nu ien s
A icle
Lac obacillus eu e i V3401 Reduces In lamma o y
Bioma ke s and Modi ies he Gas oin es inal
Mic obiome in Adul s wi h Me abolic Synd ome:
The PROSIR S udy
Ca men Teno io-Jiménez 1, Ma ía JoséMa ínez-Ramí ez 2,3, Isabel Del Cas illo-Codes 4,
Ca men A aiza-I igoyen 2, Me cedes Te ce o-Lozano 4, JoséCamacho 5, Na alia Chueca 6,7,
Fede ico Ga cía6,7, Josune Olza 8, Julio Plaza-Díaz 7,8,9,10 , Luis Fon ana 7,8,9 ,
Mónica Oli a es 11,Ángel Gil 7,8,9,10 and Ca olina Gómez-Llo en e 7,8,9,10,*
1Endoc inology and Nu i ion Clinical Managemen Uni , Uni e si y Hospi al Vi gen de las Nie es,
18014 G anada, Spain
2Endoc inology and Nu i ion Clinical Managemen Uni , Uni e si y Hospi al o Jaén, 23007 Jaén, Spain
3Depa men o Heal h Sciences, School o Heal h Sciences, Uni e si y o Jaén, 23071 Jaén, Spain
4Diges i e Diseases Clinical Managemen Uni , Uni e si y Hospi al o Jaén, 23007 Jaén, Spain
5Depa men o Signal Theo y, Ne wo king, and Communica ions, Uni e si y o G anada,
18071 G anada, Spain
6Depa men o Mic obiology, Uni e si y Hospi al Campus de la Salud, 18016 G anada, Spain
7Ins i u o de In es igación Biosani a ia ibs. GRANADA, 18012 G anada, Spain
8Depa men o Biochemis y and Molecula Biology II, School o Pha macy, Uni e si y o G anada,
18071 G anada, Spain
9Ins i u e o Nu i ion and Food Technology “JoséMa aix”, Cen e o Biomedical Resea ch,
Uni e si y o G anada, 18016 G anada, Spain
10 CIBEROBN (CIBER Physiopa hology o Obesi y and Nu i ion), Ins i u o de Salud Ca los III,
28029 Mad id, Spain
11 Biosea ch Li e, 18004 G anada, Spain
*Co espondence: gomezll@ug .es; Tel.: +34-958-241-000 (ex . 40092)
Recei ed: 27 June 2019; Accep ed: 29 July 2019; Published: 31 July 2019


Abs ac :
P e ious s udies ha e epo ed ha p obio ics may imp o e clinical and in lamma o y
pa ame e s in pa ien s wi h obesi y and me abolic synd ome (Me S). Lac obacillus (L.) eu e i V3401
has shown p omising esul s on he componen s o Me S in animal s udies. We aimed o e alua e he
e ec s o L. eu e i V3401 oge he wi h heal hy li es yle ecommenda ions on adul pa ien s wi h
Me S. Me hods: We ca ied ou a andomized, c osso e , placebo-con olled, single-cen e ial in
which we included 53 adul pa ien s newly diagnosed wi h Me S. Pa ien s we e block andomly
alloca ed by body mass index (BMI) and sex o ecei e a capsule con aining ei he he p obio ic
L. eu e i
V3401 (5
×
10
9
colony- o ming uni s) o a placebo once daily o 12 weeks. An h opome ic
a iables, biochemical and in lamma o y bioma ke s, as well as he gas oin es inal mic obiome
composi ion we e de e mined. Resul s: The e we e no di e ences be ween g oups in he clinical
cha ac e is ics o Me S. Howe e , we ound ha in e leukin-6 (IL-6) and soluble ascula cell adhesion
molecule 1 (sVCAM-1) diminished by e ec o he ea men wi h L. eu e i V3401. Analysis o
he gas oin es inal mic obiome e ealed a ise in he p opo ion o Ve ucomic obia. Conclusions:
Consump ion o L. eu e i V3401 imp o ed selec ed in lamma o y pa ame e s and modi ied he
gas oin es inal mic obiome. Fu he s udies a e needed o asce ain addi ional bene icial e ec s o
o he p obio ic s ains in Me S as well as he mechanisms by which such e ec s a e exe ed.
Nu ien s 2019,11, 1761; doi:10.3390/nu11081761 www.mdpi.com/jou nal/nu ien s
Nu ien s 2019,11, 1761 2 o 14
Keywo ds:
me abolic synd ome; gas oin es inal mic obiome; Lac obacillus eu e i V3401;
p obio ics; obesi y
1. In oduc ion
Obesi y is a ch onic disease, a ec ing de eloped and de eloping coun ies, ha has mul iple
como bidi ies and de e io a es quali y o li e. I is cha ac e ized by an inc ease o a mass, which can
consequen ly p oduce hype ophy o he adipocy es, leading o an al e ed adipose issue unc ionali y.
Indi iduals who a e obese can de elop an insulin esis ance synd ome, also called me abolic synd ome
(Me S). Me S is de ined by insulin esis ance, dyslipidemia, hype ension, and inc eased abdominal
ci cum e ence, and i is associa ed wi h he de elopmen o ype 2 diabe es (DM2), ca dio ascula
disease (CVD), and nonalcoholic a y li e disease (NAFLD). This condi ion is associa ed wi h a
wo- old inc ease in he isk o co ona y hea disease, ce eb o ascula disease, and a 1.5- old inc ease
in he isk o all-cause mo ali y [1], cons i u ing a majo public heal h challenge wo ldwide.
Nowadays, he e is sound e idence linking he me abolic dys unc ion seen in Me S o a
p oin lamma o y s a e. Adipose issue is, in pa , esponsible o his low-g ade in lamma o y
s a e h ough he inc easing elease o p oin lamma o y molecules, such as lep in and umo nec osis
ac o
α
(TNF-
α
), and he inhibi ion o adiponec in sec e ion, an an i-in lamma o y adipokine [
2
].
In ecen yea s, i has become e iden ha al e a ion o he gas oin es inal mic obiome, also called
gas oin es inal dysbiosis, may also con ibu e o he de elopmen o insulin esis ance associa ed
wi h obesi y [
3
–
5
]. Fu he mo e, di e en s udies ha e linked gas oin es inal dysbiosis wi h he
de elopmen o obesi y and o he hallma ks o Me S [
6
,
7
]. In his sense, a dec eased a io o
Bac e oide es/Fi micu es has been desc ibed in indi iduals who a e obese compa ed o no mal-weigh
indi iduals [
7
]. Likewise, indi iduals wi h a low bac e ial ichness ha e mo e dyslipidemia, insulin
esis ance, in lamma o y pheno ype, and o e all adiposi y han indi iduals wi h high bac e ial
ichness [
8
]. In addi ion,
an abe an
gas oin es inal mic obiome can p omo e subacu e sys emic
in lamma ion, insulin esis ance, and inc eased isk o CVD by mechanisms ha include exposu e o
bac e ial p oduc s, such as lipopolysaccha ide (LPS), which is esponsible o he me abolic endo oxemia
ela ed o Me S [9].
In he las yea s, ea men o he hallma ks o Me S wi h p obio ics has eme ged as a p omising
he apy. P obio ics a e li ing mic oo ganisms ha con e heal h bene i s o he hos when adminis e ed
in adequa e amoun s [
10
]. Bi idobac e ium and Lac obacillus a e he mos equen ly used gene a o
p obio ics used in humans. Some o he bene icial e ec s o p obio ics a e media ed by hei capaci y
o no malize he gas oin es inal mic obiome, ein o ce he gu ba ie unc ion composi ion [
11
,
12
],
and hei immunomodula o y ac ions [
12
,
13
]. The e o e, he addi ion o p obio ics o a heal hy die
could ep esen an in e es ing ool o igh obesi y, Me S, and associa ed in lamma ion when used
alongside die a y managemen and li es yle modi ica ions (e.g., inc eased physical ac i i y). In his
ega d, some s udies ha e ound an imp o emen o an h opome ic pa ame e s and a dec ease in
in lamma o y bioma ke s in his disease a e p obio ic adminis a ion [
14
]. Howe e , he bene icial
e ec s o p obio ics on Me S componen s a e con adic o y [
15
], p obably because o he di e en
p obio ic s ains, doses, and clinical s udy designs.
Lac obacillus (L.) eu e i V3401 s ain, deposi ed in he Spanish Type Cul u e Collec ion (CECT)
wi h accession numbe CECT 8695, was isola ed om cow’s aw milk on Ma k, Rogosa and Sha pe
(MRS) aga medium, and 16S gene sequence analysis was ca ied ou o i s iden i ica ion. In addi ion,
i s ca bohyd a e e men a ion abili y was cha ac e ized by he Analy ical P o ile Index (API) CH50
es [
16
]. This s ain has been shown o educe he abso p ion o luo es e ol, a luo escen choles e ol
analogue, by HT-29 human en e ocy es [
16
]. Fu he mo e, Wis a a s ed a hype choles e olemic die
supplemen ed wi h he p obio ic s ain o 57 days showed HDL le els simila o hose o a heal hy
con ol g oup ed a s anda d die [
16
]. Rega ding glycemic le els, hype choles e olemic animals
Nu ien s 2019,11, 1761 3 o 14
supplemen ed wi h he p obio ic s ain showed simila alues o hose o no mocholes e olemic mice,
whe eas animals unde a hype choles e olemic die wi hou he p obio ic s ain exhibi ed highe le els
han no mocholes e olemic mice [
16
]. Highe glucose le els a e ela ed o insulin esis ance, which is
no mally associa ed wi h hype choles e olemia and low HDL le els, bo h o hem componen s o Me S.
In his se ing, supplemen a ion wi h L. eu e i V3401 migh o e an addi ional me abolic ad an age
oge he wi h heal hy die and exe cise ecommenda ions in pa ien s wi h Me S.
All hings conside ed, he p esen s udy aimed o e alua e whe he he consump ion o he
p obio ic s ain L. eu e i V3401, oge he wi h heal hy li es yle (hypocalo ic die and physical ac i i y)
ecommenda ions, was capable o imp o ing Me S componen s. Fo his pu pose, we designed a
double-blind, c osso e , placebo-con olled, single-cen e , andomized clinical ial (RCT).
2. Ma e ials and Me hods
2.1. E hical S a emen
All esea ch and p ocedu es pe o med du ing he s udy complied wi h he Decla a ion o
Helsinki and he Guidelines o Good Clinical P ac ice. A e ecei ing a comple e e bal desc ip ion
o he s udy, pa ien s signed a w i en in o med consen . The s udy p o ocol was app o ed by he
local E hics Commi ee o bo h G anada and Ja
é
n ( e e ences CEI-Ja
é
n 25022016 and CEI-G anada
28022016, espec i ely).
2.2. Subjec s and Expe imen al Design
We pe o med a andomized, double-blind, c osso e , placebo-con olled, single-cen e ial in
pa ien s wi h a new diagnosis o Me S, acco ding o he c i e ia o he In e na ional Diabe es Fede a ion
(IDF). The comple e s udy design including sample size, andomiza ion, and he ial p o ocol ha e
been p e iously published [
17
] and egis e ed a www.clinical ials.go as NCT02972567. The s udy
was conduc ed in ag eemen wi h he S anda d P o ocol I ems: Recommenda ions o In e en ional
T ials (SPIRIT) guidelines.
Sample size was calcula ed based on he ange and median alue o lipopolysaccha ide (LPS) and
assuming a powe o 80% and a signi ican le el o 5% [
17
]. In b ie , a o al o 53 ou o 60 adul pa ien s
we e ec ui ed a he Endoc inology and Nu i ion Clinical Managemen Uni , Uni e si y Hospi al o
Ja
é
n (Ja
é
n, Spain) by quali ied pe sonnel. Pa ien s we e block andomly alloca ed, by BMI and sex, in a
1:1 a io o ecei e a capsule con aining ei he he p obio ic L. eu e i V3401 (5
×
10
9
colony- o ming
uni s) o he placebo (mal odex in) once daily o 12 weeks.
Bo h capsules, p obio ic and placebo, we e p o ided by Biosea ch Li e (G anada, Spain).
In addi ion,
pa icipan s ecei ed an in ensi e li es yle in e en ion p og am ha included nu i ional
and physical counseling o achie e and main ain a 7% loss o ini ial body weigh and inc ease
mode a e-in ensi y physical ac i i y o a leas 150 min/week. In Figu e 1we summa ized he
expe imen al design o he s udy.
Nu ien s 2019,11, 1761 4 o 14
Nu ien s 2019, 11, x FOR PEER REVIEW 4 o 15
Figu e 1. Illus a ion o he expe imen al design: A andomized, double-blind, c osso e , placebo-
con olled, single-cen e ial compa ing he e ec o consump ion o Lac obacillus eu e i V3401 o 12
weeks on a ious clinical, biochemical, and in lamma o y bioma ke s and gas oin es inal mic obio a.
w: weeks; : ime; x: sample collec ion.
2.3. An h opome ic, Biochemical, In lamma o y, and Ca dio ascula Da a
We pe o med a sys ema ic symp om e alua ion on each isi , wi h special emphasis on
gas oin es inal symp oms, and a physical examina ion. Body weigh (kg), heigh (cm), and wais
ci cum e ence (cm) we e measu ed by he same pe son using s anda dized p ocedu es. Blood
p essu e was aken 3 imes by he same pe son, and he mean o he h ee alues was included. The
biochemical analyses, including lipid and glucose me abolism, we e pe o med a he Uni e si y
Hospi al o Jaén ollowing in e na ionally accep ed quali y con ol p o ocols. Homeos asis
assessmen model o insulin esis ance (HOMA-IR) was calcula ed using as ing plasma glucose and
insulin alues.
Blood samples we e collec ed om each pa ien and a e 12 h o as ing, a he beginning and
he end o each in e en ion pe iod. Se um and plasma samples we e collec ed by cen i uga ion o
blood samples and kep a −80 °C un il analysis.
Plasma adipokines as well as ca dio ascula and in lamma o y bioma ke s—adiponec in,
lep in, esis in, IL-6, IL-8, TNF-α, o al plasminogen ac i a o inhibi o -1 (PAI-1), hepa ocy e g ow h
ac o (HGF), monocy e chemoa ac an p o ein 1 (MCP-1), soluble in acellula adhesion molecule
1 (sICAM-1), soluble ascula cell adhesion molecule 1 (sVCAM-1), and myelope oxidase (MPO)—
we e analyzed on a Luminex 200 sys em (Luminex Co po a ion, Aus in, Texas, USA) wi h human
monoclonal an ibodies (EMD Millipo e Co p, Bille ica, MA, USA) using MILLIplex™ ki s
(HADK1MAG-16K, HSTCMAG-28SK, HAD2MAG-61K, HCVD2MAG-67K) acco ding o he
manu ac u e ’s ecommenda ions.
LPS and LPS-binding p o ein (LBP) we e de e mined in se um samples using CEB526GE and
SEB406 HU ELISA ki s (Cloud-Clone Co p, TX, USA), espec i ely, ollowing he manu ac u e ’s
ins uc ions.
2.4. Fecal Samples, DNA Ex ac ion, and Nex -Gene a ion Sequencing
Fecal samples we e collec ed om each pa ien a each ime ( 1, 2, 2, 4, 5, and 6). Fecal
samples we e placed inside o a s e ile plas ic bo le and kep a −80 °C un il analysis. DNA was
ex ac ed using a QIAamp DNA s ool Mini Ki (QIAGEN, Ba celona, Spain) acco ding o he
manu ac u e ’s ins uc ions, wi h he excep ion ha samples we e incuba ed wi h he lysis bu e a
Figu e 1.
Illus a ion o he expe imen al design: A andomized, double-blind, c osso e , placebo-
con olled, single-cen e ial compa ing he e ec o consump ion o Lac obacillus eu e i V3401 o
12 weeks
on a ious clinical, biochemical, and in lamma o y bioma ke s and gas oin es inal mic obio a.
w: weeks; : ime; x: sample collec ion.
2.3. An h opome ic, Biochemical, In lamma o y, and Ca dio ascula Da a
We pe o med a sys ema ic symp om e alua ion on each isi , wi h special emphasis on
gas oin es inal symp oms, and a physical examina ion. Body weigh (kg), heigh (cm), and wais
ci cum e ence (cm) we e measu ed by he same pe son using s anda dized p ocedu es. Blood p essu e
was aken 3 imes by he same pe son, and he mean o he h ee alues was included. The biochemical
analyses, including lipid and glucose me abolism, we e pe o med a he Uni e si y Hospi al o Ja
é
n
ollowing in e na ionally accep ed quali y con ol p o ocols. Homeos asis assessmen model o insulin
esis ance (HOMA-IR) was calcula ed using as ing plasma glucose and insulin alues.
Blood samples we e collec ed om each pa ien and a e 12 h o as ing, a he beginning and he
end o each in e en ion pe iod. Se um and plasma samples we e collec ed by cen i uga ion o blood
samples and kep a −80 ◦C un il analysis.
Plasma adipokines as well as ca dio ascula and in lamma o y bioma ke s—adiponec in,
lep in, esis in, IL-6, IL-8, TNF-
α
, o al plasminogen ac i a o inhibi o -1 (PAI-1), hepa ocy e
g ow h ac o (HGF), monocy e chemoa ac an p o ein 1 (MCP-1), soluble in acellula adhesion
molecule 1 (sICAM-1), soluble ascula cell adhesion molecule 1 (sVCAM-1), and myelope oxidase
(MPO)—we e analyzed on a Luminex 200 sys em (Luminex Co po a ion, Aus in, TX, USA) wi h
human monoclonal an ibodies (EMD Millipo e Co p, Bille ica, MA, USA) using MILLIplex
™
ki s (HADK1MAG-16K, HSTCMAG-28SK, HAD2MAG-61K, HCVD2MAG-67K) acco ding o he
manu ac u e ’s ecommenda ions.
LPS and LPS-binding p o ein (LBP) we e de e mined in se um samples using CEB526GE and SEB406
HU ELISA ki s (Cloud-Clone Co p, TX, USA), espec i ely, ollowing he manu ac u e ’s ins uc ions.
2.4. Fecal Samples, DNA Ex ac ion, and Nex -Gene a ion Sequencing
Fecal samples we e collec ed om each pa ien a each ime ( 1, 2, 2, 4, 5, and 6). Fecal samples
we e placed inside o a s e ile plas ic bo le and kep a
−
80
◦
C un il analysis. DNA was ex ac ed
using a QIAamp DNA s ool Mini Ki (QIAGEN, Ba celona, Spain) acco ding o he manu ac u e ’s
ins uc ions, wi h he excep ion ha samples we e incuba ed wi h he lysis bu e a 95
◦
C ins ead o
Nu ien s 2019,11, 1761 5 o 14
70
◦
C o gua an ee he lysis o bo h G am-posi i e and G am-nega i e bac e ia. Ex ac ed DNA samples
we e sequenced a acili ies o he Depa men o Mic obiology, Uni e si y Hospi al Campus de la Salud
(G anada, Spain). A 16S me agenomics sequencing was pe o med ollowing he Illumina p o ocol.
In summa y, he V3-V4 egion o he bac e ial 16S RNA gene was ampli ied using he p ime s
desc ibed by Klindwo h e al., 2013 [
18
]. The PCR mix u e was composed o 5
µ
L o each o wa d
and e e se p ime s (1
µ
M, Mac ogen, Seoul, Ko ea), 2.5
µ
L o DNA empla e samples, and 12.5
µ
L
o 1x Ho Mas e Mix (KAPA HiFi HS RM, Roche, Basilea, Swi ze land) o a inal olume o 25
µ
L.
Fi e mic oli e s o elu ion solu ion was used o he nega i e con ol. The PCR condi ions we e:
ini ial dena u a ion a 95
◦
C o 3 min, ollowed by 25 cycles o dena u a ion a 95
◦
C o 30 s, p ime
annealing a 55
◦
C o 30 s, ex ension a 72
◦
C o 30 s, and a inal elonga ion a 72
◦
C o 5 min.
The PCR
p oduc s we e demons a ed by elec opho esis on a 2% aga ose gel. No ampli ica ion p oduc was
obse ed in he nega i e con ol. The ampli ica ions we e subjec ed o pu i ica ion using Ampu e
beads (Agencou Bioscience, La Jolla, CA, USA), he elu ed DNA p oduc was quan i ied using he
assays o he Qubi ki (In i ogen, Li e Technologies, Wal ham, Massachise s, USA), and hen all
samples we e pooled in equal concen a ions o sequencing. Bioanalyze 2100 was used wi h he
DNA 1000 Chip ki (Agilen , Palo Al o, CA, USA) o e alua e he quali y o he inal p oduc s o each
sample indi idually. Sequencing was ca ied ou using Illumina MiSeq pai ed-end sequencing in an
Illumina MiSeq de ice (Illumina Inc., San Diego, CA, USA) wi h 600 cycles (300 cycles o each pai ed
eading and 12 cycles o he sequence o ba codes) acco ding o he manu ac u e ’s ins uc ions.
Sequence analysis was pe o med using he me agenomic wo k low based on 16S o MiSeq Repo e
2.3 (Illumina Inc., San Diego, CA, USA).
2.5. Taxonomic Analysis
The “Quan i a i e Insigh s In o Mic obial Ecology 2” (QUIIME 2) package was used o analyze
sequence da a [
19
]. Denoising quali y, chime a check, and clus e ing we e pe o med using he DADA2
plugins implemen ed in QUIIME 2. Amplicon sequence a ian s (ASVs) wi h a ela i e p opo ion
lowe han 0.1% we e elimina ed; as a esul , he o al numbe s o ASV we e educed o 2015 bu wi h
a e y low impac on he o al da a. The G eenGenes da abase ( e sion 13.8), oge he wi h he naï e
Bayes algo i hm, was used as he e e ence 16S da abase.
2.6. S a is ical Analysis
Fo he an h opome ic, biochemical, and in lamma o y bioma ke s, esul s a e p esen ed
as he mean alues
±
s anda d de ia ion (SD), unless o he wise indica ed. Fo hose a iables
no ollowing a no mal dis ibu ion, we applied he loga i hmic ans o ma ion (insulin, HOMA
index, glyca ed hemoglobin, o al choles e ol, iacylglyce ols, alanine amino ans e ase (GPT),
gamma glu amil ans e ase (
γ
GT), C eac i e p o ein (CRP), IL-6, IL-8, adiponec in, esis in, HGF,
sICAM, sVCAM and LBP) o he in e se ans o ma ion (high-densi y lipop o ein (HDL), aspa a e
amino ans e ase (GOT).
Only pa ien s wi h less han 5 missing da a we e conside ed, esul ing in a inal numbe o
34 pa ien s
. Missing da a in hese pa ien s we e impu ed using p incipal componen analysis (PCA)
and immed sco e eg ession (TSR) [
20
]. The ea men e ec in an h opome ic, biochemical, and
in lamma o y bioma ke s was e alua ed acco ding o he app oach desc ibed by
Wellek e al. [21]
. Two
es s we e ca ied ou : (i) a p e es o signi icance o ca yo e e ec s, and (ii) a es o signi icance o
ea men e ec s. The ea men e ec s we e conside ed signi ican o hose bioma ke s o which he
null hypo hesis o he p e es was no ejec ed and he null hypo hesis o he es was ejec ed
(p<0.05)
,
con i ming ha he bioma ke p esen ed s a is ically signi ican di e ences only due o ea men
e ec s. p- alue compu a ions we e con i med wi h di e en s a e-o - he-a mul i a ia e app oaches,
including mul i a ia e analysis o a iance (MANOVA) [
22
], pa ial leas -squa es disc iminan analysis
(PLS-DA) [
23
], and ANOVA simul aneous componen analysis (ASCA) [
24
]. TheMEDA oolbox
(h ps://gi hub/josecamachop/MEDA-Toolbox) and he MANCOVAN oolbox (h p://www.ma hwo ks.

Nu ien s 2019,11, 1761 6 o 14
com/ma labcen al/ ileexchange/27014-manco an) in Ma lab (Ma hwo ks) we e used o pe o m he
s a is ical analysis.
Fo he gas oin es inal mic obio a analysis, he gene a ed sequences, ASV, we e no malized by
means o he a e ac ion me hod (Figu e S1). The alpha di e si y was measu ed by means o he
Shannon index, whe eas he unique ac ion me ic (Uni ac), bo h weigh ed and unweigh ed,
was used
o de e mine he be a di e si y. When compa ing he inc emen al o ela i e bac e ia p opo ions
be o e and a e ea men (del a), a pai wise Wilcoxon signed- ank es was used. p- alues we e
adjus ed by False disco e y a e-FDR (q- alues).
3. Resul s
3.1. An h opome ic, Biochemical, and In lamma o y Da a
An h opome ic and biochemical cha ac e is ics o he subjec s a e desc ibed in Table 1, whe eas
in Table 2 he in lamma o y bioma ke s de e mined in blood samples a e desc ibed.
In he case o BMI, dias olic blood p essu e, GOT, and LBP, we ound ha he washou pe iod
was no long enough o a oid he ca yo e e ec s. We ound signi ican di e ences o Il-6, sVCAM
(Figu e 2), and insulin le els (Table 1); howe e , we did no ind any signi ican esul s o HOMA
index (Table 1).
Nu ien s 2019, 11, x FOR PEER REVIEW
9 o 15
In he case o BMI, dias olic blood p essu e, GOT, and LBP, we ound ha he washou pe iod
was no long enough o a oid he ca yo e e ec s. We ound signi ican di e ences o Il-6, sVCAM
(Figu e 2), and insulin le els (Table 1); howe e , we did no ind any signi ican esul s o HOMA
index (Table 1).
Figu e 2. In lamma o y bioma ke s h oughou he s udy. The le els o in e leukin 6 (IL-6) and
soluble ascula cell adhesion molecule 1 (sVCAM) we e modi ied by he p obio ic consump ion (p <
0.05). Con inuous line: p obio ic g oup. Discon inuous line: placebo g oup.
3.2. Gas oin es inal Mic obiome Composi ion
We cha ac e ized he gas oin es inal mic obiome composi ion o he pa icipan s a he
beginning, middle, and end o each in e en ion pe iod (Table S1). Howe e , we we e unable o
de e mine he speci ic p esence o he L. eu e i V3401 s ain in ecal samples due o he lack o speci ic
p ime s o his s ain. As shown in Figu e 3, a he beginning o he in e en ion, he mos abundan
phyla we e Fi micu es and Bac e oide es ollowed by P o eobac e ia, Ac inobac e ia, Ve ucomic obia, and
Cyanobac e ia.
Figu e 2.
In lamma o y bioma ke s h oughou he s udy. The le els o in e leukin 6 (IL-6) and soluble
ascula cell adhesion molecule 1 (sVCAM) we e modi ied by he p obio ic consump ion (p<0.05).
Con inuous line: p obio ic g oup. Discon inuous line: placebo g oup.
3.2. Gas oin es inal Mic obiome Composi ion
We cha ac e ized he gas oin es inal mic obiome composi ion o he pa icipan s a he beginning,
middle, and end o each in e en ion pe iod (Table S1). Howe e , we we e unable o de e mine he
speci ic p esence o he L. eu e i V3401 s ain in ecal samples due o he lack o speci ic p ime s o
his s ain. As shown in Figu e 3, a he beginning o he in e en ion, he mos abundan phyla we e
Fi micu es and Bac e oide es ollowed by P o eobac e ia,Ac inobac e ia,Ve ucomic obia, and Cyanobac e ia.
Nu ien s 2019,11, 1761 7 o 14
Table 1. An h opome ic and biochemical cha ac e is ics o he pa ien s.
G oup 1 G oup 2
Placebo P obio ic P obio ic Placebo
1 3 4 6 1 3 4 6
Weigh (kg)
109.02
±
26.7 105.70
±
26.2 101.50
±
24.5 101.08
±
24.2 103.49
±
15.2
96.56 ±16.2 93.91 ±16.9 92.0 ±17.3
BMI (kg/m2)38.76 ±7.2 37.57 ±7.1 36.77 ±6.8 36.56 ±6.6 38.30 ±7.3 35.69 ±7.1 34.57 ±6.9 33.80 ±6.6
SBP (mm Hg)
137.68
±
16.9 133.28
±
15.4 133.11
±
20.4 132.21
±
14.6
139 ±22.6
129.95
±
16.0 131.30
±
20.0 131.40
±
18.6
DBP (mm Hg) 84.28 ±9.6 81.96 ±7.7 81.68 ±11.0 82.11 ±10.5 87.95 ±14.3 78.18 ±10.4 78.85 ±12.4 81.60 ±11.2
Glucose (mg/dL)
103.29
±
11.0 108.08
±
11.5
106.74 ±8.9
105.53
±
10.5
101.0 ±13.9
103.68
±
13.4 101.22
±
11.8 103.78
±
16.5
Insulin (mU/mL) 17.50 ±10.6 16.18 ±11.3 22.44 ±10.3 21.74 ±11.7 14.24 ±8.5 12.42 ±10.9 14.04 ±6.1 17.47 ±7.8
HOMA index 4.48 ±2.8 4.41 ±3.3 5.91 ±2.8 5.66 ±3.5 3.71 ±2.7 3.52 ±3.6 3.64 ±1.7 4.46 ±2.2
Glyca ed Hemoglobin (%) 5.59 ±0.4 6.04 ±2.2 5.44 ±0.3 5.49 ±0.3 5.68 ±0.4 5.90 ±1.9 5.46 ±0.3 5.44 ±0.3
To al choles e ol (mg/dL)
232.42
±
43.0 207.08
±
36.0 202.79
±
45.8 224.16
±
45.5 233.41
±
46.5 203.64
±
37.9 209.56
±
58.0 220.89
±
53.8
T iacylglyce ols (mg/dL)
119.25
±
47.6 122.46
±
59.9 109.00
±
47.3 118.89
±
52.2 130.55
±
47.5 128.23
±
57.6 112.89
±
42.7 100.56
±
62.6
LDL (mg/dL)
156.79
±
35.7 131.71
±
30.0 128.11
±
32.5 144.42
±
39.0
161.0 ±41.6
132.91
±
32.1 136.22
±
47.7 145.72
±
44.5
HDL (mg/dL) 50.54 ±14.6 50.46 ±12.2 52.47 ±13.3 54.11 ±10.2 45.95 ±9.5 44.68 ±7.9 50.44 ±10.3 54.61 ±11.5
GOT (U/L) 25.75 ±7.7 22.83 ±6.3 22.63 ±6.8 22.32 ±5.7 23.55 ±12.4 25.50 ±14.9 20.56 ±6.1 21.33 ±6.2
GPT (U/L) 34.79 ±17.4 28.17 ±14.3 22.42 ±11.9 27.89 ±12.7 24.38 ±8.9 22.24 ±8.4 18.00 ±7.5 22.50 ±11.0
γ-GT (U/L) 36.29 ±13.6 37.08 ±16.7 38.42 ±18.9 36.74 ±21.2 26.05 ±12.7 24.91 ±14.2 24.89 ±14.3 24.00 ±15.4
Values a e exp essed as means
±
SD. BMI: body mass index; SBP: sys olic blood p essu e; DBP: dias olic blood P essu e; HOMA-IR: Homeos asis assessmen model o insulin esis ance;
LDL: low-densi y lipop o ein; HDL: high-densi y lipop o ein; GOT: aspa a e amino ans e ase; GPT: alanine amino ans e ase; γ-GT: gamma glu amyl ans e ase.
Nu ien s 2019,11, 1761 8 o 14
Table 2. In lamma o y bioma ke s.
G oup 1 G oup 2
Placebo P obio ic P obio ic Placebo
1 2 3 5 1 2 3 5
CRP (mg/dL) 5.13 ±3.7 5.78 ±4.8 6.97 ±7.4 5.52 ±4.4 5.88 ±4.4 6.12 ±6.3 3.66 ±2.7 4.24 ±3.8
IL-6 (pg/mL) 2.91 ±1.8 3.33 ±2.4 3.12 ±1.9 2.62 ±2.0 2.07 ±1.2 1.79 ±1.2 1.37 ±0.8 1.72 ±1.0
IL-8 (pg/mL) 2.86 ±1.7 2.80 ±1.2 4.11 ±7.1 4.23 ±9.4 2.66 ±1.1 2.73 ±1.2 2.27 ±1.0 2.28 ±1.1
TNF-α(pg/mL) 4.70 ±2.5 4.91 ±2.6 4.59 ±2.1 3.51 ±1.7 4.05 ±1.9 4.15 ±2.1 3.05 ±1.2 3.28 ±2.0
Adiponec in (mg/L) 6.55 ±5.1 5.95 ±4.7 5.82 ±3.8 6.56 ±3.4 5.69 ±3.7 6.91 ±6.5 7.20 ±4.4 8.26 ±6.1
PAI1 (µg/L) 9.24 ±4.9 9.55 ±4.3 10.55 ±4.8 11.56 ±7.1 9.31 ±3.2 8.36 ±2.6 9.08 ±3.2 9.51 ±3.6
P-selec in (ng/mL) 46.78 ±19.7 46.70 ±21.2 49.26 ±22.8 63.47 ±38.3 48.06 ±16.7 40.51 ±11.4 60.73 ±35.4 58.09 ±21.6
Resis in (µg/L) 17.71 ±8.1 17.70 ±13.7 16.99 ±5.2 17.45 ±10.4 15.33 ±7.2 11.89 ±5.2 11.08 ±4.0 12.60 ±5.2
HGF (pg/mL) 161.12 ±97.9 155.73 ±93.6 131.45 ±65.9 162.45 ±88.3 175.06 ±75.1 170.17 ±72.7 160.47 ±68.8 157.95 ±57.5
Lep in (µg/L) 28.56 ±14.8 24.07 ±12.3 23.71 ±13.8 18.42 ±10.5 21.97 ±11.8 17.68 ±10.9 13.67 ±9.3 14.24 ±10.4
MCP-1 (pg/mL) 107.53 ±39.3 106.57 ±31.7 120.07 ±60.1 118.20 ±46.8 108.86 ±39.3 114.61 ±51.5 116.31 ±43.6 112.51 ±41.2
sICAM (ng/mL) 73.65 ±37.2 73.71 ±41.1 67.86 ±35.2 65.47 ±32.2 74.80 ±26.9 71.50 ±33.1 75.17 ±40.2 73.0 ±41.2
MPO (ng/mL) 17.69 ±5.9 19.96 ±10.7 20.70 ±13.2 30.53 ±21.2 15.56 ±7.8 18.14 ±13.0 17.31 ±12.4 19.46 ±10.2
sVCAM (ng/mL) 494.22 ±125.1 511.04 ±154.8 516.47 ±149.1 507.61 ±138.7 489.68 ±80.9 472.72 ±71.4 491.62 ±99.6 527.43 ±74.6
LPS (ng/mL) 285.81 ±107.2 277.90 ±116.3 312.22 ±126.0 326.19 ±166.0 321.82 ±105.4 316.83 ±124.0 309.91 ±136.7 308.70 ±131.4
LBP (ng/mL) 731.26 ±512.2 782.55 ±323.0 635.96 ±294.2 747.39 ±272.6 837.47 ±423.5 742.81 ±349.2 833.63 ±560.5 855.78 ±663.1
Values a e exp essed as means
±
SD. CRP: C eac i e p o ein; IL: in e leukin; TNF-
α
: umo nec osis ac o alpha; PAI1: plasminogen ac i a o inhibi o -1; HGF: hepa ocy e g ow h
ac o ; MCP-1: monocy e chemoa ac an p o ein 1; sICAM: soluble in acellula adhesion molecules 1; sVCAM: soluble ascula cell adhesion molecule 1; MPO: myelope oxidase;
LPS: lipopolysaccha ide; LBP: lipopolysaccha ide-binding p o ein.
Nu ien s 2019,11, 1761 9 o 14
Nu ien s 2019, 11, x FOR PEER REVIEW
10 o 15
Figu e 3. Baseline gas oin es inal mic obiome composi ion. Taxonomic composi ion o he
gas oin es inal communi ies a he beginning o he in e en ion. The igu e shows ba cha s o he
ela i e abundance o bac e ia a he phylum le el. Each column ep esen s a pa icipan .
Rega ding he bac e ial di e si y, we did no ind signi ican di e ences in he alpha di e si y
h oughou he s udy, measu ed as he Shannon index (H) (Figu e S2), o in he be a di e si y (Figu e
S3). The e o e, ou nex analysis was o de e mine he e olu ion o he ela i e p opo ion o speci ic
axa, namely Fi micu es, Bac e oide es, Ve ucomic obia, Ac inobac e ia, P o eobac e ia, Fusobac e ia,
Cyanobac e ia, Elusimic obia, Tene icu es, and Len isphae ae. I is wo h men ioning he e was an
inc ease in he ela i e p opo ion o he Ve ucomic omia phylum in he pa icipan s ha consumed
he p obio ic s ain (Figu e 4A). The same esul s we e ound in he Akke mansia genus (Figu e 4B).
Figu e 3.
Baseline gas oin es inal mic obiome composi ion. Taxonomic composi ion o he
gas oin es inal communi ies a he beginning o he in e en ion. The igu e shows ba cha s o
he ela i e abundance o bac e ia a he phylum le el. Each column ep esen s a pa icipan .
Rega ding he bac e ial di e si y, we did no ind signi ican di e ences in he alpha di e si y
h oughou he s udy, measu ed as he Shannon index (H) (Figu e S2), o in he be a di e si y (Figu e S3).
The e o e, ou nex analysis was o de e mine he e olu ion o he ela i e p opo ion o speci ic axa,
namely Fi micu es,Bac e oide es,Ve ucomic obia,Ac inobac e ia,P o eobac e ia,Fusobac e ia,Cyanobac e ia,
Elusimic obia,Tene icu es, and Len isphae ae. I is wo h men ioning he e was an inc ease in he ela i e
p opo ion o he Ve ucomic omia phylum in he pa icipan s ha consumed he p obio ic s ain
(Figu e 4A). The same esul s we e ound in he Akke mansia genus (Figu e 4B).
Based on he esul s desc ibed abo e, we decided o de e mine whe he he e we e signi ican
di e ences in he ela i e abundance o hese axa due o he ea men (p obio ic e sus placebo).
We, he e o e, pe o med a pai wise compa ison [
25
]. Du ing he i s in e en ion ( 1, 2, and 3) we
obse ed a signi ican inc ease in he del a alues ( 3– 1) in he Ve ucomic obia phylum due o he
ea men (p obio ic e sus placebo). Howe e , du ing he c osso e in e en ion, he di e ences
( 6– 4) we e no s a is ically signi ican , al hough we ound a signi ican end (FDR p=0.07) (Figu e 5).