Food Resea ch In e na ional 156 (2022) 111156
A ailable online 17 Ma ch 2022
0963-9969/© 2022 The Au ho (s). Published by Else ie L d. This is an open access a icle unde he CC BY-NC-ND license (h p://c ea i ecommons.o g/licenses/by-
nc-nd/4.0/).
E alua ion o he po en ial p ebio ic e ec o Himan halia elonga a, an
A lan ic b own seaweed, in an in i o model o he human dis al colon
A oa Lopez-San ama ina, Alejand a Ca delle-Cobas
*
, Alicia del Ca men Mond agon ,
Lau a Sinis e a-Loaiza , Jose Manuel Mi anda , Albe o Cepeda
Labo a o io de Higiene Inspecci´
on y Con ol de Alimen os, Depa amen o de Química Analí ica, Nu ici´
on y B oma ología, Uni e sidade de San iago de Compos ela,
27002 Lugo, Spain
ARTICLE INFO
Keywo ds:
Gu mic obio a
In i o
Fe men a ion
Fib e
H. elonga a
Algae
Diges ion
16S RNA
ABSTRACT
Un il now, al hough di e en s udies ha e shown he po en ial p ebio ic e ec o seaweed ca bohyd a es, no
s udies wi h he whole seaweeds ha e been ca ied ou . In addi ion, he p ebio ic e ec h oughpu sequencing
emains poo ly in es iga ed since mos o he published wo ks used qPCR o FISH o es ima e bac e ial changes.
In his wo k, an in i o model o he human dis al colon was used o de e mine, o he i s ime, he po en ial
p ebio ic e ec o a b own whole seaweed Himan halia elonga a. The whole seaweed was cha ac e ized in basis o
i s nu i ional and mine al composi ion and submi ed o he en i e gas oin es inal diges ion. The p ebio ic
e ec was e alua ed by he mic obial modula ion h ough 16S RNA amplicon sequencing, qPCR and sho -chain
a y acid analysis. The ob ained esul s indica ed ha he colonic ac ion o H. elonga a was used selec i ely by
he Bac e oides genus, mo e speci ically by he specie Bac eoides o a us, whe eas inulin was used mainly by he
Pa abac e oides genus, being Pa abac e oides dis asonis he mos abundan iden i ied specie. Selec i e use o inulin
by P. dis asonis is, he e o e, epo ed by he i s ime. qPCR analysis shown no signi ica i e di e ences in
Bi idobac e ium popula ion and a dec ease in Lac obacillus along he e men a ion assays wi h bo h subs a es.
Rega ding o he sho - a y acid p oduc ion, maximal concen a ion, 56.11 ±20.48 mM, was achie ed o
H. elonga a, a 24 h o e men a ion whe eas o inulin o al acid p oduc ion was 93.66 ±21.82 mM a 48 h o
assay. The me abolic pa hways associa ed wi h bac e ial gene a we e no signi ican ly di e en be ween he wo
es ed subs a es. Al hough mo e s udies a e necessa y o elucida e he p ebio ic cha ac e o H. elonga a, he
esul s p esen ed in his wo k a e p omisso y and could open new oppo uni ies o esea ch and applica ion in
he a ea o Nu i ion and Food Chemis y.
1. In oduc ion
Die a y ibe (DF) is subs an ially composed o non-diges ible poly-
saccha ides, whose consump ion in adequa e amoun s has nume ous
bene i s o human heal h (C onin, Joyce, O’Toole, & OConno , 2021).
In his sense, an inc ease in DF consump ion has been ecommended
pe sis en ly in Wes e n socie ies since he 1970 s, wi h he aim o ach-
ie e an a e age daily in ake o 25–35 g/day in adul s (S ephen, Champ,
Clo an, Flei h, an Lieshou, Mejbo n e al., 2017). Ne e heless, he
a e age consump ion by people in Wes e n coun ies has con inuously
emained below hese ecommenda ions (S ephen e al., 2017).
Cu en ly, mos o he non-diges ible polysaccha ides included in he
Wes e n die come om he cell walls o e es ial plan s (Lopez-
San ama ina, Mi anda, Mond agon, Lamas, Ca delle-Cobas e al., 2020).
Howe e , due o in ense popula ion g ow h wo ldwide in ecen de-
cades, eshwa e , an essen ial commodi y o ag icul u e, is becoming
inc easingly sca ce (Cosg o e & Loucks, 2015). Acco ding o he Food
and Ag icul u e O ganiza ion (FAO, 2017a,b), global ag icul u al p o-
duc i i y is declining a a ound 1% pe yea . Because o his decline, i is
easonable o expec ha he demand o ood om o he esou ces will
inc ease in he coming yea s.
Among nu i ional componen s ha can modula e he gu mic obio a
(GM) composi ion and unc ion, DF can play a cen al ole since i may
selec i ely p omo e he g ow h o ce ain bac e ia, such as Lac obacillus
o Bi idobac e ium, and exe bene icial e ec s o he hos ’s heal h
(Lopez-San ama ina e al., 2020). This phenomenon, when occu s, is
* Co esponding au ho a : Labo a o io de Higiene Inspecci´
on y Con ol de Alimen os, Facul ad de Ve e ina ia, pabell´
on 4 p.b., Campus Uni e si a io, 27002 Lugo,
Spain.
E-mail add ess: [email p o ec ed] (A. Ca delle-Cobas).
Con en s lis s a ailable a ScienceDi ec
Food Resea ch In e na ional
jou nal homepage: www.else ie .com/loca e/ ood es
h ps://doi.o g/10.1016/j. ood es.2022.111156
Recei ed 17 Janua y 2022; Recei ed in e ised o m 13 Ma ch 2022; Accep ed 15 Ma ch 2022
Food Resea ch In e na ional 156 (2022) 111156
2
known as he p ebio ic e ec (Salminen, Collado, Endo, Hill, Lebee ,
Quigley e al., 2021). Howe e , i is impo an o highligh ha he
cu en de ini ion o p ebio ic does no e e o any bac e ial genus, since
di e en genus can exe bene icial e ec s o he hos and e en, di e en
genus can exe he same unc ionali y. Thus, p ebio ics a e “selec i ely
e men ed ing edien s ha allow speci ic changes, bo h in he compo-
si ion and/o ac i i y in he GM ha con e bene i s upon hos wellbeing
and heal h” (Gibson, Sco , Ras all, Tuohy, Ho chkiss, Dube -Fe andon
e al., 2010). Cu en ly di e en bac e ial gene a such En e ococcus,
S ep ococcus, Pediococcus, Leuconos oc, Bacillus o speci ic s ains o
Esche ichia coli a e conside ed bene icial and e en a e used as p obio ics
in di e en comme cial p epa a ions.
An adequa e DF in ake p o ides he hos wi h subs a es o
e men a ion by gu mic obes ha possess he necessa y enzyma ic
machine y o deg ade complex ca bohyd a es (C onin e al., 2021) and
he e o e, depending on he DF s uc u e di e en bac e ia can be
s imula ed.
Rega ding seaweed composi ion, DF ep esen s up o 76% exp essed
on a d y ma e basis, in some seaweed species (Cha oensiddhi, Conlon,
Vua an, F anco, & Zhang, 2017). In addi ion, seaweeds con ain phenolic
compounds and p o eins ha has been p oposed as po en ial bene icial
ing edien s o human heal h (Cha oensiddhi e al., 2017). Among all
he po en ial bene i s o hese ing edien s, he p ebio ic ac i i y has been
one o he mos s udied and b own seaweeds he g oup seaweeds mo e
employed (Lopez-San ama ina e al., 2020). This po en ial p ebio ic
e ec has been e alua ed mainly in in i o assays simula ing he human
la ge in es ine using aeces as inoculum and di e en ca bohyd a e
ac ions, wi hou a p e ious s ep o diges ion in he uppe gas oin-
es inal ac (Cha oensiddhi e al., 2017; Chen e al., 2018; Fu e al.,
2018; S ain e al., 2020). In all hese assays an inc ease in SCFAs was
obse ed, howe e , ega ding he bac e ial popula ion, he esul s a e
con adic o y, in some cases Bi idobac e ium and Lac obacillus inc eased
whe eas in o he s, dec eased, depending on he ca bohyd a e ex ac
es ed. Howe e , all hese s udies indica e he po en iali y o he ca -
bohyd a e seaweeds as p ebio ics. Se e al in i o s udies wi h mice has
also been ca ied ou (Kim, Yu, Kim, Choi, Lee, Hong e al., 2016;
Nguyen, Kim, Gue a a, Lee, Kim e al., 2016). F om hese s udies, using
mainly ex ac s o he b own seaweed Ascophyllum nodosum, i is no
possible o ex ac clea conclusions because he esul s a e con adic-
o y. Some au ho s indica e no mic obial changes a e he ex ac
consump ion, whe eas o he epo ed an inc ease in he mic obio a di-
e si y and an inc ease in Bac e oides and Pa abac e oides species. I is
impo an o indica e ha he condi ions o he s udies, no ably a y
om one o ano he in e ms o ex ac s composi ion, dosage and ime o
exposu e.
These in e es ing p e ious esul s, posi i e o no , oge he wi h he
ac ha seaweed consump ion has inc eased in Spain and he Eu opean
Union makes he seaweeds an in e es ing p oduc o esea ch abou i s
unc ionali y. Thus, in he p esen wo k, he e alua ion o he po en ial
p ebio ic e ec o a whole b own oceanic seaweed, Himan halia elonga a,
was ca ied ou , o he i s ime, in an in i o model o he human
colon. This specie, also known as sea spaghe i, was selec ed because i is
p edominan in he Rias Baixas coas o Galicia (no hwes Spain), which
is he Spanish egion wi h he highes seaweed p oduc ion (Des, Ma -
inez, de Cas o, Viejo, Sousa, Gomez-Ges ei a e al., 2020), only in he
yea 2021a o al o 38,149 kg o b own seaweed we e collec ed.
2. Ma e ials and me hods
2.1. Seaweed
H. elonga a was ob ained, dehyd a ed, om Po omui˜
nos (Ce ceda, A
Co u˜
na, Spain). Two hund ed i y g ams o seaweed we e sampled,
c ushed, and eeze-d ied p io o es ing. The sample was s o ed a
oom empe a u e o u he analysis.
2.2. P oxima e and mine al composi ion o H. elonga a
The nu i ional composi ion o H. elonga a was de e mined p io o
and a e in i o diges ion. The nu i ional analysis o he seaweed was
ca ied ou ollowing o icial me hodologies es ablished by he Associ-
a ion o O icial Analy ical Chemis s (AOAC, 2002). The mois u e con-
en (g/100 g o p oduc ) was de e mined by d ying in a po celain
capsule a 100–105 ◦C in a mu le u nace (SNOL 8.2/1100-1, Umega
G oup AB, Li huania). The p o ein con en was de e mined by
measu ing he ni ogen con en (g/100 g o d y ma e ) by he Dumas
me hod. The a con en (g/100 g o d y ma e ) was de e mined by he
Soxhle ex ac ion me hod using pe oleum e he . The DF con en (g/
100 g o d y ma e ) was de e mined by using an enzyma ic–g a ime ic
me hod ia he Megazyme® o al die a y ibe assay ki (Megazyme,
Wicklow, I eland). The ash con en (g/100 g o d y ma e ) was de e -
mined by mu le u nace (SNOL 8.2/1100-1, Umega G oup AB,
Li huania) incine a ion a 500 ◦C The sodium con en (mg/100 g o d y
ma e ) was de e mined by a omic abso p ion spec ome y (AES, Agi-
len 5900, Agilen Technologies, San a Cla a, Cali o nia, USA). The
ca bohyd a e (g/100 g o d y ma e ) and calo ic con en s (kcal/100 g o
p oduc ) we e de e mined h ough calcula ions.
Rega ding mine als, seaweed was analyzed o
75
As,
43
Ca,
111
Cd,
63
Cu,
56
Fe,
127
I,
208
Pb, and
66
Zn (mg/kg) by induc i ely coupled plasma-
mass spec ome y (ICP-MS, Agilen 7700x, Agilen Technologies, San a
Cla a, CA, USA). Sample blanks we e p epa ed in he labo a o y in a
simila manne o he seaweed samples. P oxima e and mine al
composi ion analyses we e pe o med in iplica e bo h be o e and a e
diges ion.
2.3. In i o simula ion o o al, gas ic, and small in es inal diges ion
In i o o al, gas ic, and small in es inal diges ion was ca ied ou in
acco dance wi h he INFOGEST p o ocol (B odko b, Egge , Alminge ,
Al i o, & Assunçao, 2019). All chemicals we e pu chased om Sigma-
Ald ich (San Louis, MO, USA). Calcula ions o he di e en luids
and enzymes using he p o ocol we e made o 10 g o sample.
A he end o he in i o diges ion p ocess, he beake s wi h he
diges ed seaweed solu ion we e cooled in an ice-ba h o s op he enzy-
ma ic eac ions. A e ha , small in es inal abso p ion was simula ed by
dialysis (molecula weigh cu o o 1000 Da, Spec a/Po ®, Wal ham,
MA, USA) agains dis illed wa e a 4 ◦C o 2 days wi h agi a ion.. This
p ocess allows sepa a e molecules in solu ion by he di e ence in hei
a es o di usion h ough he semipe meable memb ane. Then, he
memb ane con en was ozen a −18 ◦C o subsequen eeze-d ying.
The eeze-d ying p ocess was pe o med by using a Vacuum F eeze
D ie (Labconco
TM
77560-LYPM-LOCK6), unde a acuum p essu e ≤
140 ×10
-3
Mba and a condense empe a u e o −46 ◦C. The en i e in
i o diges ion p ocess, including dialysis, was pe o med in iplica e.
2.4. Volun ee s and p epa a ion o s ool samples
S ool samples we e ob ained om h ee heal hy human olun ee s
(one male and wo emales, 32–50 yea s old), pa icipa ing in a ial
au ho ized by he Galician Bioe hics Commi ee ( ial 270/2018). These
olun ee s did no inges an ibio ics o pha maceu ical p epa a ions o
p e/p o/pos bio ics in he 6 mon hs p io o sample collec ion, and hey
had no gas oin es inal diso de . All o hem signed an in o med consen
documen in which hey we e in o med how hei samples would be
used, abou compliance o he s udy wi h he Decla a ion o Helsinki,
and abou he Spanish law abou pe sonal da a p o ec ion.
S ool samples (be ween 10 and 30 g each) we e collec ed by olun-
ee s in s e ile con aine s and gi en o he labo a o y wi hin 2 h o hei
collec ion. Once ecei ed, he s ool samples we e dilu ed 1:10 wi h
phospha e-bu e ed saline (PBS; 0.1 M, pH 7.0) and hen homogenized
in a s e ile bag using a paddle homogenize (MIX2, AES, Combou g,
F ance) o 5 min. The dilu ed eces we e s o ed in s e ile ja s and ozen
A. Lopez-San ama ina e al.
Food Resea ch In e na ional 156 (2022) 111156
3
a −20 ◦C un il use.
2.5. In i o human colonic simula ion
The in i o human colonic simula ion was ca ied ou acco ding o
Ca delle-Cobas, Olano, Co zo, Villamiel, Collins, Kolida e al. (2012).
B ie ly, a s e ilized e men a ion essel wi h a capaci y o 300 mL con-
aining a basal cul u e medium wi hou any sou ce o ca bon was used o
simula e human dis al colonic e men a ion. Th ee expe imen s we e
ca ied ou simul aneously: one o H. elonga a, one o inulin om
chico y (Sigma-Ald ich, S Louis, USA, p oduc numbe I2255), which
was selec ed as posi i e con ol, and one wi hou a ca bon sou ce,
wi hou subs a e (nega i e con ol).
Di e en pa ame e s we e adjus ed o simula e he condi ions o he
human dis al colon. Thus, anae obiosis was achie ed by using a
con inuous supply o O
2
- ee N
2
(Nippon Gases, Mad id, Spain) h ough
a 0.2
μ
m poly e a luo e hylene il e (Sa o ius S edim Bio ech GmbH,
Go ingen, Ge many). A he mos a ic ba h (Pha macia Bio ech,
Ams e dam, Ne he lands) was used o eci cula e wa e a 37 ◦C
h oughou he essel’s wa e jacke s o simula e he human in e nal
body empe a u e. The pH o he colon was se a 6.8 because ha is he
pH o he colon in a si ua ion o eubiosis. This pH was con olled by a pH
egula o (Hanna Ins umen s, Eiba , Spain), adding 1 M NaOH o HCl as
app op ia e.
Each e men a ion essel was illed, unde asep ic condi ions, wi h
200 mL o au ocla ed nu ien basal medium, p epa ed acco ding o
Ca delle-Cobas e al. (2012). All included chemicals we e pu chased
om Sigma-Ald ich, Me ck (Da ms ad , Ge many), o Pan eac (Ba ce-
lona, Spain). Then, he medium was adjus ed o pH 6.8 and le o e -
nigh wi h a s eam o O
2
- ee N
2
wi h s i ing. Nex , subs a es we e
s e ilized by boiling a ecipien con aining hem du ing 10–15 min (H
elonga a o inulin) and dissol ed in 52 mL o he same au ocla ed me-
dium and added o he essels a a inal concen a ion o 1% (w/ ). This
concen a ion was used based on p e ious s udies abou he e alua ion
o he p ebio ic e ec in i o, whe e he subs a e o be es ed we e used
a 1-2% (Baju y, Rawi, Sazali, Abdullah, & Sa dini, 2017; Ca delle-
Cobas e al., 2012). Finally, he essels we e inocula ed wi h 10% ( /
) (28 mL) o he p e iously p epa ed dilu ed eces.
Aliquo samples (3 mL) we e emo ed om each essel a e 0, 10,
24, and 48 h o incuba ion o eal- ime polyme ase chain eac ion
(qPCR) analysis, 16S ibosomal RNA ( RNA) amplicon sequencing, and
sho -chain a y acid (SCFA) analysis.
2.6. Bac e ial DNA ex ac ion om e men a ion samples
Bac e ial DNA was ex ac ed om he e men a ion samples by using
he DNA Realpu e Spin Food-S ool Ki ® (Real, Du iz S.L, Valencia,
Spain) ollowing he ins uc ions p o ided by he manu ac u e o ecal
samples. The pelle ob ained a e cen i uga ion (Cen i uge 5415D,
Eppendo , Hambu g; Ge many; 6100 g) o 1.2 mL o sample ( e men-
a ion essels) was eco e ed and used o DNA ex ac ion.
The ob ained DNA was quan i ied by using a Qubi ™4 luo ome e
(In i ogen, The mo Fishe Scien i ic, Ca lsbad, CA, USA), and he DNA
HS Assay Ki (In i ogen, The mo Fishe Scien i ic, Eugene, OR, USA).
DNA samples we e s o ed a −20 ◦C un il u he analysis.
2.7. Bac e ial quan i ica ion by qPCR
qPCR assays we e ca ied o cha ac e ize ecal bac e ia using phylum-
and species-speci ic p ime s o o al bac e ia, Fi micu es, Bac e oide es,
Ac inobac e ia, P o eobac e ia, Lac obacillus, and Bi idobac e ium, p e-
iously desc ibed (Mu i, Lei a, Gomez-Zumaque o, Tinahones, Ca -
dona, So igue e al., 2013).
B ie ly, qPCR expe imen s we e pe o med in a Quan S udio 12 K
Flex (Applied Biosys ems, Li e Technologies Holding, Singapo e,
Singapo e) equipmen using he as SYBR
TM
g een mas e mix (Applied
Biosys ems, Vilnius, Li huania). All PCR es s we e ca ied ou in ip-
lica e, wi h a inal olume o 10
μ
L con aining 1
μ
L o each sample DNA,
p ime s (0.4
μ
L) added a a concen a ion o 200 nM ( o each p ime ), 5
μ
L o as SYBR
TM
g een mas e mix (Applied Biosys ems), and 3.2 µL o
molecula biology g ade wa e . The he mal cycling condi ions used
we e as ollows: an ini ial DNA dena u a ion s ep a 95 ◦C o 10 min,
ollowed by 45 cycles o dena u a ion a 95 ◦C o 10 s, p ime annealing
a an op imal empe a u e o 20 s, and ex ension a 72 ◦C o 15 s.
Finally, mel cu e analysis was pe o med by slowly cooling he e-
ac ions om 95 ◦C o 60 ◦C (0.05 ◦C pe cycle) wi h simul aneous
measu emen o he SYBR g een signal in ensi y. Mel ing-poin -
de e mina ion analysis allowed con i ma ion o he speci ici y o he
ampli ica ion p oduc s.
The bac e ial concen a ion (copies/mL) om each sample was
calcula ed by compa ing he h eshold cycle (C ) alues ob ained om
he s anda d cu es wi h he Quan S udio 12 K Flex So wa e (Applied
Biosys ems). S anda d cu es we e cons uc ed o each expe imen by
using 10- old se ial dilu ions o bac e ial genomic DNA (o known
concen a ion) om pu e cul u es, co esponding o 10
1
o 10
10
copies/
mL o e men a ion media.
The pu e cul u es used o cons uc he s anda d cu es we e ob-
ained om di e en collec ions o ype cul u es— om he Spanish
Collec ion (CECT), he Belgian Co-o dina ed Collec ions o Mic o-
o ganims (LMG), and he Ge man Collec ion o Mic oo ganisms and cell
Cul u es GmbH (DSM)—as ollows: En e obac e cloacae CECT 194,
Clos idium pe ingens CECT 376, Bi idobac e ium longum CECT 4503,
Bac e oides ulga us LMG 17767, and Lac obacillus eu e i DSM 20016.
Each bac e ial s ain was g own in i s equi ed cul u e media and g ow h
condi ions and hen, DNA was ex ac ed and dilu ed o cons uc he
s anda d cu es.
The inal da a a e exp essed as an a e age o he duplica e alues
ob ained in he analyses. The e iciency o he eac ion o all pai s o
p obes was de e mined by using he slope o he calib a ion cu e ob-
ained o each o he bac e ial g oups analyzed, namely E =10^(-1/
slope). Fo he p ime pai s used in his s udy, he e iciency anged om
95% (E =1.90) o 104% (E =2.07), wi h slopes in he ange o −3.59 o
−3.16.
2.8. 16S RNA amplicon sequencing
Fo 16S RNA amplicon sequencing, 12
μ
L o DNA ex ac ed om
each sample was used o cons uc he lib a ies and he Ion GeneS u-
dio
TM
S5 Sys em (Li e Technologies, Ca lsbad, CA, USA) was used. Fo
his pu pose, he 16S hype a iable egions we e ampli ied wi h wo se s
o p ime s, 2-4–8 and 3-6,7–9, and he lib a ies we e p epa ed by
using he Ion 16S
TM
Me agenomics Ki (Li e Technologies) and he Ion
Xp ess
TM
Plus F agmen Lib a y Ki (Li e Technologies). Lib a ies con-
aining equal amoun s o PCR p oduc s pooled wi h a ba code we e
p epa ed by using he Ion Xp ess
TM
Ba code Adap e s Ki (Li e Tech-
nologies). Then, hese lib a ies we e quan i ied by using he Ion Uni-
e sal Lib a y Quan i a ion Ki (Li e Technologies). Nex , 10 pM o each
lib a y was pooled and loaded on an Ion OneTouch™ 2 Sys em (Li e
Technologies), which au oma ically pe o ms empla e p epa a ion and
en ichmen . Templa e-posi i e ion sphe e pa icles we e en iched wi h
Dynabeads™ MyOne™ S ep a idin C1 magne ic beads (In i ogen,
Ca lsbad, CA, USA) by using an Ion One Touch ES ins umen . Finally,
an Ion 520
TM
chip (Li e Technologies) was loaded wi h he samples on
an Ion GeneS udio
TM
S5 Sys em sequence using he Ion 520™ & Ion
530™ Loading Reagen s supplied in he OT2-Ki (Li e Technologies).
2.9. Sho -chain a y acids analysis
SCFA analysis was ca ied ou ollowing he p o ocol o Gull´
on,
Gull´
on, Sanz, Alonso, and Pa aj´
o (2011). One mL o e men a ion sam-
ples ob ained a e 0, 10, 24, and 48 h we e cen i uged o 7 min a 6100
g. The supe na an s we e emo ed and il e ed h ough 0.2 µm cellulose
A. Lopez-San ama ina e al.
Food Resea ch In e na ional 156 (2022) 111156
4
ace a e memb anes. Then, 20
μ
L o sample was injec ed in an Agilen
1200 se ies HPLC ins umen equipped ins umen wi h a e ac i e
index de ec o (Agilen , Waldb onn, Ge many). Sepa a ions we e ca -
ied ou on an Aminex HPX-87H column (Bio-Rad, He cules, Cali o nia,
USA). The elu ion sys em consis ed o sul u ic acid (0.003 M) ope a ing
isoc a ically wi h a low a e o 0.6 mL/min. Samples we e un a 50 ◦C.
S anda ds o o ganic acids (lac ic, o mic, ace ic, bu y ic, p opionic,
isobu y ic, ale ic, iso ale ic and succinic acids) we e ob ained om
Sigma (Poole, Do se , UK). Peaks we e iden i ied by compa ison wi h he
e en ion imes o he s anda ds and quan i ied by eg ession o mula
ob ained wi h he s anda ds.
2.10. S a is ical and bioin o ma ic analysis
A wo-way ANOVA was used o de e mine signi ica i e di e ences
o ime and subs a es as he wo co a ia es in he gene al linea model
using Tukey’s analysis. Fo signi ican di e ences (p <0.05) a one-way
ANOVA was conduc ed o each subs a e compa ing 0, 5, 10 and 24 h.
simila ly, each subs a e was compa ed i wo-way ANOVA esul s we e
signi ican (p <0.05). SPSS® .27 o Windows (SPSS Inc., Chicago, IL,
USA) was used o hese analyses.
Fo he analysis o 16S RNA amplicon sequencing, he aw
sequencing eads we e ob ained om he To en Sui e so wa e
( .5.12.2.) as BAM iles, which we e con e ed o as q iles wi h BED-
Tools w apped in o he Public Galaxy Se e (h ps://usegalaxy.eu/, .
21.05) (A gan, Bake , Ba u , an den Beek, Bou ie , Cech e al., 2018) by
G uening (2014) Galaxy w appe (h ps://gi hub.
com/bg uening/galaxy ools). The as q iles we e p ocessed wi h
QIIME 2 so wa e . 2021.8 (Bolyen, Rideou , Dillon, Bokulich, Abne ,
Al-Ghali h e al., 2019). To p oduce amplicon sequence a ian s (ASVs),
he DADA2 me hod was used o quali y il a ion (Q sco e >30),
imming, denoising, and de eplica ion. Samples wi h ea u es ( axa)
wi h a o al abundance (summed ac oss all samples) o <10 we e
emo ed. Then, ASVs we e aligned wi h ma and used o cons uc a
phylogene ic ee wi h as ee.
α
- and β- di e si y me ics we e es i-
ma ed by using q2-di e si y co e-me ics-phylogene ic a e samples
we e a e ied o a sequencing dep h o 33,000 eads. Taxonomy was
assigned o ASVs by using he q2- ea u e-classi ie classi y-sklea n naï e
Bayes axonomy classi ie agains he G eengenes 13_8 99% ope a ional
axonomic uni (OTU) e e ence sequences. The PICRUS online Galaxy
e sion on he Hu enhowe Lab ( 1.0.0) se e was used o p edic he
me agenome unc ional con en om ma ke gene su eys and ull ge-
nomes. Func ional me agenomes we e ca ego ized based on he Kyo o
Encyclopedia o Genes and Genomes (KEGG) pa hway da abase a hi-
e a chy le el 3.
STAMP so wa e ( 2.1.3) o he “S a is ical Analysis o Taxonomic
and Func ional P o iles” (Pa ks, Tyson, Hugenhol z, & Beiko, 2014) was
used o de e mina e di e ences in me abolic unc ions. Welch’s - es s
wi h Bon e oni co ec ion we e used o de e mine signi ican di e -
ences in he ela i e abundance o 20 selec ed KEGG pa hways (le el 3).
In addi ion, di e ences in he ela i e abundance o he mos common
species we e de e mined by using a G- es (wi h Ya es’ co ec ion) +
Fishe ’s exac es wi h Bon e oni co ec ion.
3. Resul s and discussion
3.1. P oxima e and mine al composi ion o H. elonga a
The p oxima e composi ion o H. elonga a can be ound in Table 1.
Al hough some ac o s such as seasonali y can al e he nu i ional
composi ion o seaweeds, in global e ms, he esul s ob ained we e
close o hose p e iously epo ed by Fe n´
andez-Sego ia, Le ma-Ga cía,
Fuen es, & Ba a , 2018 o he same seaweed species collec ed a he
same loca ion (p o ein: 7.50% s. 6.8%; DF: 36% s. 39%; a : <0.5% s.
0.1%; ash: 33% s. 30%).
A e o al, gas ic, and small in es inal diges ion, including dialysis,
he weigh o he seaweed was educed by 51.82 ±1.58% (w/w), which
would be equi alen in an in i o assay o he loss due o ood hyd olysis
and abso p ion (Asensio-G au, Cal o-Le ma, He edia, & And ´
es, 2021).
Namely, only a ound a 48% o he seaweed would each he colon o
exe i s e ec on GM. A e diges ion, he seaweed exhibi ed a di e en
nu ien composi ion compa ed wi h he aw seaweed. I is well known
ha du ing diges ion, se e al key ac o s con ibu e o he p og essi e
al e a ion o he ood ma ix, including he enzymes in ol ed
h oughou he gas oin es inal ac , as well as he acidic condi ions a
each s age (Asensio-G au e al., 2021). Because o hese ac ions, he a ,
ca bohyd a e, and ash con en s signi ican ly dec eased du ing his
diges ion p ocess, whe eas he p o ein, DF, and calo ic con en s signi -
ican ly inc eased. One o he easons because he p o ein con en
inc eased in ela ion o he o he mac onu ien s is due o he o ma ion
o o ganic complexes in he seaweed, o example wi h iodine, as i has
been indica ed in p e ious wo ks (Roma í-Ho as, Bianga, Mo eda-
Pi˜
nei o, Be mejo-Ba e a, & Szpuna , 2014; Domínguez-Gonz´
alez,
Chiocche i, He bello-He melo, V´
elez, De esa, Be mejo-Ba e a e al.,
2017). F om hese s udies, i is possible o indica e ha in seaweeds as
wakame an impo an p opo ion o iodine is unde he o m o o ganic
iodine complexes (Shah, Wuilloud, Kannamkuma a h, & Ca uso, 2005),
which can each molecula weigh s g ea e han 10 kDa wha can lead,
in consequence, o a low dialyzabili y o iodine and p o ein (Domínguez-
Gonz´
alez e al., 2017).
Thus, om an ini ial H. elonga a composi ion in which he wo main
mac onu ien s a e ibe and ca bohyd a es, a e diges ion, p o ein and
ibe a e he main componen s and, he e o e, ac a he colonic le el on
GM.
Table 1 shows he mine al con en in H. elonga a be o e and a e he
diges ion p ocess. Wi h espec o he maximum esidue limi s o
mine als, in he Eu opean Union (EU) only a speci ic limi o iodine in
seaweed was es ablished (20 mg I/kg d y seaweed, Commission
Recommenda ion [EU] 2018/464), especially o popula ions wi h
endemic iodine de iciency. Table 1 shows ha he iodine con en o
d ied H. elonga a is below his limi (4.49 mg/100 g). Conside ing ha in
2006 he EU es ablished an uppe limi o iodine in ake o 600 µg/day
o adul s, consump ion o 15 g o d ied H. elonga a would each his
amoun . Iodine is essen ial o human heal h; i may be he apeu ic in
he case o nu i ional de iciencies, bu high alues due o excessi e
consump ion could cause ad e se heal h e ec s. Hence, hese kind o
seaweed p oduc s mus be consumed wi h cau ion. In his wo k, he
Table 1
Compa ison o he nu i ional composi ion (g/100 g) and mine al con en (mg/
100 g) o sea spaghe i (Himan halia elonga a) aw and a e in i o uppe in-
es inal diges ion. The calo ic con en is exp essed in kcal/100 g. The esul s a e
exp essed as he mean ±s anda d de ia ion. Di e en le e s indica e signi ican
di e ences (p <0.05).
Nu i ional
composi ion
Raw H. elonga a H. elonga a a e uppe in i o
diges ion
Fa 1.00
a
±0.20 0.64
b
±0.12
P o ein 7.50
b
±1.43 12.26
a
±2.44
Ca bohyd a es 15.00
a
±2.56 9.87
b
±2.17
Suga s <0.5 <0.5
Die a y ibe 36.00
b
±3.71 63.6
a
±4.31
Ash 33.20
a
±3.22 16.63
b
±2.89
Calo ic con en 170.00
b
±10.22 221.48
a
±15.67
Mine als
Ca 1,512.10
a
±332.80 1,2980
a
±185.72
Fe 4.30
a
±3.76 11.73
b
±4.58
Cu 0.21
a
±0.19 0.22
a
±0.03
Zn 4.37
a
±3.34 5.94
a
±0.77
As 0.60
a
±0.25 0.45
a
±0.15
Cd 0.03
a
±0.01 0.02
a
±0.02
Pb 0.02
a
±0.02 0.05
a
±0.06
I 4.49
a
±6.77 194.67
b
±9.41
A. Lopez-San ama ina e al.
Food Resea ch In e na ional 156 (2022) 111156
5
objec i e was o show ha small amoun s o hese p oduc s (speci ically
H. elonga a) could exe a bene icial e ec on human heal h by modu-
la ing GM and ac ing as a po en ial p ebio ic. Mos p ebio ics o he gu
equi e an o al dose o a ound 3 g pe day o mo e o con e a bene i .
Typically, a ound 5 g is he a ge o uc ooligosaccha ides (FOS) and
galac ooligosaccha ides (GOS) in he daily die , and his includes plan
sou ces o p ebio ics (Salminen, e al., 2021). Consump ion o 5 g o
H. elonga a would be equi alen o consuming 200
μ
g o I, no exceeding
he es ablished limi s.
A senic in biological ma ices exis s ei he in o ganic o ms (e.g.,
a senobe aine and a senosuga s) o as ino ganic a senic (iAs) (Edmonds
& F ancesconi, 2003). While o ganoa senic o ms a e conside ed o be
non- oxic o o low oxici y, iAs is ega ded as he mos oxic o m o As
o li ing o ganisms. Thus, he p esence o As in seaweeds has sa e y
implica ions o hei use as ood. Howe e , egula ions on As in ood a e
cu en ly limi ed in he EU, and no maximum allowed le els o As
(ei he o al As o iAs) in ege ables o ood supplemen s exis (Pe u s-
do i , Slo h, & Feldmann, 2015). Al hough As specia ion in ish and
sea ood has a ac ed a lo o a en ion because o he high o al As
le els, hese commodi ies a e no gene ally impo an con ibu o s o
iAs die a y exposu e, excep o shell ish and seaweed in he egions
whe e consump ion le els o hese i ems a e app eciable (Pe u sdo i ,
Slo h, & Feldmann, 2015).
Maximum limi s o o he hea y me als ha e been es ablished by
Eu opean Commission egula ion (EC) 1881/2006, howe e , none a e
speci ic o seaweeds. Rega ding he ole able uppe in ake le els (UL)
o adul s, ou da a a e a below: Ca (UL: 2500 mg/day), Fe (no UL), Cu
(5 mg/day), Zn (25 mg/day) (Eu opean Food Sa e y Au ho i y, EFSA,
2006), Pb (25
μ
g/kg body weigh [bw]/week) (EFSA, 2010), and Cd
(2.5 mg/kg bw/weekly) (EFSA, 2011).
Compa ed wi h p e ious wo k, he Ca composi ion in he cu en
wo k (1.5 g/100 g) is like he alues ob ained by Fe n´
andez-Sego ia
e al. (2018) o he same seaweed. Rega ding Cd and Pb, he ob ained
concen a ions a e lowe han hose ound by Filippini, Baldisse o o,
Meno a, Fed izzi, Rubini, Giglio i e al. (2021) (0.03 s. 0.07 mg/kg Cd;
0.02 s. 0.06 mg/kg Pb) o he same seaweed in I aly.
One o he main isks ha seaweed consump ion can con e o
humans is he po en ial excess o hea y me als and iodine. Elemen s
such as Fe, Ca, Cu, and Zn a e equi ed o g ow h, enzyma ic eac ions,
and me abolic ac i i ies o ma ine o ganisms (Velusamy, Sa heesh
Kuma , Ram, & Chinnadu ai, 2014; Fa ias e al., 2021). Howe e , hey
can also be oxic o humans a ele a ed concen a ions, especially when
seaweeds g ow in pollu ed wa e s (Sudha san, Seede i, Ramasamy,
Subhap adha, Vai a, & Shanmugam, 2012). High concen a ions o
iodine ha e been ound in se e al coun ies o ce ain seaweed species
(Cha oensiddhi e al., 2017). O he elemen s such as As, Hg, Cd, and Pb
can be oxic e en a low concen a ions, causing cellula damage,
educed ep oduc ion and g ow h a es, o e en dea h (Velusamy e al.,
2014). No all e ec s o hea y me als a e ha m ul, because some min-
e als (such as Zn o Cu) a e essen ial o many enzymes ha p omo e
main enance, o ma ion, and homeos asis o body issues and pe o m
me abolic ac i i ies (Fa ias e al., 2021). Mo eo e , GM equi es ce ain
hea y me als such as Fe, Cu, o Zn o hei g ow h and me abolism
(Sizen so , Sizen so , K an, Salniko a, & Salniko a, 2019).
The e we e no signi ican changes in he mine al con en o
H. elonga a be o e and a e diges ion in he uppe in es inal ac , excep
o iodine and Fe, which a e concen a ed a e diges ion and dialysis. I
is impo an o no e ha some s udies ha e shown he limi a ions o
dialysis memb anes in he s udy o he bioa ailabili y o elemen s
(Domínguez-Gonz´
alez e al., 2017). On he one hand, he simula ed
gas oin es inal diges ion may cause he b eakdown o iodine complexes
in mac omolecules p esen in he seaweed ma ix, inc easing he soluble
iodine ac ion (Domínguez-Gonz´
alez e al., 2017). On he o he hand,
iodine complexes canno c oss he dialysis memb anes when he po e is
oo small, a ac o ele an o he p esen wo k gi en ha he dialysis
memb ane has a 1 kDa cu o . A p e ious s udy ca ied ou in es iga ing
Table 2
Bac e ial popula ion (log
10
DNA copies/mL) in he in i o colon model a 0, 10, 24, and 48 h o e men a ion. The esul s a e exp essed as he mean ±s anda d e o (n =6). nd, no de ec ed. A wo-way ANOVA using
gene al lineal model, pe o med wi h ime and subs a es as wo co a ia es o each bac e ial g oup, indica ed signi ican di e ences be ween bo h ime and subs a es, p <0.05. Then, a one-way ANOVA wi h Tukey’s es
was pe o med o de e mine a signi ican inc ease/dec ease o bac e ial popula ions wi h ime o he same subs a e (di e en lowe case le e s indica e signi ican di e ences), also a one-way ANOVA analysis was
ca ied ou o compa e subs a es a 10, 24 and 48 h (indica ed wi h capi al le e s).
H. elonga a Inulin Nega i e con ol
0 h 10 h 24 h 48 h 10 h 24 h 48 h 10 h 24 h 48 h
All bac e ia 9.45
a
±0.18 10.08
a,A
±0.01 10.05
a,A
±0.01 11.78
b,A
±0.01 9.74
a,B
±0.02 8.39
b,B
±0.01 11.79
c,A
±0.01 10.66
b,C
±0.01 10.69
b,A
±0.03 10.62
b,B
±0.01
Fi micu es 6.29
a
±0.18 6.66
a,b,A
±0.01 7.80
b,c,A
±0.02 7.90
c,A
±0.01 6.46
a,B
±0.03 5.57
a,B
±0.02 8.02
b,A
±0.06 7.21
a, b ,C
±0.01 7.24
A
±0.01 7.12
a,b,B
±0.01
Lac obacillus 1.38
a
±0.03 nd nd nd 0.91
b,A
±0.01 0.26
c,A
±0.03 0.23
c,A
±0.02 0.46
b,B
±0.01 0.23
c,A
±0.01 0.01
c,B
±0.01
Ac inobac e ia 6.11
a
±0.34 5.87
a,A
±0.02 6.13
a,B,C
±0.08 5.77
a,A
±0.04 6.11
a,A
±0.14 5.65
a,B
±0.15 6.75
a,A
±0.01 7.37
a,B
±0.01 6.98
a,C
±0.02 6.65
a,B
±0.02
Bi idobac e ium 5.11
a
±0.30 4.09
a,A
±0.14 5.94
aA
±0.21 4.94
a,A
±0.08 5.52
a,B
±0.01 5.22
a,A
±0.01 5.51
a,B
±0.04 4.66
a,A
±0.01 4.37ª
,B
±0.02 4.05
a,C
±0.01
Bac e oide es 6.92
a
±0.12 4.83
b,A
±0.01 7.87
c,A
±0.02 7.41
a,c,A
±0.02 5.15
b,A
±0.05 5.58
b,B
±0.04 8.38
c,A
±0.01 6.03
b,B
±0.09 6.00
b,C
±0.01 5.87
b,B
±0.18
P o eobac e ia 5.44
a
±0.28 8.62
b,A
±0.01 7.90
b,A
±0.02 7.60
b,A
±0.45 8.63
b,c,A
±0.02 7.41
b,B
±0.04 9.36
c,A
±0.02 9.77
b,B
±0.02 9.84
b,C
±0.06 9.57
b,A
±0.02
A. Lopez-San ama ina e al.

Food Resea ch In e na ional 156 (2022) 111156
6
he in es inal bioa ailabili y and bioa ailabili y o wakame seaweed
con i med his ac (Domínguez-Gonz´
alez e al., 2017). The e is a simila
si ua ion o Fe: his me al is pa o high-molecula -weigh complexes
such as e i in and canno c oss he dialysis memb anes, al hough
humans abso b i on a he in es inal le el (Fai wea he -Tai e al.,
2005).
3.2. qPCR analysis
Quan i ica ion o he main phyla as well as o Bi idobac e ium and
Lac obacillus species was ca ied ou o all samples by using qPCR. The
ob ained esul s a e shown in Table 2. The e was an inc ease in o al
bac e ia o e ime, wi h he maximum a e 48 h o e men a ion o all
cases. O he wo ks in es iga ing he e ec s o o al bac e ia ound a
maximum a e 24 h o e men a ion (V´
azquez-Rod íguez, San os-Zea,
He edia-Olea, Ace edo-Pacheco, San ac uz, Gu i´
e ez-U ibe e al.,
2021). P o eobac e ia was he phylum wi h he g ea es inc ease. Com-
pa ison o he inulin and H. elonga a assays a e 24 and 48 h did no
show signi ican changes. Fi micu es also inc eased wi h ime. Fo
H. elonga a, i eached he maximum alues a e 24 and 48 h. Fo inulin,
howe e , he e was only a signi ican change a e 48 h compa ed wi h
0 h. A sligh inc ease was obse ed o he con ol. When compa ing
bo h subs a es, signi ican changes we e ound a 24 h, being he highe
alue o H. elonga a.
Fo Bac e oide es, he e was a sligh dec ease du ing he i s 10 h o
assay may be due o an es ablishmen o adap a ion o he media o he
bac e ia p esen in he ecal inoculum. This dec ease occu s in all cases,
con ol, seaweed and inulin. A e his 10 h, a signi ican inc ease in
Bac e oide es occu ed a 24 and 48 h o he assay wi h H. elonga a
compa ed wi h 0 and 10 h. Simila esul s we e ound o he case o
Bac e oides genus using di e en polysaccha ides om seaweeds (Seong,
Bae, Seo, Kim, Kim, & Han, 2019). Fo he assay wi h inulin, he
maximum (and signi ican ) alue was obse ed a 48 h. On he con a y,
he e was a signi ican dec ease in Bac e oide es o he con ol assay o
all e men a ion imes.
Compa ing he e men a ion assays o inulin and H. elonga a a e
24 h, he e was a signi ican ly highe alue o H. elonga a a e 24 h and
a signi ican ly highe alue o inulin a e 48 h. This could indica e a
as e consump ion o ca bohyd a es in he seaweed compa ed wi h
inulin.
The e was a signi ican dec ease in Lac obacillus, whe eas he e we e
no signi ican changes o Bi idobac e ium. O he au ho s, con a iwise,
also ound signi ican inc ease in Bi idobac e ium coun s a e seaweeds
e men a ion, such as Ecklonia adia a (Cha oensiddhi e al., 2017) o
di e en seaweed polysaccha ides ex ac s (Seong e al., 2019).
3.3. Amplicon 16 RNA sequencing
The GM composi ion was e alua ed a he ela i e le el (16S RNA
amplicon sequencing; Fig. 1) and he absolu e le el (quan i ica ion o
he main phylum and Bi idobac e ium and Lac obacillus species by qPCR,
Table 3) be o e ( ime 0) and a a ious imes du ing he e men a ion
assays wi h he subs a es (H. elonga a and inulin). The ela i e e-
quency (Fig. 1) a he phylum le el showed ha bo h inulin and
H. elonga a modi ied he GM composi ion compa ed wi h he con ol.
Whe eas a ime 0 Fi micu es and Bac e oide es we e he p edominan
phyla (67% o Fi micu es and 29% o Bac e oide es), a e 10 h o
e men a ion, he e was an inc ease in he phylum P o eobac e ia o
inulin (75%) and H. elonga a (54%). This inc ease was also obse ed in
he con ol (no sou ce o ca bon added) bu a a lowe pe cen age (47%).
The e was a dec ease in Fi micu es compa ed wi h ime 0 o bo h
subs a es; i was mo e p onounced o H. elonga a, whe e only 10% o
he o al bac e ia belonged o Fi micu es phylum.
A e 24 and 48 h o e men a ion wi h H. elonga a, he Bac e oide es
phylum inc eased no ably, while bo h Fi micu es and P o eobac e ia
phyla dec eased. Fo inulin, a e 24 h o e men a ion, he Bac e oide es
phylum inc eased wi h espec o 10 h o incuba ion (19% s. 4%), bu
he majo change occu ed a e 48 h, whe e his phylum ep esen ed
62% o he o al bac e ia. O no e, Bac e oide es con ains Bac e oides
species wi h a wide ange o glycoside hyd olases and ca bohyd a e
me abolic pa hways (Mahowald, Hamil on, Mackey, Moo e, Bake ,
Scanza e al., 2019).
The esul s ob ained a ime 0 a e in acco dance wi h he li e a u e:
he human GM is mos ly composed o Fi micu es and Bac e oide es,
which ep esen mo e han 90% o he o al communi y (Lopez-San a-
ma ina e al., 2020). The changes caused by he subs a es a e also in
Fig. 1. Rela i e abundance o di e en bac e ial phyla and gene a. Bac e ial composi ion ( ela i e abundance, %) de e mined using 16S RNA amplicon
sequencing a he phylum (a) and genus (b) le els. The x axis shows he di e en subs a es e alua ed a he di e en assay imes (10, 24, and 48 h). 0 h indica es he
bac e ial composi ion be o e subs a e addi ion. Due o he la ge numbe o epo ed amilies, only he op 15 mos abundan genus a e included in he legend. INU,
inulin; CONT, nega i e con ol (no addi ion o subs a e); SW, seaweed (H. elonga a).
A. Lopez-San ama ina e al.
Food Resea ch In e na ional 156 (2022) 111156
7
ag eemen wi h ecen simila s udies. V´
azquez-Rod íguez, e al., (2021)
s udied he e ec o polysaccha ide ac ions o a b own seaweed (Sil-
e ia comp essa) on human GM by using a di e en in i o colonic model.
They ound ha he ela i e abundance o P o eobac e ia inc eased in
all ea men s wi h he e men a ion ime o he con ol, inulin, and
polysaccha ide ac ions, eaching simila pe cen ages o hose ound in
he p esen wo k. Those au ho s ound an inc ease in Bac e oide es
phylum a e 2 h o incuba ion wi h a la e dec ease wi h ime. In his
wo k, he maximum ela i e abundance o Bac e oide es occu ed a
la e imes (24 and 48 h). This could be due o he di e ences in he
subs a es including inulin, which can possess di e en deg ees o
polyme iza ion, o o he assay condi ions, because V´
azquez-Rod íguez
e al. (2021) used an in i o colonic model wi hou pH con ol, among
o he di e ences. S ain e al. (2020), in a simila s udy es ing poly-
saccha ide ex ac s o Lamina ia digi a a, also ound an inc ease in he
Bac e oide es phylum, whe eas con a iwise o hose ob ained in he
cu en wo k, no di e ences in P o eobac e ia we e obse ed.
Fu e al. (2018), who also e alua ed a new polysaccha ide ex ac ed
om ano he b own seaweed (Sa gassum hunbe gii), did no obse e
changes in he P o eobac e ia phylum, whe eas hey no ed a signi ican
educ ion in Fi micu es and an inc ease in Bac e oide es. As commen ed
be o e, hese di e ences could be a ibu ed o he di e ences in he in
i o colonic model and he s uc u e o he subs a es. These au ho s did
no use any o he p ebio ic ca bohyd a e as posi i e con ol and did no
con ol pH.
Conside ing he gene a o he Bac e oide es phylum, Bac e oides and
Pa abac e oides we e he mos abundan . Fo H. elonga a, he Bac e oides
genus showed a highe ela i e abundance, whe eas o inulin, Pa a-
bac e oides was he p edominan genus. P e ious s udies ha e docu-
men ed a ew examples in which symbio ic bac e ia belonging
o Bac e oides, a dominan genus in he human GM, possess genes o
deg ading seaweed-de i ed po phy an aga ose, algina e, and lamina in
(Hemswo h, Thompson, S eppe , Sobala, Coyle, La sb ink e al., 2016;
Dejean, Tamu a, Cab e a, Jain, Pe ei a e al., 2020).
Thi y-se en di e en bac e ial species we e iden i ied. The esul s
ob ained om he s a is ical analysis o he samples ob ained a e 24
and 48 h o e men a ion showed signi ican di e ences be ween inulin
and H. elonga a o 14 species (Fig. 2a) a e 24 h and 16 species a e 48
h (Fig. 2b).
A e 24 h o e men a ion, in he assay wi h H. elonga a he p e-
dominan species belong o he genus Bac e oides, speci ically Bac e -
oides o a us, Bac e oides agilis, Bac e oides plebeius, Bac e oides
uni o mis, and Bac e oides cop ophilus, con i ming he changes obse ed
a he genus le el. Mo eo e , hese species we e p esen in a signi i-
can ly highe p opo ion han in he assay wi h inulin. Fo he assay wi h
inulin, he p edominan species we e Pa abac e oides dis asonis and
Clos idium pe ingens, also p esen in he assay wi h H. elonga a bu a a
much lowe pe cen age.
A e 48 h o e men a ion, he main di e ences obse ed indica ed
an inc ease in B. o a us in he assay wi h inulin, whe eas he e was a
dec ease in C. pe ingens compa ed wi h he same assay a e 24 h. Fo
he assay wi h H. elonga a, he e was an inc ease in P. dis asonis oge he
a dec ease in he Bac e oides species excep o B. uni o mis, which
inc eased a e 48 h compa ed wi h 24 h (Fig. S1, Supplemen a y ma-
e ial). O he signi ican di e ences we e ound o mino abundan
species such as Rosebu ia aecis, Ruminococcus gna us and Faecalibac e-
ium p ausni zii being he wo mos abundan o he seaweed and he
las one o inulin, a 24 h.
The inc ease o Bac e oides species can be con o e sial because hey
ha e been epo ed in di e en clinical in ec ions (Wexle , 2007).
Howe e , i is impo an o no e ha Bac e oides species a e he mos
abundan in he human gu , gene ally main aining a bene icial ela-
ionship wi h he hos and hei abundance has been ela ed o he
consump ion o ich- ibe die s. When hey escape his en i onmen is
when hey can ac as pa hogens causing in ec ions. As iendly
commensal, Bac e oides species u ilize simple and complex suga s and
Table 3
Changes in lac a e and sho -chain a y acids (SCFA) concen a ions (mM) in he samples ob ained o each subs a e a e ecal e men a ion assays a 0, 10, 24, and 48 h. The esul s a e exp essed as he mean ±s anda d
e o (n =6). nd: no de ec ed (LOD =0.016–0.03 mM). A wo-way ANOVA, using gene al lineal model pe o med wi h ime and subs a es as wo co a ia es o each bac e ial g oup, indica ed signi ican di e ences
be ween bo h ime and subs a es, p <0.05. Then, a one-way ANOVA wi h Tukey’s es was pe o med o de e mine a signi ican inc ease/dec ease o SCFA concen a ion wi h ime o he same subs a e (di e en
lowe case le e s indica e signi ican di e ences), also a one-way ANOVA analysis was ca ied ou o compa e subs a es a 24 and 48 h (indica ed wi h capi al le e s).
H. elonga a Inulin Nega i e con ol
SCFAs 0h 10h 24h 48 h 10h 24h 48h 10h 24h 48h
Succinic 0.31
a
±0.23 0.50
a
±0.14 nd 0.33
a,A
±0.24 0.11
a
±0.08 5.53
b,A
±2.25 0.80
a,b,A
±0.56 0.28
a,
±0.04 0.50
a,C
±0.20 nd
Lac ic 0.64
a
±0.48 0.40
a
±0.28 3.62
a,A
±2.56 nd 0.09
a,b
±0.07 6.74
b,A
±2.39 7.09
b,A
±2.00 0.09
a
±0.06 0.23
a,B
±0.17 0.18
a,B
±0.13
Fo mic 1.16
a
±0.90 1.57
a
±1.11 7.09
a,A
±5.02 nd nd 21.87
b,B
±5.22 15.39
b,A
±2.92 0.35
a,
±0.25 1.25
a,B
±0.89 1.06
a,B
±0 .75
Ace ic 4.36
a
±0.66 1.59
a
±0.06 17.34
a,b,A
±2.54 25.93
b,A
±1.02 0.80
a
±0.47 21.42
b.A
±0.55 35.37
b,A
±5.84 0.55
a
±0.06 8.2
a.B
±0.46 9.67
a,B
±0.33
P opionic 2.42
a
±0.54 1.79
a
±0.05 8.19
b,A
±2.06 3.93
a,b,A
±0.82 1.06
a
±0.38 1.22
a,B
±0.44 16.61
b,B
±4.74 1.53
a,
±0.05 2.58
a,B
±0.38 2.34
a,A
±0.26
Isobu y ic nd nd 4.28
a,A
±2.57 0.81
a,A
±0.29 nd nd nd nd nd 0.06
a,B
±0.05
Bu y ic 3.55
a
±1.34 1.80
a
±0.31 8.17
a,A
±1.30 4.30
a,A
±1.52 2.34
a
±0.92 9.65
a,b,A
±0.86 18.23
b,B
±5.62 0.82
a,
±0.32 1.25
a.B
±0.11 0.79
a,C
±0.18
Iso ale ic nd nd 3.92
a,A
±2.21 0.84
a,A
±0.36 nd nd 0.18
a,A
±0.13 nd 0.18
a,B
±0.13 0.54
a,A
±0.25
Vale ic nd nd 3.51
a,A
±2.22 0.69
a,A
±0.49 nd nd nd nd 0.16
a,B
±0.11 0.17
a,A
±0.12
To al 12.53 ±4.27 7.64 ±1.95 56.11 ±20.48 36.03 ±4.73 4.41 ±1.91 66.43 ±11.71 93.66 ±21.82 3.62 ±0.78 14.37 ±2.45 14.82 ±2.07
A. Lopez-San ama ina e al.
Food Resea ch In e na ional 156 (2022) 111156
8
polysaccha ides o g ow h. In he p esen wo k, seawed ca bohyd a es
a e mainly used by Bac e oides o a us, ollowing by o he species such as
B. agilis, B. plebeius and B. uni o mis as p e iously indica e. B. o a us has
been p e iously epo ed as deg ade o seaweed ca bohyd a es
(Hemswo h e al., 2020). Al hough, in his case, he s ain was no
iden i ied, is ema kable o indica e ha a e s ains om B.o a us
conside ed bene icial as he case o B.o a us s ain ELH-B2 conside ed as
a po en ial nex -gene a ion p obio ic due o i s p e en i e e ec s on
lipopolysaccha ides-associa ed in lamma ion and in es inal mic obio a
diso de s in mice (Tan, Yu, Wang, Zhang, Zhao, Zhang e al., 2018).
Analysis o
α
-di e si y in e ms o Shannon H di e si y and he
numbe o OTUs showed ha o bo h subs a es, he di e si y dec eased
a e 24 h and hen sligh ly inc eased om 24 o 48 h. These changes in
e ms o di e si y we e signi ican in he assays a 24 and 48 h (wi h
espec o ime 0 h) o bo h subs a es. Wi h H. elonga a, he di e si y
was lowe a e 24 h and inc eased a e 48 h. By con as , o inulin his
di e si y emained cons an and simila o he
α
-di e si y eached o
H. elonga a a e 48 h. Fo β-di e si y, he B ay–Cu is analysis showed
ou di e en g oups: (1) 0 h; (2) con ol a 48 h; (3) H. elonga a a 24
and 48 h and inulin a 24 and 48 h; and (4) con ol a 10 h, con ol a 24
h, H. elonga a a 10 h, and inulin a 10 h.
Finally, s a is ical analysis o he 20 mos equen me abolic
pa hways ob ained wi h PICRUS showed signi ican di e ences be-
ween 0 and 24 h o H. elonga a and inulin (Fig. S2a and S2b espec-
i ely, Supplemen a y ma e ial). A 0 h, he mos equen me abolic
pa hways we e ela ed o spo ula ion, po e ion channels, lipopolysac-
cha ide biosyn hesis p o eins, ibosome biogenesis, and he pen ose
phospha e pa hway, among o he s. The s a is ical analysis ob ained o
he me abolic pa hways ob ained o H. elonga a and inulin a 24 and 48
h showed no signi ican di e ences be ween hem (Fig. S2c and S2d.
Supplemen a y ma e ial). The same esul was ob ained om he anal-
ysis o he me abolic unc ions o H. elonga a a e 24 and 48 h (Fig. 3a)
and inulin a e 24 and 48 h (Fig. 3b). Among he 20 mos equen
me abolic pa hways o H. elonga a we e ABC anspo e s; wo-
componen sys ems; anspo e s; DNA epai and ecombinan p o-
eins; sec e ion sys em; and di e en pa hways ela ed o ca bohyd a e
me abolism such as uc ose and mannose me abolism, pen ose and
glucu ona e in e con e sions, and o he glycan deg ada ion. Fo inulin,
he same pa hways we e p esen , wi h some excep ions ela ed o ca -
bohyd a e me abolism, such as uc ose and mannose me abolism,
which did no appea among he 20 mos equen in he assays wi h
inulin.
Fig. 3. Main me abolic pa hways. S a is ical Analysis Func ional P o ile o he main 20 me abolic pa hways o he samples ob ained om he in i o colonic
model wi h a) H. elonga a a 24 and 48 h and b) inulin a 24 and 48 h. The p- alue ob ained o all he me abolic unc ions (only he 20 mo e ep esen a i e in
g aphics) was highe han 0.05, indica ing no signi ican di e ences be ween he imes o assay.
Fig. 2. S a is ical di e ences o he mos abundan iden i ied bac e ial species. Signi ican di e ences (p <0.05) ob ained om he analysis o samples (a)
INU24H ( e men a ion assay wi h inulin o 24 h) and SW24H ( e men a ion assay wi h Himan halia elonga a o 24 h) and (b) INU48H ( e men a ion assay wi h
inulin o 48 h) and SW48H ( e men a ion assay wi h H. elonga a o 48 h) o he mos common species using a G- es (wi h Ya es’ co ec ion) +Fishe ’s exac es
wi h Bon e oni co ec ion in STAMP.
A. Lopez-San ama ina e al.
Food Resea ch In e na ional 156 (2022) 111156
9
3.4. SCFAs analysis
Table 3 shows he SCFA analysis. As expec ed, he e was a g ea e
SCFA p oduc ion o he assays wi h ei he subs a e compa ed wi h he
nega i e con ol (wi hou subs a e). Fo inulin, he highes SCFA p o-
duc ion occu ed a e 48 h, whe eas o H. elonga a, he maximum
p oduc ion occu ed a e 24 h (93.66 mM s. 56.11 mM). Howe e ,
when compa ing SCFA p oduc ion a e 24 and 48 h, he e we e no
signi ican di e ences be ween he subs a es.
Fo mic, ace ic, p opionic, and bu y ic acids domina ed when p o-
duc ion was maximum. These esul s a e simila o hose ob ained by
Chen e al. (2018), whe e he concen a ion o SCFA inc eased in he
g oup ea ed wi h he seaweed polysaccha ide (p edominan ly ace ic
and p opionic acids). Baju y, Rawi, Sazali, Abdullah, & Sa dini, 2017
also epo ed an inc ease in ace ic acid o e he cou se o seaweed
e men a ion.
The SCFA esul s a e in conco dance wi h he esul s ob ained o he
GM analysis. Fo he e men a ion assays wi h inulin a e 48 h and
H. elonga a a e 24 h (highes SCFA p oduc ion), he e was also a highe
ep esen a ion o he phylum Bac e oide es in GM. This phylum is known
o be he main p oduce o SCFA (Fu e al., 2018). In he case o
H. elonga a, p opionic acid inc eased conside ably a e 24 h, a which
ime he e was g ea e ep esen a ion o di e en Bac e oides species.
This may be because Bac e oide es can me abolize a ious ca bohy-
d a es o p oduce p opiona e h ough he succina e pa hway (Chen
e al., 2018).
4. Conclusions
This is he i s ime ha whole b own seaweed (H. elonga a) has been
es ed in an in i o model o he human colon o de e mine i s po en ial
p ebio ic ac i i y. The ob ained esul s in e ms o iodine and As
composi ion indica e he sui abili y and sa e y o using a simila amoun
o he p oduc o o he known and ma ke ed p ebio ic compounds.
H. elonga a was selec i ely used by some membe s om GM, causing an
inc ease in Bac e oides species; on he con a y, inulin led o a signi ican
inc ease in he ela i e abundance o P. dis asonis, which has been
desc ibed o he i s ime. While axonomic di e ences in GM we e
ound o he wo subs a es employed, he me abolic pa hways associ-
a ed wi h he GM unc ion was no signi ican ly di e en be ween he
subs a es, indica ing he po en iali y o he use o he whole seaweed as
p ebio ic. Al hough hese esul s ha e been ob ained by means an in i o
assay wi h i s limi a ions, he p esen wo k includes indings ha
encou age o con inue wi h he esea ch on he po en ial use o
H. elonga a (and maybe, o he seaweeds) as po en ial p ebio ic ing e-
dien in supplemen s o ood. Ou u u e p ospec s include a comple e
chemical cha ac e iza ion o H.elonga a, mainly polyphenols and ca -
bohyd a es, in i o s udies wi h animals and humans as well as iden i-
ica ion o he bac e ial s ain B. o a us, he main iden i ied specie ha
use he seaweed og ow and elucida e i s ole.
Funding
The au ho s hank he Xun a de Galicia and Eu opean Regional
De elopmen Funds (FEDER), g an ED431C 2018/05, o co e ing he
cos o he wo k.
CRediT au ho ship con ibu ion s a emen
A oa Lopez-San ama ina: Me hodology, Fo mal analysis, W i ing –
o iginal d a . Alejand a Ca delle-Cobas: Me hodology, Fo mal anal-
ysis, W i ing – o iginal d a , Visualiza ion. Alicia del Ca men Mon-
d agon: Me hodology. Lau a Sinis e a-Loaiza: Me hodology. Jose
Manuel Mi anda: Concep ualiza ion, In es iga ion, W i ing – e iew &
edi ing, Visualiza ion, Supe ision, P ojec adminis a ion. Albe o
Cepeda: Concep ualiza ion, Funding acquisi ion, In es iga ion, W i ing
– e iew & edi ing, Visualiza ion, Supe ision, P ojec adminis a ion.
Decla a ion o Compe ing In e es
The au ho s decla e ha hey ha e no known compe ing inancial
in e es s o pe sonal ela ionships ha could ha e appea ed o in luence
he wo k epo ed in his pape .
Acknowledgemen
Au ho s would like o hank he use o RIAIDT-USC analy ical
acili ies.
Appendix A. Supplemen a y ma e ial
Supplemen a y da a o his a icle can be ound online a h ps://doi.
o g/10.1016/j. ood es.2022.111156.
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