LWT - Food Science and Technology 187 (2023) 115362
A ailable online 29 Sep embe 2023
0023-6438/© 2023 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/).
Fecal mic obio a coope a i e me abolism o pec ins de i ed om apple
pomace: A unc ional me agenomic s udy
Ines Cal e e-To e
a
,
b
,
1
, Ca los Saba e
a
,
b
,
1
, Abela do Ma golles
a
,
b
, Lo ena Ruiz
a
,
b
,
*
a
Depa men o Mic obiology and Biochemis y o Dai y P oduc s, Ins i u o de P oduc os L´
ac eos de As u ias-Consejo Supe io de In es igaciones Cien í icas (IPLA-CSIC),
Paseo Río Lina es s/n, 33300, Villa iciosa, As u ias, Spain
b
Func ionali y and Ecology o Bene icial Mic obes (Mic oHeal h) G oup, Ins i u o de In es igaci´
on Sani a ia del P incipado de As u ias (ISPA), O iedo, As u ias, Spain
ARTICLE INFO
Keywo ds:
P ebio ics
Pec in
Mic obio a
Apple pomace
ABSTRACT
Complex polysaccha ides such as pec in a e ecei ing inc easing a en ion as eme gen p ebio ics which could be
o in e es o human heal h. Pec in comp ises a wide a ay o highly complex and di e se polysaccha ide
s uc u es, which a e abundan in ag icul u al by-p oduc s. Pec in me aboliza ion by he gu mic obio a equi es
coope a i e me abolism and c oss- eeding ne wo ks among mic obial species. He ein we ha e deepened in o he
gu modula o y p ope ies o apple pomaces and pec ins de i ed om his by p oduc , h ough a sho gun
me agenomics app oach coupled o ad anced unc ional anno a ion and p edic ion o coope a ing ne wo ks o
me agenome-assembled genomes (MAGs). Fo his pu pose, we ha e used samples om ba ch in i o ecal
e men a ions pe o med wi h samples om heal hy dono s and C ohn’s disease pa ien s, in he p esence o apple
pomace and pec ins exhibi ing di e en s uc u al p ope ies. Ou esul s con i m he capaci y o apple pomace/
pec ins o p omo e key axa gene ally unde ep esen ed in C ohn’s disease pa ien s bu ha ha e been a ibu ed
an i-in lamma o y p ope ies, Faecalibac e ium, Ruminococcaceae membe s and Akke mansia being e y
ema kable. Besides, some Ruminococcus and Akke mansia species ha e been iden i ied as key symbion s,
exhibi ing complemen a y me abolic ai s o ully me abolise pec ins om apple pomace. These esul s will aid
de eloping mic obiome- a ge ed p ebio ic and/o synbio ic s a egies o speci ic popula ion g oups.
1. In oduc ion
The p ebio ic concep has changed o e ime, in pa , because all he
ad ances in mic obiome esea ch. The In e na ional Scien i ic Associa-
ion o P obio ics and P ebio ics (ISAPP) p oposes he de ini ion “a
subs a e ha is selec i ely u ilized by hos mic oo ganisms con e ing a
heal h bene i ” (Gibson e al., 2017). Wi h his de ini ion he doo is
opened o he sea ch o new chemical compounds and subs a es wi h
p ebio ic capabili y. No ewo hy, ege able by-p oduc s ha e gene ally
been ecognized a good sou ce o ing edien s wi h p ebio ic p ope ies
(Albuque que e al., 2021). Hence, ege able was e could ha e g ea e
economic and en i onmen al a ac i eness wi hin he bio e ine y
concep due o i s unc ional and/o heal h bene i s.
Apple pomace is one o he mos abundan by-p oduc s ob ained
ac oss se e al coun ies and consis s on he solid le o e ob ained a e
apple p ocessing du ing cide manu ac u ing. I is a good sou ce o
pec in, and is ich in mine als, die a y ibe and polyphenols (An onic
e al., 2020; Cal e e-To e e al., 2021; Skinne e al., 2018), some o
which ha e been a ibu ed heal h p omo ing p ope ies. High le els o
ibe in he die ha e been epo ed o p o ec agains he de elopmen
o in lamma o y bowel diseases (IBD) and a long- e m die wi h die a y
ibe lowe s he isk o C ohn’s disease (CD). This e ec migh be
possibly linked o he sho chain a y acids (SCFAs) ha a e p oduced
upon ibe e men a ion by he in es inal mic obio a, a well-known
con ibu o o he in e play be ween die and heal h (Ishisono e al.,
2019). Hence, unde s anding how he mic obio a coope a i ely
me abolise p ebio ic ibe s is c i ical o de ine hei heal h e ec s. In
i o ecal e men a ions ha e been conduc ed o in es iga e he p ebi-
o ic po en ial o he pec in o apple pomaces (Cal e e-To e e al., 2021).
The s udies wi h pec in, ha is a he e opolysaccha ide composed o
pa ly me hyl-es e i ied galac u onic acid (GalA) and ami ied domains
wi h neu al suga s, ha e demons a ed ha pec in has good
* Co esponding au ho . Depa men o Mic obiology and Biochemis y o Dai y P oduc s, Ins i u o de P oduc os L´
ac eos de As u ias-Consejo Supe io de
In es igaciones Cien í icas (IPLA-CSIC), Paseo Río Lina es s/n, 33300, Villa iciosa, As u ias, Spain.
E-mail add ess: [email p o ec ed] (L. Ruiz).
1
These au ho s ha e equally con ibu ed o his wo k.
Con en s lis s a ailable a ScienceDi ec
LWT
jou nal homepage: www.else ie .com/loca e/lw
h ps://doi.o g/10.1016/j.lw .2023.115362
Recei ed 31 July 2023; Recei ed in e ised o m 26 Sep embe 2023; Accep ed 28 Sep embe 2023
LWT 187 (2023) 115362
2
e men a ion cha ac e is ics and ca dio ascula p o ec ion ac i i y, as
well as an i-in lamma o y and an ioxidan p ope ies (Cal e e-To e
e al., 2021; Gull´
on e al., 2013; Ribei o e al., 2022). Ne e heless,
pec in e men a ion abili y is no widely p esen in all mic obial axa
ound in he in es inal mic obio a (Saba e , Cal e e-To e, e al., 2021),
hence hei e men a ion pa e n in a ge popula ions migh be s ongly
condi ioned by he me abolic capabili ies o he axa p esen in hei gu
mic obio a, as well as by hei capaci y o es ablish c oss- eeding e-
la ionships. Unde s anding he me abolic ai s and ne wo ks equi ed
in a gu mic obio a communi y o achie ing e icien e men a ion o
pec in- ich ma e ials, and p omo ion o heal hy communi ies, is essen-
ial o a ionally design pec in-based p ebio ic ing edien s.
La es ad ances in nex -gene a ion sequencing echniques like me -
agenomics in combina ion wi h ad anced bioin o ma ic me hods ha e
e ealed use ul o unde s and he e ec o die a y ing edien s on he
in es inal mic obio a, and ha e been used o in es iga e he ibe
me abolism o heal h-associa ed mic obial gene a (Saba e ,
Cal e e-To e, e al., 2021). These me hodologies could be o u mos
in e es o ailo nex gene a ion p obio ics and p ebio ics ha could
a ge speci ic disease condi ions such as CD (Lin e al., 2019; Lo dan
e al., 2020). In his ega d, sho gun me agenomics allows he iden i i-
ca ion o he mic oo ganisms a species and s ain le els and he e-
co e y o whole me agenomic assembled genomes (MAG) om complex
mic obial communi ies o complex unc ional analysis o hei coop-
e a i e me abolism (Culp & Goodman, 2023; F ioux e al., 2020;
Tomioka e al., 2022). Howe e unc ional analysis o MAGs om
mic obiome samples ha e been sca cely used o he pu pose o p e-
bio ics cha ac e iza ion and design.
To his end, ad anced me abolic modelling so wa e ools ha e been
de eloped o cha ac e ise he me abolic po en ial and o simula e syn-
e gis ic in e ac ions be ween MAGs (Belcou e al., 2020; Saba e e al.,
2022). Me abolic coope a ion o gu mic obes, o ins ance h ough
es ablishmen o c oss- eeding ne wo ks, educes compe i ion be ween
coexis ing axa enhancing hei g ow h. These mechanisms can be
elucida ed in silico o de e mine he minimal se o key mic oo ganisms
equi ed in a communi y o coope a i ely exe a gi en unc ion, iden-
i ying essen ial and al e na i e symbion s. In his sense, essen ial
symbion s comp ise key mic oo ganisms ha a e equi ed in e e y
minimal communi y o mic oo ganisms o conduc one speci ic unc ion
h ough me abolic coope a ion, such as me aboliza ion o die a y ibe
and po en ially p ebio ic polysaccha ides like pec in (Saba e e al.,
2022). Al e na i e symbion s, on he o he hand, occu only in some o
hese minimal communi ies o in e ac ing mic oo ganisms. The e o e,
hese ools can be used o design minimal mic obial conso ia equi ed
o coope a i ely me abolise a gi en subs a e, such a complex p ebio ic
s uc u e. I should be no ed ha his in o ma ion canno be ob ained by
assembly- ee me hods o analysis o me agenomic da a.
Thei applica ion o in silico analyze me aboliza ion o complex
ca bohyd a e s uc u es equi es de ailed anno a ion o ca bohyd a e
ac i e enzymes (CAZy) in MAGs eco e ed om mic obio a samples,
and p edic ion o syne gis ic in e ac ion among coexis ing MAGs, wha
may help elucida e p ebio ic e men a ion pa e ns in he mic obio a.
Fo ins ance, in he con ex o pec in e men a ion, such app oaches
ha e enabled in es iga ion o he po en ial o pec in e men a ion o
amelio a e CD symp oms (Saba e e al., 2022). In his ega d, pec in is a
e y complex polysaccha ide and equi es many di e en enzymes o
i s enzyma ic deg ada ion including: glycoside-hyd olases (GH)
(Nguyen e al., 2019), polysaccha ide-lyases (PL) (Su he land, 1995),
ca bohyd a e-es e ases (CE) (A menda iz-Ruiz e al., 2018), and no el
ca bohyd a e-binding modules (CBM) (Bo as on e al., 2004), as
e iewed elsewhe e (Bonin & Pelloux, 2020; Ndeh e al., 2017). How-
e e , mos s udies epo ed o da e in his ega d comp ise in silico ap-
p oaches using publicly a ailable sequences and ew s udies epo MAG
eco e y om in i o ecal e men a ion expe imen s using samples
om he a ge popula ion ha is pa ien s o CD. Besides, o ou
knowledge, no p e ious a emp s o de ine mic obial conso ia o gu
commensal mic oo ganisms necessa y o coope a i ely me abolise
pec in ha e been epo ed.
In p io in es iga ions, we ha e p edic ed he po en ial o pec in
s uc u es o modula e he gu mic obio a o CD pa ien s (Saba e e al.,
2022) and ha e demons a ed in i o, h ough ecal e men a ions and
16S RNA sequencing he capabili y o apple pomaces and pec ins
de i ed he e om o modula e key commensal axa in he mic obio a o
CD pa ien s (Cal e e-To e e al., 2021). In his wo k we ha e u he
del ed in o he e men a i e p ope ies o apple pomaces de i ed om
p oduc ion o mono a ie al cide s ha ha e been epo ed o ep esen a
p omising sou ce o pec in wi h a ied s uc u al cha ac e is ics
depending on he apple a ie ies hey o igina e om: Sola ina a ie y
ich in homogalac u onan (HG) and Pe ico and Regona a ie ies ich in
hamnogalac u onan (RG)-I (Cal e e-To e e al., 2021). To his aim we
ha e cha ac e ized me abolic capabili ies om MAGs eco e ed om
ba ch ecal e men a ions pe o med wi h apple pomace and wi h hei
pec ins, by using ecal samples om dono s wi h C ohn’s disease (CD)
and heal hy dono s. Mic obial in e ac ions wi hin he modula ed com-
muni ies we e simula ed using bioin o ma ic me hods o de ine mic o-
bial conso ia o gu commensal species equi ed o me abolise pec in in
he con ex o CD as a a ional p ebio ic design s a egy.
2. Ma e ials and me hods
2.1. Fecal e men a ion expe imen s and sho gun me agenomics
sequencing
Fecal e men a ion expe imen s analyzed in his epo we e p e i-
ously desc ibed pa ially (Cal e e-To e e al. (2022)). B ie ly, ba ch
ecal e men a ions we e pe o med wi h ecal samples p o ided by
h ee heal hy male dono s (aged be ween 28 and 50 yea s) and h ee
male CD pa ien s (aged be ween 24 and 60 yea s), by using apple
pomace and pec in de i ed om h ee apple a ie ies (Pe ico, Regona
and Sola ina). In Cal e e-To e e al. (2022), 16S RNA p o iling o he
ecal e men a ions a se e al imepoin s was conduc ed and epo ed.
He ein, we u he deepen in o he unc ional capaci y o he mic obial
popula ions modula ed in he e men a ions by pe o ming sho gun
me agenomics sequencing in a selec ion o samples, coupled o unc-
ional analysis o MAGs and p edic ion o me abolic syne gies among he
modula ed popula ions.
To al samples (n =42) selec ed o me agenomic sequencing
included: i) ecal homogena es om heal hy (n =3) and CD (n =3)
dono s, ii) ecal e men a ions o apple pomace (n =9) and pec in (n =
9) de i ed om Pe ico, Regona and Sola ina apple a ie ies collec ed a
8h using heal hy dono s, iii) ecal e men a ions o apple pomace (n =9)
and pec in (n =9) de i ed om Pe ico, Regona and Sola ina apple a-
ie ies collec ed a 8h using CD dono s. Each expe imen was pe o med
in iplica e. Then, mic obial DNA was isola ed by using he Powe Soil
P oKi (Qiagen) acco ding o ou p e ious wo k (Cal e e-To e e al.,
2022).
To al DNA ex ac ed was submi ed o an ex e nal sequencing se ice
(www.BaseClea .com) o gene a e pai ed-end sequence eads (2 ×
150bp) showing an a e age minimum o 25 million eads pe sample
using an Illumina No aSeq sys em. Reads we e demul iplexed o
gene a e FASTQ ead sequence iles using bcl2 as q2 ( 2.18) and eads
con aining (pa ial) adap e s we e clipped (up o a minimum ead leng h
o 50 bp). Addi ional quali y-con ol s eps included he emo al o
con aminan low-quali y eads using Kneadda a ( 0.7.4) and T immo-
ma ic ( 0.39) so wa e. Fo his pu pose, Bow ie2 ( 4.5) was used o
map me agenomic eads agains he e e ence da abases "Homo sapiens
hg37 and human con amina ion Bow ie2" ( 0.1), o emo e hos
con amina ion.
The aw sequences da a we e deposi ed in he Sequence Read
A chi e (SRA) o he NCBI (h ps://www.ncbi.nlm.nih.go /s a,
accessed on July 18, 2023) unde BioP ojec PRJNA995911.
I. Cal e e-To e e al.
LWT 187 (2023) 115362
3
2.2. Assembly- ee analysis o me agenomes
To cha ac e ise mic obio a composi ion and me abolic unc ions o
ecal e men a ion samples, an assembly- ee analysis was i s pe -
o med. This assembly- ee analysis allows he de e mina ion o axa
abundances in he co e mic obio a composi ion and he iden i ica ion o
low-abundance axa ha migh be los du ing he me agenome assembly
p ocess. Decon amina ed eads we e analyzed ollowing Me aPhlAn 3.0
( 3.0.4) and HUMAnN 3.0 ( 3.0.0) pipelines o elucida e axonomic and
unc ional p o iles, espec i ely (F anzosa e al., 2018; T uong e al.,
2017). Wi h his aim, ChocoPhlAn da abase ( e sion "mpa_ 30_Choco-
PhlAn_201,901
″
) con aining clade-speci ic ma ke genes and UniRe 90
( e sion "uni e 90_201,901
″
) p o ein da abase, we e used o pe o m
axonomic and unc ional analyses. The abundances o gene amilies and
me abolic pa hways we e e-no malized and exp essed in uni s o copies
pe million.
2.3. Reco e y o me agenome-assembled genomes (MAGs)
To be e cha ac e ise pec in-deg ading ac i i ies o mic obial com-
muni ies ound in ecal e men a ion samples, me agenome-assembled
genomes (MAGs) we e eco e ed ollowing an assembly-based pipe-
line desc ibed in a p e ious wo k o ou esea ch g oup (Saba e e al.,
2022). B ie ly, he assembly o quali y- il e ed eads was pe o med wi h
MEGAHIT ( 1.2.9) conside ing a maximum k-me size o 127 o
gene a e he ollowing se ies o k-me s: k-21, k-31, k-41, k-51, k-61,
Fig. 1. P incipal coo dina es analysis (PCoA) o axonomic p o iles (A), gene amilies (B) and me abolic pa hways (C) ound in he mic obio a o each g oup o
samples: apple pomace (Pom) and pec in (Pec ) de i ed om di e en apple a ie ies (Pe ico, Regona and Sola ina) ha we e subjec ed o ecal e men a ion using
ecal homogena es (Homog) om heal hy and C ohn’s disease (CD) dono s. PC: p incipal coo dina e. The pe cen age o a iance explained by each PC is indica ed in
he axis. Numbe s iden i y samples o igina ing om di e en ecal dono s.
I. Cal e e-To e e al.
LWT 187 (2023) 115362
4
k-71, k-81, k-91, k-101, k-111, k-121, k-127. Me agenome eads we e
mapped agains he assembly using Bow ie2 ( .4.5) and ou pu bam iles
gene a ed we e so ed and indexed. Con igs la ge han 1.5 kilobases
we e binned sepa a ely using Me aba 2 ( .2.2.15). MAGs comple eness
and con amina ion we e assessed using CheckM ( .1.1.3)
lineage-speci ic wo k low o selec MAGs showing comple eness highe
han 50% and con amina ion lowe han 5%. Taxonomic iden i ica ion
o MAGs was pe o med using GTDB-Tk ( 2.1.1) pipeline and
ca bohyd a e-ac i e enzymes (CAZy) p o iles o MAGs we e anno a ed
ollowing “ un_dbcan” so wa e de eloped by Zhang e al. (2018).
2.4. Me abolic modelling
Once me abolic unc ions o MAGs we e anno a ed, po en ial me a-
bolic in e ac ions be ween MAGs we e calcula ed. Fo his pu pose,
MAGs we e anno a ed using P okka ( 1.14.6) o gene a e s anda d
Genbank iles (.gbk) iles con aining sequences and anno a ions ha
we e used as inpu o me age2me abo ( 1.5.0) so wa e. To simula e
mic obial me abolic in e ac ions in he p esence o pec in, a “seeds” ile
con aining pec in and di e en nu ien s ha may be ound in human
gu acco ding o p e ious s udies was p o ided (Belcou e al., 2020;
Saba e e al., 2022). Di e en simula ions we e compu ed o MAGs
eco e ed om ecal e men a ions o apple pomace and pec in using
heal hy and CD dono s.
2.5. S a is ical analysis o samples
S a is ical analysis o assembly- ee analysis ou pu s was pe o med
on R ( .4.2.3). B ie ly, alpha and be a di e si y es ima o s we e
compu ed using “Phyloseq” and “Mic obiome” R packages (McMu die &
Holmes, 2013; Lah i & She y, 2017) and P incipal Coo dina e Analysis
(PCoA) and composi ion ba plo s we e gene a ed using “Mic obiome” R
package o illus a e majo di e ences in he mic obio a p o iles. S a-
is ically signi ican di e ences (p <0.05 and p
adj
<0.25) in mic o-
biome composi ion and unc ion we e calcula ed using se e al
mic obio a-speci ic s a is ical me hods (ANCOM, LE Se and DESeq2)
implemen ed in “mic obiomeMa ke ” R package (Cao e al., 2022; Lo e
e al., 2014; Mandal e al., 2015; Sega a e al., 2011). Me agenomic eads
we e no malized p io di e en ial analysis. Taxonomic clades and
unc ions classi ied as signi ican ly di e en ial mic obes (mic obiome
ma ke s) by any o hese me hods we e selec ed o u he analysis. To
in es iga e he associa ions be ween mic obial clades and me aboli e
p oduc ion, signi ican co ela ions (p <0.05) be ween mic obial
composi ion and sho -chain a y acids (SCFAs) le els epo ed by
Cal e e-To e e al. (2022) we e calcula ed and exp essed as Pea son
co ela ion coe icien s.
CAZy domains in ol ed in pec in me abolism we e compa ed
h ough hie a chical clus e ing compu ed using R ( .4.2.3) basic unc-
ion “hclus ”.
3. Resul s
3.1. Assembly- ee analysis o me agenomes
Mic obial p o iles analysis o ecal e men a ions by assembly- ee
me hods was i s ca ied ou allowing a p ope es ima ion o mic obi-
al abundances including low-abundance mic obial eads axa ha migh
be disca ded du ing me agenome assembly. Fi s , alpha di e si y mea-
su es including Chao1, Shannon, Simpson and In e se Simpson indices
we e calcula ed o es ima e a iabili y o gene a wi hin samples. Mean
Chao1 index conside ing all samples unde s udy was 93.0 ±14.1 ye
Chao1 indexes showed no s a is ically signi ican (p >0.05) di e ences
among sample subse s including: i) ecal homogena es om heal hy
(91.7 ±10.1) and CD (90.0 ±20.4) dono s, ii) apple pomace e men-
a ion using samples om heal hy (95.3 ±9.8) and CD (91.9 ±18.5)
dono s, iii) apple pec in e men a ion using samples om heal hy (96.0
±9.7) and CD (89.6 ±18.5) dono s (Supplemen a y Ma e ial Fig. S1).
The di e en alpha di e si y coe icien s showed simila ends, indi-
ca ing a high in e indi idual a iabili y wi hin g oups: ecal e men a-
ions pe o med using samples om heal hy dono 1 showed lowe
alpha-di e si y alues han hose ob ained o he es o heal hy do-
no s, while ecal e men a ions pe o med using samples om CD dono
4 showed highe alpha-di e si y alues han hose ob ained o he es
o CD dono s.
Be a di e si y analysis o measu e mic obial di e si y be ween
g oups o samples was hen calcula ed. No signi ican (p >0.05) di -
e ences in B ay-Cu is dis ances be ween apple and pec in ac ions and
be ween heal hy and CD dono s we e ound. This migh be a ibu ed o
in ag oup a iabili y, in acco dance o he alpha di e si y analysis
p e iously desc ibed. Clus e ing based on B ay-Cu is dis ances g ouped
samples mainly acco ding o he heal h s a us o he dono (i.e. samples
om heal hy and CD dono s) al hough se e al samples co esponding o
he same g oup (i.e. apple pomace o pec in) we e clus e ed in o
di e en b anches o he hie a chical model (Supplemen a y Ma e ial
Fig. S2A). This model could no comple ely disc imina e be ween sam-
ples om di e en g oups due o in e indi idual a iabili y. In ag ee-
men wi h his obse a ion, P incipal Coo dina es Analysis (PCoA)
be e disc imina ed samples om heal hy and CD dono s (Fig. 1A). In
addi ion, se e al subg oups o samples co esponding o he same
heal hy o CD dono we e gene a ed. Howe e , samples could no be
Fig. 2. Mos abundan gene a ound in he mic obio a o each g oup o samples: apple pomace (Pom) and pec in (Pec ) de i ed om di e en apple a ie ies (Pe ico,
Regona and Sola ina) ha we e subjec ed o ecal e men a ion using ecal homogena es (Homog) om heal hy and C ohn’s disease (CD) dono s. Da a a e exp essed
as abundance pe cen ages (%).
I. Cal e e-To e e al.
LWT 187 (2023) 115362
5
Table 1
Mic obial axa showing he highes abundances in each g oup o samples: apple apple pomace (Pom) and pec in (Pec ) de i ed om di e en apple a ie ies (Pe ico, Regona and Sola ina) ha we e subjec ed o ecal
e men a ion using ecal homogena es (Homog) om heal hy and C ohn’s disease (CD) dono s. Mean abundances and s anda d de ia ions (SD) o axonomic clades a e shown.
Highe in: Taxa Taxonomic
le el:
Heal hy -
Homog
(Mean)
Heal hy -
Homog (SD)
Heal hy -
Pom (Mean)
Heal hy -
Pom (SD)
Heal hy -
Pec (Mean)
Heal hy -
Pec (SD)
CD -
Homog
(Mean)
CD -
Homog
(SD)
CD - Pom
(Mean)
CD -
Pom
(SD)
CD - Pec
(Mean)
CD -
Pec
(SD)
Heal hy -
Homog
Ruminococcaceae Family 17.75 11.16 15.48 9.84 12.64 8.76 8.71 6.04 8.54 3.47 7.46 3.78
Heal hy -
Homog
Lachnospi aceae
unclassi ied
Genus 11.85 11.19 10.87 10.25 8.02 9.12 1.23 2.13 3.25 4.89 1.58 2.93
Heal hy -
Homog
Eubac e ium ec ale Species 11.85 11.19 10.87 10.25 8.02 9.12 1.23 2.13 3.25 4.89 1.58 2.93
Heal hy -
Pom
Faecalibac e ium Genus 7.58 4.57 8.31 3.72 7.06 3.85 2.02 1.89 2.95 2.85 2.16 2.41
Heal hy -
Pom
Faecalibac e ium
p ausni zii
Species 7.58 4.57 8.31 3.72 7.06 3.85 2.02 1.89 2.95 2.85 2.16 2.41
Heal hy -
Pec
Ac inobac e ia Class 19.00 26.24 14.66 15.90 28.05 31.08 15.20 24.06 7.47 10.39 6.49 10.96
Heal hy -
Pec
Bi idobac e iales O de 18.97 26.25 14.63 15.92 28.00 31.12 15.08 24.06 7.36 10.41 6.37 10.98
Heal hy -
Pec
Bi idobac e iaceae Family 18.97 26.25 14.63 15.92 28.00 31.12 15.08 24.06 7.36 10.41 6.37 10.98
Heal hy -
Pec
Bi idobac e ium Genus 18.97 26.26 14.63 15.92 27.99 31.13 15.08 24.06 7.36 10.41 6.37 10.97
CD -
Homog
Ac inobac e ia Phylum 25.48 34.83 17.01 17.68 30.03 31.98 30.71 27.66 16.13 11.52 16.31 12.05
CD -
Homog
Co iobac e iia Class 6.48 8.59 2.34 1.96 1.97 1.16 15.51 5.52 8.66 6.08 9.83 6.80
CD -
Homog
Co iobac e iales O de 6.28 8.54 2.19 2.02 1.82 1.27 14.54 5.35 8.33 5.89 9.48 6.62
CD -
Homog
Co iobac e iaceae Family 6.23 8.45 2.19 2.02 1.81 1.27 14.54 5.35 8.33 5.89 9.48 6.62
CD -
Homog
Collinsella Genus 6.21 8.43 2.18 2.01 1.81 1.26 14.53 5.35 8.33 5.89 9.48 6.62
CD - Pec Ve ucomic obia Phylum 0.15 0.25 0.27 0.40 0.26 0.40 8.97 12.91 9.72 12.55 11.18 13.55
CD - Pec P o eobac e ia Phylum 0.19 0.17 0.49 0.38 0.70 0.53 0.74 0.47 1.47 1.37 2.00 1.62
CD - Pec Ve ucomic obiae Class 0.15 0.25 0.27 0.40 0.26 0.40 8.97 12.91 9.72 12.55 11.18 13.55
CD - Pec Ve ucomic obiales O de 0.15 0.25 0.27 0.40 0.26 0.40 8.97 12.91 9.72 12.55 11.18 13.55
CD - Pec Akke mansiaceae Family 0.15 0.25 0.27 0.40 0.26 0.40 8.97 12.91 9.72 12.55 11.18 13.55
CD - Pec Akke mansia Genus 0.15 0.25 0.27 0.40 0.26 0.40 8.97 12.91 9.72 12.55 11.18 13.55
CD - Pec Akke mansia
muciniphila
Species 0.15 0.25 0.27 0.40 0.26 0.40 8.97 12.91 9.72 12.55 11.18 13.55
I. Cal e e-To e e al.

LWT 187 (2023) 115362
6
disc imina ed acco ding o he apple a ie y (Pe ico, Regona and
Sola ina), hence sugges ing ha in e indi idual a iabili y is a s onge
sou ce o a ia ion han he speci ic apple a ie y.
Taxonomic composi ion analysis e ealed ha , among bac e ial
gene a wi h majo abundances, Bi idobac e ium, Bac e oides, Faecali-
bac e ium and Ruminococcus exhibi highe abundances in samples om
heal hy dono s han hose o CD dono s (Fig. 2). In con as , P e o ella,
Collinsella and an unclassi ied P e o ellaceae gene a showed highe
abundances in CD samples han heal hy samples. S a is ically signi ican
(p <0.05 and p
adj
<0.25) di e ences in he abundances o indi idual
axa be ween g oups we e also de e mined (Table 1). Acco ding o he
esul s ob ained, Ruminococcaceae, an unclassi ied Lachnospi aceae
genus and Eubac e ium ec ale showed he highes abundances in ecal
homogena es o heal hy dono s whe eas Collinsella was highe in ecal
homogena es om CD pa ien s. Faecalibac e ium p ausni zii and Bi ido-
bac e ium showed he highes abundances a e ecal e men a ion o
apple pomace and pec in, espec i ely, using samples om heal hy do-
no s. Simila ly, Akke mansia muciniphila showed he highes abundances
a e ecal e men a ion o pec in using samples om CD pa ien s
(Table 1). In e es ingly, no s a is ically signi ican di e ences (p <0.05
and p
adj
<0.25) we e obse ed in he axonomic p o iles o samples
om di e en apple a ie ies in ag eemen wi h PCoA whe e samples
om di e en a ie ies could no be disc imina ed (Fig. 1). Co ela ion
analysis e ealed posi i e associa ions be ween se e al SCFAs p oduce s
like Bi idobac e ium and ibe -deg ading species like A. muciniphila and
Cop ococcus eu ac us, and isobu y ic, ale ic, iso ale ic and cap oic acids
le els (Fig. 3).
Wi h ega d o he unc ional analysis o me agenomes, he gene
coun (i.e., o al numbe o di e en mic obial genes) showed no s a-
is ically signi ican (p >0.05) di e ences be ween g oups: i) ecal ho-
mogena es om heal hy (76054.7 ±21812.7) and CD (61188.0 ±
40925.8) dono s, ii) apple pomace e men a ion using samples om
heal hy (78391.1 ±23745.9) and CD (64005.3 ±38706.4) dono s, iii)
apple pec in e men a ion using samples om heal hy (77197.1 ±
29959.8) and CD (60732.3 ±39541.0) dono s. No signi ican (p >0.05)
di e ences in be a di e si y es ima o s be ween apple and pomace
ac ions and be ween heal hy and CD dono s we e ound. Clus e
analysis o unc ional p o iles led o simila clus e s o hose ob ained in
he axonomic analysis, whe e samples we e classi ied mainly acco ding
o hei heal h s a us (i.e. samples om heal hy and CD dono s) and
simila g oups o samples (i.e. apple pomace o pec in ac ions) we e
clus e ed in he same b anches (Supplemen a y Ma e ial Figs. S2B and
C). PCoA o unc ional p o iles disc imina ed samples om heal hy and
CD dono s and samples om he same heal hy o CD dono we e
g ouped oge he (Fig. 1B and C). This beha iou was obse ed o bo h
mic obial gene amilies and me abolic pa hways.
Mic obial gene amilies and me abolic pa hways showing he highes
(p <0.05 and p
adj
<0.25) abundances in ecal homogena e and e -
men a ions o apple pomace samples om heal hy dono s co esponded
o F. p ausni zii and E. ec ale while hose genes and pa hways inc eased
in e men a ion o pec in samples co esponded o B. adolescen is and
F. p ausni zii (Table 2). These pa hways we e in ol ed in amino acid,
nucleo ide, i amin and ca bohyd a e me abolism (Supplemen a y
Ma e ial Table S1). In con as , CD ecal homogena es we e en iched in
C. ae o aciens genes. In e es ingly, pec in samples om CD dono s was
en iched in a wide ange o A. muciniphila gene amilies and me abolic
pa hways (Table 2), in ol ed in nucleo ide and amino acid me abolism
(Supplemen a y Ma e ial Table S1). These esul s ag ee wi h he axo-
nomic analysis o samples (Fig. 1, Table 1).
3.2. Reco e y o me agenome-assembled genomes (MAGs)
Assembly-based analyses allowed o eco e a o al 1080 MAGs om
me agenome samples including: i) ecal homogena es om heal hy (n =
237) and CD (n =314) dono s, ii) apple pomace e men a ion using
samples om heal hy (n =117) and CD (n =134) dono s, iii) apple
pec in e men a ion using samples om heal hy (n =142) and CD (n =
136) dono s (Supplemen a y Ma e ial Table S2). Mos ep esen ed axa
among MAGs eco e ed om samples ob ained using heal hy dono s
included Aga hobac e ec alis, C. eu ac us, Pa abac e oides dis asonis,
Ruminococcus species and no el Ch is ensenellales UBA11524
sp000437595 species, whe eas A. muciniphila, P. dis asonis and Rumi-
nococcus species a e he mos ep esen ed among MAGs eco e ed om
samples ob ained om CD pa ien s (Supplemen a y Ma e ial Table S2).
Func ional anno a ion o CAZy domains o MAGs e ealed simila -
i ies in enzyme domains ound in all e men a ion g oups (Fig. 4). I
should be no ed ha pec in is mainly composed o linea chains o
α
-1,4-
D- GalA chains and se e al ami ied domains ha comp ise al e na e
sequences o GalA and
α
-(1, 2) linked
α
-L- hamnosyl esidues which may
be subs i u ed a O-4 wi h linea o b anched oligosaccha ides (Saba e ,
Fig. 3. Co elog ams showing he associa ions be ween mic obial composi ion and sho -chain a y acids (SCFAs) le els. Blue and ed do s indica e posi i e and
nega i e co ela ions exp essed as Pea son co ela ion coe icien s. Colou in ensi y and do size a e in p opo ion o magni ude. These co ela ions we e s a is ically
signi ican (p <0.05). (Fo in e p e a ion o he e e ences o colou in his igu e legend, he eade is e e ed o he Web e sion o his a icle.)
I. Cal e e-To e e al.
LWT 187 (2023) 115362
7
Cal e e-To e, e al., 2021; Tan & Nie, 2020). The e o e, CAZy domains
ha may be in ol ed in pec in me abolism comp ise a wide ange o
ac i i ies including a abino u anosidases, xylosidases, xylanases, poly-
galac u onases, hamnosidases, pec in es e ases and hamnogalac u -
onan lyases, among o he s (Fig. 4).
Wi h ega d o pec in es e ases, pec in me hyles e ase domains we e
anno a ed in Selenomonas and no el Ch is ensenellales UBA11524 MAGs
eco e ed om pomace and pec in samples e men ed using aecal
samples om heal hy dono s (Fig. 4A). Simila ly, pec in ace yles e ase
domains we e anno a ed in Gemmige and Ch is ensenellales UBA11524
eco e ed om hese e men a ions. These axa and o he gene a
(Akke mansia, Allop e o ella, Fimenecus, Pa abac e oides, Ruminococcus
and Selenomonas) eco e ed om ecal e men a ions o bo h subs a es
(apple pomace and pec in) showed he highes numbe o pec in-
deg ading ac i i ies including a abinases, galac u onases, galac u -
onidases and enzymes ac ing on complex hamnogalac u onan-II ami-
ied domains like ace ic acid and KDO (2-ke o-3-deoxy-D-manno-
oc ulosonic acid) hyd olases (Fig. 4A). In e es ingly, hamnogalac u -
onan endolyase was cha ac e is ic o Ruminococcus species (Fig. 4A).
In e men a ions wi h aecal samples om CD pa ien s, pec in es-
e ases we e anno a ed in En e oclos e , Gemigge , Lachnospi a and P e-
o ella MAGs eco e ed om pomace and pec in samples (Fig. 4B).
These axa and o he gene a (Akke mansia, C yp obac e oides,
Eisenbe gella and no el species Acu alibac e aceae CAG-177 and
UBA1417) showed he highes numbe o pec in-deg ading domains in
MAGs eco e ed om apple pomace and pec in samples (Fig. 4B).
Simila CAZy p o iles we e obse ed o MAGs eco e ed om bo h
pomace and pec in samples e men ed using CD dono s (Fig. 4B).
3.3. Me abolic modelling
Once pec in-deg ading ac i i ies o MAGs we e anno a ed, mic obial
in e ac ions we e simula ed compu a ionally, wi h he aim o de ining
gu mic obial conso ia capable o me abolise pec in in he con ex o
CD, as a a ional p ebio ic o synbio ic design s a egy. Essen ial sym-
bion s, needed o sa is y speci ic me abolic unc ions on each minimal
communi y o in e ac ing mic obes, and al e na i e symbion s, occu
only in some o hese minimal communi ies, we e de e mined acco ding
o Belcou e al. (2020). Di e ences in he po en ial me abolic in-
e ac ions o MAGs we e obse ed acco ding o mul iple c i e ia: heal h
s a us (heal hy and CD dono s), subs a e (apple pomace and pec in) and
apple a ie y (Pe ico, Regona and Sola ina) (Figs. 5 and 6; Supple-
men a y Ma e ial Fig. S3).
Key symbion s de e mined o each apple a ie y comp ise Akke -
mansia, Bi idobac e ium and Ruminococcus species among o he s (Figs. 5
and 6). In gene al, Bi idobac e ium and Ruminococcus MAGs comp ise
essen ial symbion s in all e men a ion expe imen s while A. muciniphila
MAGs comp ise al e na i e symbion s in e men a ion expe imen s
ca ied ou using CD samples. No ably, essen ial and al e na i e sym-
bion s p edic ed a e di e en acco ding o he heal h s a us and sub-
s a e used o he e men a ions. Fo ins ance, in apple pomace om
Pe ico a ie y e men ed using heal hy dono s, essen ial Ruminococcus
symbion s (Ruminococcus sp000433635 and R. bici culans) es ablish
mul iple in e ac ions wi h wo di e en communi ies o al e na i e
symbion s comp ising o he Ruminococcus and P e o ella species: i)
Ruminococcus sp000980705, ii) R. b omii and P e o ella sp002251385
(Supplemen a y Ma e ial Fig. S3A). Howe e , pec in samples om his
a ie y e men ed using heal hy dono s showed a di e en in e ac ion
p o ile: essen ial symbion s P. dis asonis and Anae o ib io sp900548165
in e ac wi h al e na i e symbion s P e o ella sp000434975 and Fi mi-
cu es bac e ium CAG-41 sp900066215, while essen ial symbion Fi mi-
cu es bac e ium CAG-83 sp000431575 in e ac s wi h al e na i e
symbion P e o ella sp003447235 (Supplemen a y Ma e ial Fig. S3B).
On he o he hand, apple pomace ac ions om his a ie y e men ed
using CD dono s e ealed in e ac ions be ween essen ial symbion s
A. muciniphila and P. cop i and al e na i e symbio ic communi ies
including Odo ibac e splanchnicus, and P. dis asonis among o he s
(Supplemen a y Ma e ial Fig. S3C). In con as , in apple pec in om he
same a ie y e men ed using CD dono s, Akke mansia muciniphila
s ains comp ise al e na i e symbion s ha show po en ial in e ac ions
wi h all essen ial symbion s (Supplemen a y Ma e ial Fig. S3D). These
esul s highligh he ole o Ruminococcus in symbio ic in e ac ions o all
subs a es ega dless heal h s a us, as well as A. muciniphila in CD
mic obio a.
Di e ences be ween apple a ie ies we e also in es iga ed (Figs. 5
and 6). Po en ial in e ac ions be ween R. callidus and Ruminococcus
CAG-177 wi h A. muciniphila we e iden i ied in apple pomace om
Regona a ie y e men ed using CD dono s (Fig. 5B). In e ac ions be-
ween A. muciniphila s ains and all essen ial symbion s we e also ound
in ecal e men a ions o apple pomace om Sola ina a ie y (Fig. 5C).
Simila ly, A. muciniphila and R. bici culans showed po en ial c oss-
eeding mechanisms wi h all essen ial symbion s in e men a ion ex-
pe imen s o pec in om hese a ie ies (Fig. 6B and C).
4. Discussion
This wo k p o ides a comp ehensi e cha ac e iza ion o he ca bo-
hyd a e me abolism o MAGs eco e ed om ecal e men a ion wi h
apple pomace and pec in and de ines no el mic obial conso ia capable
Table 2
Numbe o mic obial gene amilies and me abolic pa hways showing he highes
abundances in each g oup o samples: apple apple pomace (Pom) and pec in
(Pec ) de i ed om di e en apple a ie ies (Pe ico, Regona and Sola ina) ha
we e subjec ed o ecal e men a ion using ecal homogena es (Homog) om
heal hy and C ohn’s disease (CD) dono s. These gene amilies and pa hways a e
summa ised by bac e ial species.
Mic obial gene amilies
Highe in: Taxa n genes/pa hways
Heal hy - Homog Faecalibac e ium p ausni zii 2104
Heal hy - Homog Eubac e ium ec ale 1534
Heal hy - Homog Collinsella ae o aciens 91
Heal hy - Homog Bi idobac e ium adolescen is 4
Heal hy - Homog Bi idobac e ium longum 3
Heal hy - Pom Faecalibac e ium p ausni zii 899
Heal hy - Pom Eubac e ium ec ale 546
Heal hy - Pec Bi idobac e ium adolescen is 1267
Heal hy - Pec Faecalibac e ium p ausni zii 542
Heal hy - Pec Eubac e ium ec ale 34
Heal hy - Pec Bi idobac e ium longum 11
CD - Homog Collinsella ae o aciens 932
CD - Homog Akke mansia muciniphila 14
CD - Homog Bi idobac e ium longum 10
CD - Homog Faecalibac e ium p ausni zii 3
CD - Homog Bi idobac e ium adolescen is 2
CD - Homog Eubac e ium ec ale 2
CD - Pom Akke mansia muciniphila 8
CD - Pom Faecalibac e ium p ausni zii 6
CD - Pom Bi idobac e ium longum 1
CD - Pom Eubac e ium ec ale 1
CD - Pec Akke mansia muciniphila 1433
CD - Pec Faecalibac e ium p ausni zii 8
CD - Pec Bi idobac e ium longum 2
CD - Pec Collinsella s e co is 1
Mic obial me abolic pa hways
Highe in: Taxa n genes/pa hways
Heal hy - Homog Faecalibac e ium p ausni zii 50
Heal hy - Homog Eubac e ium ec ale 41
Heal hy - Homog Collinsella ae o aciens 1
Heal hy - Pom Faecalibac e ium p ausni zii 12
Heal hy - Pom Eubac e ium ec ale 11
Heal hy - Pec Bi idobac e ium adolescen is 49
Heal hy - Pec Eubac e ium ec ale 1
Heal hy - Pec Faecalibac e ium p ausni zii 1
CD - Homog Collinsella ae o aciens 47
CD - Pec Akke mansia muciniphila 33
I. Cal e e-To e e al.
LWT 187 (2023) 115362
8
o me abolise pec in in he con ex o CD. In his ega d, A. muciniphila
and se e al Ruminococcus clades including no el species showed a wide
ange o pec in-deg ading enzymes and po en ial syne gis ic in-
e ac ions wi h o he gu mic obes ound in CD mic obio a. I should be
no ed ha A. muciniphila and Ruminococcus species a e eme ging p o-
bio ics. P e ious esea ch highligh s he in e es o de elop nex gen-
e a ion p obio ics o p omo e hese axa and o a ge speci ic disease
condi ions such as CD (Lin e al., 2019; Lo dan e al., 2020). Resul s he e
p esen ed ag ee wi h p e ious bioin o ma ic app oaches whe e a wide
ange o polysaccha ide-deg ading enzymes we e anno a ed in MAGs o
A. muciniphila, leading o po en ial coope a ion mechanisms be ween
hese axa and o he gu mic obes in he p esence o pec in (Saba e
e al., 2022). In ac , i has been p oposed ha pec in adminis a ion may
amelio a e gu dysbiosis in CD by p omo ing me abolic in e ac ions
be ween key species such as A. muciniphila (Saba e e al., 2022).
Expe imen al s udies epo c oss- eeding mechanisms be ween
A. muciniphila and o he gu commensals like E. hallii (F i s e al., 2021).
Simila ly, me abolic coope a ion mechanisms be ween di e en
Ruminococcus species in he con ex o ibe me abolism ha e been e-
po ed (C os e al., 2018), in ag eemen wi h ou esul s. P e ious in-
es iga ions om ou esea ch g oup epo an inc emen in
Akke mansia, no el Lachnospi aceae and Ruminococcaceae gene a a e
ecal e men a ion o apple pomace and pec in (Cal e e-To e e al.,
2022), in ag eemen wi h esul s ob ained he e h ough a sho gun
me agenomics app oach. In addi ion, o he au ho s epo ed ha
adminis a ion o a abinoxylan oligosaccha ides and high me hoxyla ed
pec in p omo e A. muciniphila (Zhai e al., 2019), while Lachnospi a and
Ruminococcaceae we e inc eased a e ecal e men a ion o some ci us
and suga bee pec in ac ions (La sen e al., 2019). Fecal e men a ion
o o he unc ional polysaccha ides such as so ghum a abinoxylans led
o an inc emen in Bac e oides and Bi idobac e ium and Lachnospi aceae
(Yao e al., 2023). The majo i y o pec in e men a ion s udies epo
16S RNA gene sequence da a al hough sho gun me agenomics analysis
desc ibed in his wo k allows axonomic iden i ica ion a species and
s ain le el as well as complex me abolic s udies o design no el con-
so ia o in e ac ing mic obes (Bikel e al., 2015; V´
azquez-Cas ellanos
Fig. 4. Hea map showing he p esence o di e en glycosidases (indica ed as black cells) in me agenome-assembled genomes (MAGs) eco e ed om each g oup o
samples: apple pomace (Pom) and pec in (Pec ) de i ed om di e en apple a ie ies ha we e subjec ed o ecal e men a ion using ecal homogena es om heal hy
(A) and C ohn’s disease (CD) (B) dono s. The pe cen age (%) o MAGs con aining each unc ional domain is shown. Codes co esponding o he Ca bohyd a e-Ac i e
enZYmes Da abase (CAZy) amily o each enzyme ha e been assigned.
I. Cal e e-To e e al.
LWT 187 (2023) 115362
9
e al., 2019).
Mic obial abundances o Bi idobac e ium, Bac e oides, Ruminococcus
and Faecalibac e ium we e highe in me agenomes om heal hy dono s
han in hose om CD pa ien s in ag eemen wi h p e ious s udies
(Clooney e al., 2021; Saba e e al., 2022). I has been sugges ed ha
ch onic in lamma o y diseases lead o a dec ease o bac e ia wi h
an i-in lamma o y capaci ies like F. p ausni zii (Nishida e al., 2018;
Sokol e al., 2008). Simila ly, Ruminococcus abundances migh be
dec eased in pa ien s wi h CD, including child en, adolescen s and
adul s (Nishida e al., 2018; Vacca e a., 2020), in ag eemen wi h ou
esul s, al hough some speci ic clades wi hin his g oup, ha e been
associa ed wi h p o-in lamma o y p ope ies (Henke e al., 2019).
Associa ions be ween mic obial composi ion and SCFAs le els we e
also epo ed in he li e a u e. In his ega d, posi i e co ela ions be-
ween Akke mansia and majo SCFAs ace ic, p opanoic and bu y ic acids
ha e been epo ed (Cal e e-To e e al., 2022), in ag eemen wi h ou
esul s. Pec in u iliza ion by he gu mic obio a may lead o he p o-
duc ion o bu y ic acid, mainly h ough u iliza ion o galac ose and
galac u onic acid o pec in chains (Fu e al., 2018). Speci ically,
A. muciniphila and F. p ausni zii comp ise specialis gu anae obic bac-
e ia in ol ed in he b eakdown o die a y ibe (Duncan e al., 2016),
leading o SCFAs p oduc ion and nume ous heal h bene i s (Lin e al.,
2019).
In conclusion, ou esul s sugges ha he s uc u e o he coope -
a ing mic obial conso ia a y depending on he pa icula a ie y o
apple pomace/pec in. This obse a ion ag ees wi h p io wo ks ha
epo ed ha ine polysaccha ide s uc u e de e mines he selec ion o
dis inc e men ing conso ia (Yao e al., 2023). Mos s udies up o da e
epo changes in he mic obio a axonomic p o iles induced by pec in
adminis a ion. Howe e , ew s udies o da e ha e explo ed he po en-
ial in e ac ions be ween mic obial species modula ed by pec in in he
con ex o in lamma o y diseases. In his ega d, esul s he e p esen ed
sugges ha A. muciniphila and Ruminococcus species a e key mic obes
p esen in CD mic obio a ha migh be in ol ed in mul iple
c oss- eeding mechanisms o me abolise pec in. Bioin o ma ic pipelines
employed in his wo k could be o special in e es o assis he a ional
design o p ebio ic o symbio ic in e en ions o modula e he gu
mic obio a o speci ic popula ion g oups. In addi ion, hese me hods
allow he selec ion o key species and mic obial conso ia o me abolise
a gi en subs a e including biologically ac i e polysaccha ides (Shakh-
ma o e al., 2019). In i o demons a ion o he coope a i e me a-
bolism o complex pec in by he mic obial conso ia he ein de ined, will
undoub edly aid owa ds he de elopmen o mic obiome- a ge ed
p ebio ic and/o symbio ic s a egies o speci ic popula ion g oups.
CRediT au ho ship con ibu ion s a emen
Ines Cal e e-To e: In es iga ion, Fo mal analysis, W i ing – e iew
& edi ing. Ca los Saba e : Me hodology, Fo mal analysis, W i ing –
o iginal d a . Abela do Ma golles: Concep ualiza ion, W i ing – e-
iew & edi ing. Lo ena Ruiz: Concep ualiza ion, Resou ces, W i ing –
e iew & edi ing.
Decla a ion o compe ing in e es
Au ho s ha e no con lic o in e es o decla e.
Fig. 5. Me abolic ne wo k illus a ing po en ial c oss- eeding mechanisms be ween me agenome-assembled genomes (MAGs) eco e ed om apple pomace om
Pe ico (A), Regona (B) and Sola ina (C) a ie ies e men ed using C ohn’s disease pa ien s samples. Ne wo k nodes (i. e. ci cles con aining di e en mic obial
communi ies showing equi alen me abolic unc ions) a e connec ed by black lines indica ing syne gis ic ela ionships be ween communi ies and complemen a y
me abolic. MAGs inside he same node play he same ole and could be eplaced by o he membe s om he same communi y wi h he excep ion o essen ial
symbion s included in he g een node. Blue and ed nodes co espond o Akke mansia and Ruminococcus species, espec i ely. MAGs om ne wo k A) include: 1:
Clos idium AP scindens, 2: Bi idobac e ium longum, 3: E epia gaba o ous, 4: Campylobac e B sp905373215, 5: CAJFUH01, 6: Cop omonas sp900066535, 7: Eisenbe giella
po ci, 8: BX12 sp014333425, 9: Ruminococcus D bici culans, 10: Ruminococcus C callidus, 11: Mailhella exc emen igallina um, 12: CAG-170 sp000432135, 13: Phasco-
la c obac e ium A succina u ens, 14: En e oclos e sp001517625, 15: UBA1394 sp900538575, 16: Duodenibacillus in es ina ium, 17: UBA1417 sp003531055, 18:
Me hanob e ibac e A smi hii, 19: Akke mansia muciniphila, 20: Alis ipes A ihumii, 21: Fimi icinus sp900544375, 22: Angelakisella sp900547385, 23: Odo ibac e
splanchnicus, 24: C yp obac e oides sp000433355, 25: P e o ella cop i, 26: Pa abac e oides dis asonis, 27: Lachnospi a eligens A, 28: Akke mansia muciniphila. MAGs om
ne wo k B) include: 1: Ace a i ac o sp003447295, 2: Cop omonas sp900066535, 3: Campylobac e B sp905373215, 4: BX12 sp014333425, 5: En e oclos e clos-
idio o mis, 6: Clos idium AP scindens, 7: Gemmige qucibialis, 8: Ruminococcus D bici culans, 9: Ru henibac e ium lac a i o mans, 10: Pa abac e oides dis asonis, 11: ER4
sp000765235, 12: Bilophila sp902373525, 13: Phascola c obac e ium aecium, 14: CAG-217 sp000436335, 15: Duodenibacillus in es ina ium, 16: Phascola c obac e ium A
succina u ens, 17: Lachnospi a eligens A, 18: UBA1417 sp003531055, 19: CAG-170 sp000432135, 20: Akke mansia muciniphila, 21: Ruminococcus C callidus, 22:
Akke mansia muciniphila, 23: Akke mansia muciniphila, 24: CAG-177 sp003514385, 25: UBA1394 sp900538575, 26: Angelakisella sp900547385. MAGs om ne wo k
C) include: 1: Cop omonas sp900066535, 2: Bi idobac e ium longum, 3: Clos idium AP scindens, 4: CAJFUH01, 5: Eisenbe giella po ci, 6: BX12 sp014333425, 7:
Ruminococcus C callidus, 8: CAG-217 sp000436335, 9: Phascola c obac e ium aecium, 10: Ru henibac e ium lac a i o mans, 11: Aga hobac e ec alis, 12:
Lachnospi a eligens A, 13: Phascola c obac e ium A succina u ens, 14: Me hanob e ibac e A smi hii, 15: Akke mansia muciniphila, 16: Campylobac e B
sp905373215, 17: Akke mansia muciniphila, 18: Collinsella. (Fo in e p e a ion o he e e ences o colou in his igu e legend, he eade is e e ed o he Web
e sion o his a icle.)
I. Cal e e-To e e al.