Ci a ion: Doblado, L.; Díaz, L.E.;
No a, E.; Ma cos, A.; Monsal e, M.
In es inal E ec s o Fil e ed
Alkalinized Wa e in Lean and Obese
Zucke Ra s. Mic oo ganisms 2024,12,
316. h ps://doi.o g/10.3390/
mic oo ganisms12020316
Academic Edi o : Ma La osa
Recei ed: 5 Decembe 2023
Re ised: 22 Decembe 2023
Accep ed: 30 Janua y 2024
Published: 2 Feb ua y 2024
Copy igh : © 2024 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and
condi ions o he C ea i e Commons
A ibu ion (CC BY) license (h ps://
c ea i ecommons.o g/licenses/by/
4.0/).
mic oo ganisms
A icle
In es inal E ec s o Fil e ed Alkalinized Wa e in Lean and
Obese Zucke Ra s
Lau a Doblado 1, Ligia Espe anza Díaz 2, Es he No a 2, Ascensión Ma cos 2and Ma ía Monsal e 1,*
1
Ins i u o de In es igaciones Biomédicas Sols-Mo eale (CSIC-UAM), A u o Dupe ie 4, 28029 Mad id, Spain;
[email p o ec ed]
2Ins i u e o Science, Food Technology and Nu i ion (ICTAN), Spanish Na ional Resea ch Council (CSIC),
JoséAn onio No áis 6, 28040 Mad id, Spain; [email p o ec ed] (L.E.D.); [email p o ec ed] (E.N.);
[email p o ec ed] (A.M.)
*Co espondence: [email p o ec ed]
Abs ac : This s udy e alua ed he in es inal e ec s o alkalinized il e ed wa e in lean and obese
adul Zucke a s. Fo 3 mon hs, 12-week-old a s consumed ei he ap wa e o il e ed alkalinized
ap wa e om Mad id ci y. Weigh gain was moni o ed, changes in me abolism we e e alua ed
by indi ec calo ime y, and o al an ioxidan capaci y and le els o in lamma o y media o s we e
measu ed in plasma. Feces we e collec ed, hei mic obial composi ion was analyzed and his ological
analysis o he small and la ge in es ine was pe o med, assessing he gene al s a e o he mucosa
(MUC2), he in lamma o y s a e (F4/80) and he p esence o oxida i e modi ica ions in p o ein 4-
Hyd oxynonenal (4-HNE) by immuno luo escence (IF) and immunohis ochemis y (IHC). The esul s
ob ained showed ha he consump ion o alkalinized il e ed wa e imp o ed he composi ion o he
in es inal mic obiome and he s a e o he in es inal mucosa, educing bo h local and sys emic in lam-
ma ion and he le el o oxida i e s ess. These changes we e accompanied by a be e main enance o
he oxida i e s a us in a s. No di e ences we e obse ed in an ioxidan capaci y no in weigh gain.
The inco po a ion o p obio ics in he die had a signi ican impac on he mic obiome. These e ec s
we e indica i e o an imp o emen in gene al me abolic, oxida i e and in lamma o y s a us.
Keywo ds: alkalized il e ed wa e ; p obio ics; in es ine; in lamma ion; oxida i e me abolism;
oxida i e s ess
1. In oduc ion
Gas oin es inal (GI) diseases ha e an es ima ed p e alence o 2276.27 million [
1
].
Among hem, unc ional dyspepsia and acid e lux a e pa icula ly common, wi h p e a-
lence es ima ions eaching up o 30%, wi h abou 10% eaching Rome IV unc ional dys-
pepsia (FD), which incu s conside able associa ed heal h impai men s [
2
]. Cu en pha -
macological ea men s o FD aim o ei he neu alize acid o block i s p oduc ion [
3
].
Howe e , eme ging e idence o impo an side e ec s associa ed wi h long- e m ea men s
ha e aised conce ns and he boos ed sea ch o al e na i e app oaches [
4
]. Among he
al e na i e pallia i e in e en ions, d inking alkalinized wa e has gained conside able
a en ion, especially in Eas e n coun ies. The mos commonly used and s udied is he
elec olyzed alkaline- educed wa e (EARW), he consump ion o which has been ela ed o
an imp o emen in FD symp oms [
5
]. The a ailable da a also sugges ha he consump ion
o alkalinized wa e has an i-in lamma o y e ec s, h ough s ill-unclea mechanisms ha
may be o ele ance o o he pa hologies [
6
]. O pa icula in e es , in his con ex , he e
a e o he common diso de s o he in es inal ack such as in es inal dysbiosis.
The indus ial e olu ion undamen ally al e ed he in es inal mic obiome ecosys em,
d as ically educing i s complexi y and inc easing i s unc ional ins abili y [7]. A la ge se
o unc ional s udies ha e demons a ed he key ole played by nu i ional pa e ns on
he p ese a ion o a heal hy mic obiome [
8
] and he ma ked associa ion o obesi y wi h
Mic oo ganisms 2024,12, 316. h ps://doi.o g/10.3390/mic oo ganisms12020316 h ps://www.mdpi.com/jou nal/mic oo ganisms
Mic oo ganisms 2024,12, 316 2 o 16
in es inal dysbiosis [
9
]. Hence, his e idence has also d i en he s udy o he e ec on
di e en wa e sou ces, on in es inal dysbiosis and me abolic homeos asis [
10
]. While i is
gene ally ag eed ha he pH o d inking wa e can impac he mic obiome, i s na u e and
he e ec o EARW consump ion a e s ill a ma e o con o e sy. A wo-week in e en ion
s udy wi h EARW in heal hy human olun ee s concluded ha wa e pH had no impac
on he composi ion o he gu mic obio a o glucose egula ion in young male adul s [
11
].
In con as , in ano he s udy, mice we e ea ed o ou weeks wi h EARW, and he au ho s
showed ha in he ecal mic obiome, he ela i e abundances o 20 axa di e ed signi ican ly
om con ols. Howe e , he signi icance o hese changes was no s ablished [12].
Al e na i e alkaliniza ion p ocedu es, ha do no in ol e elec olysis bu make use
o il e -based sys ems, a e also in he ma ke , bu scien i ic s udies on hei p esumed
heal h e ec s a e s ill a hei in ancy. We ecen ly e alua ed he ascula e ec s o il e
alkalinized wa e consump ion in oden s and ound an imp o ed ascula eac i i y ha
was associa ed wi h educed sys emic in lamma ion [
13
], a esul consis en wi h simila
indings on EARW.
Ou s udy aimed o es he in es inal e ec s o alkalinized il e ed wa e , he possible
di e en ial ole in he lean heal hy s. obese, using, as a es model, Zucke a s, as well
as o assess whe he he in ake o p obio ics, a common ea men o in es inal p oblems,
modi ies i s in es inal e ec s. Obese Zucke a s a e a widely used model o gene ic
obesi y caused by a mu a ion ( a) in he gene ha encodes he ecep o o lec in, an
ano exigenic ho mone. These a s, which su e om insulin esis ance, glucose in ole ance,
and me abolic synd ome, a e he bes exis ing a model o s udies ela ed o obesi y and
diabe es [
14
]. Ra s and mice a e he mos equen ly used animal models o biomedical
s udies, wi h a s being physiologically, mo phologically and gene ically close o humans
han mice, which makes hem an ideal model o biomedical and pha macological s udies.
We ound ha il e ed wa e educed in es inal in lamma ion and oxida i e s ess
ma ke s, imp o ed he gu mucosa s a us, and posi i ely impac ed he mic obiome p o ile
ollowing h ee mon hs o ea men .
2. Ma e ials and Me hods
Ra s. Male Zucke lean (lean) and obese (obese) a s we e used. The animals we e
pu chased om Cha les Ri e and housed a he Ins i u o de In es igaciones Biomédicas
Sols-Mo eale (IIBM) animal acili y unde con olled empe a u e (20–22
◦
C) and humidi y
(50
±
10%) condi ions. The animals we e unde 12 h ligh /12 h da k cycles and had ad
libi um access o ood and wa e . The animal expe imen a ion p o ocols we e app o ed by
he IIBM Ins i u ional Animal Ca e and Use Commi ee, he IIBM Biosa e y Commi ee,
he Consejo Supe io de In es igaciones Cien í icas (CSIC) Bioe hics Commi ee and inal
app o al was g an ed by he En i onmen Depa men o he Communi y o Mad id (Re .
PROEX 117/20). All p ocedu es we e in acco dance wi h he Decla a ion o Helsinki. All
animals ecei ed humane ca e in acco dance wi h he c i e ia desc ibed in he “Guide o
he Ca e and Use o Labo a o y Animals” p epa ed by he Na ional Academy o Sciences
and published by he Na ional Ins i u es o Heal h (No. 86-23 e ised in 1985). The used 16-
week-old a s we e di ided in o h ee expe imen al g oups: one g oup was gi en ap wa e
(no il e ed, no s e ilized) a ailable a he ins i u e o d ink; he second and hi d g oups
we e gi en he same wa e bu p e iously il e ed wi h he Alkana u
®
alkalinizing wa e
il a ion sys em. The wa e - il e ing sys em was analyzed by an au ho ized labo a o y
(Oli e Rodés) and ound o con o m wi h AENOR Spanish No m UNE 149101:2015 o
human consump ion [
15
]. The il e ing sys em adds 15 mg/L o Mg
2+
, has been shown o
emo e T ihalome hanes (THMs) and Cl
−
, o p o ide wa e ee o mic oo ganisms and o
no elease Na
+
. The plas ic ja has been app o ed o con ac wi h ood and demons a ed
o be ee o Bisphenol A (BPA), epoxidized soybean oil (ESBO) and ph hala e es e s [
16
].
The wa e was changed daily. The hi d g oup o animals was ed a die supplemen ed
wi h 3 g/kg o he p obio ic Mega lo a 9 EVO (Solchem
®
, Ba celona, Spain). Mega lo a
9 EVO con ains 2
×
10
9
colony- o ming uni s (c u)/g o Bi idobac e ium lac is W51 and
Mic oo ganisms 2024,12, 316 3 o 16
W52, En e ococcus aecium W54,Lac obacillus acidophilus W22, Lac obacillus pa acasei W20,
Lac obacillus plan a um W1 and W21, Lac obacillus sali a ius W24 and Lac ococcus lac is W19.
In humans, he ecommended dose is 1–2 g [
17
]. Each expe imen al g oup included
6 animals. Weigh gain was moni o ed e e y 2 weeks. F esh eces we e collec ed a
= 0
, 6 weeks (6w) and h ee mon hs (3mo) o ea men . The animals we e ea ed o
3mo and hen sac i iced by decapi a ion a e in ape i oneal injec ion wi h pen oba bi al
sodium (100 mg/kg). Blood was collec ed, and plasma and pe iphe al blood mononuclea
cells (PBMC) we e sepa a ed using a Ficoll-Paque
TM
PLUS (Cy i a Cy i a Eu ope GmbH
Sucu sal en España, Ba celona, Spain) g adien and s o ed a
−
80
◦
C. The in es ines we e
collec ed, washed in phospha e-bu e ed saline (PBS) and cu in o sec ions. Tissue sec ions
we e snap ozen and s o ed a
−
80
◦
C o ixed in 10% bu e ed o malin, hen embedded
in pa a in, and 4
µ
m sec ions we e cu wi h a mic o ome, depa a inized and hyd a ed
p io o s aining.
Indi ec calo ime y. The PhenoMas e indi ec calo ime y sys em (TSE Sys ems
GmbH, Bad Hombu g o de Höhe, Ge many) was used o analyze me abolism in li e,
eely mo ing animals, au oma ically and con inuously, o e h ee ligh –da k cycles in
a dedica ed, con olled-en i onmen acili y a he IIBM. Ra s we e i s acclima ized o
he PhenoMas e oom o h ee days be o e he expe imen began. Each a was hen
assigned o an indi idual cage wi h a sepa a e gas collec ion and deli e y sys em, which
was con olled by TSE PhenoMas e 5.1 so wa e. The ollowing pa ame e s we e examined:
olume (V) O
2
, VCO
2
, RER, ene gy expendi u e (H1+), ac i i y (XT + YT), weigh o ood
in ake and wa e olume consumed. Da a we e egis e ed e e y hou o e 72 h. Animals
we e exposed o 12 h ligh and 12 h da k cycles. Da a we e ex ac ed and analyzed using
TSE PhenoMas e 5.1 so wa e. Da a p esen ed co espond o he mean alues o O
2
consumed o CO
2
p oduced pe hou and egis e ed du ing he day (12 h) o nigh (12 h)
pe iods o he second 24 h cycle (2nd day), when measu emen s we e mo e s able.
Mic obiome. Feces samples we e collec ed, and bac e ial DNA was ex ac ed using he
QIAamp
®
Fas DNA S ool Mini Ki (Quiagen, Hilden, Ge many). Bac e ial quan i ica ion
was pe o med by qPCR using speci ic p ime s o each o he es ed bac e ial g oups:
Bac e oides,Blau ia coccoides-Eubac e ium ec ale g oup, Clos idium clus e IV, Bi idobac e ium
spp., Lac obacillus spp., En e obac e iaceae, En e ococcus spp., Faecaibac e ium p aus nizii and
Akke mansia muciniphila di ec ed agains he genes ha encode 16S RNA.
En e obac e iaceae
Eco1457 Fo wa d 5′-CATTGACGTTACCCGCAGAAGAAGC-3′
Eco1652 Re e se 5′-CTCTACGAGACTCAAGCTTGC-3′
Blau ia coccoides-Eubac e ium ec ale G oup
gCcoc Fo wa d 5′-AAATGACGGTACCTGACTAA-3′
gCcoc Re e se 5′-CTTTGAGTTTCATTCTTGCGAA-3′
Clos idium clus e IV
sg-Clep Fo wa d 5′-GCACAAGCAGTGGAGT-3′
sg-Clep Re e se 5′-CTTCCTCCGTTTTGTCAA-3′
En e ococcus spp.
En e oc Fo wa d 5′-CCCTTATTGTTAGTTGCCATCATT-3′
En e oc Re e se 5′-ACTCGTTGTACTTCCCATTGT-3′
Lac obacillus spp.
Lac o Fo wa d 5′-CACCGCTACACATGGAG-3′
Lac o Re e se 5′-AGCAGTAGGGAATCTTCCA-3′
Bi idobac e ium spp.
Bi Fo wa d 5′-TCGCGTC(C/T)GGTGTGAAAG-3′
Bi Re e se 5′-CCACATCCAGC(A/G)TCCAC-3′
Bac e oides agilis G oup
B a Fo wa d 5′-ATAGCCTTTCGAAAGRAAGAT-3′
B a Re e se 5′-CCAGTATCAACTGCAATTTTA-3′
Mic oo ganisms 2024,12, 316 4 o 16
Faecalibac e ium p aus nizii
Fp au223 Fo wa d 5′-GATGGCCTCGCGTCCGATTAG-3′
Fp au420 Re e se 5′-CCGAAGACCTTCTTCCTCC-3′
Akke mansia muciniphila
S-S -Muc-1437-a-A-20 Fo wa d 5′- CCTTGCGGTTGGCTTCAGAT-3′
S-S -Muc-1129-a-a-20 Re e se 5′-CAGCACGTGAAGGTGGGGAC-3′
Immunohis ochemis y (IHC). Tissue ixa ion and s aining we e pe o med using he
Vec as ain ABC ki and he DAB pe oxidase subs a e ki (bo h om Vec o Labo a o ies,
Bu lingame, CA, USA) ollowing he manu ac u e ’s ins uc ions. Samples we e incuba ed
wi h an i-F4/80 p ima y an ibody (MCA497; AbD Se o ec, Ox o d, UK) and hen wi h
an i- a seconda y an ibody (NA935 GE Heal hca e, Bos on, MA, USA) linked o alkaline
phospha ase (AP), exposed o he AP subs a e and, once s aining de eloped, moun ed.
Images we e acqui ed wi h a Nikon E90i mic oscope equipped wi h a DS-Fi1 came a
(Nikon, Tokyo, Japan).
Immuno luo escence (IF). Fixa ion, s aining, and analysis p ocedu es we e as p e-
iously desc ibed [
13
]. P ima y an ibodies used we e an i-Mucin 2 (sc-7314; San a C uz
Bio echnology Inc., Dallas, TX, USA) and an i-4-HNE (AB5605, Me k, Da ms ad , Ge -
many). Immuno luo escence was de ec ed by incuba ion wi h a luo escen seconda y
an ibody (FITC; Me ck, Da ms ad , Ge many). The samples we e hen coun e s ained wi h
DAPI (In i ogen Co p., Ca lsbad, CA, USA), moun ed, and isualized using a luo escence
mic oscope, Zeiss LSM 700.
Image analysis. IHC and IF images we e analyzed using Fiji-ImageJ 2.0.0- c-69/1.52p
so wa e (NIH, Be hesda, MD, USA) o de e mine he a io o posi i e a ea o issue a ea
(IHC) o o al in eg a ed luo escence signal o issue a ea (IF).
Gene exp ession analysis. In es inal issue samples we e homogenized in he p esence
o 1 mL o T izol
TM
eagen (The moFishe Sci., Wal ham, MA, USA), and o al RNA was
isola ed ollowing he manu ac u e ’s ins uc ions. Complemen a y DNA (cDNA) was
syn hesized, om o al RNA p epa a ions, by e e se ansc ip ion o 1
µ
g o RNA, using
Moloney Mu ine Leukemia Vi us (M-MLV) e e se ansc ip ase (RT) (P omega Bio ech
Ibé ica SL, Alcobendas, Mad id, Spain), in a inal olume o 20
µ
. The mix u e was incu-
ba ed a 37
◦
C o 45 min and hen cooled o 2 min a 4
◦
C. The esul ing cDNA was used
as a empla e o subsequen quan i a i e polyme ase chain eac ion (qPCR). The speci ic
p ime s used a e lis ed below. Each 10
µ
L PCR eac ion included 1
µ
L cDNA, 5
µ
L qPCR-
BIO SyG een Mas e mix (Cul ek SL, Du chche G oup, San Fe nando de Hena es, Mad id,
Spain) and p ime s (0.3
µ
M). Samples we e analyzed in iplica e on a Mas e cycle
®
Re-
alPlex2, Eppendo Ibe ica SLU (San Sebas ian de los Reyes, Mad id, Spain). 36B4 was
used as loading con ol.
36B4 Fo wa d 5′-GCGACCTGGAGTCCAACTA-3′
36B4 Re e se 5′: ATCTGCTGCATCTGCTTGG-3′
IL-1B Fo wa d 5′-GCCAACAAGTGGTATTCTCCATGAGC-3′
IL-1B Re e se 5′-TTGTCACCCCGGATGGAATG-3′
IL-10 Re e se 5′-TTGTCACCCCGGATGGAATG-3′
IL-10 Fo wa d 5′-GCTCAGCACTGCTATGTTGC-3′
Cy okines. Ci cula ing le els o IL-1
β
, IL-4, IL-6, TNF
α
and IL-10 we e analyzed
by cellula cy ome y in plasma samples using Mul iplex Cy okine Assays (P o-ca aPlex
Immunoassays (The moFishe Scien i ic, Wal ham, MA, USA)) a he Cen o Nacional de
Bio ecnología (CNB, CSIC, Mad id, Spain) Flow Cy ome y Uni . The lean a samples we e
pooled in pai s (each analyzed sample/da a poin is he mix u e o wo plasma samples,
om wo a s); obese a plasma samples we e no pooled. The alues ob ained we e
co ec ed by he concen a ion o p o eins in he sample de e mined by he Low y me hod
ia colo ime y.
Mic oo ganisms 2024,12, 316 5 o 16
An ioxidan capaci y. An ioxidan capaci y was de e mined in plasma samples us-
ing he e-BQC elec ochemical analy ical sys em (BioQuoChem, O iedo, Spain), which
measu es o al (QT), as (Q1) and slow-ac ing (Q2) an ioxidan capaci y.
S a is ical analysis. Mic oso Excel 16.16.27 (Mic oso , Redmond, WA, USA) was
used o da a p ocessing. G aphPad P ism 9 (Do ma ics, Bos on, MA, USA) was used
o s a is ical analysis and g aph p epa a ion. Da a a e exp essed as mean
±
s anda d
de ia ion (SD) o s anda d e o o he mean (SEM), as indica ed in he igu e legends.
The no mali y o he da a was assessed using he Kolmogo o –Smi no es . S a is ical
signi icance o di e ences be ween g oups was assessed using wo- ailed unpai ed o
pai ed - es o compa isons be ween wo g oups; one-way and wo-way ANOVA was
used o he analysis o ime-dependen a ia ion and he di e en ial esponse o ea men s
o wo g oups, espec i ely. Le ene’s es was used o equali y o a iances. The alues
we e conside ed s a is ically signi ican when p< 0.05. No alues we e disca ded; alues
no included could no be ob ained due o acquisi ion p oblems o echnical p oblems wi h
he samples o animals.
3. Resul s
In o de o es he in es inal e ec s o alkalinized il e ed wa e and he di e en ial
impac in obesi y, lean and obese Zucke a s we e ea ed o 3mo wi h ap wa e o
il e ed wa e . The p obio ic Mega lo a 9 EVO was included in he die o a g oup o a s
dinking il e ed wa e , adjus ing he dose o ha ecommended o humans. The decision
o pe o m a 3mo ea men (3mo) was based on ou p e ious s udies, showing signi ican
changes in a s ea ed o 3mo. We also collec ed blood and s ool samples a 6 weeks o a
be e ollow-up o he ea men esponses.
3.1. Weigh Gain
The a s’ weigh gain was moni o ed e e y wo weeks, and bo h absolu e and ela i e
weigh gain alues we e de e mined (Figu e 1). The esul s did no show signi ican
di e ences associa ed wi h he ea men s excep a ou weeks o ea men , when he
ela i e weigh gain (%) o he il e ed wa e and p obio ic g oups was signi ican ly g ea e
han ha o he il e ed wa e g oup, bu his di e ence was no main ained a longe
ea men imes. Simila ly, he a eas unde he cu e (AUC) o he absolu e alues did no
show signi ican di e ences be ween ea men g oups. The e o e, we concluded ha he
ea men s did no ha e a ele an e ec on he animals’ weigh gain.
Mic oo ganisms 2024, 12, x FOR PEER REVIEW 6 o 16
Figu e 1. Absolu e (uppe panel) and ela i e (lowe panel) weigh gain de e mined e e y 2 weeks
o 3mo. In he bo om panel, weigh alues a = 0 we e conside ed 100%. The g aphs show mean
+/− SD.
3.2. Ci cula ing Cy okines
Nex , in o de o de e mine he gene al in lamma o y s a us o he a s, he ci cula -
ing le els o cy okines (IL-1β, IL-4, IL-6, TNFα and IL-10) we e e alua ed in blood plasma
samples. Signi ican changes we e ound in he le els o IL-1β and IL-10, cy okines gene -
ally conside ed as ma ke s o ype M1 and M2 mac ophages, espec i ely. A e 6w o
ea men , highe le els o IL-1β we e obse ed in lean a s ha consumed ap wa e han
in a s ha consumed il e ed wa e , and lowe le els o IL-10 in a s ha consumed ap
wa e han in a s ha consumed il e ed wa e (Figu e 2), sugges ing ha d inking alka-
linized wa e a o s an immunosupp essi e and an i-in lamma o y p o ile.
Figu e 2. Changes in plasma le els o IL-1β and IL-10 (le els a 6w—le els a = 0) ollowing 6w o
ea men . Tap, il e , il e + p obio ic. The g aph shows mean +/− SEM. * p < 0.05 ( es ).
3.3. An ioxidan Capaci y
Then, we e alua ed how he ea men s affec ed he o al an ioxidan capaci y in
blood plasma samples. To his end, as (Q1), slow (Q2) and o al (QT) an ioxidan capac-
i ies we e measu ed using he e-BQC elec ochemical eade (BioQuoChem). No signi i-
can diffe ences we e obse ed be ween he ea men g oups a ei he 6w o 3mo (Figu e
Figu e 1. Absolu e (uppe panel) and ela i e (lowe panel) weigh gain de e mined e e y 2 weeks o
3mo. In he bo om panel, weigh alues a = 0 we e conside ed 100%. The g aphs show mean +/
−
SD.

Mic oo ganisms 2024,12, 316 6 o 16
3.2. Ci cula ing Cy okines
Nex , in o de o de e mine he gene al in lamma o y s a us o he a s, he ci cula ing
le els o cy okines (IL-1
β
, IL-4, IL-6, TNF
α
and IL-10) we e e alua ed in blood plasma
samples. Signi ican changes we e ound in he le els o IL-1
β
and IL-10, cy okines gen-
e ally conside ed as ma ke s o ype M1 and M2 mac ophages, espec i ely. A e 6w
o ea men , highe le els o IL-1
β
we e obse ed in lean a s ha consumed ap wa e
han in a s ha consumed il e ed wa e , and lowe le els o IL-10 in a s ha consumed
ap wa e han in a s ha consumed il e ed wa e (Figu e 2), sugges ing ha d inking
alkalinized wa e a o s an immunosupp essi e and an i-in lamma o y p o ile.
Mic oo ganisms 2024, 12, x FOR PEER REVIEW 6 o 16
Figu e 1. Absolu e (uppe panel) and ela i e (lowe panel) weigh gain de e mined e e y 2 weeks
o 3mo. In he bo om panel, weigh alues a = 0 we e conside ed 100%. The g aphs show mean
+/− SD.
3.2. Ci cula ing Cy okines
Nex , in o de o de e mine he gene al in lamma o y s a us o he a s, he ci cula -
ing le els o cy okines (IL-1β, IL-4, IL-6, TNFα and IL-10) we e e alua ed in blood plasma
samples. Signi ican changes we e ound in he le els o IL-1β and IL-10, cy okines gene -
ally conside ed as ma ke s o ype M1 and M2 mac ophages, espec i ely. A e 6w o
ea men , highe le els o IL-1β we e obse ed in lean a s ha consumed ap wa e han
in a s ha consumed il e ed wa e , and lowe le els o IL-10 in a s ha consumed ap
wa e han in a s ha consumed il e ed wa e (Figu e 2), sugges ing ha d inking alka-
linized wa e a o s an immunosupp essi e and an i-in lamma o y p o ile.
Figu e 2. Changes in plasma le els o IL-1β and IL-10 (le els a 6w—le els a = 0) ollowing 6w o
ea men . Tap, il e , il e + p obio ic. The g aph shows mean +/− SEM. * p < 0.05 ( es ).
3.3. An ioxidan Capaci y
Then, we e alua ed how he ea men s affec ed he o al an ioxidan capaci y in
blood plasma samples. To his end, as (Q1), slow (Q2) and o al (QT) an ioxidan capac-
i ies we e measu ed using he e-BQC elec ochemical eade (BioQuoChem). No signi i-
can diffe ences we e obse ed be ween he ea men g oups a ei he 6w o 3mo (Figu e
Figu e 2. Changes in plasma le els o IL-1
β
and IL-10 (le els a 6w—le els a = 0) ollowing 6w o
ea men .
Mic oo ganisms 2024, 12, x FOR PEER REVIEW 13 o 16
pe o med a p e ious s udy ha es ed he effec s o he consump ion o il e ed alka-
linized wa e in mice and a s o 3mo and ound e idence sugges ing a educ ion in oxi-
da i e s ess and in lamma ion [13]. The p esen s udy, hus, aimed o e alua e he in es-
inal impac o il e ed alkalinized wa e in lean and obese a s. The esul s ob ained sup-
po he hypo hesis ha il e ed alkalinized wa e , when compa ed o un il e ed–uns e i-
lized ap wa e om Mad id ci y, has a posi i e effec on bo h sys emic and in es inal
in lamma ion, be e p ese ing he in es inal mucin laye and p e en ing in es inal
dysbiosis. These changes we e linked o a be e main enance o oxida i e me abolism,
sugges ing a be e me abolic s a us o he a s consuming il e ed wa e (Figu e 4). O
no e, he imp o ed me abolic p o ile is no ela ed o any de ec able changes in weigh
gain (Figu e 1). Taking in o conside a ion he de ec ed changes in he mic obiome, wi h
inc eased Akke mansia muciniphila and Clos idium clus e IV and dec eased En e obac e i-
aceae in esponse o ea men wi h il e ed alkalinized wa e (Figu e 9), i is emp ing o
specula e ha hese changes a e likely he main d i e s o he p ese a ion o he in es inal
mucin laye , leading, in u n, o educed in es inal and sys emic in lamma ion, wi h a gen-
e al posi i e impac on whole body me abolism. Akke mansia muciniphila is a mucin-de-
g ading bac e ium ha has also been shown o s imula e mucin p oduc ion wi h a ne
posi i e impac on he hickness o he mucin laye , he p ese a ion o gu ba ie unc-
ion and in es inal immune homeos asis [33]. Clos idium clus e IV is well known o effi-
cien ly e men ing he plan polysaccha ides composing die a y ibe . Clos idium species
ha e been epo ed o a enua e in lamma ion, and i s me aboli es, including bu y a e,
seconda y bile acids and indolep opionic acid, ha e been shown o play a p obio ic ole,
p ima ily h ough hei effec s on in es inal epi helial cells, s eng hening he in es inal
ba ie [34].
En e obac e iaceae a e acul a i e anae obes ha , in no mal condi ions, play
a key ole in in es inal oxygen consump ion, allowing o he p oli e a ion o anae obes,
necessa y o polysaccha ide e men a ion. Nu ien o e load and exposu e o in lamma-
o y media o s ha e been p oposed o educe hei oxida i e capaci y and d i e hei p o-
li e a ion, hus p omo ing obesi y- ela ed dysbiosis [35].
Figu e 9. qPCR analysis o mic obial con en in eces. The g aphs show he analysis o bac e ial
g oups analyzed a he indica ed imes. Tap, il e , il e + p obio ic. The ep esen ed al-
ues in he g aphs a e he diffe ences in he numbe o bac e ia be ween he alues ob ained a he
indica ed ime and = 0 o each indi idual a analyzed, mean alues +/− SD. S a is ical compa ison
Tap,
Mic oo ganisms 2024, 12, x FOR PEER REVIEW 13 o 16
pe o med a p e ious s udy ha es ed he effec s o he consump ion o il e ed alka-
linized wa e in mice and a s o 3mo and ound e idence sugges ing a educ ion in oxi-
da i e s ess and in lamma ion [13]. The p esen s udy, hus, aimed o e alua e he in es-
inal impac o il e ed alkalinized wa e in lean and obese a s. The esul s ob ained sup-
po he hypo hesis ha il e ed alkalinized wa e , when compa ed o un il e ed–uns e i-
lized ap wa e om Mad id ci y, has a posi i e effec on bo h sys emic and in es inal
in lamma ion, be e p ese ing he in es inal mucin laye and p e en ing in es inal
dysbiosis. These changes we e linked o a be e main enance o oxida i e me abolism,
sugges ing a be e me abolic s a us o he a s consuming il e ed wa e (Figu e 4). O
no e, he imp o ed me abolic p o ile is no ela ed o any de ec able changes in weigh
gain (Figu e 1). Taking in o conside a ion he de ec ed changes in he mic obiome, wi h
inc eased Akke mansia muciniphila and Clos idium clus e IV and dec eased En e obac e i-
aceae in esponse o ea men wi h il e ed alkalinized wa e (Figu e 9), i is emp ing o
specula e ha hese changes a e likely he main d i e s o he p ese a ion o he in es inal
mucin laye , leading, in u n, o educed in es inal and sys emic in lamma ion, wi h a gen-
e al posi i e impac on whole body me abolism. Akke mansia muciniphila is a mucin-de-
g ading bac e ium ha has also been shown o s imula e mucin p oduc ion wi h a ne
posi i e impac on he hickness o he mucin laye , he p ese a ion o gu ba ie unc-
ion and in es inal immune homeos asis [33]. Clos idium clus e IV is well known o effi-
cien ly e men ing he plan polysaccha ides composing die a y ibe . Clos idium species
ha e been epo ed o a enua e in lamma ion, and i s me aboli es, including bu y a e,
seconda y bile acids and indolep opionic acid, ha e been shown o play a p obio ic ole,
p ima ily h ough hei effec s on in es inal epi helial cells, s eng hening he in es inal
ba ie [34].
En e obac e iaceae a e acul a i e anae obes ha , in no mal condi ions, play
a key ole in in es inal oxygen consump ion, allowing o he p oli e a ion o anae obes,
necessa y o polysaccha ide e men a ion. Nu ien o e load and exposu e o in lamma-
o y media o s ha e been p oposed o educe hei oxida i e capaci y and d i e hei p o-
li e a ion, hus p omo ing obesi y- ela ed dysbiosis [35].
Figu e 9. qPCR analysis o mic obial con en in eces. The g aphs show he analysis o bac e ial
g oups analyzed a he indica ed imes. Tap, il e , il e + p obio ic. The ep esen ed al-
ues in he g aphs a e he diffe ences in he numbe o bac e ia be ween he alues ob ained a he
indica ed ime and = 0 o each indi idual a analyzed, mean alues +/− SD. S a is ical compa ison
il e ,
Mic oo ganisms 2024, 12, x FOR PEER REVIEW 13 o 16
pe o med a p e ious s udy ha es ed he effec s o he consump ion o il e ed alka-
linized wa e in mice and a s o 3mo and ound e idence sugges ing a educ ion in oxi-
da i e s ess and in lamma ion [13]. The p esen s udy, hus, aimed o e alua e he in es-
inal impac o il e ed alkalinized wa e in lean and obese a s. The esul s ob ained sup-
po he hypo hesis ha il e ed alkalinized wa e , when compa ed o un il e ed–uns e i-
lized ap wa e om Mad id ci y, has a posi i e effec on bo h sys emic and in es inal
in lamma ion, be e p ese ing he in es inal mucin laye and p e en ing in es inal
dysbiosis. These changes we e linked o a be e main enance o oxida i e me abolism,
sugges ing a be e me abolic s a us o he a s consuming il e ed wa e (Figu e 4). O
no e, he imp o ed me abolic p o ile is no ela ed o any de ec able changes in weigh
gain (Figu e 1). Taking in o conside a ion he de ec ed changes in he mic obiome, wi h
inc eased Akke mansia muciniphila and Clos idium clus e IV and dec eased En e obac e i-
aceae in esponse o ea men wi h il e ed alkalinized wa e (Figu e 9), i is emp ing o
specula e ha hese changes a e likely he main d i e s o he p ese a ion o he in es inal
mucin laye , leading, in u n, o educed in es inal and sys emic in lamma ion, wi h a gen-
e al posi i e impac on whole body me abolism. Akke mansia muciniphila is a mucin-de-
g ading bac e ium ha has also been shown o s imula e mucin p oduc ion wi h a ne
posi i e impac on he hickness o he mucin laye , he p ese a ion o gu ba ie unc-
ion and in es inal immune homeos asis [33]. Clos idium clus e IV is well known o effi-
cien ly e men ing he plan polysaccha ides composing die a y ibe . Clos idium species
ha e been epo ed o a enua e in lamma ion, and i s me aboli es, including bu y a e,
seconda y bile acids and indolep opionic acid, ha e been shown o play a p obio ic ole,
p ima ily h ough hei effec s on in es inal epi helial cells, s eng hening he in es inal
ba ie [34].
En e obac e iaceae a e acul a i e anae obes ha , in no mal condi ions, play
a key ole in in es inal oxygen consump ion, allowing o he p oli e a ion o anae obes,
necessa y o polysaccha ide e men a ion. Nu ien o e load and exposu e o in lamma-
o y media o s ha e been p oposed o educe hei oxida i e capaci y and d i e hei p o-
li e a ion, hus p omo ing obesi y- ela ed dysbiosis [35].
Figu e 9. qPCR analysis o mic obial con en in eces. The g aphs show he analysis o bac e ial
g oups analyzed a he indica ed imes. Tap, il e , il e + p obio ic. The ep esen ed al-
ues in he g aphs a e he diffe ences in he numbe o bac e ia be ween he alues ob ained a he
indica ed ime and = 0 o each indi idual a analyzed, mean alues +/− SD. S a is ical compa ison
il e + p obio ic. The g aph shows mean +/
−
SEM. * p< 0.05 ( es ).
3.3. An ioxidan Capaci y
Then, we e alua ed how he ea men s a ec ed he o al an ioxidan capaci y in blood
plasma samples. To his end, as (Q1), slow (Q2) and o al (QT) an ioxidan capaci ies
we e measu ed using he e-BQC elec ochemical eade (BioQuoChem). No signi ican
di e ences we e obse ed be ween he ea men g oups a ei he 6w o 3mo (Figu e 3),
sugges ing ha he ea men s did no globally a ec he an ioxidan capaci y o he a s.
Mic oo ganisms 2024, 12, x FOR PEER REVIEW 7 o 16
3), sugges ing ha he ea men s did no globally affec he an ioxidan capaci y o he
a s.
Figu e 3. Elec ochemical analysis o he o al an ioxidan capaci y (QT) in plasma samples om a s
ea ed o 6w (le panel) o 3mo ( igh panel). Tap, il e , il e + p obio ic. The g aph
shows he elec ochemical uni s p o ided by he e-BQC eade and includes mean alues +/− SD.
3.4. Indi ec Calo ime y
In o de o analyze he impac o he ea men s on sys emic me abolism, an indi ec
calo ime y sys em was used o de e mine he a s’ O
2
consump ion and CO
2
p oduc ion
and how hey changed in esponse o ea men s a 6w (1.5mo) and 3mo (Figu e 4). The
g oup o ap wa e a s showed a g adual dec ease in me abolic ac i i y, affec ing bo h O
2
consump ion and CO
2
p oduc ion. These changes we e signi ican in he nigh pe iod in
no mal a s a 3mo and in obese a s bo h du ing he nigh and day pe iods a 3mo. The
dec ease in CO
2
p oduc ion was also signi ican in he obese ap wa e g oup o a s a 6w.
These educ ions we e no obse ed o did no each s a is ical signi icance in he g oups
ea ed wi h il e ed wa e , bo h wi h and wi hou p obio ics. In ac , in obese a s, he
magni ude o he diffe ence was signi ican ly smalle in a s ea ed wi h il e ed wa e
and p obio ics han in con ol a s, in he day and nigh pe iods, o bo h O
2
consump ion
and CO
2
p oduc ion. These esul s sugges ha ea men wi h il e ed wa e , pa icula ly
in combina ion wi h p obio ics, main ains a highe me abolic ac i i y han ap wa e , es-
pecially in obese a s.
Figu e 4. Analysis o O
2
consump ion (uppe panels) and CO
2
p oduc ion olumes (lowe panels)
by indi ec calo ime y. The a s we e e alua ed be o e ea men , a 6w and 3mo o ea men .
Tap, Fil e , Fil e + P obio ic. The alues shown in he g aphs a e he diffe ence o O
2
olume
(V) consumed (uppe panel) o CO
2
p oduced (lowe panel) be ween he indica ed ime and = 0
o each indi idual a analyzed. De e mina ions we e aken e e y hou o 72 h, he p esen ed da a
Figu e 3. Elec ochemical analysis o he o al an ioxidan capaci y (QT) in plasma samples om a s
ea ed o 6w (le panel) o 3mo ( igh panel).
Mic oo ganisms 2024, 12, x FOR PEER REVIEW 13 o 16
pe o med a p e ious s udy ha es ed he effec s o he consump ion o il e ed alka-
linized wa e in mice and a s o 3mo and ound e idence sugges ing a educ ion in oxi-
da i e s ess and in lamma ion [13]. The p esen s udy, hus, aimed o e alua e he in es-
inal impac o il e ed alkalinized wa e in lean and obese a s. The esul s ob ained sup-
po he hypo hesis ha il e ed alkalinized wa e , when compa ed o un il e ed–uns e i-
lized ap wa e om Mad id ci y, has a posi i e effec on bo h sys emic and in es inal
in lamma ion, be e p ese ing he in es inal mucin laye and p e en ing in es inal
dysbiosis. These changes we e linked o a be e main enance o oxida i e me abolism,
sugges ing a be e me abolic s a us o he a s consuming il e ed wa e (Figu e 4). O
no e, he imp o ed me abolic p o ile is no ela ed o any de ec able changes in weigh
gain (Figu e 1). Taking in o conside a ion he de ec ed changes in he mic obiome, wi h
inc eased Akke mansia muciniphila and Clos idium clus e IV and dec eased En e obac e i-
aceae in esponse o ea men wi h il e ed alkalinized wa e (Figu e 9), i is emp ing o
specula e ha hese changes a e likely he main d i e s o he p ese a ion o he in es inal
mucin laye , leading, in u n, o educed in es inal and sys emic in lamma ion, wi h a gen-
e al posi i e impac on whole body me abolism. Akke mansia muciniphila is a mucin-de-
g ading bac e ium ha has also been shown o s imula e mucin p oduc ion wi h a ne
posi i e impac on he hickness o he mucin laye , he p ese a ion o gu ba ie unc-
ion and in es inal immune homeos asis [33]. Clos idium clus e IV is well known o effi-
cien ly e men ing he plan polysaccha ides composing die a y ibe . Clos idium species
ha e been epo ed o a enua e in lamma ion, and i s me aboli es, including bu y a e,
seconda y bile acids and indolep opionic acid, ha e been shown o play a p obio ic ole,
p ima ily h ough hei effec s on in es inal epi helial cells, s eng hening he in es inal
ba ie [34].
En e obac e iaceae a e acul a i e anae obes ha , in no mal condi ions, play
a key ole in in es inal oxygen consump ion, allowing o he p oli e a ion o anae obes,
necessa y o polysaccha ide e men a ion. Nu ien o e load and exposu e o in lamma-
o y media o s ha e been p oposed o educe hei oxida i e capaci y and d i e hei p o-
li e a ion, hus p omo ing obesi y- ela ed dysbiosis [35].
Figu e 9. qPCR analysis o mic obial con en in eces. The g aphs show he analysis o bac e ial
g oups analyzed a he indica ed imes. Tap, il e , il e + p obio ic. The ep esen ed al-
ues in he g aphs a e he diffe ences in he numbe o bac e ia be ween he alues ob ained a he
indica ed ime and = 0 o each indi idual a analyzed, mean alues +/− SD. S a is ical compa ison
Tap,
Mic oo ganisms 2024, 12, x FOR PEER REVIEW 13 o 16
pe o med a p e ious s udy ha es ed he effec s o he consump ion o il e ed alka-
linized wa e in mice and a s o 3mo and ound e idence sugges ing a educ ion in oxi-
da i e s ess and in lamma ion [13]. The p esen s udy, hus, aimed o e alua e he in es-
inal impac o il e ed alkalinized wa e in lean and obese a s. The esul s ob ained sup-
po he hypo hesis ha il e ed alkalinized wa e , when compa ed o un il e ed–uns e i-
lized ap wa e om Mad id ci y, has a posi i e effec on bo h sys emic and in es inal
in lamma ion, be e p ese ing he in es inal mucin laye and p e en ing in es inal
dysbiosis. These changes we e linked o a be e main enance o oxida i e me abolism,
sugges ing a be e me abolic s a us o he a s consuming il e ed wa e (Figu e 4). O
no e, he imp o ed me abolic p o ile is no ela ed o any de ec able changes in weigh
gain (Figu e 1). Taking in o conside a ion he de ec ed changes in he mic obiome, wi h
inc eased Akke mansia muciniphila and Clos idium clus e IV and dec eased En e obac e i-
aceae in esponse o ea men wi h il e ed alkalinized wa e (Figu e 9), i is emp ing o
specula e ha hese changes a e likely he main d i e s o he p ese a ion o he in es inal
mucin laye , leading, in u n, o educed in es inal and sys emic in lamma ion, wi h a gen-
e al posi i e impac on whole body me abolism. Akke mansia muciniphila is a mucin-de-
g ading bac e ium ha has also been shown o s imula e mucin p oduc ion wi h a ne
posi i e impac on he hickness o he mucin laye , he p ese a ion o gu ba ie unc-
ion and in es inal immune homeos asis [33]. Clos idium clus e IV is well known o effi-
cien ly e men ing he plan polysaccha ides composing die a y ibe . Clos idium species
ha e been epo ed o a enua e in lamma ion, and i s me aboli es, including bu y a e,
seconda y bile acids and indolep opionic acid, ha e been shown o play a p obio ic ole,
p ima ily h ough hei effec s on in es inal epi helial cells, s eng hening he in es inal
ba ie [34].
En e obac e iaceae a e acul a i e anae obes ha , in no mal condi ions, play
a key ole in in es inal oxygen consump ion, allowing o he p oli e a ion o anae obes,
necessa y o polysaccha ide e men a ion. Nu ien o e load and exposu e o in lamma-
o y media o s ha e been p oposed o educe hei oxida i e capaci y and d i e hei p o-
li e a ion, hus p omo ing obesi y- ela ed dysbiosis [35].
Figu e 9. qPCR analysis o mic obial con en in eces. The g aphs show he analysis o bac e ial
g oups analyzed a he indica ed imes. Tap, il e , il e + p obio ic. The ep esen ed al-
ues in he g aphs a e he diffe ences in he numbe o bac e ia be ween he alues ob ained a he
indica ed ime and = 0 o each indi idual a analyzed, mean alues +/− SD. S a is ical compa ison
il e ,
Mic oo ganisms 2024, 12, x FOR PEER REVIEW 13 o 16
pe o med a p e ious s udy ha es ed he effec s o he consump ion o il e ed alka-
linized wa e in mice and a s o 3mo and ound e idence sugges ing a educ ion in oxi-
da i e s ess and in lamma ion [13]. The p esen s udy, hus, aimed o e alua e he in es-
inal impac o il e ed alkalinized wa e in lean and obese a s. The esul s ob ained sup-
po he hypo hesis ha il e ed alkalinized wa e , when compa ed o un il e ed–uns e i-
lized ap wa e om Mad id ci y, has a posi i e effec on bo h sys emic and in es inal
in lamma ion, be e p ese ing he in es inal mucin laye and p e en ing in es inal
dysbiosis. These changes we e linked o a be e main enance o oxida i e me abolism,
sugges ing a be e me abolic s a us o he a s consuming il e ed wa e (Figu e 4). O
no e, he imp o ed me abolic p o ile is no ela ed o any de ec able changes in weigh
gain (Figu e 1). Taking in o conside a ion he de ec ed changes in he mic obiome, wi h
inc eased Akke mansia muciniphila and Clos idium clus e IV and dec eased En e obac e i-
aceae in esponse o ea men wi h il e ed alkalinized wa e (Figu e 9), i is emp ing o
specula e ha hese changes a e likely he main d i e s o he p ese a ion o he in es inal
mucin laye , leading, in u n, o educed in es inal and sys emic in lamma ion, wi h a gen-
e al posi i e impac on whole body me abolism. Akke mansia muciniphila is a mucin-de-
g ading bac e ium ha has also been shown o s imula e mucin p oduc ion wi h a ne
posi i e impac on he hickness o he mucin laye , he p ese a ion o gu ba ie unc-
ion and in es inal immune homeos asis [33]. Clos idium clus e IV is well known o effi-
cien ly e men ing he plan polysaccha ides composing die a y ibe . Clos idium species
ha e been epo ed o a enua e in lamma ion, and i s me aboli es, including bu y a e,
seconda y bile acids and indolep opionic acid, ha e been shown o play a p obio ic ole,
p ima ily h ough hei effec s on in es inal epi helial cells, s eng hening he in es inal
ba ie [34].
En e obac e iaceae a e acul a i e anae obes ha , in no mal condi ions, play
a key ole in in es inal oxygen consump ion, allowing o he p oli e a ion o anae obes,
necessa y o polysaccha ide e men a ion. Nu ien o e load and exposu e o in lamma-
o y media o s ha e been p oposed o educe hei oxida i e capaci y and d i e hei p o-
li e a ion, hus p omo ing obesi y- ela ed dysbiosis [35].
Figu e 9. qPCR analysis o mic obial con en in eces. The g aphs show he analysis o bac e ial
g oups analyzed a he indica ed imes. Tap, il e , il e + p obio ic. The ep esen ed al-
ues in he g aphs a e he diffe ences in he numbe o bac e ia be ween he alues ob ained a he
indica ed ime and = 0 o each indi idual a analyzed, mean alues +/− SD. S a is ical compa ison
il e + p obio ic. The g aph
shows he elec ochemical uni s p o ided by he e-BQC eade and includes mean alues +/−SD.
3.4. Indi ec Calo ime y
In o de o analyze he impac o he ea men s on sys emic me abolism, an indi ec
calo ime y sys em was used o de e mine he a s’ O
2
consump ion and CO
2
p oduc ion
and how hey changed in esponse o ea men s a 6w (1.5mo) and 3mo (Figu e 4). The
Mic oo ganisms 2024,12, 316 7 o 16
g oup o ap wa e a s showed a g adual dec ease in me abolic ac i i y, a ec ing bo h
O
2
consump ion and CO
2
p oduc ion. These changes we e signi ican in he nigh pe iod
in no mal a s a 3mo and in obese a s bo h du ing he nigh and day pe iods a 3mo.
The dec ease in CO
2
p oduc ion was also signi ican in he obese ap wa e g oup o
a s a 6w. These educ ions we e no obse ed o did no each s a is ical signi icance
in he g oups ea ed wi h il e ed wa e , bo h wi h and wi hou p obio ics. In ac , in
obese a s, he magni ude o he di e ence was signi ican ly smalle in a s ea ed wi h
il e ed wa e and p obio ics han in con ol a s, in he day and nigh pe iods, o bo h O
2
consump ion and CO
2
p oduc ion. These esul s sugges ha ea men wi h il e ed wa e ,
pa icula ly in combina ion wi h p obio ics, main ains a highe me abolic ac i i y han ap
wa e , especially in obese a s.
Mic oo ganisms 2024, 12, x FOR PEER REVIEW 7 o 16
3), sugges ing ha he ea men s did no globally affec he an ioxidan capaci y o he
a s.
Figu e 3. Elec ochemical analysis o he o al an ioxidan capaci y (QT) in plasma samples om a s
ea ed o 6w (le panel) o 3mo ( igh panel). Tap, il e , il e + p obio ic. The g aph
shows he elec ochemical uni s p o ided by he e-BQC eade and includes mean alues +/− SD.
3.4. Indi ec Calo ime y
In o de o analyze he impac o he ea men s on sys emic me abolism, an indi ec
calo ime y sys em was used o de e mine he a s’ O
2
consump ion and CO
2
p oduc ion
and how hey changed in esponse o ea men s a 6w (1.5mo) and 3mo (Figu e 4). The
g oup o ap wa e a s showed a g adual dec ease in me abolic ac i i y, affec ing bo h O
2
consump ion and CO
2
p oduc ion. These changes we e signi ican in he nigh pe iod in
no mal a s a 3mo and in obese a s bo h du ing he nigh and day pe iods a 3mo. The
dec ease in CO
2
p oduc ion was also signi ican in he obese ap wa e g oup o a s a 6w.
These educ ions we e no obse ed o did no each s a is ical signi icance in he g oups
ea ed wi h il e ed wa e , bo h wi h and wi hou p obio ics. In ac , in obese a s, he
magni ude o he diffe ence was signi ican ly smalle in a s ea ed wi h il e ed wa e
and p obio ics han in con ol a s, in he day and nigh pe iods, o bo h O
2
consump ion
and CO
2
p oduc ion. These esul s sugges ha ea men wi h il e ed wa e , pa icula ly
in combina ion wi h p obio ics, main ains a highe me abolic ac i i y han ap wa e , es-
pecially in obese a s.
Figu e 4. Analysis o O
2
consump ion (uppe panels) and CO
2
p oduc ion olumes (lowe panels)
by indi ec calo ime y. The a s we e e alua ed be o e ea men , a 6w and 3mo o ea men .
Tap, Fil e , Fil e + P obio ic. The alues shown in he g aphs a e he diffe ence o O
2
olume
(V) consumed (uppe panel) o CO
2
p oduced (lowe panel) be ween he indica ed ime and = 0
o each indi idual a analyzed. De e mina ions we e aken e e y hou o 72 h, he p esen ed da a
Figu e 4. Analysis o O
2
consump ion (uppe panels) and CO
2
p oduc ion olumes (lowe panels)
by indi ec calo ime y. The a s we e e alua ed be o e ea men , a 6w and 3mo o ea men .
Mic oo ganisms 2024, 12, x FOR PEER REVIEW 13 o 16
pe o med a p e ious s udy ha es ed he effec s o he consump ion o il e ed alka-
linized wa e in mice and a s o 3mo and ound e idence sugges ing a educ ion in oxi-
da i e s ess and in lamma ion [13]. The p esen s udy, hus, aimed o e alua e he in es-
inal impac o il e ed alkalinized wa e in lean and obese a s. The esul s ob ained sup-
po he hypo hesis ha il e ed alkalinized wa e , when compa ed o un il e ed–uns e i-
lized ap wa e om Mad id ci y, has a posi i e effec on bo h sys emic and in es inal
in lamma ion, be e p ese ing he in es inal mucin laye and p e en ing in es inal
dysbiosis. These changes we e linked o a be e main enance o oxida i e me abolism,
sugges ing a be e me abolic s a us o he a s consuming il e ed wa e (Figu e 4). O
no e, he imp o ed me abolic p o ile is no ela ed o any de ec able changes in weigh
gain (Figu e 1). Taking in o conside a ion he de ec ed changes in he mic obiome, wi h
inc eased Akke mansia muciniphila and Clos idium clus e IV and dec eased En e obac e i-
aceae in esponse o ea men wi h il e ed alkalinized wa e (Figu e 9), i is emp ing o
specula e ha hese changes a e likely he main d i e s o he p ese a ion o he in es inal
mucin laye , leading, in u n, o educed in es inal and sys emic in lamma ion, wi h a gen-
e al posi i e impac on whole body me abolism. Akke mansia muciniphila is a mucin-de-
g ading bac e ium ha has also been shown o s imula e mucin p oduc ion wi h a ne
posi i e impac on he hickness o he mucin laye , he p ese a ion o gu ba ie unc-
ion and in es inal immune homeos asis [33]. Clos idium clus e IV is well known o effi-
cien ly e men ing he plan polysaccha ides composing die a y ibe . Clos idium species
ha e been epo ed o a enua e in lamma ion, and i s me aboli es, including bu y a e,
seconda y bile acids and indolep opionic acid, ha e been shown o play a p obio ic ole,
p ima ily h ough hei effec s on in es inal epi helial cells, s eng hening he in es inal
ba ie [34].
En e obac e iaceae a e acul a i e anae obes ha , in no mal condi ions, play
a key ole in in es inal oxygen consump ion, allowing o he p oli e a ion o anae obes,
necessa y o polysaccha ide e men a ion. Nu ien o e load and exposu e o in lamma-
o y media o s ha e been p oposed o educe hei oxida i e capaci y and d i e hei p o-
li e a ion, hus p omo ing obesi y- ela ed dysbiosis [35].
Figu e 9. qPCR analysis o mic obial con en in eces. The g aphs show he analysis o bac e ial
g oups analyzed a he indica ed imes. Tap, il e , il e + p obio ic. The ep esen ed al-
ues in he g aphs a e he diffe ences in he numbe o bac e ia be ween he alues ob ained a he
indica ed ime and = 0 o each indi idual a analyzed, mean alues +/− SD. S a is ical compa ison
Tap,
Mic oo ganisms 2024, 12, x FOR PEER REVIEW 13 o 16
pe o med a p e ious s udy ha es ed he effec s o he consump ion o il e ed alka-
linized wa e in mice and a s o 3mo and ound e idence sugges ing a educ ion in oxi-
da i e s ess and in lamma ion [13]. The p esen s udy, hus, aimed o e alua e he in es-
inal impac o il e ed alkalinized wa e in lean and obese a s. The esul s ob ained sup-
po he hypo hesis ha il e ed alkalinized wa e , when compa ed o un il e ed–uns e i-
lized ap wa e om Mad id ci y, has a posi i e effec on bo h sys emic and in es inal
in lamma ion, be e p ese ing he in es inal mucin laye and p e en ing in es inal
dysbiosis. These changes we e linked o a be e main enance o oxida i e me abolism,
sugges ing a be e me abolic s a us o he a s consuming il e ed wa e (Figu e 4). O
no e, he imp o ed me abolic p o ile is no ela ed o any de ec able changes in weigh
gain (Figu e 1). Taking in o conside a ion he de ec ed changes in he mic obiome, wi h
inc eased Akke mansia muciniphila and Clos idium clus e IV and dec eased En e obac e i-
aceae in esponse o ea men wi h il e ed alkalinized wa e (Figu e 9), i is emp ing o
specula e ha hese changes a e likely he main d i e s o he p ese a ion o he in es inal
mucin laye , leading, in u n, o educed in es inal and sys emic in lamma ion, wi h a gen-
e al posi i e impac on whole body me abolism. Akke mansia muciniphila is a mucin-de-
g ading bac e ium ha has also been shown o s imula e mucin p oduc ion wi h a ne
posi i e impac on he hickness o he mucin laye , he p ese a ion o gu ba ie unc-
ion and in es inal immune homeos asis [33]. Clos idium clus e IV is well known o effi-
cien ly e men ing he plan polysaccha ides composing die a y ibe . Clos idium species
ha e been epo ed o a enua e in lamma ion, and i s me aboli es, including bu y a e,
seconda y bile acids and indolep opionic acid, ha e been shown o play a p obio ic ole,
p ima ily h ough hei effec s on in es inal epi helial cells, s eng hening he in es inal
ba ie [34].
En e obac e iaceae a e acul a i e anae obes ha , in no mal condi ions, play
a key ole in in es inal oxygen consump ion, allowing o he p oli e a ion o anae obes,
necessa y o polysaccha ide e men a ion. Nu ien o e load and exposu e o in lamma-
o y media o s ha e been p oposed o educe hei oxida i e capaci y and d i e hei p o-
li e a ion, hus p omo ing obesi y- ela ed dysbiosis [35].
Figu e 9. qPCR analysis o mic obial con en in eces. The g aphs show he analysis o bac e ial
g oups analyzed a he indica ed imes. Tap, il e , il e + p obio ic. The ep esen ed al-
ues in he g aphs a e he diffe ences in he numbe o bac e ia be ween he alues ob ained a he
indica ed ime and = 0 o each indi idual a analyzed, mean alues +/− SD. S a is ical compa ison
Fil e ,
Mic oo ganisms 2024, 12, x FOR PEER REVIEW 13 o 16
pe o med a p e ious s udy ha es ed he effec s o he consump ion o il e ed alka-
linized wa e in mice and a s o 3mo and ound e idence sugges ing a educ ion in oxi-
da i e s ess and in lamma ion [13]. The p esen s udy, hus, aimed o e alua e he in es-
inal impac o il e ed alkalinized wa e in lean and obese a s. The esul s ob ained sup-
po he hypo hesis ha il e ed alkalinized wa e , when compa ed o un il e ed–uns e i-
lized ap wa e om Mad id ci y, has a posi i e effec on bo h sys emic and in es inal
in lamma ion, be e p ese ing he in es inal mucin laye and p e en ing in es inal
dysbiosis. These changes we e linked o a be e main enance o oxida i e me abolism,
sugges ing a be e me abolic s a us o he a s consuming il e ed wa e (Figu e 4). O
no e, he imp o ed me abolic p o ile is no ela ed o any de ec able changes in weigh
gain (Figu e 1). Taking in o conside a ion he de ec ed changes in he mic obiome, wi h
inc eased Akke mansia muciniphila and Clos idium clus e IV and dec eased En e obac e i-
aceae in esponse o ea men wi h il e ed alkalinized wa e (Figu e 9), i is emp ing o
specula e ha hese changes a e likely he main d i e s o he p ese a ion o he in es inal
mucin laye , leading, in u n, o educed in es inal and sys emic in lamma ion, wi h a gen-
e al posi i e impac on whole body me abolism. Akke mansia muciniphila is a mucin-de-
g ading bac e ium ha has also been shown o s imula e mucin p oduc ion wi h a ne
posi i e impac on he hickness o he mucin laye , he p ese a ion o gu ba ie unc-
ion and in es inal immune homeos asis [33]. Clos idium clus e IV is well known o effi-
cien ly e men ing he plan polysaccha ides composing die a y ibe . Clos idium species
ha e been epo ed o a enua e in lamma ion, and i s me aboli es, including bu y a e,
seconda y bile acids and indolep opionic acid, ha e been shown o play a p obio ic ole,
p ima ily h ough hei effec s on in es inal epi helial cells, s eng hening he in es inal
ba ie [34].
En e obac e iaceae a e acul a i e anae obes ha , in no mal condi ions, play
a key ole in in es inal oxygen consump ion, allowing o he p oli e a ion o anae obes,
necessa y o polysaccha ide e men a ion. Nu ien o e load and exposu e o in lamma-
o y media o s ha e been p oposed o educe hei oxida i e capaci y and d i e hei p o-
li e a ion, hus p omo ing obesi y- ela ed dysbiosis [35].
Figu e 9. qPCR analysis o mic obial con en in eces. The g aphs show he analysis o bac e ial
g oups analyzed a he indica ed imes. Tap, il e , il e + p obio ic. The ep esen ed al-
ues in he g aphs a e he diffe ences in he numbe o bac e ia be ween he alues ob ained a he
indica ed ime and = 0 o each indi idual a analyzed, mean alues +/− SD. S a is ical compa ison
Fil e + P obio ic. The alues shown in he g aphs a e he di e ence o O
2
olume (V) consumed (uppe panel) o CO
2
p oduced (lowe panel) be ween he indica ed ime and
= 0 o each indi idual a analyzed. De e mina ions we e aken e e y hou o 72 h, he p esen ed
da a co esponds o he mean o bo h 12 h day and 12 h nigh pe iods o he 2nd day, when collec ed
alues we e mo e s able. The g aph shows mean alues +/−SD. * p< 0.05 ( es ).
3.5. In es inal Mucosa
3.5.1. Mucin
In o de o e alua e he in es inal impac o he ea men s, we s udied he in es inal
mucosa o he small in es ine and colon h ough he IF labeling o in es inal issue sec ions
wi h an ibodies di ec ed agains mucin (MUC2), an oligome ic p o ein ha is a undamen al
componen o he in es inal mucus. The educ ion in MUC2 le els is gene ally associa ed
wi h he loss o he in es inal ba ie , inc easing he isk o in lamma o y p ocesses and
in es inal in ec ions [18]. In lean a s, ea men wi h il e ed wa e signi ican ly inc eased
mucin le els in he colon. Likewise, he global compa ison o all he lean a s ea ed wi h
il e ed wa e (wi h o wi hou p obio ic), wi h espec o he ap wa e g oup, showed a
signi ican inc ease in mucin le els in he colon. Fu he mo e, in obese a s, a signi ican
inc ease in mucin le els was obse ed in a s ea ed wi h il e ed wa e and p obio ics
compa ed o he ap wa e g oup. The di e ence was also signi ican when a s ea ed wi h
p obio ics we e included in he analysis (Figu e 5). These esul s sugges ha ea men wi h
il e ed wa e , wi h o wi hou p obio ics, esul s in a be e main enance o he in es inal
mucosa han ap wa e .
Mic oo ganisms 2024,12, 316 8 o 16
Mic oo ganisms 2024, 12, x FOR PEER REVIEW 8 o 16
co esponds o he mean o bo h 12 h day and 12 h nigh pe iods o he 2nd day, when collec ed
alues we e mo e s able. The g aph shows mean alues +/− SD. * p < 0.05 ( es ).
3.5. In es inal Mucosa
3.5.1. Mucin
In o de o e alua e he in es inal impac o he ea men s, we s udied he in es inal
mucosa o he small in es ine and colon h ough he IF labeling o in es inal issue sec ions
wi h an ibodies di ec ed agains mucin (MUC2), an oligome ic p o ein ha is a unda-
men al componen o he in es inal mucus. The educ ion in MUC2 le els is gene ally as-
socia ed wi h he loss o he in es inal ba ie , inc easing he isk o in lamma o y p o-
cesses and in es inal in ec ions [18]. In lean a s, ea men wi h il e ed wa e signi ican ly
inc eased mucin le els in he colon. Likewise, he global compa ison o all he lean a s
ea ed wi h il e ed wa e (wi h o wi hou p obio ic), wi h espec o he ap wa e g oup,
showed a signi ican inc ease in mucin le els in he colon. Fu he mo e, in obese a s, a
signi ican inc ease in mucin le els was obse ed in a s ea ed wi h il e ed wa e and
p obio ics compa ed o he ap wa e g oup. The diffe ence was also signi ican when a s
ea ed wi h p obio ics we e included in he analysis (Figu e 5). These esul s sugges ha
ea men wi h il e ed wa e , wi h o wi hou p obio ics, esul s in a be e main enance
o he in es inal mucosa han ap wa e .
Figu e 5. De e mina ion o MUC2 le els by IF analysis o issue sec ions om he small in es ine
and colon. Tap, il e , il e + p obio ic. Collec ed da a co espond o he in eg a ed luo-
escence signal di ided by he issue a ea. The g aphs show he x old change ela i e o he mean
o he lean o obese con ol g oup ( ap), mean +/− SD. * p < 0.05 ( es ). Each da a poin co esponds
o he mean alues pe a . Rep esen a i e images o he g oups aken wi h a 20x objec i e a e in-
cluded in he le panels.
Figu e 5. De e mina ion o MUC2 le els by IF analysis o issue sec ions om he small in es ine and
colon.
Mic oo ganisms 2024, 12, x FOR PEER REVIEW 13 o 16
pe o med a p e ious s udy ha es ed he effec s o he consump ion o il e ed alka-
linized wa e in mice and a s o 3mo and ound e idence sugges ing a educ ion in oxi-
da i e s ess and in lamma ion [13]. The p esen s udy, hus, aimed o e alua e he in es-
inal impac o il e ed alkalinized wa e in lean and obese a s. The esul s ob ained sup-
po he hypo hesis ha il e ed alkalinized wa e , when compa ed o un il e ed–uns e i-
lized ap wa e om Mad id ci y, has a posi i e effec on bo h sys emic and in es inal
in lamma ion, be e p ese ing he in es inal mucin laye and p e en ing in es inal
dysbiosis. These changes we e linked o a be e main enance o oxida i e me abolism,
sugges ing a be e me abolic s a us o he a s consuming il e ed wa e (Figu e 4). O
no e, he imp o ed me abolic p o ile is no ela ed o any de ec able changes in weigh
gain (Figu e 1). Taking in o conside a ion he de ec ed changes in he mic obiome, wi h
inc eased Akke mansia muciniphila and Clos idium clus e IV and dec eased En e obac e i-
aceae in esponse o ea men wi h il e ed alkalinized wa e (Figu e 9), i is emp ing o
specula e ha hese changes a e likely he main d i e s o he p ese a ion o he in es inal
mucin laye , leading, in u n, o educed in es inal and sys emic in lamma ion, wi h a gen-
e al posi i e impac on whole body me abolism. Akke mansia muciniphila is a mucin-de-
g ading bac e ium ha has also been shown o s imula e mucin p oduc ion wi h a ne
posi i e impac on he hickness o he mucin laye , he p ese a ion o gu ba ie unc-
ion and in es inal immune homeos asis [33]. Clos idium clus e IV is well known o effi-
cien ly e men ing he plan polysaccha ides composing die a y ibe . Clos idium species
ha e been epo ed o a enua e in lamma ion, and i s me aboli es, including bu y a e,
seconda y bile acids and indolep opionic acid, ha e been shown o play a p obio ic ole,
p ima ily h ough hei effec s on in es inal epi helial cells, s eng hening he in es inal
ba ie [34].
En e obac e iaceae a e acul a i e anae obes ha , in no mal condi ions, play
a key ole in in es inal oxygen consump ion, allowing o he p oli e a ion o anae obes,
necessa y o polysaccha ide e men a ion. Nu ien o e load and exposu e o in lamma-
o y media o s ha e been p oposed o educe hei oxida i e capaci y and d i e hei p o-
li e a ion, hus p omo ing obesi y- ela ed dysbiosis [35].
Figu e 9. qPCR analysis o mic obial con en in eces. The g aphs show he analysis o bac e ial
g oups analyzed a he indica ed imes. Tap, il e , il e + p obio ic. The ep esen ed al-
ues in he g aphs a e he diffe ences in he numbe o bac e ia be ween he alues ob ained a he
indica ed ime and = 0 o each indi idual a analyzed, mean alues +/− SD. S a is ical compa ison
Tap,
Mic oo ganisms 2024, 12, x FOR PEER REVIEW 13 o 16
pe o med a p e ious s udy ha es ed he effec s o he consump ion o il e ed alka-
linized wa e in mice and a s o 3mo and ound e idence sugges ing a educ ion in oxi-
da i e s ess and in lamma ion [13]. The p esen s udy, hus, aimed o e alua e he in es-
inal impac o il e ed alkalinized wa e in lean and obese a s. The esul s ob ained sup-
po he hypo hesis ha il e ed alkalinized wa e , when compa ed o un il e ed–uns e i-
lized ap wa e om Mad id ci y, has a posi i e effec on bo h sys emic and in es inal
in lamma ion, be e p ese ing he in es inal mucin laye and p e en ing in es inal
dysbiosis. These changes we e linked o a be e main enance o oxida i e me abolism,
sugges ing a be e me abolic s a us o he a s consuming il e ed wa e (Figu e 4). O
no e, he imp o ed me abolic p o ile is no ela ed o any de ec able changes in weigh
gain (Figu e 1). Taking in o conside a ion he de ec ed changes in he mic obiome, wi h
inc eased Akke mansia muciniphila and Clos idium clus e IV and dec eased En e obac e i-
aceae in esponse o ea men wi h il e ed alkalinized wa e (Figu e 9), i is emp ing o
specula e ha hese changes a e likely he main d i e s o he p ese a ion o he in es inal
mucin laye , leading, in u n, o educed in es inal and sys emic in lamma ion, wi h a gen-
e al posi i e impac on whole body me abolism. Akke mansia muciniphila is a mucin-de-
g ading bac e ium ha has also been shown o s imula e mucin p oduc ion wi h a ne
posi i e impac on he hickness o he mucin laye , he p ese a ion o gu ba ie unc-
ion and in es inal immune homeos asis [33]. Clos idium clus e IV is well known o effi-
cien ly e men ing he plan polysaccha ides composing die a y ibe . Clos idium species
ha e been epo ed o a enua e in lamma ion, and i s me aboli es, including bu y a e,
seconda y bile acids and indolep opionic acid, ha e been shown o play a p obio ic ole,
p ima ily h ough hei effec s on in es inal epi helial cells, s eng hening he in es inal
ba ie [34].
En e obac e iaceae a e acul a i e anae obes ha , in no mal condi ions, play
a key ole in in es inal oxygen consump ion, allowing o he p oli e a ion o anae obes,
necessa y o polysaccha ide e men a ion. Nu ien o e load and exposu e o in lamma-
o y media o s ha e been p oposed o educe hei oxida i e capaci y and d i e hei p o-
li e a ion, hus p omo ing obesi y- ela ed dysbiosis [35].
Figu e 9. qPCR analysis o mic obial con en in eces. The g aphs show he analysis o bac e ial
g oups analyzed a he indica ed imes. Tap, il e , il e + p obio ic. The ep esen ed al-
ues in he g aphs a e he diffe ences in he numbe o bac e ia be ween he alues ob ained a he
indica ed ime and = 0 o each indi idual a analyzed, mean alues +/− SD. S a is ical compa ison
il e ,
Mic oo ganisms 2024, 12, x FOR PEER REVIEW 13 o 16
pe o med a p e ious s udy ha es ed he effec s o he consump ion o il e ed alka-
linized wa e in mice and a s o 3mo and ound e idence sugges ing a educ ion in oxi-
da i e s ess and in lamma ion [13]. The p esen s udy, hus, aimed o e alua e he in es-
inal impac o il e ed alkalinized wa e in lean and obese a s. The esul s ob ained sup-
po he hypo hesis ha il e ed alkalinized wa e , when compa ed o un il e ed–uns e i-
lized ap wa e om Mad id ci y, has a posi i e effec on bo h sys emic and in es inal
in lamma ion, be e p ese ing he in es inal mucin laye and p e en ing in es inal
dysbiosis. These changes we e linked o a be e main enance o oxida i e me abolism,
sugges ing a be e me abolic s a us o he a s consuming il e ed wa e (Figu e 4). O
no e, he imp o ed me abolic p o ile is no ela ed o any de ec able changes in weigh
gain (Figu e 1). Taking in o conside a ion he de ec ed changes in he mic obiome, wi h
inc eased Akke mansia muciniphila and Clos idium clus e IV and dec eased En e obac e i-
aceae in esponse o ea men wi h il e ed alkalinized wa e (Figu e 9), i is emp ing o
specula e ha hese changes a e likely he main d i e s o he p ese a ion o he in es inal
mucin laye , leading, in u n, o educed in es inal and sys emic in lamma ion, wi h a gen-
e al posi i e impac on whole body me abolism. Akke mansia muciniphila is a mucin-de-
g ading bac e ium ha has also been shown o s imula e mucin p oduc ion wi h a ne
posi i e impac on he hickness o he mucin laye , he p ese a ion o gu ba ie unc-
ion and in es inal immune homeos asis [33]. Clos idium clus e IV is well known o effi-
cien ly e men ing he plan polysaccha ides composing die a y ibe . Clos idium species
ha e been epo ed o a enua e in lamma ion, and i s me aboli es, including bu y a e,
seconda y bile acids and indolep opionic acid, ha e been shown o play a p obio ic ole,
p ima ily h ough hei effec s on in es inal epi helial cells, s eng hening he in es inal
ba ie [34].
En e obac e iaceae a e acul a i e anae obes ha , in no mal condi ions, play
a key ole in in es inal oxygen consump ion, allowing o he p oli e a ion o anae obes,
necessa y o polysaccha ide e men a ion. Nu ien o e load and exposu e o in lamma-
o y media o s ha e been p oposed o educe hei oxida i e capaci y and d i e hei p o-
li e a ion, hus p omo ing obesi y- ela ed dysbiosis [35].
Figu e 9. qPCR analysis o mic obial con en in eces. The g aphs show he analysis o bac e ial
g oups analyzed a he indica ed imes. Tap, il e , il e + p obio ic. The ep esen ed al-
ues in he g aphs a e he diffe ences in he numbe o bac e ia be ween he alues ob ained a he
indica ed ime and = 0 o each indi idual a analyzed, mean alues +/− SD. S a is ical compa ison
il e + p obio ic. Collec ed da a co espond o he in eg a ed luo escence
signal di ided by he issue a ea. The g aphs show he x old change ela i e o he mean o he lean
o obese con ol g oup ( ap), mean +/
−
SD. * p< 0.05 ( es ). Each da a poin co esponds o he
mean alues pe a . Rep esen a i e images o he g oups aken wi h a 20
×
objec i e a e included in
he le panels.
3.5.2. In es inal In lamma ion
•F4/80
Conside ing ha changes in he mucosa a e usually ela ed o he in lamma o y s a e
and ha he esul s poin ed o an imp o emen in he in lamma o y s a e a he sys emic
le el, in a s ea ed wi h il e ed wa e , we es ablished he le el o in es inal in lamma ion
by de e mining he p esence o mac ophages by IHC s aining wi h an F4/80 an ibody.
Analysis o he F4/80-posi i e a ea showed, in he small in es ine o no mal a s and
in he colon o obese a s, a signi ican ly lowe p esence o mac ophages in a s ea ed
wi h il e ed wa e han in a s ha consumed ap wa e . Ra s ea ed wi h il e ed wa e
and p obio ics showed a simila end, bu , in his case, he di e ences did no each
s a is ical signi icance (Figu e 6). These da a a e consis en wi h he imp o emen obse ed
in he in es inal mucosa and he educ ion in in lamma ion a he sys emic le el, obse ed
p e iously, suppo ing ha a s ea ed wi h il e ed wa e had a be e in es inal s a us.
Mic oo ganisms 2024,12, 316 9 o 16
Mic oo ganisms 2024, 12, x FOR PEER REVIEW 9 o 16
3.5.2. In es inal In lamma ion
• F4/80
Conside ing ha changes in he mucosa a e usually ela ed o he in lamma o y s a e
and ha he esul s poin ed o an imp o emen in he in lamma o y s a e a he sys emic
le el, in a s ea ed wi h il e ed wa e , we es ablished he le el o in es inal in lamma ion
by de e mining he p esence o mac ophages by IHC s aining wi h an F4/80 an ibody.
Analysis o he F4/80-posi i e a ea showed, in he small in es ine o no mal a s and
in he colon o obese a s, a signi ican ly lowe p esence o mac ophages in a s ea ed
wi h il e ed wa e han in a s ha consumed ap wa e . Ra s ea ed wi h il e ed wa e
and p obio ics showed a simila end, bu , in his case, he diffe ences did no each s a-
is ical signi icance (Figu e 6). These da a a e consis en wi h he imp o emen obse ed
in he in es inal mucosa and he educ ion in in lamma ion a he sys emic le el, obse ed
p e iously, suppo ing ha a s ea ed wi h il e ed wa e had a be e in es inal s a us.
Figu e 6. IHC s aining o mac ophages in issue sec ions om he small in es ine and colon using
an ibodies di ec ed agains F4/80. Tap, il e , il e + p obio ic. Collec ed da a co espond
o he posi i e a ea di ided by he issue a ea. The g aphs show he % ela i e o he mean o he
con ol g oup ( ap), mean +/− SD. * p < 0.05 ( - es ). Each da a poin co esponds o he mean o he
alues ob ained o each a . Rep esen a i e images o he g oups aken wi h a 20x objec i e a e
included in he le panels.
• Cy okine’s gene exp ession
Subsequen ly, he exp ession le els o IL-1B and IL-10 in he in es inal issue we e
analyzed. The mRNA le els o IL-1B in he small in es ine o obese a s ea ed wi h il-
e ed wa e and p obio ics we e lowe han hose o ap-wa e - ea ed a s. Howe e , e-
ga ding he o he g oups, no signi ican changes we e ound, al hough obese a s ea ed
wi h il e ed wa e showed a end owa ds lowe le els o IL-1B and highe le els o IL-
10 han a s ea ed wi h ap wa e , bo h in he small and la ge in es ine sec ions, an ob-
se a ion consis en wi h he da a de i ed om plasma samples (Figu e 7).
Figu e 6. IHC s aining o mac ophages in issue sec ions om he small in es ine and colon using
an ibodies di ec ed agains F4/80.
Mic oo ganisms 2024, 12, x FOR PEER REVIEW 13 o 16
pe o med a p e ious s udy ha es ed he effec s o he consump ion o il e ed alka-
linized wa e in mice and a s o 3mo and ound e idence sugges ing a educ ion in oxi-
da i e s ess and in lamma ion [13]. The p esen s udy, hus, aimed o e alua e he in es-
inal impac o il e ed alkalinized wa e in lean and obese a s. The esul s ob ained sup-
po he hypo hesis ha il e ed alkalinized wa e , when compa ed o un il e ed–uns e i-
lized ap wa e om Mad id ci y, has a posi i e effec on bo h sys emic and in es inal
in lamma ion, be e p ese ing he in es inal mucin laye and p e en ing in es inal
dysbiosis. These changes we e linked o a be e main enance o oxida i e me abolism,
sugges ing a be e me abolic s a us o he a s consuming il e ed wa e (Figu e 4). O
no e, he imp o ed me abolic p o ile is no ela ed o any de ec able changes in weigh
gain (Figu e 1). Taking in o conside a ion he de ec ed changes in he mic obiome, wi h
inc eased Akke mansia muciniphila and Clos idium clus e IV and dec eased En e obac e i-
aceae in esponse o ea men wi h il e ed alkalinized wa e (Figu e 9), i is emp ing o
specula e ha hese changes a e likely he main d i e s o he p ese a ion o he in es inal
mucin laye , leading, in u n, o educed in es inal and sys emic in lamma ion, wi h a gen-
e al posi i e impac on whole body me abolism. Akke mansia muciniphila is a mucin-de-
g ading bac e ium ha has also been shown o s imula e mucin p oduc ion wi h a ne
posi i e impac on he hickness o he mucin laye , he p ese a ion o gu ba ie unc-
ion and in es inal immune homeos asis [33]. Clos idium clus e IV is well known o effi-
cien ly e men ing he plan polysaccha ides composing die a y ibe . Clos idium species
ha e been epo ed o a enua e in lamma ion, and i s me aboli es, including bu y a e,
seconda y bile acids and indolep opionic acid, ha e been shown o play a p obio ic ole,
p ima ily h ough hei effec s on in es inal epi helial cells, s eng hening he in es inal
ba ie [34].
En e obac e iaceae a e acul a i e anae obes ha , in no mal condi ions, play
a key ole in in es inal oxygen consump ion, allowing o he p oli e a ion o anae obes,
necessa y o polysaccha ide e men a ion. Nu ien o e load and exposu e o in lamma-
o y media o s ha e been p oposed o educe hei oxida i e capaci y and d i e hei p o-
li e a ion, hus p omo ing obesi y- ela ed dysbiosis [35].
Figu e 9. qPCR analysis o mic obial con en in eces. The g aphs show he analysis o bac e ial
g oups analyzed a he indica ed imes. Tap, il e , il e + p obio ic. The ep esen ed al-
ues in he g aphs a e he diffe ences in he numbe o bac e ia be ween he alues ob ained a he
indica ed ime and = 0 o each indi idual a analyzed, mean alues +/− SD. S a is ical compa ison
Tap,
Mic oo ganisms 2024, 12, x FOR PEER REVIEW 13 o 16
pe o med a p e ious s udy ha es ed he effec s o he consump ion o il e ed alka-
linized wa e in mice and a s o 3mo and ound e idence sugges ing a educ ion in oxi-
da i e s ess and in lamma ion [13]. The p esen s udy, hus, aimed o e alua e he in es-
inal impac o il e ed alkalinized wa e in lean and obese a s. The esul s ob ained sup-
po he hypo hesis ha il e ed alkalinized wa e , when compa ed o un il e ed–uns e i-
lized ap wa e om Mad id ci y, has a posi i e effec on bo h sys emic and in es inal
in lamma ion, be e p ese ing he in es inal mucin laye and p e en ing in es inal
dysbiosis. These changes we e linked o a be e main enance o oxida i e me abolism,
sugges ing a be e me abolic s a us o he a s consuming il e ed wa e (Figu e 4). O
no e, he imp o ed me abolic p o ile is no ela ed o any de ec able changes in weigh
gain (Figu e 1). Taking in o conside a ion he de ec ed changes in he mic obiome, wi h
inc eased Akke mansia muciniphila and Clos idium clus e IV and dec eased En e obac e i-
aceae in esponse o ea men wi h il e ed alkalinized wa e (Figu e 9), i is emp ing o
specula e ha hese changes a e likely he main d i e s o he p ese a ion o he in es inal
mucin laye , leading, in u n, o educed in es inal and sys emic in lamma ion, wi h a gen-
e al posi i e impac on whole body me abolism. Akke mansia muciniphila is a mucin-de-
g ading bac e ium ha has also been shown o s imula e mucin p oduc ion wi h a ne
posi i e impac on he hickness o he mucin laye , he p ese a ion o gu ba ie unc-
ion and in es inal immune homeos asis [33]. Clos idium clus e IV is well known o effi-
cien ly e men ing he plan polysaccha ides composing die a y ibe . Clos idium species
ha e been epo ed o a enua e in lamma ion, and i s me aboli es, including bu y a e,
seconda y bile acids and indolep opionic acid, ha e been shown o play a p obio ic ole,
p ima ily h ough hei effec s on in es inal epi helial cells, s eng hening he in es inal
ba ie [34].
En e obac e iaceae a e acul a i e anae obes ha , in no mal condi ions, play
a key ole in in es inal oxygen consump ion, allowing o he p oli e a ion o anae obes,
necessa y o polysaccha ide e men a ion. Nu ien o e load and exposu e o in lamma-
o y media o s ha e been p oposed o educe hei oxida i e capaci y and d i e hei p o-
li e a ion, hus p omo ing obesi y- ela ed dysbiosis [35].
Figu e 9. qPCR analysis o mic obial con en in eces. The g aphs show he analysis o bac e ial
g oups analyzed a he indica ed imes. Tap, il e , il e + p obio ic. The ep esen ed al-
ues in he g aphs a e he diffe ences in he numbe o bac e ia be ween he alues ob ained a he
indica ed ime and = 0 o each indi idual a analyzed, mean alues +/− SD. S a is ical compa ison
il e ,
Mic oo ganisms 2024, 12, x FOR PEER REVIEW 13 o 16
pe o med a p e ious s udy ha es ed he effec s o he consump ion o il e ed alka-
linized wa e in mice and a s o 3mo and ound e idence sugges ing a educ ion in oxi-
da i e s ess and in lamma ion [13]. The p esen s udy, hus, aimed o e alua e he in es-
inal impac o il e ed alkalinized wa e in lean and obese a s. The esul s ob ained sup-
po he hypo hesis ha il e ed alkalinized wa e , when compa ed o un il e ed–uns e i-
lized ap wa e om Mad id ci y, has a posi i e effec on bo h sys emic and in es inal
in lamma ion, be e p ese ing he in es inal mucin laye and p e en ing in es inal
dysbiosis. These changes we e linked o a be e main enance o oxida i e me abolism,
sugges ing a be e me abolic s a us o he a s consuming il e ed wa e (Figu e 4). O
no e, he imp o ed me abolic p o ile is no ela ed o any de ec able changes in weigh
gain (Figu e 1). Taking in o conside a ion he de ec ed changes in he mic obiome, wi h
inc eased Akke mansia muciniphila and Clos idium clus e IV and dec eased En e obac e i-
aceae in esponse o ea men wi h il e ed alkalinized wa e (Figu e 9), i is emp ing o
specula e ha hese changes a e likely he main d i e s o he p ese a ion o he in es inal
mucin laye , leading, in u n, o educed in es inal and sys emic in lamma ion, wi h a gen-
e al posi i e impac on whole body me abolism. Akke mansia muciniphila is a mucin-de-
g ading bac e ium ha has also been shown o s imula e mucin p oduc ion wi h a ne
posi i e impac on he hickness o he mucin laye , he p ese a ion o gu ba ie unc-
ion and in es inal immune homeos asis [33]. Clos idium clus e IV is well known o effi-
cien ly e men ing he plan polysaccha ides composing die a y ibe . Clos idium species
ha e been epo ed o a enua e in lamma ion, and i s me aboli es, including bu y a e,
seconda y bile acids and indolep opionic acid, ha e been shown o play a p obio ic ole,
p ima ily h ough hei effec s on in es inal epi helial cells, s eng hening he in es inal
ba ie [34].
En e obac e iaceae a e acul a i e anae obes ha , in no mal condi ions, play
a key ole in in es inal oxygen consump ion, allowing o he p oli e a ion o anae obes,
necessa y o polysaccha ide e men a ion. Nu ien o e load and exposu e o in lamma-
o y media o s ha e been p oposed o educe hei oxida i e capaci y and d i e hei p o-
li e a ion, hus p omo ing obesi y- ela ed dysbiosis [35].
Figu e 9. qPCR analysis o mic obial con en in eces. The g aphs show he analysis o bac e ial
g oups analyzed a he indica ed imes. Tap, il e , il e + p obio ic. The ep esen ed al-
ues in he g aphs a e he diffe ences in he numbe o bac e ia be ween he alues ob ained a he
indica ed ime and = 0 o each indi idual a analyzed, mean alues +/− SD. S a is ical compa ison
il e + p obio ic. Collec ed da a co espond
o he posi i e a ea di ided by he issue a ea. The g aphs show he % ela i e o he mean o he
con ol g oup ( ap), mean +/
−
SD. * p< 0.05 ( - es ). Each da a poin co esponds o he mean o he
alues ob ained o each a . Rep esen a i e images o he g oups aken wi h a 20
×
objec i e a e
included in he le panels.
•Cy okine’s gene exp ession
Subsequen ly, he exp ession le els o IL-1B and IL-10 in he in es inal issue we e
analyzed. The mRNA le els o IL-1B in he small in es ine o obese a s ea ed wi h il e ed
wa e and p obio ics we e lowe han hose o ap-wa e - ea ed a s. Howe e , ega ding
he o he g oups, no signi ican changes we e ound, al hough obese a s ea ed wi h
il e ed wa e showed a end owa ds lowe le els o IL-1B and highe le els o IL-10 han
a s ea ed wi h ap wa e , bo h in he small and la ge in es ine sec ions, an obse a ion
consis en wi h he da a de i ed om plasma samples (Figu e 7).
Mic oo ganisms 2024, 12, x FOR PEER REVIEW 10 o 16
Figu e 7. qRT-PCR analysis o he exp ession le els o IL-1B and IL-10 in small in es ine and colon
issue samples. Tap, il e , il e + p obio ic. The g aphs show he diffe ences in ΔΔC o he
indi idual alues pe a and he mean alue o he co esponding con ol g oup ( ap), mean +/−
SD. Fo cla i y’s sake, −ΔΔC alues a e p esen ed, wi h highe alues indica ing highe mRNA ex-
p ession. * p < 0.05 ( es ).
• Oxida i e s ess (OS)
To de e mine i hese changes we e ela ed o he le els o oxida i e s ess p esen in
he issue, he p esence o p o eins modi ied wi h 4-HNE was analyzed by IF, using spe-
ci ic an ibodies. Ra s ea ed wi h il e ed wa e had, o e all, signi ican ly less modi ied
p o eins in he small in es ine han ap wa e a s. The same end was obse ed in he
colon, as well as in a s ea ed wi h il e ed wa e and p obio ics, al hough diffe ences
did no each s a is ical signi icance (Figu e 8). The e o e, hese da a suppo he idea ha
ea men wi h il e ed wa e educes oxida i e s ess a he in es inal le el, an effec ha
may be ela ed o an imp o emen in he in lamma o y s a us and he p ese a ion o he
mucosa.
Figu e 8. De e mina ion o 4-HNE-modi ied p o ein le els by IF analysis o issue sec ions om he
small in es ine and colon. Tap, il e , il e + p obio ic. Collec ed da a co espond o he in-
eg a ed luo escence signal di ided by he issue a ea. The g aphs show he % ela i e o he mean
o he con ol g oup ( ap), mean +/− SD. *** p < 0.001 ( es ). Each da a poin co esponds o he mean
Figu e 7. qRT-PCR analysis o he exp ession le els o IL-1B and IL-10 in small in es ine and colon
issue samples.
Mic oo ganisms 2024, 12, x FOR PEER REVIEW 13 o 16
pe o med a p e ious s udy ha es ed he effec s o he consump ion o il e ed alka-
linized wa e in mice and a s o 3mo and ound e idence sugges ing a educ ion in oxi-
da i e s ess and in lamma ion [13]. The p esen s udy, hus, aimed o e alua e he in es-
inal impac o il e ed alkalinized wa e in lean and obese a s. The esul s ob ained sup-
po he hypo hesis ha il e ed alkalinized wa e , when compa ed o un il e ed–uns e i-
lized ap wa e om Mad id ci y, has a posi i e effec on bo h sys emic and in es inal
in lamma ion, be e p ese ing he in es inal mucin laye and p e en ing in es inal
dysbiosis. These changes we e linked o a be e main enance o oxida i e me abolism,
sugges ing a be e me abolic s a us o he a s consuming il e ed wa e (Figu e 4). O
no e, he imp o ed me abolic p o ile is no ela ed o any de ec able changes in weigh
gain (Figu e 1). Taking in o conside a ion he de ec ed changes in he mic obiome, wi h
inc eased Akke mansia muciniphila and Clos idium clus e IV and dec eased En e obac e i-
aceae in esponse o ea men wi h il e ed alkalinized wa e (Figu e 9), i is emp ing o
specula e ha hese changes a e likely he main d i e s o he p ese a ion o he in es inal
mucin laye , leading, in u n, o educed in es inal and sys emic in lamma ion, wi h a gen-
e al posi i e impac on whole body me abolism. Akke mansia muciniphila is a mucin-de-
g ading bac e ium ha has also been shown o s imula e mucin p oduc ion wi h a ne
posi i e impac on he hickness o he mucin laye , he p ese a ion o gu ba ie unc-
ion and in es inal immune homeos asis [33]. Clos idium clus e IV is well known o effi-
cien ly e men ing he plan polysaccha ides composing die a y ibe . Clos idium species
ha e been epo ed o a enua e in lamma ion, and i s me aboli es, including bu y a e,
seconda y bile acids and indolep opionic acid, ha e been shown o play a p obio ic ole,
p ima ily h ough hei effec s on in es inal epi helial cells, s eng hening he in es inal
ba ie [34].
En e obac e iaceae a e acul a i e anae obes ha , in no mal condi ions, play
a key ole in in es inal oxygen consump ion, allowing o he p oli e a ion o anae obes,
necessa y o polysaccha ide e men a ion. Nu ien o e load and exposu e o in lamma-
o y media o s ha e been p oposed o educe hei oxida i e capaci y and d i e hei p o-
li e a ion, hus p omo ing obesi y- ela ed dysbiosis [35].
Figu e 9. qPCR analysis o mic obial con en in eces. The g aphs show he analysis o bac e ial
g oups analyzed a he indica ed imes. Tap, il e , il e + p obio ic. The ep esen ed al-
ues in he g aphs a e he diffe ences in he numbe o bac e ia be ween he alues ob ained a he
indica ed ime and = 0 o each indi idual a analyzed, mean alues +/− SD. S a is ical compa ison
Tap,
Mic oo ganisms 2024, 12, x FOR PEER REVIEW 13 o 16
pe o med a p e ious s udy ha es ed he effec s o he consump ion o il e ed alka-
linized wa e in mice and a s o 3mo and ound e idence sugges ing a educ ion in oxi-
da i e s ess and in lamma ion [13]. The p esen s udy, hus, aimed o e alua e he in es-
inal impac o il e ed alkalinized wa e in lean and obese a s. The esul s ob ained sup-
po he hypo hesis ha il e ed alkalinized wa e , when compa ed o un il e ed–uns e i-
lized ap wa e om Mad id ci y, has a posi i e effec on bo h sys emic and in es inal
in lamma ion, be e p ese ing he in es inal mucin laye and p e en ing in es inal
dysbiosis. These changes we e linked o a be e main enance o oxida i e me abolism,
sugges ing a be e me abolic s a us o he a s consuming il e ed wa e (Figu e 4). O
no e, he imp o ed me abolic p o ile is no ela ed o any de ec able changes in weigh
gain (Figu e 1). Taking in o conside a ion he de ec ed changes in he mic obiome, wi h
inc eased Akke mansia muciniphila and Clos idium clus e IV and dec eased En e obac e i-
aceae in esponse o ea men wi h il e ed alkalinized wa e (Figu e 9), i is emp ing o
specula e ha hese changes a e likely he main d i e s o he p ese a ion o he in es inal
mucin laye , leading, in u n, o educed in es inal and sys emic in lamma ion, wi h a gen-
e al posi i e impac on whole body me abolism. Akke mansia muciniphila is a mucin-de-
g ading bac e ium ha has also been shown o s imula e mucin p oduc ion wi h a ne
posi i e impac on he hickness o he mucin laye , he p ese a ion o gu ba ie unc-
ion and in es inal immune homeos asis [33]. Clos idium clus e IV is well known o effi-
cien ly e men ing he plan polysaccha ides composing die a y ibe . Clos idium species
ha e been epo ed o a enua e in lamma ion, and i s me aboli es, including bu y a e,
seconda y bile acids and indolep opionic acid, ha e been shown o play a p obio ic ole,
p ima ily h ough hei effec s on in es inal epi helial cells, s eng hening he in es inal
ba ie [34].
En e obac e iaceae a e acul a i e anae obes ha , in no mal condi ions, play
a key ole in in es inal oxygen consump ion, allowing o he p oli e a ion o anae obes,
necessa y o polysaccha ide e men a ion. Nu ien o e load and exposu e o in lamma-
o y media o s ha e been p oposed o educe hei oxida i e capaci y and d i e hei p o-
li e a ion, hus p omo ing obesi y- ela ed dysbiosis [35].
Figu e 9. qPCR analysis o mic obial con en in eces. The g aphs show he analysis o bac e ial
g oups analyzed a he indica ed imes. Tap, il e , il e + p obio ic. The ep esen ed al-
ues in he g aphs a e he diffe ences in he numbe o bac e ia be ween he alues ob ained a he
indica ed ime and = 0 o each indi idual a analyzed, mean alues +/− SD. S a is ical compa ison
il e ,
Mic oo ganisms 2024, 12, x FOR PEER REVIEW 13 o 16
pe o med a p e ious s udy ha es ed he effec s o he consump ion o il e ed alka-
linized wa e in mice and a s o 3mo and ound e idence sugges ing a educ ion in oxi-
da i e s ess and in lamma ion [13]. The p esen s udy, hus, aimed o e alua e he in es-
inal impac o il e ed alkalinized wa e in lean and obese a s. The esul s ob ained sup-
po he hypo hesis ha il e ed alkalinized wa e , when compa ed o un il e ed–uns e i-
lized ap wa e om Mad id ci y, has a posi i e effec on bo h sys emic and in es inal
in lamma ion, be e p ese ing he in es inal mucin laye and p e en ing in es inal
dysbiosis. These changes we e linked o a be e main enance o oxida i e me abolism,
sugges ing a be e me abolic s a us o he a s consuming il e ed wa e (Figu e 4). O
no e, he imp o ed me abolic p o ile is no ela ed o any de ec able changes in weigh
gain (Figu e 1). Taking in o conside a ion he de ec ed changes in he mic obiome, wi h
inc eased Akke mansia muciniphila and Clos idium clus e IV and dec eased En e obac e i-
aceae in esponse o ea men wi h il e ed alkalinized wa e (Figu e 9), i is emp ing o
specula e ha hese changes a e likely he main d i e s o he p ese a ion o he in es inal
mucin laye , leading, in u n, o educed in es inal and sys emic in lamma ion, wi h a gen-
e al posi i e impac on whole body me abolism. Akke mansia muciniphila is a mucin-de-
g ading bac e ium ha has also been shown o s imula e mucin p oduc ion wi h a ne
posi i e impac on he hickness o he mucin laye , he p ese a ion o gu ba ie unc-
ion and in es inal immune homeos asis [33]. Clos idium clus e IV is well known o effi-
cien ly e men ing he plan polysaccha ides composing die a y ibe . Clos idium species
ha e been epo ed o a enua e in lamma ion, and i s me aboli es, including bu y a e,
seconda y bile acids and indolep opionic acid, ha e been shown o play a p obio ic ole,
p ima ily h ough hei effec s on in es inal epi helial cells, s eng hening he in es inal
ba ie [34].
En e obac e iaceae a e acul a i e anae obes ha , in no mal condi ions, play
a key ole in in es inal oxygen consump ion, allowing o he p oli e a ion o anae obes,
necessa y o polysaccha ide e men a ion. Nu ien o e load and exposu e o in lamma-
o y media o s ha e been p oposed o educe hei oxida i e capaci y and d i e hei p o-
li e a ion, hus p omo ing obesi y- ela ed dysbiosis [35].
Figu e 9. qPCR analysis o mic obial con en in eces. The g aphs show he analysis o bac e ial
g oups analyzed a he indica ed imes. Tap, il e , il e + p obio ic. The ep esen ed al-
ues in he g aphs a e he diffe ences in he numbe o bac e ia be ween he alues ob ained a he
indica ed ime and = 0 o each indi idual a analyzed, mean alues +/− SD. S a is ical compa ison
il e + p obio ic. The g aphs show he di e ences in
∆∆
C o
he indi idual alues pe a and he mean alue o he co esponding con ol g oup ( ap), mean
+/
−
SD. Fo cla i y’s sake,
−∆∆
C alues a e p esen ed, wi h highe alues indica ing highe mRNA
exp ession. * p< 0.05 ( es ).
Mic oo ganisms 2024,12, 316 16 o 16
34.
Guo, P.; Zhang, K.; Ma, X.; He, P. Clos idium Species as P obio ics: Po en ials and Challenges. J. Anim. Sci. Bio echnol. 2020,11,
24. [C ossRe ] [PubMed]
35.
Baldelli, V.; Scalda e i, F.; Pu ignani, L.; Del Chie ico, F. The Role o En e obac e iaceae in Gu Mic obio a Dysbiosis in
In lamma o y Bowel Diseases. Mic oo ganisms 2021,9, 697. [C ossRe ] [PubMed]
Disclaime /Publishe ’s No e: The s a emen s, opinions and da a con ained in all publica ions a e solely hose o he indi idual
au ho (s) and con ibu o (s) and no o MDPI and/o he edi o (s). MDPI and/o he edi o (s) disclaim esponsibili y o any inju y o
people o p ope y esul ing om any ideas, me hods, ins uc ions o p oduc s e e ed o in he con en .