Cha ac e iza ion o Rugulop e yx okamu ae algae: A sou ce o bioac i e
pep ides, omega-3 a y acids, and ola ile compounds
Fe nando Ri e o-Pino
a,b
, Te esa Gonzalez-de la Rosa
a,b
, Ma ia To ecillas-Lopez
a,b
,
Luna Ba e a-Chamo o
a,b
, Jose Luis del Rio-Vazquez
a
, El i a Ma quez-Pa adas
a,b
,
A ica Fe nandez-P io
a,b
, Ma co Ga cia-Vaque o
c
, Jose Ca los Ga cia-Gomez
d
,
Se gio Mon se a -de la Paz
a,b,*
, Ca men Ma ia Cla o-Cala
b,e
a
Depa men o Medical Biochemis y, Molecula Biology, and Immunology, School o Medicine, Uni e si y o Se ille, Spain
b
Ins i u o de Biomedicina de Se illa, IBiS/Hospi al Uni e si a io Vi gen del Rocio/CSIC/Uni e sidad de Se illa, Se ille 41013, Spain
c
School o Ag icul u e and Food Science, Uni e si y College Dublin, Bel ield, D04V1W8 Dublin, I eland
d
Depa men o Zoology, Facul y o Biology, Uni e si y o Se ille, Spain
e
Depa men o Pha macology, Pedia ics, and Radiology, School o Medicine, Uni e si y o Se ille, Spain
ARTICLE INFO
Keywo ds:
Algae
Bioac i e pep ides
Molecula docking
Vi amin A
ABSTRACT
This s udy p o ides a de ailed cha ac e iza ion o he in asi e algae Rugulop e yx okamu ae, highligh ing i s
nu i ional composi ion, mine al con en , and po en ial bioac i e compounds. This biomass con ains 14.18 %
p o ein, 21.29 % lipids (wi h a high omega-3 con en ), ib e (31.32 %), and signi ican amoun s o mine als like
calcium, sodium, po assium, sulphu , and i on. Phenolic compounds (0.74 %) and ola ile compounds, such as
e inol, we e also iden i ied. Pep idome analysis e ealed 626 unique pep ides, wi h 21 low molecula weigh
pep ides showing po en ial ac i i y agains angio ensin con e ing enzyme and dipep idyl pep idase IV when
assessed using in silico ools and using molecula docking. Addi ionally, he an ioxidan capaci y o he alga was
demons a ed wi h a signi ican ee adical inhibi ion (EC
50
: 2.09 mg/mL). O e all, his s udy p o ides ini ial
e idence on he nu i ional po en ial o R. okamu ae, which may ha e po en ial o u u e applica ions in ood
and bio echnology ields.
1. In oduc ion
The in asion o Rugulop e yx okamu ae (R. okamu ae), a b own
mac oalgae species, in o Eu opean wa e s and he S ai o Gib al a
p esen s a p essing conce n (Ma le a e al., 2024). Howe e , consid-
e ing he huge amoun o his algae which is being collec ed, and wi h
he pu pose o adding new esea ch lines aligned wi h he new policies
o p omo ing an en i onmen ally iendly ood sys em, ecen esea ch
is ocusing on cha ac e iza ion o his mul icellula algae, which could
be employed as sou ce o mac o- and mic onu ien s. The e lies a po-
en ial oppo uni y o i s u iliza ion as a aluable ood and eed
esou ce (Kammle e al., 2024). E o s o ha ness i s biomass o hese
pu poses could no only mi iga e he ecological h ea s posed by i s
p esence bu also con ibu e o add essing ood secu i y conce ns and
educing dependency on adi ional sou ces o p o ein (Ba cellos e al.,
2023; Go dalina e al., 2021).
I mus be no ed ha he composi ion o b own algae depends on
a ious ac o s, including en i onmen al condi ions, such as wa e
empe a u e, ligh in ensi y, nu ien a ailabili y, and salini y. On op o
ha , gene ic ac o s and he species-speci ic me abolic pa hways o
b own algae can in luence hei biochemical composi ion. Algae syn-
hesize a di e se a ay o compounds including polysaccha ides (such as
algina e, lamina in, and ucoidan), polyphenols, pigmen s (like uco-
xan hin), p o eins, and lipids. The ela i e p opo ions o hese con-
s i uen s can a y signi ican ly be ween di e en species o b own algae
and e en wi hin he same species unde di e en en i onmen al con-
di ions, including season and place o g ow h, as pe o he ma ine o -
ganisms (Deepika e al., 2022; Go dalina e al., 2021; Mo ales-Medina
e al., 2016). The po en ial o explo ing new species o e s a huge op-
po uni y o disco e no el compounds—s uc u es ne e iden i ied
* Co esponding au ho a : Depa men o Medical Biochemis y, Molecula Biology, and Immunology. School o Medicine, Uni e sidad de Se illa, A . D . Fed iani
s/n, 41009 Se ille, Spain.
E-mail add ess: [email p o ec ed] (S. Mon se a -de la Paz).
Con en s lis s a ailable a ScienceDi ec
Food Chemis y
jou nal homepage: www.else ie .com/loca e/ oodchem
h ps://doi.o g/10.1016/j. oodchem.2025.143084
Recei ed 20 Decembe 2024; Recei ed in e ised o m 14 Janua y 2025; Accep ed 24 Janua y 2025
Food Chemis y 473 (2025) 143084
A ailable online 25 Janua y 2025
0308-8146/© 2025 The Au ho s. Published by Else ie L d. This is an open access a icle unde he CC BY license ( h p://c ea i ecommons.o g/licenses/by/4.0/ ).
be o e—wi h po en ial applica ions ac oss a ious indus ies, i biolog-
ical o echno- unc ional ac i i ies a e demons a ed.
The po en ial o R. okamu ae in ood applica ions has been sca cely
explo ed, highligh ing he po en ial o e penes, educing suga s, pig-
men s, ib es, wi h po en ial an i-in lamma o y,
α
-glucosidase inhibi o y
o an ibac e ial ac i i y, among o he s, bu mos ly h ough in i o
s udies. On he o he hand, i has been epo ed he high oxici y o his
species a ibu ed o hei high con en o sesqui e penes, no ound on
o he algae (Ba cellos e al., 2023). I in ended o be used o human
consump ion, a p ope isk assessmen should be pe o med, ensu ing
ha no sa e y conce ns a ise om i s consump ion.
U ilizing in asi e mac oalgae o p oducing high- alue p oduc s
p esen s a p omising s a egy o mi iga e he nega i e en i onmen al
e ec s o hese species. Mac oalgae o e dis inc economic ad an ages
as hey do no equi e eshwa e o a able land o cul i a ion, hus
a oiding compe i ion wi h con en ional ood sou ces (Agus ín e al.,
2023). Howe e , de e mina ion o how season and place o g ow h o
he ma ine o ganisms is a ec ing i s composi ion is s ill in a e y ea ly
s age o R. okamu ae, as sca ce li e a u e is a ailable. In addi ion, he
p ocessing o he samples collec ed highly a ec s he p oduc s in he
ex ac s (De la Lama-Cal en e e al., 2024). The con en o speci ic
pep ides o phenolic compounds, in o he sou ces, has been associa ed
wi h an ioxidan and an i-in lamma o y p ope ies, due o he s uc u e
o hese molecules. In he case o R. okamu ae, o he au ho s’ knowl-
edge, li le has been desc ibed abou he bioac i e po en ial o i s
compounds.
This wo k p o ides a comple e cha ac e iza ion o eeze-d ied
R. okamu ae collec ed om he coas o Spain du ing Sp ing 2024. The
cha ac e iza ion o he biomass included mac onu ien s, a y acids
p o ile, phenolic and ola ile compounds, amino acids, and mine al
con en s. The in i o an ioxidan ac i i y and ul as uc u al cha ac-
e iza ion was also pe o med oge he wi h pep idome iden i ica ion, in
silico analyses and molecula docking o he iden i ied bioac i e
pep ides.
2. Ma e ials and me hods
2.1. Biomass and chemical eagen s
F esh R. okamu ae was collec ed in Ta i a (Cadiz, Spain) on he 11 h
Ap il 2024. The samples we e s o ed (−20 ◦C) and eeze-d ied (C yodos
−80 ◦C, Tels a ), esul ing in yields o d ied biomass o 18.10 %. The
biomass was milled using a comme cial cu e mill (HR-2172/00; Phi-
lips, he Ne he lands), and s o ed in polyp opylene ubes unde e ig-
e a ion (4 ◦C) o u he analyses.
2.2. Chemical cha ac e iza ion
All chemical measu emen s we e pe o med in iplica e excep in
he case o o al phenolic con en ha we e pe o med in quad uplica e.
2.2.1. Mac onu ien composi ion
P o ein concen a ion was analysed by elemen al mic oanalysis,
using a LECO TRUSPEC MICRO analyse (Leco Co po a ion, S . Joseph,
MI, USA), mul iplying he ni ogen con en by a con e sion ac o o 5
(Ceb i´
an-Llo e e al., 2024). The a con en o he samples was de e -
mined a e ex ac ion wi h a sol en mix u e hexane/isop opanol (1:1;
: ) (D usch e al., 2012). To al die a y ib e (p edominan ly hemicel-
lulose, cellulose, and lignin) was de e mined by diges ion in a de e gen
solu ion wi h a neu al de e gen solu ion, hea -s able bac e ial
α
-amylase, and sodium sul i e using ANKOM
2000
echnology simila ly o
Coblen z e al. (2019). Mois u e and ash we e calcula ed ollowing he
UNE-EN14774–3 and UNE-EN14775 me hods.
2.2.2. De e mina ion o o al amino acids
To al amino acids (TAA) we e de e mined as desc ibed by Gonzalez-
de la Rosa e al. (2024). Samples we e hyd olysed wi h 6 N HCl (110 ◦C,
one day). The analyses we e pe o med using a The mo Scien i ic liquid
ch oma og aphy sys em consis ing o a bina y UHPLC Dionex Ul ima e
3000 RS, connec ed o a quad upole-O bi ap QExac i e hyb id mass
spec ome e (The moFishe Scien i ic, USA) wi h hea ed elec osp ay
ioniza ion (HESI) p obe. Sepa a ion was ca ied ou using an Acqui y
UPLC BEH Amide column (1.7
μ
m pa icle size, 100 ×2.1 mm) (Wa e s)
a 35 ◦C a a low a e o 0.4 mL/min. A bina y g adien , consis ing o (A)
ACN:H2O (85:15, / ) and (B) H2O bo h con aining 10 mM ammonium
o ma e and 0.15 % o mic acid, was used wi h he ollowing elu ion
p o ile: 0 % B (6 min), linea g adien om 5.9 % B o 17.6 % B (4 min),
linea g adien o 29.4 %B (2 min) and inally 0 % B (6 min). The in-
jec ion olume was 5
μ
L.
To al essen ial amino acid (EAA) alues we e calcula ed by adding
he concen a ions o his idine, isoleucine, leucine, lysine, me hionine,
phenylalanine, h eonine, and aline; non-essen ial amino acids (NEAA)
alues we e calcula ed by adding he concen a ions o aspa ic and
glu amic acid, se ine, glycine, a ginine, alanine, y osine, and p oline in
he samples. The a io EAA/TAA (%) e e s o he p opo ion o essen ial
amino acids o o al amino acids.
2.2.3. De e mina ion o a y acid p o ile
Fa y acid me hyl es e s (FAME) we e analysed using he me hod as
desc ibed by Sukhija & Palmquis , 1988; Gonzalez-de la Rosa e al.,
2024. FAME we e p epa ed acco ding o Ju´
a ez e al. (2008). Sho ly, 1
mL o n-hexane and 3 mL o me hanolic HCl we e added o samples and
hea ed o 90 min a 70 ◦C; when he samples e u ned o oom em-
pe a u e, 5 mL K
2
CO
3
and 2 mL o n-hexane we e added, ollowed by a
cen i uga ion, and he uppe o ganic phase was collec ed and dissol ed
in 1 mL o n-hexane. FAME we e analysed using an Agilen 6890 N gas
ch oma og aph equipped wi h a lame ioniza ion de ec o , and a HP-88
capilla y column (Agilen Technologies Spain, S.L., Mad id, Spain) (100
m leng h, 0.25 mm in e nal diame e , and 0.2
μ
m phase hickness). The
ini ial o en empe a u e was 100 ◦C, wi h an inc ease o 3 ◦C pe minu e
un il 158 ◦C was eached, hen he inc ease was 1.5 ◦C o 190 ◦C, and
his empe a u e was main ained o 15 min. The empe a u e was hen
aised o 200 ◦C wi h an inc ease o 2 ◦C pe minu e. Finally, a apid ise
was made a 10 ◦C pe minu e o 240 ◦C. This empe a u e was main-
ained o 10 min. This empe a u e was main ained o 10 min. The
injec o empe a u e was se a 300 ◦C and he de ec o empe a u e was
se a 320 ◦C. Spli mode was used o he injec ion. The iden i ica ion o
he a y acids was ca ied ou by compa ison o hei e en ion imes
wi h he e en ion imes in a mix u e o s anda ds. Resul s o he a y
acid p o ile a e exp essed as a pe cen age o o al iden i ied a y acids
and a e shown as mean ±SD.
2.2.4. De e mina ion o he o al phenolic con en and iden i ica ion
To al phenolic con en was de e mined as desc ibed by Single on
e al. (1965). B ie ly, 20
μ
L o sample (10 mg/mL) p e iously dissol ed
in a me hanol:wa e solu ion o ex ac he phenols, and s anda d (gallic
acid, om o 25 o 250 mg/L) we e mixed wi h 80
μ
L o 0.7 M Na
2
CO
3
and 100
μ
L o Folin−Ciocal eu phenol eagen (0.2 M). The mix u es
we e incuba ed o 10 min and he abso bance o he eac ion was ead a
655 nm using a In ini y M-NANO spec opho ome e (Tecan, Un e s-
be gs , Aus ia). The esul s we e exp essed as mg o gallic acid
equi alen (GAE) pe 100 mg o sample.
Phenolic compounds we e hen iden i ied by UHPLC Dionex Ul i-
ma e 3000 RS, connec ed o a quad upole-O bi ap QExac i e hyb id
mass spec ome e (The moFishe Scien i ic, San Jose, CA, USA)
equipped wi h hea ed elec osp ay ioniza ion sou ce (HESI-II) ope a ed
in nega i e ioniza ion mode. Sepa a ion was ca ied ou using an Acq-
ui y BEH C18 column (1.7
μ
m pa icle size, 100 ×2.1 mm) (Wa e s, MA,
USA) kep a 40 ◦C using a low a e o 0.5 mL/min. A bina y g adien
consis ing o (A) wa e and (B) me hanol bo h con aining 0.1 % o mic
acid was used wi h he ollowing elu ion p o ile: 5 % B (1 min), linea
g adien o 100 % B (9 min), 100 % B (2 min) and inally 5 % B (3 min).
F. Ri e o-Pino e al.
Food Chemis y 473 (2025) 143084
2
The injec ion olume was 5
μ
L. Xcalibu so wa e was used o ins u-
men con ol and da a acquisi ion. A da a dependen acquisi ion me hod
(Top5) was used in nega i e mode a esolu ion 70,000 and 17,500 a m/
z 200 FWHM o Full Scan and P oduc Ion Scan, espec i ely. HESI
sou ce pa ame e s we e: sp ay ol age, −3.0 kV; S-lens RF le el, 50;
capilla y empe a u e, 320 ◦C; shea h and auxilia y gas low, 60 and 25
espec i ely (a bi a y uni s); and p obe hea e empe a u e, 400 ◦C.
T ace Finde 5.1 so wa e was used o da a ea men . The iden i ica-
ion was made by compa ing e en ion ime, he exac masses o he
pseudomolecula ion, and hei agmen ions (maximum de ia ion o 5
ppm) wi h he da a con ained in a phenolic compounds da abase wi h 87
compounds. Iso opic pa e n sco es highe han 80 % we e also equi ed.
2.2.5. Quan i ica ion o mine al con en s
The analysis was pe o med using induc i ely coupled plasma op i-
cal emission spec oscopy (ICP-OES), a e a mic owa e-assis ed diges-
ion p ocedu e wi h HNO
3
and H
2
O
2
a 200 ◦C (San os e al., 2019).
2.2.6. Quan i ica ion o ola ile compounds
Vola ile compounds we e quan i ied acco ding o Guzm´
an e al.
(2020). The ex ac ion o he ola ile compounds o he seaweed was
ca ied ou by headspace solid-phase mic oex ac ion (SPME). A ib e o
di inylbenzene/ca boxene/polydime hylsiloxane (DVB-CAR-PDMS; 1
cm long ×110
μ
m diame e ; Supelco, Belle on e, PA, USA) was se in he
headspace o he ial o 10 min a 40 ◦C while shaking he sample. The
adso bed compounds we e deso bed in o he spli -spli less injec o o a
gas ch oma og aph (GC) a 250 ◦C o 5 min. The analysis was con-
duc ed using a The mo Scien i ic T ace 1300 GC (Milan, I aly) sys em
coupled o an ion ap mass spec ome e (The moScien i ic ISQ QD
Single Quad upole Mass Spec ome e (MS), Milan, I aly) using a VF-42
WAXms column (30 m ×250
μ
m i.d. ×0.50
μ
m ilm hickness, Agilen
Technologies, Inc.2012, San a Cla a, CA, USA) whe e helium was he
ca ie gas. The condi ions we e an ini ial hold a 45 ◦C o 4 min, hen
inc eased o 150 ◦C a 5 ◦C/min and main ained o 3 min, a subsequen
amp o 250 ◦C a 6 ◦C/min, and emained a 250 ◦C o 5 min. The
ans e line was a 280 ◦C. The MS ope a ed in elec on impac mode
wi h an ioniza ion ene gy o 70 eV, eco ding da a a a scan a e o 1
scan pe second. The ela i e abundance o ola ile compounds was
calcula ed based on peak a ea in eg a ion in he ch oma og ams.
Re en ion indices (RI) we e de e mined using a se ies o n-alkanes unde
iden ical ch oma og aphic condi ions. Ten a i e compound iden i ica-
ion was achie ed by compa ing he mass spec a wi h en ies in he
Na ional Ins i u e o S anda ds and Technology (NIST; Gai he sbu g,
MD, USA) lib a y o p e iously published da a.
2.3. Ul as uc u al cha ac e iza ion
Ul as uc u al cha ac e iza ion o he d ied R. okamu ae was done
using scanning elec on mic oscopy (SEM) as desc ibed in Mon se a -de
la Paz e al. (2023). Images we e ob ained wi h a Zeiss C ossbeam 550
scanning elec on mic oscope (Zeiss, Mad id, Spain) a an accele a ing
ol age o 2.00 kV a di e en magni ica ions (50×, 200×, 500×, and
981×).
2.4. An ioxidan ac i i y
The 2,2-diphenyl-1-pic ylhyd azyl (DPPH) adical sca enging ac-
i i y o he samples was analysed acco ding o Pico e al. (2010). The
samples we e ho oughly mixed wi h 0.1 mM DPPH in me hanol (1:1, /
), incuba ed in da k condi ions (30 min) and hei abso bance was ead
a 515 nm. Thei hal -maximum e ec i e concen a ion (EC
50
) alue
was de e mined as he concen a ion o sample ha educes he DPPH
ac i i y by 50 % (Gonzalez-de la Rosa e al., 2024).
2.5. Pep ide ex ac ion, pu i ica ion, and sequence iden i ica ion
Samples we e acidi ied wi h 0.5 % i luo oace ic acid ( / ). The
desal ing and concen a ion s ep was pe o med wi h ZipTip C18 (Me ck
Millipo e, Da ms ad , Ge many) and he samples we e d ied in a speed-
acuum. LC-TIMS-MS/MS was pe o med wi h a nanoElu e nano low
ul a-high p essu e LC sys em (B uke Dal onics, B emen, Ge many)
coupled o a imsTOF P o 2 mass spec ome e , equipped wi h a Cap i-
eSp ay nanoelec osp ay ion sou ce (B uke Dal onics) acco ding o he
p ocedu e desc ibed in he pa en P202230873. Fu he de ails on he
me hodology can be ound in Mon se a -de la Paz e al. (2023). In his
case, he e e ence lib a y was acqui ed om he axonomic le el
Phaephyaceae_2024-05-10. Unique p o ein pep ides we e se o la ge
han 1 and a high con idence sco e o −10lgP >20 was applied o
indica e a p o ein accu a ely iden i ied.
2.6. Bioac i i y p edic ion in silico analysis
All he pep ides below 1000 Da we e subjec ed o in silico analyses
(p edic ion ools aiming o cha ac e ize di e en p ope ies): a) Tox-
inP ed so wa e o physio-chemical p ope ies (Gup a e al., 2013); b)
Pep ideRanke , AHT-Pin, S ack-DPPIV, P eAIP, AnOxPeP ed-1.0, o
gene al bioac i i y, inhibi ion o angio ensin con e ing enzyme (ACE)
and dipep idyl pep idase IV (DPP-IV), an i-in lamma o y and an ioxi-
dan ac i i y, espec i ely; (Cha oenkwan e al., 2022; Kha un e al.,
2019; Kuma e al., 2015; Mooney e al., 2012; Olsen e al., 2020; c)
PASTA 2.0 o es ima e seconda y s uc u e (Walsh e al., 2014); d) The
ool BIOPEP, was employed o ca y ou an in silico simula ed gas o-
in es inal diges ion (SGID) o he pep ides (using pepsin, chymo ypsin,
and ypsin), aiming o p edic he po en ial new sequences p oduced
a e diges i e deg ada ion and hei po en ial bioac i i y (Minkiewicz
e al., 2019).
2.7. Molecula docking
Molecula docking was ca ied ou o de e mine he binding a ini y
ene gy o h ee selec ed pep ides wi h di e en ecep o s, including,
DPP-IV, ACE, and TLR4/MD2. The X- ay c ys al s uc u es o DPP-IV
(PDB: 5Y7H), ACE (PDB: 1O8A), and TLR4/MD2 (PDB: 3FXI) we e ob-
ained om he RCSB PDB da abase (P o ein Da a Bank, h p://www.
csb.o g/). Ligands and all he wa e molecules we e emo ed om
he ecep o PDB ile, while pola hyd ogen a oms we e added using
UCSF Chime a so wa e (San F ancisco, Cali o nia, USA). The 3D
s uc u es o he pep ides we e ob ained, and hei s uc u e was mini-
mized, by USCF Chime a. The molecula s uc u es o he ecep o s and
he pep ides we e hen con e ed o PDBQT o ma wi h Au oDock
Tools. Fo each ecep o , he AGFR p og am was used in a di e en way
o calcula e he speci ic g id boxes o he selec ed pep ides. Fo he DPP-
IV enzyme, i s h ee ac i e si e pocke s (S1, S2, S3) we e selec ed om
he A-chain o he pep ides docking box. S1 includes Ty 547, Se 630,
Ty 631, Val656, T p659, Ty 662, Ty 666, Asn710, Val711 and His740;
S2 has he esidues Glu205, Glu206 and Ty 662, and S3 consis s o
Se 209, Phe357, and A g358 (Gui e al., 2022). Fo he ACE, a g id box
coo dina e X =37.89, Y =37.57, and Z =47.93 wi h dimensions 31.5 ×
52.5 ×29.25 was gene a ed wi h AGFR, compu ing pocke s wi h
Au oSi e 1.1, speci ic o pep ides. Fo he TLR4/MD2, AGFR p og am
was used o calcula e he posi ions and sizes o he speci ic docking
boxes o each pep ide. Au oDock C ank Pep was employed o pe o m
docking analysis. Finally, he po en ial bes docking sco e de e mined
was selec ed o each ecep o and isualized ia Bio ia Disco e y S udio
Visualize isualized ia Bio ia Disco e y S udio Visualize , as well as
he 2-dimensional (2D) and su ace anno a ion o bo h ligand in-
e ac ions wi h he p o ein.
F. Ri e o-Pino e al.
Food Chemis y 473 (2025) 143084
3
3. Resul s and discussion
3.1. Chemical cha ac e iza ion
3.1.1. P oxima e composi ion
The composi ion and p ope ies o he mac oalgae R. okamu ae ha e
been sca cely epo ed in li e a u e, and only ecen ly, hei ull cha -
ac e iza ion has been desc ibed (Ceb i´
an-Llo e e al., 2024) o he i s
ime, acco ding o hese au ho s. In Table 1, he p oxima e analysis o
he sample collec ed o his s udy is depic ed. The p o ein con en was
14.18 %, whe eas he a con en was 21.29 %, and he neu al ib e was
31.32 %. On op o ha , he mois u e was 4.90 % and he ashes co e-
sponded o he highes ac ion, 31.90 %. I mus be no ed ha he
p o ein con en migh be no exac ly es ima ed, since he ni ogen- o-
p o ein ac o employed o he calcula ions was 5, as ecen ly done
by o he au ho s (Ceb i´
an-Llo e e al., 2024) o o he b own seaweeds,
so he con en s o p o ein epo ed he e may be unde es ima ed when
compa ed o o he s using he con en ional con e sion ac o o 6.25.
The a iabili y in he composi ion desc ibed o his alga in he
li e a u e is huge, as o p o ein and lipids con en alues ange om
12.20 o 49.05 % and 4.02–17.30 %, espec i ely. P e ious esea ch
explo ed he in luence o he en i onmen al condi ions on he compo-
si ion o b own mac oalgae (Ga cia-Vaque o e al., 2021; Kons an in
e al., 2023) and hus, hese a e likely o play a s ong in luence on he
concen a ions o all he compounds epo ed in his s udy, e en i he
samples we e collec ed om simila geog aphical loca ions. The eby,
Fe ei a-An a e al. (2023) epo ed a simila p o ein con en ha he
one epo ed o he he eby assessed sample, wi h a alue o 16.43 %,
whe eas he lipid ac ion was lowe , wi h a con en o 6.17 %. The
sample e alua ed by hese au ho s (Fe ei a-An a e al., 2023) was
collec ed in July 2021 – when empe a u e was anging om 19 ◦C o
27 ◦C app oxima ely - in he same a ea as he one epo ed in his s udy.
A mean p o ein con en o 18.7 % was epo ed by Nunes e al. (2024)
wi h a neu al de e gen ib e o 54.00 % and acid de e gen ib e o
41.96 %, o h ee samples collec ed in Decembe 2023 om h ee
di e en a eas wi hin he P ainha ba hing a ea.
Acco ding o Ceb i´
an-Llo e e al. (2024), hei sample (collec ed in
G anada, Spain, al hough no in o ma ion abou he season), showed as
well as con en o ca bohyd a es o 60.4 % and 4.5 % o polyphenols. In
addi ion o ha , Agus ín e al. (2023) epo ed ha soluble die a y ib e,
insoluble die a y ib e wi hou lignin, Klason lignin, and o al die a y
ib e wi hou lignin in d y basis (% w/w) o R. okamu ae was 27.3, 13.6,
13.7, and 4.5 %, espec i ely. In ac , Miyashi a e al. (2013) indica ed
ha b own seaweeds can con ain a a ac ion anging om 10 o 20 %
pe d y weigh , in line wi h he esul s ob ained o R. okamu ae in his
s udy. I mus be no ed, in he analysis o ib e, ha he de e gen
me hods do no accoun o he whole ac ion o soluble ib es, and
consequen ly, he alue epo ed is unde es ima ed (Jensen e al., 2010).
The i adia ion and high oxygen concen a ions can lead o he o -
ma ion o ee adicals and o he s ong oxidizing agen s. Consequen ly,
ma ine species migh p oduce essen ial an ioxidan compounds, such as
i amin C and phenolics, o p o ec hemsel es agains oxida i e s ess,
including UV adia ion. Addi ionally, and simila ly o o he pa ame e s
ela ed o seaweed composi ion, i has been epo ed ha he i amin
composi ion o seaweed a ies and is in luenced by he algal species,
g ow h s age, geog aphic loca ion, salini y, season, a ailabili y o ligh ,
and seawa e empe a u e (P aiboon e al., 2018). O e all, he
R. okamu ae sample con ains a balanced composi ion including p o eins,
a s, and ib e, which seems p omising o be used as ood o eed in-
g edien s. Howe e , u he cha ac e iza ion s udies shall be done in
o de o p o ide a mo e de ailed iden i ica ion o he sample.
3.1.2. Amino acid composi ion
The amino acid p o ile o R. okamu ae is epo ed in Table 2, oge he
he quan i ica ion o essen ial and non-essen ial amino acids, and a
compa ison wi h he nu i ional ecommenda ions in adul s p oposed by
he FAO/WHO/UNU.
No ably, aline was he amino acid p esen a he highes concen-
a ion (254.29 ±5.26 mg/g), ollowed by leucine (157.86 ±2.07 mg/
g) and phenylalanine (119.76 ±1.16 mg/g). These le els a e signi i-
can ly highe han he o he essen ial amino acids measu ed, and bo h
a e ele an o p o ein syn hesis and muscle me abolism. Thus, hese
alues sugges ha he unique p o ile o amino acid composi ion o his
mac oalgae could be o nu i ional ele ance.
Ceb i´
an-Llo e e al. (2024) epo ed ha essen ial amino acids
(mainly leucine, phenylalanine, and aline) cons i u ed 32 % o he o al
amino acids in R. okamu ae, compa able o adi ional ood sou ces, such
as eggs o casein. Howe e , he diges ibili y o hese p o eins should be
add essed in o de o accoun o he p o ein quali y o his biomass.
In his case, he limi ing amino acids a e his idine (1.46 ±0.09 mg/
g) and lysine (1.43 ±0.11 mg/g). Howe e , gi en ha he use o his
algae as ood would no imply ha i is he sole die a y p o ein sou ce o
humans, bu ins ead is inco po a ed in o a di e se and balanced die , i s
consump ion is unlikely o ad e sely a ec p o ein nu i ion.
3.1.3. Fa y acid composi ion
The a y acid p o ile o R. okamu ae is epo ed in Table 3, and ac-
coun s o he o al amoun o a o he biomass o 22.3 % as men ioned
Table 1
Chemical composi ion o R. okamu ae. Da a a e exp essed as a
pe cen age o d y weigh and a e shown as mean ±SD (n =3).
P oxima e composi ion
(g/100 g o d y weigh )
R. okamu ae
P o ein con en 14.18 ±0.06
Fa con en 21.29 ±0.93
Fib e 31.32 ±0.31
Phenols 0.74 ±0.98
Mois u e 4.90 ±0.28
Ash 31.90 ±0.28
Table 2
Amino acid composi ion o R. okamu ae. Da a a e exp essed as mg amino acids
pe g o al p o ein and a e shown as mean ±SD (n =3).
Amino acids
(mg/g o p o ein)
R. okamu ae 2013 FAO/WHO/UNU
a
Essen ial amino acids
His idine 1.46 ±0.09 15
Isoleucine 98.11 ±1.22 30
Leucine 157.86 ±2.07 59
Lysine 1.43 ±0.11 45
Me hionine 19.22 ±0.56 16
Cys ine n.d. 6
Phenylalanine 119.76 ±1.16 38
Th eonine 20.75 ±0.31 23
Valine 254.29 ±5.26 39
Non-essen ial amino acids
Aspa ic acid 53.19 ±0.25 –
Glu amic acid 93.21 ±1.84 –
Se ine 20.37 ±0.43 –
Glycine 22.81 ±0.36 –
A ginine 10.23 ±0.15 –
Alanine 99.26 ±1.07 –
Ty osine 11.46 ±1.24
Glu amine n.d.
P oline 16.59 ±0.38 –
To al essen ial amino acids 672.87 ±4.56
To al non-essen ial amino acids 327.13 ±3.15
EAA/TAA
b
(%) 67.29 %
NEAA/TAA
b
(%) 32.71 %
a
FAO/WHO/UNU 2013. Die a y p o ein quali y e alua ion in human nu i-
ion. FAO FOOD and Nu i ion Documen NO. 92. Sco ing pa e n mg/g p o ein
equi emen s o adul s. n.d.; no de ec ed.
b
EAA: o al essen ial amino acids; TAA: o al amino acids; NEAA: o al non-
essen ial amino acids.
F. Ri e o-Pino e al.
Food Chemis y 473 (2025) 143084
4
p e iously. The main a y acids de ec ed we e C16:0, C18:0, and C14:0,
all o hem sa u a ed a y acids, ep esen ing a ound 62 % o he sam-
ple. Then, oleic acid (C18:1n-9c) ep esen ed 8.43 % o he sample.
Mo eo e , he sample was also high in C20:5n-3 (eicosapen aenoic acid,
EPA) wi h alues o a ound 7.7 %, as well as o C20:4n-6 (a achidonic
acid, ARA) and C18:3n-3 (
α
-linolenic acid) ha ep esen ed app oxi-
ma ely 5.1 and 3.4 %, espec i ely. Monounsa u a ed a y acids ep-
esen ed 13.3 % o he o al a y acids. In he case o polyunsa u a ed
a y acids, omega-3 we e 12.1 % and omega-6 ep esen ed a 9 % o he
o al a y acids, making an omega-6/omega-3 a io o 0.74:1. This a io
indica es ha he R. okamu ae-de i ed lipid ac ion con ains mo e
omega-3 han omega-6 a y acids, which is posi i e since i is gene ally
ecommended o main ain a balance whe e omega-3 is a leas equal o
o g ea e han omega-6 (Ka ageo gou e al., 2023). This esul in highly
compa able o he esul s ob ained by Ceb i´
an-Llo e e al. (2024),
which epo ed sa u a ed a y acids as he mos abundan ac ion
(a ound 61 %), ollowed by polyunsa u a ed a y acids, a ound 24 %
and monounsa u a ed a y acids, a ound 15 %. Howe e , hese au ho s
epo ed di e en con en o speci ic a y acids compa ed o his s udy,
such as lowe con en o C16:0 and C18:0, and mo e o C14:0 and
C18:1n-9. In ela ion o EPA, ARA, and
α
-linolenic acid, R. okamu ae
om his s udy had highe con en o all o hem compa ed o o he ood
sou ces such as oli e oil, ishes such as Tilapia (Chepki ui e al., 2021) o
some legume seeds (Kh isanapan e al., 2019). These di e ences migh
be a ibu ed o bo h he loca ion whe e he aw ma e ial was collec ed
om, and he season, since o ins ance, o o he sou ces o lipids, i has
been demons a ed ha he con en o unsa u a ed a y acids could be
highe in win e .
Acco ding o Aussan e al. (2018), he empe a u e is one o he key
ac o s in de e mining he con en o EPA and docosahexaenoic acid
(DHA) in mic oalgae, in ela ion o he luidi y o cell memb anes. The
in oduc ion o hese polyunsa u a ed a y acids in o he memb anes is
educed when empe a u es ise, and consequen ly, he p opo ion o
polyunsa u a ed a y acids in algae is epo ed o inc ease in win e .
The samples he eby analysed we e collec ed in Ta i a (Spain) du ing he
sp ing in 2024. The a e age empe a u e eco ded by he Ta i a s a ion
in Ap il 2024 was 16.96 ◦C, anging be ween a minimum o 9.1 ◦C and a
maximum o 28.3 ◦C. In o de o unde s and seasonal a ia ion, samples
aken in di e en mon hs should be analysed. In he same line, he
R. okamu ae sample collec ed in summe , as men ioned abo e (Fe ei a-
An a e al., 2023) con ained palmi ic acid (50.1 %), my is ic acid (22 %),
9-hexadecenoic acid (11 %), 9-oc adecenoic (12 %), s ea ic acid (2.6 %),
and eicosanoic acid (2.3 %), di e en om he cu en esul s. The a y
acid p o ile o an ing edien should be also assessed in e ms o he egio-
Table 3
Fa y acid composi ion o R. okamu ae. Da a a e exp essed as a pe cen age
o o al iden i ied a y acids and a e shown as mean ±SD (n =3).
Fa y acid
( ela i e amoun , %)
R. okamu ae
Cap ic acid (C10:0) 0.08 ±0.01
Lau ic acid (C12:0) 0.21 ±0.02
T idecylic acid (C13:0) 0.10 ±0.01
My is ic acid (C14:0) 8.74 ±0.08
My is oleic acid (C14:1n-9) 0.17 ±0.04
Pen adecylic acid (C15:0) 0.95 ±0.02
10-Pen adecenoic acid (C15:1n-5) 0.26 ±0.07
Palmi ic acid (C16:0) 40.03 ±0.28
Palmi oleic acid (C16:1n-9c) 3.14 ±0.00
Ma ga ic acid (C17:0) 0.35 ±0.13
Ma ga oleic acid (C17:1n-9c) 0.13 ±0.04
S ea ic acid (C18:0) 13.37 ±0.08
Elaidic acid (C18:1n-9 ) 0.12 ±0.01
Oleic acid (C18:1n-9c) 8.43 ±0.04
Linoelaidic acid (C18:2n-6 ) 0.10 ±0.02
Linoleic acid (C18:2n-6c) 2.38 ±0.07
γ-Linolenic acid (C18:3n-6 g) 0.61 ±0.05
α
-Linolenic acid (C18:3n-3
α
)3.41 ±0.01
A achidic acid (C20:0) 0.54 ±0.05
Gondoic acid (C20:1n-9) 0.62 ±0.17
Eicosadienoic acid (C20:2n-6) 0.14 ±0.05
Dihomo γ-linolenic acid (C20:3n-6) 0.48 ±0.08
A achidonic acid (C20:4n-6) 5.06 ±0.07
Eicosapen aenoic acid (C20:5n-3) 7.75 ±0.31
Eicosa ienoic acid (20:3n-3) 0.16 ±0.02
Heneicosanoic acid (C21:0) 0.42 ±0.04
Behenic acid (C22:0) 0.42 ±0.13
E ucic acid (C22:1n-9) 0.18 ±0.07
Docosadienoic acid (C22:2n-6) 0.24 ±0.11
Clupanodonic acid (C22:5n-3) 0.50 ±0.04
Docosahexaenoic acid (C22:6n-3) 0.24 ±0.03
T icosanoic acid (C23:0) 0.19 ±0.06
Lignoce ic acid (C24:0) 0.27 ±0.14
Ne onic acid (C24:1n-9) 0.26 ±0.03
To al sa u a ed a y acids 65.6 %
To al monounsa u a ed a y acids 13.3 %
To al polyunsa u a ed a y acids 21.1 %
Omega-3 12.1 %
Omega-6 9 %
Omega-6/omega-3 a io 0.74:1
Table 4
Cha ac e iza ion o he phenolic p o ile o R. okamu ae. Da a a e exp essed as
ppb and a e shown as mean ±SD (n =4).
Phenol (Con .)
a
(ppb)
b
Fo mula R. okamu ae
3-hyd oxy y osol (1/3) C
8
H
10
O
3
2.51 ±0.04
2,4-dihyd oxybenzoic acid (1/3) C
7
H
6
O
4
7.97 ±0.11
4-hyd oxybenzoic acid (2/3) C
7
H
6
O
3
11.88 ±0.25
Benzoic acid (1/3) C
7
H
6
O
2
83.28 ±0.96
Ca eic acid (1/3) C
9
H
8
O
4
5.19 ±0.09
Dihyd oca eic acid (2/3) C
9
H
10
O
4
8.19 ±0.12
Ellagic acid (3/3) C
14
H
6
O
8
15.61 ±0.15
Flo e ic acid (1/3) C
9
H
10
O
3
41.50 ±0.20
Gen isic acid (1/3) C
7
H
6
O
4
7.97 ±0.07
Homo anillic acid (1/3) C
9
H
10
O
4
8.19 ±0.09
Indoleace ic acid (2/3) C
10
H
9
NO
2
12.02 ±0.10
Indolac ic acid (2/3) C
11
H
11
NO
3
2.91 ±0.00
P-couma ic acid (1/3) C
9
H
8
O
3
91.87 ±0.84
P o oca echic acid (1/3) C
7
H
6
O
4
7.97 ±0.07
Quinic acid (1/3) C
7
H
12
O
6
2.99 ±0.01
Salicylic acid (3/3) C
7
H
6
O
3
11.88 ±0.04
T ans-cinnamic acid (1/3) C
9
H
8
O
2
2.65 ±0.02
Ca echol (2-hyd oxyphenol) (1/3) C
6
H
6
O
2
5.30 ±0.06
Kaemp e ol-3-O-Glc (0/3) C
21
H
20
O
11
3.81 ±0.04
Lu eolin-4’-O-Glc (0/3) C
21
H
20
O
11
3.81 ±0.03
Lu eolin-7-O-Glc (0/3) C
21
H
20
O
11
3.81 ±0.03
Que ci in (Que ce in-3-O- hamnoside) (0/3) C
21
H
20
O
11
3.81 ±0.03
Taxi olin (0/3) C
15
H
12
O
7
16.03 ±0.11
Vanillin (2/3) C
8
H
8
O
3
31.74 ±0.18
3,4-Dihyd oxyxyphenylglycol (1/3) C
8
H
10
O
4
3.26 ±0.03
4-O-Ca eoylquinic acid (3/3) C
16
H
18
O
9
5.93 ±0.06
Abscisic acid (1/3) C
15
H
20
O
4
15.92 ±0.12
Chlo ogenic Acid (3-O-Ca eoylquinic Acid) (2/3) C
16
H
18
O
9
5.93 ±0.02
Gibbe ellic Acid (GA3) (1/3) C
19
H
22
O
6
18.04 ±0.19
Iso anillic acid (1/3) C
8
H
8
O
4
4.02 ±0.06
Jasmonic acid (1/3) C
12
H
18
O
3
4.14 ±0.05
Sy ingic acid (1/3) C
9
H
10
O
5
2.84 ±0.03
Vanillic acid (1/3) C
8
H
8
O
4
4.02 ±0.03
A omadend in (0/3) C
15
H
12
O
6
9.31 ±0.10
B e i olin (1/3) C
10
H
12
O
4
2.72 ±0.04
Ca echin (0/3) C
15
H
14
O
6
3.69 ±0.04
Epica echin (0/3) C
15
H
14
O
6
3.69 ±0.04
E iodic yol (0/3) C
15
H
12
O
6
9.31 ±0.11
E hyl galla e (1/3) C
9
H
10
O
5
2.84 ±0.02
Gibbe ellin A12 (2/3) C
20
H
28
O
4
265.55 ±1.21
Gibbe ellin A19 (2/3) C
20
H
26
O
6
67.60 ±0.51
Gibbe ellin A20 (1/3) C
19
H
24
O
5
15.80 ±0.14
Gibbe ellin A44 (2/3) C
20
H
26
O
5
76.57 ±0.48
Gibbe ellin A53 (1/3) C
20
H
28
O
5
23.67 ±0.21
Oleu opein (1/3) C
25
H
32
O
13
9.55 ±0.12
Pino esinol (1/3) C
20
H
22
O
6
2.60 ±0.03
a
Con : Con idence based on e en ion ime, ionic agmen a ion, and iso opes;
b
ppb has been es ima ed wi h he a ea and concen a ion o he in e nal s an-
da d o 2,4-dihyd oxybenzoic acid.
F. Ri e o-Pino e al.
Food Chemis y 473 (2025) 143084
5
dis ibu ion o he a y acid, as he bioa ailabili y o he a y acids in a
iacylglyce ide depends on i s posi ion on he backbone, so u he
s udies should go owa ds ha di ec ion.
3.1.4. Phenolic compounds
O e all, he o al polyphenol con en s o his mac oalgae we e
0.0074 ±0.0010 mg gallic acid/mg o sample. In Table 4, he phenolic
compounds iden i ied by mass spec ome y a e shown. When analysing
he ype o compounds iden i ied, i was obse ed ha some o hese
phenols a e common componen s in o he plan -de i ed sou ces, such as
oli e, be ies, ee ba k, anilla pods, and a ious plan s, aligning wi h
he p o iles epo ed in simila s udies (Ri e o-Pino e al., 2024; Tanase
e al., 2019; Zhang e al., 2023). I has been epo ed ha , o example,
ex ac ion wi h e hanol inc eases polyphenol yields by 4.59–10.78 imes
when compa ed wi h wa e , whe eas on he con a y, wi h wa e , he
con en o educed suga is highe (doi:h ps://doi.o g/10.1016/j.
jen man.2024.122504). Consequen ly, p ocessing o aw ma e ials is a
key s ep in o de o concen a e speci ic compounds which may be o
in e es . Ceb ian-Llo e e al., (2024) epo ed a con en o 4.5 % o o al
phenols, employing he same me hodology as he one in his s udy, bu
no u he iden i ica ion o phenolic compounds was pe o med by hese
au ho s. Howe e , phlo o annins ha e been epo ed o be he mos
impo an class o polyphenols p esen in b own seaweeds. Nume ous
s udies ha e demons a ed ha he po en an ioxidan quali ies o
phlo o annins de i ed om di e en species o b own seaweeds may be
a ibu ed o hei dis inc molecula s uc u e (Duan e al., 2023).
3.1.5. Mine al con en s
The con en o mine als o R. okamu ae a e epo ed in Table 5. In
his s udy, he ash con en s we e highe han 30 %, whe eas o he au-
ho s epo ed lowe alues (e.g., 11 % by Ceb i´
an-Llo e e al., 2024).
The mine als ound a he highes concen a ions we e po assium, so-
dium, calcium, and sulphu , wi h alues o e 19 g/kg. The con en o
sal is high, hus, po en ially limi ing he use le els o his sample as ood
when used as ing edien . Ceb ian e al., (2024) epo ed high con en o
magnesium (a ound 7 g/kg), calcium (5.5 g/kg), po assium and sodium
(2.5 g/kg), s on ium (1.5 g/kg), chlo ine (1.1 g/kg), and aluminium (1
g/kg). Howe e , o he au ho s did no de ec magnesium o calcium, o
ins ance, while inding a high con en o sodium (Agabo-Ga cía e al.,
2023). Simila ly o o he composi ion pa ame e s analysed in his s udy,
he a iabili y in he concen a ions epo ed be ween s udiues can be
explained by he di e ences in he en i onmen in which he algae
g ow. The high con en o sodium and calcium has also been epo ed o
o he b own algae (Meng e al., 2022), and e en when no being among
he mos abundan ones, he i on con en o R. okamu ae (463.6 mg/kg)
was subs an ial, as has been also highligh ed in o he species. The high
Table 5
Mine al con en o R. okamu ae. Da a a e exp essed as mg/kg
and a e shown as mean ±SD (n =3).
Mine al (mg/kg) R. okamu ae
Ca 20,482.11 ±11.30
Co n.d.
C 0.35 ±0.00
Cu 7.02 ±0.00
Fe 463.62 ±0.00
K45,696.54 ±426.42
Mg 7985.80 ±0.12
Mn 15.38 ±0.00
Na 36,157.94 ±0.00
Ni 5.55 ±0.00
P2011.24 ±0.14
S19,816.62 ±1.82
Se n.d.
V2.58 ±0.00
Zn 31.13 ±0.00
n.d., no de ec ed.
Table 6
Vola ile compounds iden i ied in R. okamu ae. Da a a e exp essed as ela i e
alue and a e shown as mean ±SD (n =3).
Compounds
( ela i e amoun , %)
R. okamu ae
Ca boxylic acids 7.26 ±0.01
Ace ic acid 6.00 ±0.33
Bu anoic acid 0.02 ±0.00
Bu anoic acid, 3-me hyl-(Iso ale ic acid) 0.19 ±0.01
2-pen enoic acid 0.16 ±0.04
Hexanoic acid 0.11 ±0.01
Oc anoic acid 0.64 ±0.04
Nonanoic acid 0.08 ±0.00
Decanoic acid 0.11 ±0.01
Dodecanoic acid 0.06 ±0.00
Hexadecanoic acid 0.05 ±0.00
Oc adecanoic acid 0.04 ±0.00
Alcohols 12.33 ±0.01
E hanol 1.37 ±0.06
2-bu yl-1-oc anol 0.05 ±0.00
3-bu en-2-ol-2-me hyl (
α
,
α
-dime hyl-allylalcohol) 0.10 ±0.02
Pinacol (2,3bu anediiol-2,3-dime hyl) 0.01 ±0.00
1-pen anol 0.13 ±0.01
1-pen en-3-ol 1.02 ±0.06
2-pen en-1-ol, (E)- 0.23 ±0.01
2-pen en-1-ol, (Z)- 1.29 ±0.07
1-hexanol 0.10 ±0.01
Phenol 0.04 ±0.00
Benzeneme hanol 0.04 ±0.00
Benzenee hanol 0.06 ±0.00
3-hexen-1-ol 0.08 ±0.00
3-me hyl-1-hexen-1-ol/1-me hylcyclohexanol 0.56 ±0.03
2,4-hexadien-1-ol 0.34 ±0.02
2-e hylhexanol 3.73 ±0.21
Cyclohexanol, 2,4-dime hyl- 0.10 ±0.01
2,4-die hyl-1-hep anol/ Ca anol 0.04 ±0.00
2-bu yl-1-oc anol 0.04 ±0.00
Ci onellol-dihyd o (1-oc anol,3,7-dime hyl) 0.36 ±0.02
Ci anellol (6-oc en-1-ol) 0.25 ±0.01
(Z)-oc -2-en-1-ol 0.06 ±0.00
(E)-2-nonen-1-ol 0.19 ±0.01
2-e hyl-2-me hyl- idecanol 0.03 ±0.00
1-oc adecanol 0.04 ±0.00
1-oc en-3-ol 0.15 ±0.01
2,4-decadien-1-ol 0.04 ±0.00
1-hexadecanol 0.05 ±0.00
E hyl-linalool 0.05 ±0.00
Lonol 0.04 ±0.00
Linalool 1.12 ±0.06
Aldehydes 5.43 ±0.01
Bu anal, 3-me hyl- 0.12 ±0.01
2-bu enal 0.01 ±0.00
2-pen enal,
€
-0.42 ±0.02
Hexanal 0.07 ±0.00
2-hexenal,
€
-0.07 ±0.00
Sa baldehyde (2,4-hexadienal) 0.04 ±0.00
Benzaldehyde 0.75 ±0.04
Benzaldehyde,2-me hyl 0.17 ±0.01
€
-2-hep enal 0.10 ±0.01
(Z)-4-hep enal 0.07 ±0.00
2,4-hep adienal, (E,Z)- 0.44 ±0.02
2,4-Hep adienal, (E,E)- 0.71 ±0.04
2,4-oc adienal 0.02 ±0.00
Ci onellal (6-oc enal-3,7-dime hyl) 0.07 ±0.00
€
-oc -2-enal 0.12 ±0.01
Nonanal 0.06 ±0.00
(E,E)-2,4-nonadienal 1.67 ±0.09
€
-2-decenal 0.13 ±0.01
(E,Z)-2,4-decadienal 0.02 ±0.00
2-dodecenal 0.09 ±0.00
(E,Z)-2,4-dodecadienal 0.44 ±0.00
Pho oci al 0.04 ±0.00
Ke ones 7.57 ±0.04
2-p opanone 4.21 ±0.18
Ace oin (2-bu anone,3hyd oxy) 0.13 ±0.01
2-pen anone 0.52 ±0.02
4-hyd oxy-4-me hyl-2-pen anone 0.02 ±0.00
(con inued on nex page)
F. Ri e o-Pino e al.
Food Chemis y 473 (2025) 143084
6
Table 6 (con inued)
Compounds
( ela i e amoun , %)
R. okamu ae
5-hep en-2-one, 6-me hyl- 0.50 ±0.03
3,5-hep adien-2-ona, 6-me hyl- 1.82 ±0.10
6-oc en-2-one 0.02 ±0.00
2-oc anone 0.07 ±0.00
3-oc en-2-one 0.03 ±0.00
3,5-oc adien-2-one (E,E)- 0.08 ±0.00
3-hexen-2-one/4-me hyl-4-oc enal 0.05 ±0.00
(E,E)-2,4-dodecadienal 0.30 ±0.02
Vale ophenone 0.02 ±0.00
Alipha ic hyd oca bons 34.66 ±0.02
2-hexene-3,5,5- ime hyl- 0.11 ±0.01
Hep ane 0.38 ±0.02
Hep ane, 2-me hyl- 0.35 ±0.02
Hep ane, 2,3-dime hyl- 0.84 ±0.04
1,6-dime hylhep a-1,3,5- iene 0.18 ±0.01
7-hexyl-eicosane 0.08 ±0.00
3-e hyl-1,5-Oc adiene 0.51 ±0.02
Decane 1.01 ±0.04
Dodecane 0.54 ±0.03
T idecane 0.13 ±0.01
Te adecane 0.48 ±0.03
Pen adecane 29.28 ±1.62
Hep adecane 0.56 ±0.03
8-Hep adecene 0.40 ±0.02
9-nonadecene 0.26 ±0.01
1,4,6,9-nonadeca e aene 0.03 ±0.00
Fa nesan (Dodecane,2,6,10- ime hyl) 0.03 ±0.00
Phy one/Phy ol 0.06 ±0.00
T icosane 0.14 ±0.01
Te acosane 0.03 ±0.00
Pen acosane 0.04 ±0.00
Hep acosane 0.06 ±0.00
Oc acosane 0.06 ±0.00
A oma ic hyd oca bons 1.20 ±0.00
p-xilene (1,4-dime hyl-benzene) 0.25 ±0.01
o-xilene (1,2-dime hyl-benzene) 0.06 ±0.00
1,3,5- ime hyl-Benzene 0.03 ±0.00
S y ene 0.65 ±0.04
o-cymene (benzene,1-me hyl-2-(1-me hyle hyl) 0.05 ±0.00
Cumene (benzene 1-me hyle hyl-) 0.01 ±0.00
p-cymene (benzene,1-me hyl-4-(1-me hyle hyl) 0.01 ±0.00
Dime hyls y ene 0.04 ±0.00
Benzene, 1,3-bis(1,1-dime hyle hyl)- 0.13 ±0.01
Lac ones 0.13 ±0.00
δ-decalac one 0.07 ±0.00
γ-dodecalac one 0.07 ±0.00
Es e s 2.00 ±0.00
Linalyl o ma e 0.85 ±0.05
bu anoic acid, bu yles e /Isop opyl my is a e 0.09 ±0.01
Bu anoic acid, decyles e 0.05 ±0.00
(2Z)-2-pen enylace a e 0.09 ±0.00
Hexanoic acid, hexyles e 0.02 ±0.00
2-e hylbu ylhexanoa e 0.03 ±0.00
Vale ic anhyd ide (pen anoic acid,1,1-anhyd ide)/
4-hep anone, 3,5-dime hyl
0.03 ±0.00
Hep adecanoic acid e hyl es e 0.07 ±0.00
Me hyl olea e 0.56 ±0.03
Me hyl palmi a e 0.07 ±0.00
E hyl olea e 0.03 ±0.00
E hyl palmi a e 0.16 ±0.01
Te penes 27.71 ±0.04
Limonene 0.63 ±0.04
Eucalyp ol 0.58 ±0.03
Cymen-7-ol 0.05 ±0.00
ß- hujone 0.02 ±0.00
T ans-3-ca en-2-ol 0.02 ±0.00
Ge mac ene 0.17 ±0.18
Bou bonene 1.86 ±0.10
Selinene/Elemene 1.14 ±0.06
β-copaene/
α
-cubebene/Ge mac ene 0.37 ±0.02
α
-cubebene/Ylangene 0.33 ±0.02
α
-cubebene/A mo phene/Cadina-3-5-diene 0.05 ±0.00
ß-cycloci al 0.12 ±0.01
1-epi-cubenol 0.02 ±0.00
β-ca yophyllene 0.64 ±0.04
Table 6 (con inued)
Compounds
( ela i e amoun , %)
R. okamu ae
Seychellene/β-chamig ene 0.04 ±0.00
Gu junene/Guaiene/Guaia-1(5),11-diene 0.25 ±0.01
Aco adiene/Copaene/Cubenene/amo phene 0.19 ±0.01
aco adiene/ aco enol 0.31 ±0.02
Aco enol/Elemene/Neocali opsene 0.36 ±0.02
Bo neol 0.04 ±0.00
Ge mac ene-D/Copaene/Cubenene/amo phene 2.25 ±0.12
Bisabolene 0.19 ±0.01
α
- a nesene 0.03 ±0.00
Guaia-1(5),7(1)-diene 0.04 ±0.00
del a-ca dinene/Cadina-1(10),4diene 0.06 ±0.00
Ch ysan henone 0.08 ±0.00
2-pien-10-ol 0.07 ±0.00
Cumene/Benzene,1,3,5 ime hyl/ o-e hyl- oluene 0.06 ±0.00
Calamenene/Cadina-1,3,5- iene 0.03 ±0.00
α
-Ionone 0.11 ±0.01
Bisabolone, oxide 0.07 ±0.00
Ca yophyllene,oxide 0.09 ±0.01
Elemene/Hinesol 0.19 ±0.01
Bo neol 0.04 ±0.00
cis-β- e pineol/pinocamphone 0.04 ±0.00
Geije en/P egeije ene 0.02 ±0.00
San alol 0.29 ±0.02
β-ionone 0.25 ±0.01
Cemb ene 0.15 ±0.01
β-humulene 0.15 ±0.01
Elemene/Isoge mac ene/Valencene 0.08 ±0.00
Thujopsene 0.10 ±0.01
β-ionone, 5,6-epoxy 2.26 ±0.01
Humulene/Ve iciol 0.48 ±0.03
Ca yophyllene/Khusimene 3.96 ±0.22
Guaia-5-11-diene 0.21 ±0.01
Cubebene 0.81 ±0.98
Elenene 0.29 ±0.02
Guaia-5-11-diene/Elemene 0.16 ±0.01
Ve iciol/Rimuen/Rosa-5,15-diene 0.05 ±0.00
Bulnesene 0.19 ±0.01
Muu olene/ Copaene 0.03 ±0.00
Re inol 6.68 ±0.37
Re inol ace a e 0.37 ±0.02
Re inal 0.22 ±0.25
Cubi ene 0.21 ±0.01
P egeije ene 0.06 ±0.00
FoKienol 0.06 ±0.00
Re inol ace a e 0.75 ±0.36
Ced anediol 0.02 ±0.00
Bacdanol 0.27 ±0.01
Ced -8-en-13-ol 0.23 ±0.01
Cup enene 0.52 ±0.03
Me hand os enolone 0.14 ±0.01
Re inoic acic me hyles e 0.15 ±0.01
Ni ogen compounds 0.004 ±0.00
Indane 0.01 ±0.00
Sulphu compounds 0.10 ±0.01
Dime hyl sul oxide 0.10 ±0.01
Fu an compounds 0.72 ±0.00
Fu an, 2-pen yl- 0.16 ±0.01
ans-2-(2-pen enyl u an) 0.09 ±0.00
2(5H)- u anone,5,5-dime hyl 0.07 ±0.00
Benzeno hio u an 0.34 ±0.02
2(4H)-benzo u anone,5,6,7,7a e ahyd o-4,4,7a- ime hyl 0.07 ±0.01
Naph halene de i a i es 0.37 ±0.02
1,4-dime hyl e alin 0.19 ±0.01
2,7-dime hyl e alin 0.21 ±0.01
O he s 0.52 ±0.00
Dib omome hane 0.10 ±0.01
B omo o m 0.37 ±0.02
Biphenyloxide 0.03 ±0.00
Dibu hylpha ale 0.02 ±0.00
Unknows 0.81 ±0.02
F. Ri e o-Pino e al.
Food Chemis y 473 (2025) 143084
7
con en o sulphu , in spi e o he sulphu -con aining amino acids lack,
migh be due o sulpha ed polysaccha ides like ucoidan and algina e.
These compounds ha e sulphu a oms a ached o hei suga uni s,
con ibu ing signi ican ly o he o e all sulphu con en (Uso e al.,
2022).
The a iabili y o mine al con en , in e ms o o al concen a ion,
bu also in he speci ic con ibu ion o each o hem, has o be
comp ehensi ely add essed in he assessmen o no el sou ces, and i is
impo an o know he main ac o s a ec ing his composi ional da a. As
he p esence o speci ic hea y me als may pose a isk i in ended o be
used as ood ing edien , esea ch is being done o educe hei con en by
using inno a i e s a egies such as ul asounds echnologies (No iega-
Fe n´
andez e al., 2021).
3.1.6. Vola ile compounds
In Table 6, he ola ile compounds iden i ied a e indica ed, oge he
wi h he pe cen age o a ea. 114 compounds we e iden i ied wi h
alipha ic hyd oca bons as p edominan ep esen ing 34.66 % ollowed
by e penes wi h alues o 27.71 %, alcohols ep esen ing an amoun o
12.33 %, and ca boxylic acids and ke ones a ound 7 %. As epo ed in
o he b own algae, alipha ic compounds (e.g., pen adecane) we e he
majo i y as epo ed o Clados ephus spongiosus (Radman e al., 2023) o
Amphi oa igida (Cikoˇ
s e al., 2021). O e all, he analysis e ealed ha
he majo ola ile componen s we e pen adecane (29.28 %), e inol
(6.68 %), ace ic acid (6.00 %) and 2-p opanone (4.21 %).
The con en o e inol (6.68 %) could be ele an conside ing he
heal h bene i s o his compound when added o he die . I mus be
no ed ha in ood, he e m i amin A comp ises all- ans- e inol (also
called e inol), na u ally occu ing molecules associa ed wi h he bio-
logical ac i i y o e inol, and p o i amin A ca o enoids ha a e die a y
p ecu so s o e inol. Thus, R. okamu ae could be o in e es o his
indus y, since i is a o m o i amin A au ho ised o oods and ood
supplemen s (Ca azo e al., 2021). On op o ha e inol is an e ec i e
an i-aging o he skin, due o i s abili y o s imula e collagen syn hesis,
educe oxida i e s ess, and modula e gene exp ession (Quan, 2023), so,
he use o his mac oalgal species as sou ce o an i-aging compounds
dese es u he in es iga ion.
Ace one (4.21 %) can be ound as an ing edien in a a ie y o con-
sume p oduc s anging om cosme ics o p ocessed and unp ocessed
oods, i is gene ally ecognized as sa e (GRAS) subs ance when p esen
in d inks, baked oods, desse s, and p ese es a concen a ions anging
om 5 o 8 mg/L (Besinis e al., 2016). Then, 2-e hylhexan-1-ol (3.59 %)
is a na u al p oduc ound in Camellia sinensis and Alpinia chinensis,
whe eas ans, ans-2,4-nonadienal (1.61 %) is a na u al p oduc ound
in A emisia annua, P unus a ium, and Aga icus bispo us. These com-
pounds ha e been epo ed ha ha e a a y, g een a oma quali y, ac-
co ding o Hosoglu e al. (2020), a e iden i ying hem in he
mic oalgae C yp hecodinium cohnii. Simila ly, 3,5-hep adien-2-ona, 6-
me hyl- (1.75 %) was ound as well in A h ospi a pla ensis, while no
de ec ed on o he mic oalgae and cyanobac e ia (Mo an e al., 2022).
Special a en ion has o be gi en o some o hese compounds when
using his mac oalgal species as ood o eed, as a long- e m ch onic
oxici y s udy in mice and a s showed oxici y due o hujone, and
consequen ly, he p oposed uses o R. okamu ae as a new ing edien
should be assessed ca e ully o ensu e i s sa e y. Essen ial oils, such as
limonene, eucalyp ol, e inol, e c., ha e been e alua ed as componen s
o eed o ood. Thus, u u e s udies should also e alua e changes in he
composi ion o ola iles when conside ing adding his biomass in o ood
o eed, aiming o es ablish sa e le els o inclusion.
3.2. Ul as uc u al cha ac e iza ion
Fig. 1 shows di e en images ob ained by SEM, a di e en con ig-
u a ions. SEM allows o pe o m de ailed obse a ions o he su ace o
he sample pa icles a a nanome ic scale, which can help o unde s and
he mo phology and su ace s uc u es ha can in luence hei unc-
ional p ope ies and can also be used o e i y he uni o mi y and
consis ency o he p oduc be ween di e en ba ches.
Fig. 1. Su ace cha ac e is ics o R. okamu ae by SEM. Images we e aken a (A) Magni ica ion 50×(scale ma ks =100
μ
m), (B) 200×(scale ma ks =30
μ
m), (C)
500×(scale ma ks =10
μ
m), and (D) 981×(scale ma ks =10
μ
m), and AV =2.0 kV o all.
F. Ri e o-Pino e al.
Food Chemis y 473 (2025) 143084
8
3.3. An ioxidan ac i i y
The an ioxidan ac i i y o R. okamu ae was e alua ed by he DPPH
adical sca enging me hod. The samples o his s udy had an EC
50
alue
o 2.090 ±0.093 mg/mL, which is in line wi h ecen epo s e alua ing
he in i o an ioxidan ac i i y o e hanolic ex ac s achie ed om
Fig. 2. Numbe o sequences (Y-axis) wi h speci ic pep ide leng h ( om 7 o 26 amino acids leng h – X axis).
Table 7
Cha ac e iza ion o he 20 pep ides sequences wi h a molecula weigh below <1000 Da iden i ied in R. okamu ae on he basis o in silico analyses.
Pep ides ToxinP ed
a
Pas a 2.0
b
Hyd ophobici y S e ic
hind ance
Wa e
Solubili y
pI Cha ge Amphipa hici y Sel -
agg
c
Diso de
p obabili y
Seconda y s uc u e
d
α
-helix β-s and Coil
VAPEEHPV −0.06 0.5 Good 4.51 −1.50 0.5
1–4
(NI) 100 – – 100
APILVPVGK 0.17 0.58 Poo 9.11 1.00 0.41
3–7
(NI) 100 –11.11 88.89
FVKGYKY −0.12 0.69 Good 9.55 2.00 1.05
1–7
(NI) 100 – – 100
KEAKEVVE −0.34 0.67 Good 4.79 −1.00 1.39
4–8
(NI) 100 50 –50
KAALDLWK −0.12 0.59 Good 8.94 1.00 0.92
4–7
(NI) 100 50 –50
KAAIDLWK −0.1 0.61 Good 8.94 1.00 0.92
4–7
(NI) 100 37.5 –62.5
TGLFLDPKG −0.01 0.61 Good 6.19 0.00 0.41
1–5
(NI) 100 – – 100
VFTGSPGKY −0.01 0.62 Poo 8.94 1.00 0.41
1–4
(NI) 100 – – 100
TVDAKAGVKA −0.11 0.63 Good 8.94 1.00 0.73
1–4
(NI) 100 – – 100
ITDEDIKQ −0.32 0.69 Good 4.03 −2.00 0.77
4–7
(NI) 100 50 –50
VGDGIARIY 0.01 0.68 Good 6.19 0.00 0.27
5–9
(NI) 100 –22.22 77.78
ETGIKVVDL −0.01 0.66 Good 4.38 −1.00 0.55
4–7
(NI) 100 –33.33 66.67
AGFAGDDAPR −0.16 0.62 Good 4.21 −1.00 0.25
1–4
(NI) 100 – – 100
SHISTGGGASL 0.06 0.54 Poo 7.1 0.50 0.13
2–5
(NI) 100 – – 100
VAPEEHPVL 0.01 0.05 Good 4.51 −1.50 0.44
6–9
(NI) 100 – – 100
SQSEKDWD −0.5 0.64 Good 4.03 −2.00 0.77
2–5
(NI) 100 – – 100
VVIGHVDAGK 0.07 0.61 Good 7.09 0.50 0.51
1–6
(PA) 100 –40 60
EAGGITQHVS −0.03 0.57 Poo 5.25 −0.50 0.4
5–9
(NI) 100 – – 100
VLVGGSTRIP 0.04 0.61 Poo 10.11 1.00 0.25
1–4
(NI) 100 – – 100
TPDLTDPKL −0.22 0.56 Good 4.21 −1.00 0.41
3–6
(NI) 100 – – 100
a
Pep ides we e subjec ed o calcula ion ia h ps://webs.iii d.edu.in/ agha a/ oxinp ed/design.php/. whe e he hyd ophobici y, s e ic hinde ance, solubili y,
isoelec ic poin (pI), cha ge, and amphipa hici y we e calcula ed.
b
The web se e PASTA 2.0 (h p://p o ein.bio.unipd.i /pas a2/) compu es he endency o pep ide sel -agg ega ion (Sel -agg) o he possible egion a sequence.
The p obabili y o in insic diso de and po ion o es ima ed seconda y s uc u e ha complemen he agg ega ion da a we e also epo ed.
c
Sel -agg ega ion-p one egion and amyloids (Pa allel agg ega ion (PA) and Non agg ega ing esidue (NI));
d
P obabili y in seconda y s uc u e.
F. Ri e o-Pino e al.
Food Chemis y 473 (2025) 143084
9
