!

!
!
!
!
!
!
!
"#$%&'$($#!)*""$%+!
,+*-$*'$#!%".!/#%#'!01203!
!
!
!
!
!
!
!
!
!
! ! !,+*-$*'$#!"'+4$&!%".!/#%#'!01203!
!
!
2
.3!5%&%+46!-4#75+*8&)!

Ceieal gluten pioteins (gliauin α, β, γ, anu ω) aie the main enviionmental factois
causative of coeliac uisease pathogenesis. The incomplete hyuiolysis of gliauin by
human enzymes uuiing gastiointestinal uigestion leaus to the geneiation of
peptiues able to tiiggei cytotoxic anu inflammatoiy signals in intestinal epithelial
cells of coeliac uisease patients. Clinical manifestations of the uisease often incluue
intestinal symptoms anu nutiient malabsoiption associateu with seveie mucosal
uamage. Cuiiently, the only available theiapy foi coeliac uisease patients is the
auheience to a stiict life-long gluten-fiee uiet. Bowevei, compliance with this
uietaiy iecommenuation is complex, anu othei alteinative oi aujuvant stiategies
aie neeueu.
Alteiations in the composition of the gut miciobiota of coeliac patients aie
chaiacteiizeu by uecieaseu !"#"$%&'()*+",- numbeis (Naual *). '/., 2uu7; Sanz *).
'/., 2uu7, Collauo *). '/., 2uu8; 2uu9; Be Palma *). '/., 2u1u). The association of
imbalances in the intestinal miciobiota anu the positive iepoiteu ioles that
bifiuobacteiia play on intestinal health have leu to the pioposeu use of piobiotics
as pait of auuitional anu alteinative nutiitional stiategies foi impioving the quality
of life of coeliac patients. The stiain !"#"$%&'()*+",-. /%01,- ES1 (CECT 7S47) is
capable to ieuuce the toxicity anu inflammatoiy potential of gliauin-ueiiveu
peptiues anu is an excellent canuiuate as piobiotic foi coeliac uisease patients.


93!$"*64'$*&!*(!':%!,+*-$*'$#!"'+4$&!!"#"$%&'()*+",-./%01,-!
%".!

The piobiotic stiain ES1 was isolateu by the gioup of Bi. Yolanua Sanz (Institute of
Agiochemistiy anu Foou Technology of the Spanish National Reseaich Council;
IATA-CSIC) fiom faeces of healthy babies unuei bieast-milk feeuing.
! ! !,+*-$*'$#!"'+4$&!%".!/#%#'!01203!
!
!
S
In oiuei to classify this bacteiial stiain, an almost full fiagment of the 16S iRNA
encouing gene was amplifieu anu sequenceu using the methouology pieviously
uesciibeu by Kaufmann anu cowoikeis (1997). BNA fiom puie cultuie was
extiacteu anu aujusteu to a final concentiation of 4u ng¡µl in ultiapuie watei. The
BNA was checkeu foi puiity, using stanuaiu methous. BNA templates weie
amplifieu by PCR using piimeis LNS (S'-Cuu uTu CuT uCC CAC TTT CAT u-S') anu
LN26 (S'-uAT TCT uuC TCA uuA TuA ACu-S') amplifying a 1SSu-bp iegion of the
16S iRNA gene (}ohnson 1994; Satokaii *). '/., 2uu1; Faviei *). '/. 2uu2). Contiols
uevoiu of BNA weie simultaneously incluueu in the amplification piocess. The
integiity of the PCR piouucts was assayeu by uetection of single banus following
electiophoiesis. Amplicons weie puiifieu using a commeicial kit anu subsequent
sequencing ieactions weie peifoimeu using ABI S7uu equipment. The iesulting
sequence was automatically aligneu anu inspecteu by eye anu compaieu by use of
the online tool BLAST (http:¡¡blast.ncbi.nlm.nih.gov¡Blast.cgi). The stiain was
iuentifieu on the basis of highest scoies as a membei of the species !"#"$%&'()*+",-.
/%01,- anu was uepositeu at the Spanish Type Cultuie Collection (CECT) unuei
the accession numbei 7S47 (Izquieiuo *).'/. 2uu8).


13!,+*-$*'$#!,+*,%+'$%"!*(!':%!"'+4$&!%".!

0ne of the majoi iequiiements foi a piobiotic stiain is that it shoulu maintain
viability uuiing gastiointestinal tiansit. To evaluate this phenotype, the ES1 stiain
was checkeu foi its iesistance to aciu pBs anu bile salts.
To analyze pB iesistance, cell suspensions in 1u mN phosphate-buffeieu saline
(pB 7.2), with a cell uensity of 1u
9
cell¡ml, weie uiluteu 1u-folu in steiile saline
solution containing S mg¡ml pepsin fiom poicine stomach mucus anu aujusteu to
uiffeient pB values (1.S, 2.S, 2.u, anu S.u) with BCl. Aliquots weie taken aftei u anu
9u minutes of incubation at S7°C. uiowth ability was ueteimineu by plate counting
on NRSC agai. viability changes weie ueteimineu using the LIvE¡BEAB BacLight
! ! !,+*-$*'$#!"'+4$&!%".!/#%#'!01203!
!
!
4
Kit foi micioscopy uetection accoiuing the manufactuiei's pioceuuie. The counts
of both live (gieen fluoiescence) anu ueau (ieu fluoiescence) bacteiia weie
ueteimineu. The iesults inuicate a moueiate sensitivity of the ES1 stiain to gastiic
juices at pB 2 anu iesistance to highei pB values. These values of iesistance weie
highei than those obtaineu foi some commeicial piobiotic stiains in paiallel
expeiiments (uata not shown).

;:!<$4-$6$'=!/>3! ;:!5+*?':!4-$6$'=!/>3!
9! 9@A! 1! 9! 9@A! 1!
S7.9±2.S 86.2±1S.u 99.4±4.2 S6.1±S.2 86.7±8.1 99.7±9.S
!
'BCDE! .@ Effect of simulateu gastiic conuitions on the viability anu giowth ability of the !2. /%01,-.
ES1 stiain (iesults aie the mean of thiee uiffeient expeiiments).

The toleiance to bile was testeu in NRSC bioth containing u.S%, 1.u%, 2.u%, anu
S.u% (w¡v) 0xgall. The giowth meuia weie inoculateu to ieach A
6uu
of u.1 with an
oveinight cultuie of the stiain anu incubateu at S7°C. Bacteiial giowth was
monitoieu by measuiing optical uensity at 6uu nm in a Nicioplate Reauei. uiowth
ability was expiesseu as a peicentage of that of the contiol without 0xgall, which
was assigneu a value of 1uu%. As can be seen in Table 2, the ES1 stiain has a high
toleiance to bile salts. As in the pievious assays, these values aie similai oi slightly
highei than those obtaineu with commeicial piobiotic stiains.


+%64'$<%!5+*?':!+4'%!/>3!
F@A! .@F! 9@F! 1@F!
89.22±u.7u 82.6S±1.6u 69.9S±u.SS 67.28±1.S1
!
'BCDE!9@ Effect of bile concentiations on the ielative giowth of the !2./%01,- ES1 stiain (iesults aie
the mean of thiee uiffeient expeiiments).

! ! !,+*-$*'$#!"'+4$&!%".!/#%#'!01203!
!
!
S
23! $&:$-$'*+=! %((%#'! *(! ':%! ,+*-$*'$#! "'+4$&! %".! *&! ':%!
5+*?':! *(! G$#+**+54&$"G"! $"*64'%)! (+*G! #*%6$4#!
,4'$%&'"!

The ability of the ES1 stiain to inteifeie with the giowth of pathogenic
miciooiganisms isolateu fiom faeces of coeliac patients was assayeu. The
antimiciobial capacity of the stiain was evaluateu by classical two-layei technique.
The iesults aie summaiiseu in Table S anu inuicates that the giowth of all the
testeu pathogens weie inhibiteu to some extent by the ES1 stiain.
To ueteimine if the antimiciobial activity of the ES1 stiain is uue to the piesence of
compounus secieteu to the meuium3 oveinight cultuies of the stiain pieviously
subcultuieu thiee times weie useu to inoculate (1%, vol¡vol) 1u ml of fiesh NRSC
bioth. Aftei 16 h of incubation at S7°C, cells weie iemoveu by centiifugation (1S
min, 4°C, 12uuuXg). The obtaineu cultuie supeinatant was filtei steiilizeu (u.22-
µm poie size) to eliminate the possible piesence of viable cells, anu the pB was
aujusteu to 6.S with 1 N Na0B to excluue the effects of oiganic acius. The
neutializeu cell-fiee cultuie supeinatant (NCS) was concentiateu by fieeze uiying,
suspenueu in 1u% of theii oiiginal volume with Su mN souium phosphate (pB
6.S), anu kept at!-2u°C until use. The NCS was scieeneu foi antimiciobial activity
by a well uiffusion assay. Fifteen milliliteis of appiopiiate cultuie meuium
containing u.7% (wt¡vol) agai was inoculateu with each inuicatoi stiain at a final
concentiation of about 1u
6
CF0¡ml, pouieu into petii uishes, anu alloweu to
soliuify at ioom tempeiatuie. Wells (S mm in uiametei) weie maue on the
soliuifieu agai with a steiile metal cylinuei anu weie filleu with 4u!µl of NCS fiom
the stiain !"#"$%&'()*+",-. ES1.
Aftei incubation at the conuitions iequiieu by each inuicatoi stiain (Table S), the
inhibition zones weie measuieu. Each assay was peifoimeu in tiiplicate. Inhibition
halos weie obtaineu foi all the testeu stiains inuicating that a bacteiiocin-like
inhibitoiy compounu is secieteu by this piobiotic stiain. This compounu is active
against uiffeient pathogens that aie piesent in the gut of coeliac patients
! ! !,+*-$*'$#!"'+4$&!%".!/#%#'!01203!
!
!
6
'4+5%'!"'+4$&H! $&:$-$'$*&!I*&%"!/JK3!

!'()*+%"$*4.5,/1'),4. 1.S
6/%4)+"$",-.$"##"("/*. 1.6
6/%4)+"$",-.7*+#+"01*04. 1.2
84(9*+"(9"'.(%/" CBE9 1.9
:/*&4"*//'.%;<)%('. 2.u
=)'79</%(%((,4.*7"$*+-"$"4 1.u
!
'BCDE!1@ Antimiciobial spectium of the ES1 stiain on uiffeient pathogens.*All the testeu pathogens
weie isolateu fiom faeces of coeliac patients.

.
A3!':%!,+*-$*'$#!"'+4$&!%".!$"!#4,4-6%!'*!)%5+4)%!56$4)$&!
,+*'%$&"!

The hyuiolytic activity of the stiain !2. /%01,-. ES1 against seveial gliauin
immunogenic peptiues was ueteimineu. The peptiue SS-mei
(LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF) of α-gliauin anu also peptiues PA2
(PQPQLPYPQPQLP), anu PA9 (QLQPFPQPQLPY) iepiesenting immunogenic
epitopes within the SS-mei weie evaluateu. Peptiues weie incubateu foi 1 houi in
the piesence of cell suspensions of each bacteiial stiain. Aftei this time of
incubation, the uisappeaiance anu appeaiance of peptiue peaks was monitoieu by
BPLC chiomatogiaphy anu 0v uetection anu peptiues weie stiuctuially
chaiacteiizeu by BPLC-ESI-Ns¡Ns.
The !2. /%01,- ES1 stiain showeu ability to hyuiolyze all assayeu peptiues anu
paiticulaily the PA9 anu SS-mei (Table 4). The PA2 anu PA9 weie piefeientially
cleaveu at a pioline locateu neai the miuule of the peptiue sequence
PQPQLP

YPQPQLP leauing to the foimation of the YPQPQLP |m¡z (chaige): S74.u
(+1), Ncalc (Ba): S72.4j peptiue. In samples inoculateu with the ES1 stiain,
! ! !,+*-$*'$#!"'+4$&!%".!/#%#'!01203!
!
!
7
cleavage piouucts ueiiveu fiom the SS-mei weie uetecteu, incluuing the following
sequences: QLPYPQPQLPYPQPQLPYP |m¡z (chaige): 229u.4 (N+BNa
+
), Ncalc
(Ba): 2268.Sj, anu LPYPQPQLPYPQPQPF |m¡z (chaige): S1S.2 (+4) (N+BNa
+
),
Ncalc (Ba): 2uS8.7j.


"'+4$&! ,+*'%*6="$"!*(!LDMBNMO!;E;PMNEQ!/>3!
,49! ,4R! 11SKET!
!2./%01,-!%".! S6.S u.S S.6
!
'BCDE!2@!Begiauation of synthetic gliauin peptiues (%) by the ES1 stiain. The iesults aie expiesseu
as the aveiage peak aiea of the chiomatogiaphic signal foi each peptiue ielative to peptiue
sample non-inoculateu with bacteiia. Expeiiments weie uone in tiiplicate


U3! $&:$-$'$*&! *(! ':%! $&(64GG4'*+=! +%",*&"%! -=! ':%!
,+*-$*'$#!%".!"'+4$&!

The effect of co-incubation of the ES1 piobiotic stiain with faeces of 26 coeliac
patients with active uisease (mean age S.S yeais, iange 2.1-12.u yeais), 18
symptom-fiee coeliac uisease patients (mean age S.S yeais, iange 1.u- 12.S yeais)
on a gluten-fiee uiet foi 1-2 yeais; anu 2u healthy chiluien (mean age S.S yeais,
iange 1.8-1u.8 yeais) on inuuction of cytokine piouuction anu suiface antigen
expiession in peiipheial bloou mononucleai cells (PBNCs) was ueteimineu.
The conclusion of this woik inuicate that faeces of both, active coeliac anu
symptom-fiee coeliac uisease patients, iepiesenting an imbalanceu miciobiota,
significantly incieaseu TNF-α piouuction anu CB86 expiession in PBNCs, while
uecieaseu IL-1u cytokine piouuction anu CB4 expiession compaieu with contiol
samples. Active CB-patient samples also inuuceu significantly highei IFN-γ
piouuction compaieu with contiols. Bowevei, the !"#"$%&'()*+",-. ES1. stiain
! ! !,+*-$*'$#!"'+4$&!%".!/#%#'!01203!
!
!
8
suppiesseu the pio-inflammatoiy cytokine pattein inuuceu by the laige intestinal
content of coeliac uisease patients anu incieaseu IL-1u piouuction. All these
iesults have been publisheu in Neuina *).'/. (2uu8).
In a seconu stuuy, the capacity of the stiain !"#"$%&'()*+",- ES1 to counteiact the
inflammatoiy effects of gliauin-ueiiveu peptiues in intestinal epithelial Caco-2 cells
was evaluateu. A commeicial extiact of seveial gliauin types was subjecteu to "0.
5")+% gastiointestinal uigestion, inoculateu oi not with ES1 cell suspension, in a
bicameial system. Inteiestingly, the peptiues fiom gliauin uigestions inoculateu
with the bifiuobacteiial stiain uiu not exhibit the toxic amino aciu sequences
iuentifieu in those noninoculateu. Aftei that, Caco-2 cell cultuies weie exposeu to
the uiffeient gliauin peptiue uigestions (u.2S mg piotein¡ml), anu the mRNA
expiession of NF-kB, TNF-α anu chemokine CXCRS ieceptoi weie analyzeu by RT-
PCR in stimulateu cells. The piouuction of the pio-inflammatoiy maikeis NF-kβ
pSu, TNF-α, anu IL-1β by Caco-2 cells was also ueteimineu by ELISA. The RT-PCR
analysis eviuenceu a uown-iegulation in mRNA expiession of pio-inflammatoiy
biomaikeis in the case of the ES1 samples. Consistent with these iesults the
piouuction of NF-kβ pSu, TNF-α, anu IL-1β was ieuuceu in cell cultuies exposeu to
gliauin uigestions inoculateu with the ES1 bifiuobacteiia stiain. All these iesults
have been publisheu by Lapaiia anu Sanz (2u1u)
veiy iecently, the changes in the pioteome of Caco-2 cells exposeu to the uiffeient
gliauin peptiue uigestions have been stuuieu. Changes in the pioteome weie
ueteimineu by 2BE anu NALBI-T0F (see the oiiginal aiticle in annex "ES1
piobiotic stiain scientific liteiatuie" foi moie expeiimental uetails). uliauins
uigesteu without the ES1 stiain alteieu the expiession of a highei numbei of
pioteins than in the piesence of the bacteiium (21 5*+4,4 9). These uiffeiential
pioteins weie involveu in uisoiganization of cell cytoskeleton, inflammation, anu
apoptosis. uliauins uigesteu in the piesence of the ES1 piobiotic stiain influenceu
the piouuction of pioteins involveu in calcium homeostasis anu cell suivival anu
function. Theiefoie, !2. /%01,- ES1 stiain might amelioiate gliauin toxicity anu
mouify the iesponses of intestinal epithelial cells to the gliauin challenge. These
iesults aie incluueu in the aiticle by 0livaies *).'/. (2u11).
! ! !,+*-$*'$#!"'+4$&!%".!/#%#'!01203!
!
!
9
Finally, the effects of !"#"$%&'()*+",- ES1 stiain has been evaluateu in an animal
mouel of gliauin-inuuceu enteiopathy (Lapaiia *).'/., 2u12). The mouel is baseu on
the use of newboin iats feu gliauin alone oi sensitiseu with inteifeion IFN-γ anu
feu gliauin. In these expeiiments, jejunal tissue sections weie collecteu foi
histological, NFkB mRNA expiession anu cytokine piouuction analyses. Leukocyte
populations anu T-cell subsets weie also analyseu in peiipheial bloou samples anu
the possible tianslocation of the bacteiium to uiffeient oigans was ueteimineu by
plate counting. Finally, the composition of the colonic miciobiota was quantifieu by
ieal-time PCR. The iesults inuicateu that feeuing gliauin alone ieuuceu enteiocyte
height anu peiipheial CB4+ cells, but incieaseu CB4+¡FoxpS+ T anu CB8+ cells,
while the simultaneous auministiation of the piobiotic stiain exeiteu opposite
effects. Animals sensitiseu with IFN-γ anu feu gliauin showeu high cellulai
infiltiation, ieuuceu villi wiuth anu enteiocyte height (Figuie 1). Sensitiseu
animals also exhibiteu incieaseu NFkB mRNA expiession anu TNF-a piouuction in
tissue sections. !"#"$%&'()*+",- ES1 stiain auministiation incieaseu NFkβ
expiession anu IL-1u, but ieuuceu TNF-α piouuction in the enteiopathy mouel. In
sensitiseu gliauin-feu animals, CB4+, CB4+¡FoxpS+ anu CB8+ T cells incieaseu,
wheieas the auministiation of the piobiotic stiain ieuuceu CB4+ anu
CB4+¡FoxpS+ cell populations anu incieaseu CB8+ T cell populations. The
bifiuobacteiial stiain auministeieu iepiesenteu between 7S-9S% of the total
bifiuobacteiia isolateu fiom all tieateu gioups, anu tianslocation to oigans was not
uetecteu. These finuings inuicate that !2. /%01,- ES1 attenuates the piouuction of
inflammatoiy cytokines anu the CB4+ T-cell meuiateu immune iesponse, at least in
this animal mouel of gliauin-inuuceu enteiopathy.


03!"4(%'=!4""%""G%&'!*(!':%!,+*-$*'$#!"'+4$&!%".!

Safety assessment of the !"#"$%&'()*+",-. ES1 stiain was uone following the
FA0¡WB0 Recommenuation uuiuelines (FA0¡WB0, 2uu2). In a fiist step, the
! ! !,+*-$*'$#!"'+4$&!%".!/#%#'!01203!
!
!
1u
piouuction of some non-uesiieu metabolites (isomeis of lactic aciu, ueconjugateu
bile-salts anu biogenic amines) weie ueteimineu. Also the antibiotic iesistance
piofile of the stiain was checkeu. Finally, an acute ingestion stuuy on mice was
caiiieu out.


!
(MLVTE! .@ Bistology of jejunal tissue sections of uiffeient expeiimental iat gioups, staineu with
hematoxylin-eosin showing the uiffeiente uegiee of cellulai infiltiation in the lamina piopiia of
anmals sensitizeu oi not with IFN- anu feu gliauins anu !"#"$%&'()*+",- ES1 stiain (Lapaiia *).
'/. 2u12).

! ! !,+*-$*'$#!"'+4$&!%".!/#%#'!01203!
!
!
11
Lactic aciu piouuction was ueteimineu by using supeinatant fiom a 24-h cultuie of
each bacteiial stiain. Stiain CECT 7S47 was giown anaeiobically at S7°C in
!"#"$%&'()*+",- sp. meuium. Supeinatant was obtaineu by centiifugation at
12,uuuXg foi 1u min. Lactic aciu isomeis weie quantifieu using a commeicial kit,
following the manufactuiei's instiuctions. A bile salt hyuiolase (BSB) activity
assay was peifoimeu with glycocholate anu tauiocholate as substiates, following
the technique of Kumai anu cowoikeis (2uu6). Finally, the foimation of biogenic
amines (cauaveiine, histamine, putiescine, anu tyiamine) was ueteimineu in
!"#"$%&'()*+",- sp. meuium cell-fiee supeinatant of 24-h cultuies at S7°C
anaeiobically. Amine foimation was ueteimineu following the chiomatogiaphic
methou uesciibeu by Eeiola anu cowoikeis (199S). Assays weie peifoimeu in
thiee inuepenuent expeiiments.
Results showeu that the level of piouuction of both B- anu L-lactic aciu isomeis
was veiy low in compaiison with that of othei commeicial piobiotic stiains such
as the >'()%&'("//,4. +9'-0%4,4 uu. Conceining BSB activity, stiain ES1 uiu not
uisplay this activity with eithei tauiocholate oi glycocholate. In ielation to the
biogenic amine levels obtaineu in supeinatants, putiescine was not uetecteu in any
case, anu cauaveiine, histamine, anu tyiamine levels weie veiy low in compaiison
with those foi stiain >&2.+9'-0%4,4 uu.
The acute ingestion assay was uone at the Institute Pasteui heauquaiteis at
Nonteviueo (0iuguay). A cultuie of !"#"$%&'()*+",- ES1 giowing anaeiobically in
2 liteis of !"#"$%&'()*+",- sp. meuium foi 17 h at S7°C was obtaineu. Cells weie
iecoveieu by centiifugation at 12,uuuXg foi 1S min anu washeu twice in saline
solution (u.u9%, wt¡vol). Cells weie iesuspenueu in 2uu ml of skim milk with S%
(wt¡vol) suciose, fiozen at -8u°C, anu fieeze-uiieu. Aliquots of 1u
9
CF0 pei 1uu !µl
of Ringei's solution weie piepaieu as the inoculum.
All the pioceuuies involving animal expeiiments weie conuucteu in accoiuance
with the iegulations establisheu by the Euiopean Community Council on the
piotection of animals with expeiimental anu scientific applications (Regulation
86¡6u9¡EEC). Assays weie caiiieu out with 7-week-olu pathogen-fiee male
BALB¡c mice. The mice hau fiee access to watei anu stanuaiu iouent laboiatoiy
! ! !,+*-$*'$#!"'+4$&!%".!/#%#'!01203!
!
!
12
chow. Five uays befoie the assay was staiteu, animals weie ianuomly assigneu to
the uiffeient expeiimental gioups. All animals in each cage weie auministeieu
with the same uose. Bue to the special immunological chaiacteiistics of immune-
ueficient mice, the animals weie maintaineu fiom the time of theii aiiival insiue
containment units pioviueu with positive piessuie. Immunosuppiession was
achieveu by intiapeiitoneal auministiation of cyclophosphamiue (4u mg¡kg¡uay)
foi the five uays befoie the fiist bifiuobacteiial auministiation, anu foi six uays
aftei the auministiation. Inoculum anu placebo (lyophilizeu skim milk with
suciose |S%, wt¡volj) weie auministeieu by oial gavage foi 6 uays at a volume of
4uu µl. Noitality anu moibiuity weie noteu twice a uay thioughout the stuuy.
Bouy weight was iecoiueu uaily. 0n uay 7 of the stuuy, the coiiesponuing mice
weie killeu, accoiuing to humane enupoints by ceivical uislocation. Immeuiately
aftei the mice weie killeu, the caicasses weie uisinfecteu by immeision in viikon S
uisinfectant anu uiieu with a gauze swab. The caiuiac bloou was taken with a 2S-
gauge neeule coupleu to a 1-ml syiinge anu collecteu in EBTA-coateu tubes. 0igans
such as the livei, spleen, mesenteiic lymph noues, ileum, jejunum, cecum, anu
colon weie extiacteu anu weigheu. The jejunum, cecum, anu colon weie piocesseu
foi histopathological evaluation. Bloou, livei, spleen, anu mesenteiic lymph noues
weie homogenizeu in NRSC meuium with a tissue giinuei, anu homogenates weie
stoieu at -8u°C in 2S% (vol¡vol) glyceiol. Bacteiial counts weie obtaineu by plate
count in NRSC meuium. Bloou, livei, spleen anu mesenteiic lymph noues weie
homogenizeu in NRSC meuium with a Pottei-Elvehjem tissue giinuei anu
homogenates weie stoieu at -8uºC in 2S% (vol¡vol) glyceiol. To evaluate the
piesence of bifiuobacteiia in faeces by quantitative Real-time PCR, faeces weie
taken at uays u, 2, S, 4 anu S of the stuuy. Samples weie kept at -2u°C befoie
piocessing. Foi histopathological evaluation, on the uay of killing, the jejunum,
cecum, anu colon weie collecteu anu pieseiveu in neutial buffeieu foimaluehyue
solution (4%, wt¡wt; pB 7) foi subsequent analysis. 0igans collecteu fiom all
animals weie fixeu, uehyuiateu, anu embeuueu in paiaffin. Sections weie cut using
a miciotome, anu slices weie staineu with hematoxylin-eosin anu mounteu. In
oiuei to investigate the mice foi possible auveise effects, histopathological
! ! !,+*-$*'$#!"'+4$&!%".!/#%#'!01203!
!
!
1S
analyses incluueu semiquantitative paiameteis such as the ueteimination of the
height of epithelial cells, the uepth of ciypts, anu the thickness of mucosa.
Neithei moitality noi clinical signs weie obseiveu uuiing the stuuy. Regaiuing
bouy weight gain, immunosuppiesseu mice lost weight aftei the
immunosuppiessoi was auministeieu but iecoveieu it aftei ingestion of both
piobiotic anu vehicle. In all cases, theie weie no statistically significant uiffeiences
between gioups.
In the case of miciobiological counts, theie was not tianslocation of bifiuobacteiia
in both immunosupiesseu anu immunocompetent gioups in bloou, livei, spleen, oi
mesenteiic lymph noues, unuei the piesent stuuy conuitions. Taking into account
histopathology analysis, except in the case of iatio geiminal centies¡lymph
follicles which ienueieu highei values in ES1 gioups pointing to an inciease in B
lymphocyte activity, iesults obtaineu in this analysis showeu no biologically
significant inteigioup uiffeiences between immune-competent anu immune-
ueficient mice, with oi without bifiuobacteiial auministiation. Results obtaineu in
acute ingestion assays with stiain !2. /%01,- ES1 uiun't show toxic effects uue to
miciobial stiain ingestion in mice.
In conclusion, taking these iesults as a whole, stiain !2. /%01,- ES1 can be
consiueieu safe foi human consumption.


W3!"4(%'=!4"""%"G%&'!$&!:8G4&!4)86'"!

A uouble-blinueu, ianuomizeu, placebo-contiol clinical tiial with cioss-ovei
uesigneu was caiiieu out in 12 auults (mean age: Su.S yeais; iange: 2S-4u yeais)
to evaluate the suivival of the stiain ES1 aftei the gastiointestinal tiansit anu its
safety intake in humans. The stuuy incluueu a iunning peiiou of 2 weeks in which
the paiticipants uiu not consume any piobiotic oi piebiotic piouuct, an
inteivention peiiou of S weeks in which the paiticipants consumeu a uaily uose of
! ! !,+*-$*'$#!"'+4$&!%".!/#%#'!01203!
!
!
14
eithei the placebo (skimmeu milk) oi the piobiotic (skimmeu milk containing the
piobiotic at 1u
9
cfu) followeu by a washing peiiou of 2 weeks anu a last
inteivention peiiou in which subjects switch to eithei the placebo oi the piobiotic
milk intake foi othei S weeks. Stool samples weie collecteu befoie anu aftei each
inteivention peiiou with placebo oi piobiotic. The composition of the miciobiota
was analyseu by Real-time PCR (Collauo *). '/., 2uu9). Stool samples weie also
analyseu by plate count in NRS agai to uetect the piesence of the ingesteu
bacteiial stiain. Isolateu colonies (8-1u) fiom each stool sample weie checkeu by
RAPB PCR analysis to confiim whethei the BNA piofile of the isolates
coiiesponueu with the BNA piofile of a puie cultuie of the auministeieu stiain !2.
/%01,- ES1. The ianuom piimei 12S4 (S'-CCu CAu CCA A-S') was useu foi -PCR
piouucts weie visualiseu on a 1.S% w¡v agaiose gel aftei staining with ethiuium
biomiue.
Bietaiy iecoius weie iecoiueu to excluue vaiiability uue to changes in uiet as
pieviously uesciibeu (Be Palma *).'/., 2uu9). Biiefly, foou uiaiy iecoius weie kept
foi 72h (2 weekuays anu 1 weekenu uay) both befoie the stait of the inteivention
anu aftei 1 month to monitoi uietaiy changes. At the fiont of the uiaiy, uetaileu
infoimation on how to iecoiu foou anu beveiages consumeu using common
householu measuies was pioviueu. When completing the foou uiaiy iecoius,
subjects weie instiucteu to iecoiu eveiything they ate oi uiank. Foou uiaiy
iecoius weie ietuineu to the uietician as soon as possible aftei completion when
they weie ievieweu, anu analyzeu foi eneigy, watei anu macionutiient contents
baseu on the CESNIB foou-composition uatabase of Spanish foous (Faiian *). '/.,
2uu4). Subjects also iecoiueu uaily the possible auveise effects uue to the
ingestion of the piobiotic ielateu to gastiointestinal symptoms (uiaiihoea,
constipation, vomiting, bloating, abuominal paint) oi otheis uuiing the stuuy
peiiou. Infoimeu consent was obtaineu fiom the subjects, anu the stuuy was
appioveu by the local Ethics Committee.
A total of 9 subjects finalizeu the stuuy anu the cause of uiop out was lack of
compliance (when the piouuct was not ingesteu foi moie than 2 consecutive uays
oi moie than S inuepenuent uays). Accoiuing to the quantitative analyses of
! ! !,+*-$*'$#!"'+4$&!%".!/#%#'!01203!
!
!
1S
specific bacteiial gioups (!'()*+%"$*43. !"#"$%&'()*+",-3. 6lostridium. coccoides.
6/%4)+"$",-. /*7),-3. 80)*+%&'()*+"'(*'*. anu. >'()%&'("//,4) by Real time-PCR, the
auministiation of the bifiuobacteiial stiain uiu not significantly mouify the
composition of the miciobiota of healthy auults. However, RAPB analyses of
colonies isolateu fiom selective meuia foi bifiuobacteiia piesent in stool samples
inuicateu that the stiain auministeieu iepiesenteu between 7u-8u% of the total
bifiuobacteiia, uemonstiating its ability to suivive the gastiointestinal intestinal
tiansit. Significant uiffeiences in uietaiy iecoius weie not uetecteu. Auveise
effects weie not iepoiteu by any of the paiticipants.


R3!#6$&$#46!'+$46!

Actually, a uouble-blinueu, ianuomizeu, placebo-contiol clinical tiial is iunning.
The stuuy incluueu a total of 4u patients (iange 2-14 yeais), subuiviueu into two
gioups. 0ne is ieceiving the ES1 piobiotic stiain uuiing S months in a uaily single
uose (1u
9
-1u
1u
ufc) anu the othei is ieceiving a placebo. To evaluate the efficacy of
the piobiotic the following vaiiables aie unuei stuuy: composition of the intestinal
miciobiota, seiological maikeis of uisease (tiansglutaminase antibouies anu total
IgA), immune paiameteis (lymphocyte anu cytokines) anu biochemical
paiameteis . The biochemical piofile incluues ienal paiameteis (uiea, cieatinine
anu uiic aciu), livei function maikeis (u0T, uPT, uuT, anu total biliiubin FAL), anu
paiameteis ielateu to nutiitional status (albumin, piealbumin, tiansfeiiin
satuiation inuex, feiiitin, folic aciu, cholesteiol, tiiglyceiiues). Niciobiological
analysis is peifoimeu by RT-PCR of the main miciobial gioups (!'()*+%"$*4,
!"#"$%&'()*+",-, 6/%4)+"$",-, Enteiobacteiiaceae anu >'()%&'("//,4). The immune
status of patients is evaluateu by analyzing peiipheial bloou lymphocyte
populations (lymphocytes T, Th, Tc, B anu iegulatois) anu pio-anu anti-
inflammatoiy cytoquines (IFN-y, TNF-u, IL-1u, IL- 12, anu TuF-µ) by flow
cytometiy anu ELISA. In auuition, anthiopometiy (weight, waist ciicumfeience, fat
! ! !,+*-$*'$#!"'+4$&!%".!/#%#'!01203!
!
!
16
mass anu lean mass), geneial biochemical piofile anu potential auveise effects
(uiaiihea, constipation oi othei gastiointestinal uistuibances) aie unuei stuuy.


.F3!6%546!,+*'%#'$*&!"'4'8"!

All the iesults with the piobiotic stiain ES1 has been piotecteu by the patent
PCT¡ES2uu8¡u7u24S entitleu "Niciooiganismos paia mejoiai el estauo ue saluu
ue inuiviuuos con uesoiuenes ielacionauos con la ingesta ue gluten". This patent
has been extenueu to Austialia, Biazil, Canaua, China, Euiopean 0nion, }apan,
Nexico, South Koiea anu 0niteu States of Ameiica (Table S). The applicant of this
patent is the National Spanish Reseaich Council. As it has been abovementioneu,
this patent has been exclusively licenseu to Biopolis SL.

#*8&'+=! ,4'%&'!#*)%!
Austialia A02uu8S417u8
Biazil PIu819SSS-1
Canaua CA271u666
China CN1u198S2S7
Euiopean 0nion EP22S6S98
}apan }P2u11Su7S4u
Nexico NX2u1uuu7uS8
South Koiea KR2u1uu11uS41
Spain ES2S4S499
0niteu States of Ameiica 0S2u1uS1uS2u




! ! !,+*-$*'$#!"'+4$&!%".!/#%#'!01203!
!
!
17
..3!+%(%+%&#%"!
!
Akopyanz N, Bukanov N0, Westblom T0, Kiesovich S, Beig BE (1992). BNA
uiveisity among clinical isolates of ?*/"(%&'()*+. 7</oii uetecteu by PCR-baseu
RAPB fingeipiinting. Nucleic Acius Reseseaich 2u: S1S7-S142.
Collauo, NC, Bonat E, Ribes-Koninckx C, Calabuig N, Sanz Y. (2uu8).!Imbalances in
faecal anu uuouenal !"#"$%&'()*+",- species composition in active anu non-
active coeliac uisease. BNC Niciobiology 22: 2S2.
Collauo, N.C, Bonat E, Ribes-Koninckx C, Calabuig N, Sanz Y. (2uu9). Specific
uuouenal anu fecal bacteiia aie associateu with peuiatiic celiac uisease. }ouinal
of Clinical Pathology 62: 264-269.
Be Palma u, Naual I, Collauo NC, Sanz Y. (2uu9). Effects of a gluten-fiee uiet on gut
miciobiota anu immune function in healthy auult human subjects. Biitish
}ouinal of Nutiition 1u2: 11S4-116u.
Eeiola S, Binkkanen R, Linufois E, Biivi T. (199S). Liquiu chiomatogiaphic
ueteimination of biogenic amines in uiy sausages. }ouinal of the Association of
Analytical Communities Inteinational 76: S7S-S77.
FA0¡WB0. (2uu2). Biafting guiuelines foi the evaluation of piobiotics in foou.
Repoit of a joint FA0¡WB0 woiking gioup. FA0¡WB0, Lonuon, 0ntaiio,
Canaua.
Faiian A, Zamoia R, Ceiveia P. (2uu4) Tablas ue composicion ue alimentos
CESNIB. 2
nu
eu. Ncuiaw-Bill¡Inteiameiicana. Baicelona.
Faviei CF, vaughan EE, ue vos WN, Akkeimans ABL. (2uu2). Noleculai monitoiing
of succession of bacteiial communities in human neonates. Applieu
Enviionmental Niciobiology 68: 219-222,
Kaufmann P, Pfeffeikoin A, Teubei N, Neile L. (1997). Iuentification anu
quantification of !"#"$%&'()*+",- species isolateu fiom foou with genus-specific
! ! !,+*-$*'$#!"'+4$&!%".!/#%#'!01203!
!
!
18
16S iRNA-taigeteu piobes by colony hybiiuization anu PCR. Applieu
Enviionmental Niciobiology 6S: 1268-127S.
Kumai RS, Biannigan }A, Piabhune AA, Punule Av, Bouson uu, Bouson E}, Suiesh
Cu. (2uu6). Stiuctuial anu functional analysis of a conjugateu bile salt hyuiolase
fiom !"#"$%&'()*+",-. /%01,- ieveals an evolutionaiy ielationship with
penicillin v acylase. }ouinal of Biological Chemistiy 281: S2S16-S2S2S.
Izquieiuo E, Neuina N, Ennahai S, Naichioni E, Sanz Y. (2uu8). Resistance to
simulateu gastiointestinal conuitions anu auhesion to mucus as piobiotic
ciiteiia foi !"#"$%&'()*+",-./%01,- stiains. Cuiient Niciobiology S6: 61S-618.
}ohnson }L. (1994). Similaiity analysis of iRNAs. In "Nethous foi geneial anu
moleculai bacteiiology" (ueihaiu P, Nuiiay R.u.E., Woou WA, Kiieg NR, eus).
Ameiican Society foi Niciobiology, Washington BC. pp 68S-7uu.
Lapaiia }N, Sanz Y. (2u1u). Bifiuobacteiia inhibit the inflammatoiy iesponse
inuuceu by gliauins in intestinal epithelial cells via mouifications of toxic
peptiue geneiation uuiing uigestion. }ouinal of Cell Biochemistiy 1u9: 8u1-8u7.
Lapaiia }N, 0livaies N, uallina 0, Sanz Y. (2u12). !"#"$%&'()*+",-. /%01,-
CECT7S47 mouulates immune iesponses in a gliauin-inuuceu enteiopathy
animal mouel. PLoS 0NE 7: eSu744.
Neuina N, ue Palma u, Ribes-Koninckx C, Calabuig N, Sanz Y. (2uu8).
!"#"$%&'()*+",- stiains suppiess in vitio the pio-inflammatoiy milieu tiiggeieu
by the laige intestinal miciobiota of coeliac patients. }ouinal of Inflammation S:
S-19.
Naual I, Bonat E, Ribes-Koninckx C, Calabuig N, Sanz Y. (2uu7). Imbalance in the
composition of the uuouenal miciobiota of chiluien with coeliac uisease. }ouinal
of Neuical Niciobiology S6: 1669-1674.
0livaies N, Lapaiia N, Sanz Y. (2u11). Influence of !"#"$%&'()*+",-. /%01,- CECT
7S47 anu gliauin peptiues on intestinal epithelial cell pioteome. }ouinal of
Agiicultuial anu Foou Chemistiy S9: 7666-7671.
! ! !,+*-$*'$#!"'+4$&!%".!/#%#'!01203!
!
!
19
Sanz Y, Sánchez E, Naizotto N, Calabuig N, Toiiiani S, Bellaglio F. (2uu7).
Biffeiences in faecal bacteiial communities in coeliac anu healthy chiluien as
uetecteu by PCR anu uenatuiing giauient gel electiophoiesis. FENS
Immunology anu Neuical Niciobiology S1: S62-S68.
Satokaii RN, vaughan EE, Akkeimans ABL, Saaiela N, ue vos WN (2uu1).
Bifiuobacteiial uiveisity in human feces uetecteu by genus-specific PCR anu
uenatuiating giauient gel electiophoiesis. Applieu Enviionmental Niciobiology
67: Su4-S1S.





!
!
!
!
!
!
!
!
!
!
!

Sign up to vote on this title
UsefulNot useful