le e s wi hin he same mic oalgae ep esen s a is ical
di e ences (
ρ
<0.05).
Mic oalgae F/T HPH ENZ UAE pH 7 pH 10 pH 13
C. ulga is 28.0 ±0.2
a
26.6 ±0.2
b
2.2 ±0.2
c
14.3 ±0.7
d
14.4 ±3.3
d,e
12.4 ±0.3
e
13.1 ±0.3
e
N. oceanica 34.6 ±0.2
31.5 ±0.2
g
9.3 ±1.8
h
29.0 ±1.3
i
30.6 ±0.1
i
32.0 ±0.7
g
23.6 ±0.6
j
T. chui 25.6 ±0.2
k
28.1 ±0.1
l
6.5 ±0.8
m
23.5 ±0.1
n
12.3 ±2.1
o
6.8 ±1.2
m
2.8 ±0.2
p
Fig. 1. P o ein con en in mic oalgae ex ac s (g p o ein/ 100 g mic oalgae lyophilized ex ac ) unde i e p o ein ex ac ion echniques (F eeze/Thawing (F/T),
High-P essu e Homogeneize (HPH), Enzyma ic ex ac ion (ENZ), Ul asound-Assis ed Ex ac ion (UAE), and pH adjus men (pH 7, pH 10, and pH 13). Di e en
le e s wi hin he same mic oalgae ep esen s a is ical di e ences (
ρ
<0.05).
Table 4
Molecula weigh ela i e p opo ions dis ibu ion o Chlo ella ulga is ex ac s
ob ained a e F/T, HPH, Enz, UAE, pH 7, pH 10 and pH 13.
kDa F/T HPH ENZ UAE pH 7 pH 10 pH 13
<6 3 % 2 % 4 % 5 % 28 % 24 % 23 %
50–20 –––– 16 % –
100–200 ––––7 % 6 % 15 %
>337 97 % 98 % 96 % 95 % 65 % 46 % 62 %
Table 5
Molecula weigh ela i e p opo ions dis ibu ion o Nannocho opsis oceanica
ex ac s ob ained a e F/T, HPH, ENZ, UAE, pH 7, pH 10 and pH 13.
kDa F/T HPH ENZ UAE pH 7 pH 10 pH 13
<6 69 % 18 % 24 % 21 % 20 % 26 % 29 %
10–20 –59 % 55 % 59 % 59 % 46 % –
20–50 – – – – – – 43 %
>337 31 % 23 % 21 % 20 % 21 % 28 % 28 %
C. Mo ei a e al.
Algal Resea ch 86 (2025) 103958
4
a ea HPH showed highe in ensi y han he o he ou samples, and ENZ
he lowes (Fig. 3A). The second de i a i e spec a analysis indica ed he
p esence o he same seconda y s uc u e ega dless o he echniques
selec ed (Fig. 3B).
3.3.3. Te aselmis chui
The MW analysis o he T. chui p o ein indica ed he p esence o
agg ega es exceeding 337 kDa ac oss all p o ein ex ac ion echniques
(15 %). The lowes MW was obse ed a pH 7 (<6 kDa), while highe
MW we e eco ded a pH 10 and pH 13 (50 kDa). >50 % o he p o ein
size is dis ibu ed be ween 15 and 50 kDa o all he p o ein ex ac ion
echniques u ilized.
The FTIR spec a o he T. chui ex ac s as p e iously men ioned o
C. ulga is, and N. oceanica, displayed simila i ies among he i e p o ein
ex ac ion echniques, al hough in he amine I a ea HPH showed he
lowes in ensi y and pH 13 he highes (Fig. 4A). In N. oceanica, he
second de i a i e spec a analysis indica ed he p esence o he same
seconda y s uc u e ega dless o he echniques selec ed (Fig. 4B).
4. Discussion
4.1. P oximal composi ion o mic oalgae biomass
N. oceanica and T. chui we e g own in ma ine au o ophic media.
Al hough he p o ein, ca bohyd a e and lipid con en a e gene ally
aligned wi h p e ious s udies o bo h mic oalgae [32–35] high a ia-
ions a e ound in li e a u e, depending, e.g., on he g ow h condi ions
o ha es ing me hods. [34] epo 22–37 % p o ein con en , 29–40 %
ca bohyd a es con en , 15–22 % lipids con en and 8–11 % ash o
Nannochlo opsis spp., depending on he esidence ime in he pho o-
bio eac o . Liu e al., 2015 also epo ed signi ican a ia ions in he
p o ein (24–30 %) and lipid (42–28 %) con en depending on he g ow h
Fig. 2. A) FTIR spec a o Chlo ella ulga is ex ac s (4000–400 cm
−1
) B) FTIR de i a i e spec a o Chlo ella ulga is ex ac s (1710–1590 cm
−1
) unde F/T, HPH,
ENZ, UAE, pH 7, pH 10 and pH 13 ex ac s om Chlo ella ulga is. Wa enumbe s o he FTIR spec a we e de e mined wi h a peak-picking ool based on he second
de i a i e o hese cu es. The e ical lines a ibu e anges o wa enumbe s o speci ic seconda y s uc u es o p o eins.
C. Mo ei a e al.
Algal Resea ch 86 (2025) 103958
5
medium composi ion (ni ogen deple ed o eple ed), o N. oceanica.
Ne e heless, he ash con en p esen in N. oceanica and T. chui we e
highe han he ones p esen on he li e a u e. Howe e , sal con en in
he inal biomass can be s ongly in luenced by he ha es ing me hods.
Fo ins ance, Das e al. [32] ha e epo ed ash con en s o 19 % and 30 %
o Te aselmis sp. ha es ed by c oss- low il a ion o by elec o-
coagula ion, espec i ely. The biomass u ilized in his s udy was p o-
cu ed om a la ge-scale supplie based in Eu ope. The mic oalgal
species N. oceanica and T. chui we e ha es ed h ough a il a ion p o-
cess, ollowed by pas eu iza ion and sp ay d ying. In an indus ial
con ex , i is no easible o achie e comple e emo al o he g ow h
medium. Speci ically, N. oceanica and T. chui a e cul i a ed in ma ine
wa e wi h a salini y o 30 o 40 g/L. Following il a ion, he concen-
a ion o mic oalgae is an icipa ed o ise om 1 o 2 g/L o 60 g/L,
esul ing in a subs an ial inc ease in he a io o mic oalgae o he e-
sidual sal in he medium. Howe e , due o he inhe en cha ac e is ics
o he p ocessing, i is expec ed ha app oxima ely 30 % o he inal
p oduc will consis o ash, which includes sal and a ious mic o-
nu ien s, a e he sp ay-d ying s age. This phenomenon may accoun
o he obse ed highe ash con en in he mic oalgae powde compa ed
o exis ing li e a u e. In a s udy p esen ed by Magpusao e al. [36]
simila ash con en was ound ela ed o he biomass being esuspended
in bu e sal solu ion be o e o packing o deli e y as a we pas e [36].
The C. ulga is composi ion is simila o he one p esen in he
Fig. 3. A) FTIR spec a o Nannochlo opsis oceanica ex ac s (4000–400 cm
−1
) B) FTIR de i a i e spec a (1710–1590 cm
−1
) o F/T, HPH, ENZ, UAE, pH 7, pH 10 and
pH 13 ex ac s om Nannochlo opsis oceanica. Wa enumbe s o he FTIR spec a we e de e mined wi h a peak-picking ool based on he second de i a i e o hese
cu es. The e ical lines a ibu e anges o wa enumbe s o speci ic seconda y s uc u es o p o eins.
C. Mo ei a e al.
Algal Resea ch 86 (2025) 103958
6
li e a u e o s ain g ow h in a media wi h glucose [37,38].
The p o ein con en was a ound 30 % o all he s udied mic oalgae
(Table 1). P e ious s udies has been epo ed a simila composi ion in
e ms o p o eins wi h C. ulga is showing a con en o 39.4 %,
N. oceanica o 35.1 % and T. chui 31.1 % [7]. Mic oalgae’s p o ein
con en is highe han mos ege ables and pulses (e.g., 10 % - 22 % o
peas o beans), making hem a ac i e as a ood p o ein sou ce [4].
Some di e ences may be pa ly explained by geog aphical and sea-
sonal a ia ion, as well as by me hodological di e ences, g ow h con-
di ions, and quan i ica ion me hodologies [39,40].
4.2. P o ein eco e y
Fo C. ulga is, he highe ex ac ion yields we e ob ained unde F/T
and HPH me hodologies ye , HPH besides being as e han F/T and also
a mo e sus ainable p ocess. UAE did no signi ican ly imp o e he p o-
ein ex ac ion yield, since he inal alue was simila o an ex ac ion a
pH 7 (Table 3).
In T. chui’s case, HPH showed he bes p o ein ex ac ion yield, wi h
28 % o he o al p o ein being solubilized, ollowed by F/T wi h 25 %
and UAE wi h 23 %. This shows ha F/T wo ks almos as well as HPH
and UAE o p o ein ex ac ion o T. chui.
In N. oceanica, and unlike in C. ulga is and T. chui, he e appea s o
be no ad an age in using F/T, HPH, UAE, o pH 10 since he gains a e
Fig. 4. A) FTIR spec a o Te aselmis chui ex ac s (4000–400 cm
−1
) B) FTIR de i a i e spec a (1710–1590 cm
−1
) o F/T, HPH, ENZ, UAE, pH 7, pH 10 and pH 13
ex ac s om Te aselmis chui. Wa enumbe s o he FTIR spec a we e de e mined wi h a peak-picking ool based on he second de i a i e o hese cu es. The
e ical lines a ibu e anges o wa enumbe s o speci ic seconda y s uc u es o p o eins.
C. Mo ei a e al.
Algal Resea ch 86 (2025) 103958
7
ma ginal compa ed o simple ex ac ion o solubiliza ion a pH 7. The
only ins ance whe e a signi ican inc ease was obse ed was F/T, wi h
an inc ease o app oxima ely 13 %. Ne e heless, N. oceanica s ands ou
as he algae o which he p o ein ex ac ion yield was highe , p obably
due o he g ea e solubili y o i s p o eins.
Subjec ing he we biomass o eezing, ini ia es he c ys alliza ion o
in acellula wa e , and subsequen hawing p omp s he expansion o
hese c ys als, esul ing in he up u e o cellula s uc u es [41].
Al hough F/T me hods ha e adi ionally been used o hei simplici y,
hey a e labou -in ensi e, ime-consuming, and ene gy-in ensi e p o-
cesses a scale, making hem un iable o mos applica ions [13].
The ENZ ex ac ion had he lowe yields o all algae es ed, wi h a
maximum o 9 % o he mic oalgae p o ein being solubilized o
N. oceanica, 6 % o T. chui, and 2 % o C. ulga is. Ca bohyd ases, like
he ones p esen in Viscozyme® L, a e in ol ed in he disin eg a ion o
cell wall issue, acili a ing p o ein ex ac ion. The dec ease in iscosi y
caused by he deg ada ion o β-glucan was expec ed o help in he sol-
ubiliza ion and ex ac ion o p o ein. Mic oalgae, like Nannochlo opsis is
a sou ce o β-glucan, e en hough hey a e no he main cons i uen o
hei cell wall [42]. Enzyma ic ea men s we e mos e ec i e when
used as a p e- ea men o enhance p o ein ex ac ion. I concluded ha
he use o ca bohyd ases was bene icial in imp o ing he ex ac ion
yields [43]. This educed ex ac ion yield could be asc ibed o p o ein
dena u a ion (and consequen ly insolubiliza ion) due o he long pe iod
(48 h) a a mode a e empe a u e (50 ◦C), causing he p o ein o emain
in he solid phase. This could be a s a egy i he in en ion was o
concen a e he p o ein in he solid phase, while ob aining a liquid
ex ac ich in o he compounds (e.g. ca bohyd a es). Also, a s udy
pe o med by Sa i e al. [44] showed ha 35 % o he p o ein om
N. gadi ana was eleased using he ENZ app oach, a conside ably highe
alue han ou esul s. The au ho s obse ed ha he use o p o eases
was no enough o weaken he in eg i y o he cell wall, so low
cy ome y did no de ec b oken cells. These esul s can also be obse ed
in ou wo k o all he mic oalgae s udied. In addi ion, he use o en-
zymes has esul ed in he hyd olysis o he p o eins loca ed in he cell
wall o N. gadi ana, which implies ha he emaining componen s o he
cell wall emained in ac [44].
Alkaline ea men s cons i u e a widely employed non-mechanical
p ocedu e o ex ac ing and solubilising p o eins om mic oalgae.
Usually, his ype o ea men signi ican ly enhances he yield o
ex ac ed p o eins. Sodium hyd oxide is expec ed o induce he hyd o-
lysis o es e and hyd ogen bonds p esen among a ious componen s
(polysaccha ides and non-polysaccha ides) wi hin mic oalgae cell walls
[45]. N. oceanica p o ein ex ac ion was highe be ween pH 7 and 10. As
he pH inc eased, he solubili y d opped sligh ly o an a e age o 27 %
be ween pH 10–13. A pH in he ange o 5.5–11 has li le impac on he
solubili y and ex ac ion yield o p o eins om N. oceanica [46]. The
same was obse ed o C. ulga is, whe e he esul s did no show s a-
is ical di e ences in p o ein ex ac ion be ween pH 7 and 13. Highe
solubili y o p o eins in alkaline media was a ibu ed o he ne elec-
ical cha ges o he p o eins [47]. T. chui had a highe p o ein solubili y
a pH 7. Howe e , wi h an inc ease in pH, he ex ac ion e iciency
signi ican ly dec eased, eaching 46 % and 78 % a pH 10 and pH 13,
espec i ely, compa ed o pH 7 [47]. Thus, i seems be e o keep he
ini ial pH a 7 o bo h mic oalgae, educing p ocess ime and chemicals.
[48] s udied he in luence o he pH in p o ein solubiliza ion in Nan-
nochlo opsis spp, and obse ed ha a pH 13 he p o ein eco e y was
highe (be ween 10 and 12 %), al hough conside ably low compa ed o
he esul s p esen ed in ou wo k [48].
The UAE and HPH echniques ha e eme ged as inno a i e “clean
echnology” me hods ha ha e ga ne ed inc eased a en ion in ecen
yea s. They o e se e al ad an ages, including minimal sol en usage,
limi ed equipmen equi emen s, and low economic and en i onmen al
impac s. UAE employs ul asonic wa es o induce he o ma ion,
g ow h, and subsequen collapse o bubbles wi hin he wa e , acili-
a ing he elease o in acellula componen s. HPH o ces he passage o
a luid h ough a al e wi h na ow s il s, esul ing in a subs an ial in-
c ease in p essu e, and hen s iking an impac ing [41]. The esul ing
mechanical e ec s, such as u bulence, shea s ess, and ca i a ion, lead
o he ex ac ion o cellula s uc u es. HPH usually has a p onounced
impac on he mic os uc u al p ope ies o a ious mic oalgae sus-
pensions [25]. In a p e ious s udy pe o med wi h Nannochlo opsis he
p o ein ex ac ion yield using HPH was 50 % o he o al p o ein.
Howe e , his da a is no di ec ly compa able wi h he da a om he
p esen wo k, as he e is no in o ma ion abou he quan i y o he p o-
ein p esen in he ex ac . The au ho s also jus i y ha he elease o all
p o eins was no achie ed due o he insoluble na u e o some p o eins
ha emained in he pelle [44]. A ecen s udy by Rida e al. [49] on
T. suecica indica ed a o al p o ein con en in he ex ac ollowing HPH
a 300 ba s o 26.1 ±0.3 % d y weigh [49]. This inding aligns closely
wi h he concen a ions ob ained in he p esen esea ch. Howe e , he
p o ein ex ac ion yields epo ed we e signi ican ly highe (61.9 ±3.9
%). No ably, ano he in es iga ion ca ied ou by Del an e al. [25]
achie ed an ex ac ion yield o 80 % o o al p o ein a an HPH o 400
ba s, whe eas ou s udy eco ded only 28.1 % [25]. I is impo an o
no e ha Del an’s wo k implemen ed a desal ing s ep o he ini ial
biomass, which po en ially enhanced p o ein ex ac ion e iciency.
Fu he mo e, in he Rida’s wo k hey u ilized he ul a il a ion pos -
cen i uga ion s ep, a me hod ha e ec i ely elimina ed mos washed
mine als along wi h ce ain los ex acellula componen s, including
smalle p o eins and ca bohyd a es. The obse ed di e ences in
ex ac ion yields may also be a ibu able o he a ia ions in he e i-
ciency o he HPH equipmen employed and se ing condi ions. Also,
Sa i e al. [51] iden i ied he empe a u e ac o as a signi ican in luence
on ex ac ion p ocesses, demons a ing ha ex ac ions conduc ed a
46 ◦C yielded highe esul s han hose pe o med a 25 ◦C. This
enhancemen is a ibu ed o he abili y o ele a ed empe a u es o
solubilize a ious componen s, pa icula ly s a ch and p o ein [51].
The esul ing aqueous ex ac s exhibi p o ein concen a ions
consis en wi h he a o emen ioned ex ac ion yields, i.e., in he case o
C. ulga is, all ex ac s ha e a p o ein con en be ween 19 and 23 %,
excep o enzyma ic ex ac ion, hus con i ming ha o he compounds
may ha e been p e e en ially ex ac ed when Viscozyme® L was used,
and mo e p o ein emained unex ac ed in he solid phase. In he
N. oceanica and T. chui ex ac s, he same end was obse ed, and e en
wi h enzyma ic ex ac ion, a high pe cen age o p o ein was ob ained
(Fig. 1).
Despi e he low p o ein ex ac ion yield a pH 10 and 13 o
C. ulga is, he ex ac has a p o ein con en simila o F/T. This could be
a ibu ed o a educ ion in he lipid con en o he mic oalgae due o he
saponi ica ion p ocess unde alkaline ea men s [52].
C. ulga is and N. oceanica, while achie ing accep able p o ein
ex ac ion yields h ough he HPH p ocess, exhibi ed a lowe le el o
speci ici y in hei p o ein ex ac ing, leading o ex ac s wi h sligh ly
educed p o ein pe cen age. This de ia ion in p o ein con en is likely
a ibu ed o he HPH p ocess’s lowe speci ici y in e ms o solubiliza-
ion. I is p obable ha o he componen s, such as lipids, ca bohyd a es,
and ashes, we e solubilized o a g ea e ex en alongside p o eins.
This s udy ep esen s he i s a emp o compa e he pe o mance o
h ee dis inc mic oalgae a ian s cha ac e ized by di e ing cell wall
composi ions ega ding hei suscep ibili y o a ious p o ein ex ac ion
echniques o p o ein eco e y. This ma ks he pionee ing in es iga ion
in o he s uc u al analysis o he eco e ed p o ein subsequen o
applica ion o di e se eco e y echnologies. Al hough, he maximum
p o ein ex ac ion yield each has been 28 %, 35 %, and 28 % o
C. ulga is, N. oceanica, and T. chui, espec i ely using dH
2
O as a sol en
a 25 ◦C, he esul s a e in line wi h p e ious s udies. Fo ins ance,
employing mild p o ein ex ac ion echniques esul ed in p o ein yields
o 28–43 % o C. ulga is and N. ocula e [50], and 7–12 % o
T. impellucida and suecica [53,54].
The pu pose o enhanced p o ein yield h ough a ious echnological
me hods is me wi h se e al limi ing ac o s. The ex ac ion o p o eins
C. Mo ei a e al.
Algal Resea ch 86 (2025) 103958
8
om mic oalgae is impeded by he p esence o a physical ba ie c ea ed
by he cell wall, hinde ing he elease o p o eins [55]. Also, ce ain
p o eins a e bound o o he molecules h ough ionic in e ac ions, spe-
ci ically wi h polysaccha ides, impeding hei ex ac ion [4]. The high
iscosi y and ionic in e ac ions esul ing om he p esence o cell walls
and in acellula polysaccha ides u he add complexi y o he ex ac-
ion p ocess also, he use o alkaline sol en s has acili a ed inc eased
p o ein ex ac ion om selec mac oalgae species [56]. The combina-
ion o cell-dis up ing echniques, such as HPH and pH 13, may hold
po en ial o inc easing p o ein yield. Howe e , depending on he
applica ion, di e en me hods can be conside ed. Fu he wo k is
needed o s udy he p o ein ex ac om mic oalgae ob ained by
di e en cell-dis up ing echniques and i s impac on he p o ein
s uc u e and unc ionali y.
4.3. Molecula weigh and seconda y s uc u e o ex ac ed p o ein
4.3.1. Chlo ella ulga is
The ch oma og ams o C. ulga is e ealing he p esence o agg e-
ga es ac oss all ex ac ion me hods (>337 kDa) (Table 3 and Fig. 5-
supplemen a y ma e ial). The ch oma og ams a pH 10 and pH 13
highligh he p esence o MW below 120 kDa. This disc epancy MW
dis ibu ion sugges s ha he alkaline condi ions a pH 10 and 13 may
induce a speci ic b eakdown o al e a ion in he molecula s uc u e o
p o eins, esul ing in lowe MW species. U su e al. [52] ha e also e-
po ed an highe elu ed peak co esponding o MW highe han 670 kDa
and lowe peaks (<60 kDa) when exposed o alkaline pH. The p esence
o highe MW has been iden i ied as he signa u e o complex soluble
agg ega es o p o eins and chlo ophyll, ein o cing his conclusion by
he g een colou o ex ac s ye as also epo ed in his s udy, alkaline
ex ac ion modi ies he MW p o ile compa ed o o he s ex ac ion
echniques showing MW below 60 kDa [52].
FTIR spec oscopy measu es he abso p ion o IR adia ion by a
sample and p o ides da a on he wa eleng h and in ensi y o he ab-
so p ion. High polyme s’ IR spec al da a can be unde s ood in e ms o
he ib a ions o a s uc u al epea uni . The amide I band (1700–1600
cm
−1
) is he mos sensi i e spec al egion o p o ein seconda y s uc-
u al componen s, mainly ep esen ing he C
–
–
O s e ch ib a ions o
he pep ide linkages (abou 80 %). The equencies o he amide I band
componen s closely co ela e wi h each seconda y s uc u al elemen o
he p o eins. On he o he hand, he amide II band mainly o igina es
om in-plane NH bending (40–60 %) and he CN s e ching ib a ion
(18–40 %). The seconda y s uc u al composi ion is based on he
assump ion ha any p o ein can be conside ed as a linea sum o a ew
undamen al seconda y s uc u al elemen s. Co ela ions be ween IR
spec a and p o ein seconda y s uc u es ha e been es ablished, and
assignmen s o amide I band componen s o p o ein seconda y s uc u e
elemen s (such as
α
-helix, β-shee , β- u n, and andom s uc u es) a e
a ailable o p o eins in bo h H
2
O and D
2
O media [29].
A compa ison o he FTIR spec a o he C. ulga is lyophilized ex ac
(Fig. 2) showed ha only p o ein om F/T me hodology was sligh ly
shi ed o lowe wa enumbe s, indica ing an inc eased o de in sec-
onda y p o ein s uc u e, namely
α
-helix (1656 ±2.0 cm
−1
). β-Shee
s uc u es esul in cha ac e is ics wa enumbe s in he egions 1696 ±
2.0 cm
−1
o 1667 ±1.0 cm
−1
and 1642 ±1.0 cm
−1
o 1624 ±1.0 cm
−1
[30]. The seconda y s uc u e o p o eins is in luenced by he speci ic
sequence o amino acids and he in e ac ions be ween molecules. P o-
ein ex ac ion echniques induce changes in he seconda y s uc u e by
dis up ing hese in e ac ions [29]. In HPH, UAE, and alkaline pH p o-
cesses, p o ein molecules un old, leading o he dis up ion o
α
-helices
and hei con e sion in o β-shee s. Sonica ion can induced he o ma ion
o agg ega es, subsequen ly causing a dec ease in
α
-helix con en and an
inc ease in β-shee con en [57]. S ong abso p ion signals obse ed a
1110, 1118 and 1113 cm
−1
a e a ibu ed o he C–O–C band, indica ing
he p esence o polysaccha ides, which can suppo he s a emen ha
p o ein could be agg ega ed due o hei p esence [58].
4.3.2. Nannochlo opsis oceanica
Va ious condi ions such as HPH, ENZ, pH 7, and pH 10 exhibi ed
dis inc peaks, indica ing he p esence o p o eins wi h MW anging
be ween 10 and 20 kDa (Fig. 6- supplemen a y ma e ial). Con e sely, a
pH 13, p o eins appea ed o ha e MW be ween 20 and 50 kDa, wi h
lowe MW p o eins obse ed unde F/T and UAE echniques (5–10 kDa,
and <5 kDa, espec i ely) (Table 5). The use o sodium hyd oxide may
ha e led o he solubiliza ion o insoluble p o eins in wa e , esul ing in
highe MW p o eins. Alkaline condi ions a e an icipa ed o ha e a hy-
d oly ic e ec on he biomass, which may ha e libe a ed di e en p o-
eins om he mic oalgae s uc u e. This was likely due o ende ing
hese p o eins accessible o he sol en and sligh ly hyd olyzing hem,
hus enabling hei solubiliza ion [59].
Abso bance peaks we e obse ed in he ange o 3200–3500 cm
−1
,
indica ing he p esence o he OH g oup in cellulose which was he
p edominan ca bohyd a e ound in he Nannochlo opsis [60]. Ano he
peak a 1049 cm
−1
was a ibu ed o he C-O-C band, sugges ing he
p esence o polysaccha ides wi hin he 950–1200 cm
−1
ange.
The analysis o he second de i a i e o he abso p ion spec a
(Fig. 3) wi hin he ange o 1600–1700 cm
−1
indica ed ha he p e-
dominan componen s o he spec a we e consis en ly p esen in all
complexes. The samples exhibi ed an abso p ion peak a 1627–1642
cm
−1
, which was consis en ly a ibu ed o C
–
–
O s e ching (amide I)
and associa ed wi h he p o ein β-shee s s uc u e (1623–1641 cm
−1
).
This obse a ion aligns wi h he seconda y s uc u e composi ion. This
obse a ion sugges s ha he p o ein’s seconda y s uc u e emains
la gely unal e ed, con i ming he p ese a ion o he same s uc u al
cha ac e is ics in all se en ex ac s [61].
4.3.3. Te asemis chui
The T. chui p o ein analysis, simila o N. oceanica, showed a dis inc
peak ac oss a ious condi ions: F/T (10–50 kDa), HPH (10–20 kDa),
ENZ (5–20 kDa), UAE (5–20 kDa), pH 7 (5–20 kDa), pH 10 (10–50 kDa),
and pH 13 (10–50 kDa) (Table 6 and Fig. 7- supplemen a y ma e ial),
wi h smalle agg ega es p esen in all samples s udied, exceeding 337
kDa. The p esence o low MW p o eins in Te aselmis sp. Sa i e al.
s udied he ex ac ion o p o ein om Te aselmis suecica and ound he
p esence o p o eins wi h MW be ween 10 and 60 kDa o HPH aqueous
ex ac ion, which ma ches he indings o his wo k [51]. The esul s
showed ha he FTIR spec a o T. chui lyophilized ex ac s (Fig. 4) we e
e y simila , wi h small di e ences in he abso p ion in ensi y o some
cha ac e is ic peaks. I indica ed ha he seconda y s uc u e could be
he same, wi h a comple e absence o
α
-helix. The s ong abso p ion
peaks a e assigned o a C
–
–
O s e ching (amide I) and co espond o
β-shee s uc u es [58]. The peaks be ween p o ein ex ac ion ech-
niques end o ollow he same endency, which is suppo ed by he
unchanged na u e o he seconda y s uc u e.
The h ee mic oalgae species p esen ed a highe p opo ion o
β-shee s and β- u n s uc u es, sugges ing a mo e in lexible and olded
globulin con igu a ion. This s uc u al a ibu e, p ima ily associa ed
wi h he p e alence o β- u ns, in oduces cons ain s on he con o -
ma ional en opy o he pep ide chain [62]. The e o e, his heigh ened
igidi y may con ibu e o he compa a i ely lowe solubili y o mic o-
algae p o eins compa ed o p o eins om al e na i e plan sou ces.
Howe e , he p ecise mechanis ic implica ions o hese s uc u al
Table 6
Molecula weigh ela i e p opo ions dis ibu ion o T. chui ex ac s ob ained
a e F/T, HPH, ENZ, UAE, pH 7, pH 10 and pH 13 and ela i e p opo ions.
kDa F/T HPH ENZ UAE pH 7 pH 10 pH 13
<6 26 % 24 % 21 % 24 % 33 % 20 % 27 %
10–20 3 % ––6 % –6 % –
20–30 37 % 47 % 58 % 54 % 41 % 7 % 6 %
50 ––– 37 % 35 %
>337 34 % 29 % 21 % 16 % 26 % 30 % 32 %
C. Mo ei a e al.
Algal Resea ch 86 (2025) 103958
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