mic oo ganisms
A icle
S ess E ec o Food Ma ices on Viabili y o P obio ic Cells
du ing Model Diges ion
Pe a Ma ousko a 1, Julie Hoo a 1, Pe Rysa ka 2and I ana Ma o a 1,*


Ci a ion: Ma ousko a, P.; Hoo a, J.;
Rysa ka, P.; Ma o a, I. S ess E ec o
Food Ma ices on Viabili y o
P obio ic Cells du ing Model
Diges ion. Mic oo ganisms 2021,9,
1625. h ps://doi.o g/10.3390/
mic oo ganisms9081625
Academic Edi o : Alex Galanis
Recei ed: 2 July 2021
Accep ed: 27 July 2021
Published: 30 July 2021
Publishe ’s No e: MDPI s ays neu al
wi h ega d o ju isdic ional claims in
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ia ions.
Copy igh : © 2021 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
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A ibu ion (CC BY) license (h ps://
c ea i ecommons.o g/licenses/by/
4.0/).
1Facul y o Chemis y, B no Uni e si y o Technology, Pu kyno a 118, 61200 B no, Czech Republic;
[email p o ec ed] (P.M.); [email p o ec ed] (J.H.)
2
Pha maceu ical Bio echnology, L d., Slezska 949/32, 12000 P ague, Czech Republic; [email p o ec ed]
*Co espondence: ma [email p o ec ed]; Tel.: +420-541149419
Abs ac :
The aim o his s udy was o e alua e he in luence o model (alcohol, suga , sal , p o ein
and acid) and eal oods and be e ages on he iabili y o p obio ics du ing incuba ion and a i icial
diges ion. Viabili y o monocul u es Lac obacillus acidophilus CCM4833 and Bi idobac e ium b e e
CCM7825T, and a comme cial mix u e o 9 p obio ic bac e ial s ains, was es ed by cul i a ion assay
and low cy ome y. In model oods, he bes iabili y was de e mined in he p esence o 0.2 g/L
glucose, 10% albumin and 10% e hanol. As he mos sui able eal ood o p obio ic su i al, complex
p o ein and ca bohyd a e subs a es we e ound, such as bee b o h, po a o salad wi h po k, chicken
wi h ice, chocola e sp ead, po idge and yoghu . The bes liquid was milk and mea b o h, ollowed
by Coca-Cola, bee and co ee. Viabili y o p obio ics was highe when consumed wi h meals han
wi h be e ages only. Addi ion o p ebio ics inc eased he iabili y o p obio ics, especially in p esence
o ins an and as oods. Gene ally, he highes iabili y o p obio ics du ing a i icial diges ion
was obse ed in mixed cul u e in he p esence o p o ein, suga and a , o hei combina ion. The
inc ease o cell iabili y obse ed in such oods du ing model diges ion may u he con ibu e o he
posi i e e ec o p obio ics on human heal h.
Keywo ds: p obio ics; ood ma ices; ood s ess; cell iabili y; model diges ion
1. In oduc ion
P obio ics a e li ing mic oo ganisms ha , when adminis e ed in su icien quan i y
(a ound 10
6
–10
7
CFU/mL o g o ca ie ood p oduc ), p o ide a heal h bene i o he
hos , in pa icula h ough a eplacemen o inclusion p ocess bene icial bac e ia in he
gas oin es inal ac [1,2].
In many s udies, he e ec s o p obio ics and p ebio ics on a ious diseases ha e
been obse ed. Some s udies con i med ha die a y ibe and p obio ics ha e posi i e
e ec s on in ec ious diseases [
3
]. P obio ics ha e also shown posi i e esponses o clinical
ea men agains se e al diseases and diso de s, such as cons ipa ion and dia hea, ood
alle gies, in lamma o y bowel disease, p e en ion and ea men o diabe es, obesi y and
cance and diseases ela ed o pa hogenic mic obes [
4
–
6
]. The an imic obial ac i i y o
p obio ics occu s h ough (i) educed pH due o p oduc ion o ace ic and lac ic acids,
(ii) bac e iocins’ accumula ion and (iii) compounds blocking bac e ial adhesion o he ep-
i helial cells and consequen ly educing pa hogen oxins’ p oduc ion [
7
]. P obio ic bac e ia
a e also c ucial o he ma u a ion o immune cells. This in es inal mic obio a s imula es
he ma u a ion and unc ionali y o he immune cells h ough hei me aboli es [
5
]. P o-
bio ics a e in ol ed in he egula ion o in es inal heal h, imp o ed lac ose diges ion and
main aining bone heal h, and hey make unc ional componen s such as an ioxidan s and
an i-hype ensi es [
7
]. Fu he , ecen e idence and ongoing s udies sugges ha in es inal
mic obio a has a bidi ec ional e ec on mood diso de s and can hus a ec s ess and
anxie y [
4
,
8
]. Resea ch on human diseases is e ealing he i al oles played by he gu
mic obio a. Unde s anding he impac o he gu mic obio a on he hos heal h is essen ial
Mic oo ganisms 2021,9, 1625. h ps://doi.o g/10.3390/mic oo ganisms9081625 h ps://www.mdpi.com/jou nal/mic oo ganisms
Mic oo ganisms 2021,9, 1625 2 o 17
o design s a egies ocused on p obio ics’ manipula ion. The e o e, o he knowledge on
op ions which a e esponsible o inc easing he iabili y o p obio ics will allow us o
design new s a egies o imp o e he heal h o he consume [9].
The e a e se e al mechanisms by which p obio ics may bene i humans, including
p oduc ion o an imic obial subs ances, s eng hening o in es inal ba ie , modula ion
o immune esponse and an agonism o pa hogenic mic oo ganisms, ei he by p oduc-
ion o an imic obial agen s o by compe i ion o binding si es, nu ien s and g ow h
ac o s
[4,10,11]
. Mo e p ecisely, he in e ac ion be ween p obio ics and pa hogens can be
di ided in o h ee s eps: he physical in e ac ion be ween he p obio ic and he epi he-
lium, he in e ac ion be ween p obio ics and he immune sys em and, inally, he di ec
in e ac ion be ween p obio ics and pa hogens [
12
]. The in e ac ion be ween p obio ics
and pa hogens may be obse ed in he hos s, bu also in oods, whe e i can ha e a pos-
i i e e ec oo, and inco po a ing p obio ics in ood ma ices can be a new op ion o
ood sa e y [
11
,
12
]. Mo eo e , p obio ic bac e ia inco po a ed in o oods should be able
o su i e gas ic ansi and each he small in es ine in su icien numbe s o iable
cells [2,11].
In ood-deli e ed p obio ics, iabili y o p obio ics is essen ial o achie e he heal h
bene i s associa ed wi h hei consump ion. Excep o echnological s ess du ing ood
p ocessing ollowed by s ess connec ed wi h s abiliza ion, packaging and s o age, p o-
bio ics should unde go addi ional s ess du ing consump ion. The passage h ough he
unique en i onmen o he gas oin es inal ac is a sou ce o high gas ic acidi y, oxygen
s ess induced by ROS eleased om mucosal su aces, bile sal s ess, osmo ic s ess and
many o he e ec s [
2
]. Thus, ood subs a es used o ca y p obio ics can be conside ed as
one o he majo ac o s in egula ing coloniza ion o p obio ics in he gas oin es inal ac .
Food helps o bu e he bac e ia h ough he s omach and may con ain o he unc ional
ing edien s, o example p ebio ics, ha can in luence g ow h, iabili y and su i al, acid
and bile ole ance, adhesion o p obio ics o in es inal cells [
11
] and di e en unc ionali y
o p obio ics ha de e mine hei e icacy in he gas oin es inal ac [13].
A way o inc easing he e icacy o p obio ic p epa a ions may be he combina ion o
bo h p obio ics and p ebio ics as synbio ics, which p o ide an imp o ed su i al du ing he
passage o he p obio ic bac e ia h ough he in es inal ac [
13
]. De elopmen o unc ional
oods may also modula e he gu mic obio a and con ey heal h e ec s. Majo challenges in
his a ea can be he inco po a ion o p obio ics in oods, selec ion o he p ebio ic candida e
o selec ion o bac e ial s ains and encapsula ion o p obio ic bac e ia [
8
]. Howe e , he
addi ion o p ebio ics o p oduc s may nega i ely in luence he p oduc [
14
]. P obio ics
may also change he senso y quali y o he p oduc because some s ains may g ow in he
ood ma ix and p oduce me aboli es, which in e ac wi h he ood. The encapsula ion o
he p obio ic may also a ec he ood ex u e [
15
]. On he o he hand, die a y supplemen s
in he o m o capsules, able s and o he o ma s may be used. Ne e heless, a cu en
s udy on p obio ics does no p esen a de ini i e answe as o whe he he e is supe io i y
o equi alence on deli e y o p obio ics in oods o in he o m o supplemen s [10].
Howe e , p obio ics used as supplemen s may educe he unc ional e icacy o p obi-
o ics due o exclusion o he po en ial syne gis ic e ec o he ood, i hey a e no se ed
oge he wi h app op ia e ood [
13
]. Many s udies ha e ocused on es ing he e ec o
he ood ma ix on p obio ics du ing s o age. Rega ding he inco po a ion o p obio ics
in o ood p oduc s, he ood ma ix should mee hese equi emen s: low aw, neu al o
sligh ly acidic pH, p esence o ibe o p ebio ic compounds and a high bu e ing capaci y
( o example a high a con en ). Fo inco po a ion o p obio ic s ains in o he p oduc ,
he s ain selec ion is also impo an [
16
]. Howe e , he e ec o ood on p obio ics du ing
diges ion should be es ed as well, because he abili y o su i e in he gas oin es inal
en i onmen is ecognized as a undamen al equisi e o p obio ics [17,18].
The main aim o his wo k was o s udy he in luence o di e en ypes o eal ood
and be e ages on he iabili y and g ow h o p obio ic bac e ia du ing simula ed passing
h ough he gas oin es inal ac . The main goal was o mimic he mos common condi ions
Mic oo ganisms 2021,9, 1625 3 o 17
and en i onmen s used du ing p obio ics’ consump ion in a b oad popula ion, such as he
e ec o di e se eal oods, including some homemade p oduc s, ins an oods, snacks, as
ood p oduc s and some be e ages, o ind p ac ical ecommenda ions o a die a y egime
o op imum p obio ics’ in ake.
2. Ma e ials and Me hods
2.1. Ma e ial
2.1.1. P obio ic S ains
Bac e ial s ains Lac obacillus acidophilus CCM 4833 and Bi idobac e ium b e e CCM 7825T
used in his s udy we e pu chased om he Czech Collec ion o Mic oo ganisms in B no,
Czech Republic.
The comme cial p epa a ion Biop on 9 (Walma k, L d., T inec, Czech Republic) was
selec ed as an op imum sou ce o mixed p obio ic s ains. This ood supplemen con ained a
mix u e o 9 bac e ial cul u es o Bi idobac e ium bi idum,Bi idobac e ium b e e,Bi idobac e ium
longum,Lac obacillus acidophilus,Lac obacillus casei,Lac obacillus plan a um,Lac obacillus
hamnosus,Lac obacillus lac is and S ep ococcus he mophilus, 9
×
10
9
CFU (colony- o ming
uni s) in daily dose; addi ionally, he capsule con ains 240 mg o uc o-oligosaccha ides.
2.1.2. Food Ma ices
All used samples o eal oods a e summa ized in Table 1. The se o eal samples
should ep esen ypical oods and be e ages consumed daily by mos o he popula ion.
Be e ages, ins an oods (ins an soups, pas a wi h c eam sauce, po idge) and snacks
(yoghu , pudding, chocola e sp ead, pas y, poppy seed cake, chips, po a o salad) we e
ob ained om local e ail. Ins an oods we e mixed wi h ho wa e o milk acco ding o he
p oduce s’ ecommenda ions. Homemade p oduc s (mixed ege able b o h, chicken b o h,
bee b o h, boiled chicken mea wi h ice, po a o salad wi h ied po k) we e p epa ed
immedia ely be o e expe imen s om na u al ma e ials (300 g) boiled in wa e (1 L) wi h
3 g o sal . Hambu ge was ecei ed om as ood e ail. F ui s and ege ables we e used
as mix u es o oma o, cucumbe and peppe , wi h he a io o 1:1:1.
Table 1. O e iew o es ed eal oods and be e ages.
Be e ages Soups Main Cou ses Snacks F ui , Vege ables
ap wa e bee b o h ins an Hambu ge — as ood yoghu chocola e
black ea pea ins an soup wi h
c ou ons
pas a wi h c eam
sauce—ins an
pudding wi h whipped
c eam apple wi h banana
black co ee ins an soup wi h li e
dumplings
chicken wi h ice—boiled
chocola e sp ead oma o, cucumbe
and whi e peppe
bee (4.9%
alcohol)
ege able
b o h—homemade
po a o salad wi h ied
po k po idge
juice o ange
chicken b o h—homemade
pas y
milk (3.5% a ) bee b o h—homemade poppy seed cake
Coca-Cola Chips—sal ed
2.2. Me hods
2.2.1. Cul i a ion o P obio ic Bac e ia
The s ains we e inocula ed in o MRS b o h (HiMedia, Mumbai, India) and incuba ed
o 48 h a 37
◦
C. The cells we e ha es ed by cen i uga ion a 6000 pm o 4
◦
C o 10 min
(He mle Z36 HK, He mle, Ge many), and washed wi h dis illed wa e . A e wa ds, cells
we e used o inocula ion, and a e incuba ion and cul i a ion in di e en en i onmen s,
o iabili y analysis.
Be o e use, he p obio ic s ains om he comme cial Biop on capsule we e hyd a ed
by s e ile dis illed wa e o 20 min and hen used o inocula ion and cul i a ion.
Mic oo ganisms 2021,9, 1625 4 o 17
2.2.2. Model Food Ma ices
As model ood ma ices, solu ions wi h a ious concen a ions o alcohol (e hanol
5–40%), suga (0.2–20 g/L o glucose), sal (0.4–10 g/L o NaCl) and p o ein (bo ine se um
albumin 5–20 g/L) we e p epa ed in s e ile dis illed wa e . As a model o acidic ood,
s e ile dis illed wa e adjus ed o pH = 3 by HCl was used. S anda d and pu e chemicals
we e pu chased om Sigma Ald ich (Sigma-Ald ich, Me ck KGaA, Da ms ad , Ge many).
All used model oods a e summa ized in Table 2.
Table 2. O e iew o es ed model oods.
1 2 3 4
S e ile dis illed wa e pH 7 pH 3 (wi h HCl)
P o ein (Bo ine Albumin) 5 g/L 10 g/L 20 g/L
Saccha ide (Glucose) 0.2 g/L 2 g/L 10 g/L
Sal (NaCl) 0.4 g/L 1.2 g/L 4 g/L 10 g/L
Alcohol (E hanol) 5% 10% 20% 40%
2.3. Model Diges ion
A i icial s omach juice was p epa ed om 0.25 g o pepsin dissol ed in 100 mL o
dis illed wa e . To his solu ion, 0.84 mL o 35% hyd ochlo ic acid was added. Final pH
was adjus ed o 0.9. A i icial panc ea ic luid was p epa ed wi h 0.25 g o panc ea in and
1.5 g o sodium hyd ogen ca bona e in 100 mL o dis illed wa e (pH = 8.9). Bile luid was
composed o 0.4 g o bile acid sal s dissol ed in 100 mL o phospha e bu e . P obio ics we e
added o 300 g o be e age, liquid ood o solid ood homogenized in an app op ia e wa e
olume. A mix u e o p obio ic cells (1.10
10
CFU) and ood/be e age was i s incuba ed
a 37
◦
C o 15 min o each s a ing alues o cell numbe s. Then, he mix u e o p obio ics
and ood ma ix was mixed wi h s omach juice (in he a io o 3:1) and incuba ed o 20
min a 37
◦
C. Addi ionally, in es inal luids o med by a mix u e o panc ea ic luid and bile
sal s in he a io o 1:1 ( / ) we e added o a inal a io o ood and all diges ion juices o 3:2,
simila ly o GIT condi ions. Model diges ion was se as a sho ened con inuous p ocess.
2.4. De e mina ion o Cell Viabili y by Cul i a ion Me hod and Flow Cy ome y
The iabili y o p obio ic bac e ia was es ed by cul i a ion assay on Pe i dishes wi h
MRS medium by he o e low me hod. A e 48 h o cul i a ion, colony- o ming uni s,
e.g., bac e ial colonies (CFU/mL), we e coun ed. The cul i a ion me hod was used o
e alua ion o all ypes o oods and be e ages, including model oods.
The iabili y o p obio ic bac e ia exposed o model and eal liquid oods and be e -
ages was also ollowed by di ec analysis o cell iabili y using low cy ome y (Apogee
Flow Sys ems, Hemel Hemps ead, UK). As a luo escen p obe, p opidium iodide (Sigma-
Ald ich, Da ms ad , Ge many) was used. Fo analysis, 100
µ
L o liquid sample was aken,
dilu ed o 1 mL and added o p opidium iodide. A e 5 min o incuba ion in da kness,
iabili y o p obio ic cells was measu ed in a clea liquid model and eal ood ma ixes in
each s age o diges ion.
2.5. Viabili y o Monocul u es and Mixed P obio ic Cul u es in Model and Real Foods and
Be e ages in Condi ions o Model Diges ion
P obio ic bac e ial cul u es o Lac obacillus acidophilus,Bi idobac e ium b e e, o a com-
me cial mix u e o p obio ic cells (1 capsule o Biop on 9; abou 9.10
9
CFU) we e added
o he 300 g o model o eal ood/be e ages in he amoun o 1.10
10
CFU. Each o he
es ed p obio ics samples we e i s mixed wi h ood/be e age and incuba ed o 15 min
a 37
◦
C. Then, he samples we e exposed o model diges ion acco ding o Sec ion 2.3. In
egula in e als (a e o be o e incuba ion), he amoun o li ing cells was measu ed
using cul i a ion me hods and low cy ome y. The e alua ion o iabili y, CFU numbe
and, hus, he in luence o indi idual model and eal oods is complica ed by he con-
inuous g ow h o p obio ic cells in any en i onmen . Thus, he numbe o iable cells
Mic oo ganisms 2021,9, 1625 5 o 17
was measu ed g adually a he beginning o each s age o con inuously pe o med model
diges ion and a e i s comple ion. The i s e alua ion was pe o med a e he incuba ion
o p obio ics wi h ood ma ix as he esponse o dilu ion and ood s ess en i onmen . This
measu emen was ollowed by e alua ion a e acidi ica ion a he beginning o s omach
diges ion, hen a e 20 min incuba ion in s omach juice (pH 2.1), ollowed by cell coun ing
a e alkaliza ion and mixing wi h he mix u e o panc ea ic and bile juices and a he end
o he diges ion p ocess (pH 7.9, 2 h). Acidi y o he en i onmen (pH) was checked du ing
he whole diges ion p ocess.
2.6. Viabili y o Mix u e o P obio ic Cells in Di e en Types o Foods and Be e ages wi h/wi hou
Addi ion o P ebio ics
P obio ics om a comme cial capsule we e e-hyd a ed o 20 min and solubilized in
s e ile dis illed wa e . Then, cells we e mixed wi h eal ood ma ix and wi h o wi hou
he addi ion o p ebio ics (300 mg o inulin, Sigma-Ald ich, Me ck KGaA, Da ms ad ,
Ge many). The ecommended daily dose o p obio ics mix u e (1 capsule, 1.10
10
CFU) was
suspended in 300 g o homogenized ood o be e age. This p epa ed mix u e was hen
incuba ed a 37
◦
C o 15 min. Then, samples we e aken o analysis o he numbe o
iable cells a he beginning o he diges ion p ocess (1 mL). Due o he he e ogenic cha -
ac e o samples, iabili y o p obio ic cells was de e mined using cul i a ion echniques.
De e mining he numbe o cells in he samples was pe o med a e 48 h o cul i a ion in
MRS aga .
2.7. Viabili y o Monocul u es and Mixed P obio ic Cul u es in Real Foods and Be e ages in
Condi ions o Model Diges ion
P obio ic bac e ial cul u es o Lac obacillus acidophilus,Bi idobac e ium b e e, o a com-
me cial mix u e o p obio ic cells (con en o capsule Biop on 9) we e added o he 300 g o
model o eal ood/be e ages in he amoun o 1.10
10
CFU. A he beginning o incuba ion,
model ood ma ixes can be conside ed as s e ile (see Sec ion 2.2.2). Real oods we e
p epa ed immedia ely be o e incuba ion and, hus, he p esence o o he mic oo ganisms
was minimized. Each o he es ed p obio ics samples we e i s mixed wi h ood/be e age
and incuba ed o 15 min. Then, he samples we e exposed o model diges ion acco ding
o Sec ion 2.3. In egula in e als (a e o du ing incuba ion), he amoun o li ing cells
was measu ed. The iabili y o p obio ics du ing simula ed gas oin es inal condi ions
we e pe o med using cul i a ion me hods and low cy ome y (liquid samples). Cul i-
a ion me hods we e used o all he e ogenic samples, and o homogenous solu ions, i
was possible o de e mine hem di ec ly by FC. The e alua ion o iabili y and CFU in
indi idual model and eal oods a e complica ed by con inuous g ow h o p obio ic cells in
any en i onmen . Thus, he iabili y a e diges ion (o indi idual s ages) was compa ed
o he alue o CFU a he beginning o model diges ion, a e sho - ime incuba ion wi h
he ood.
3. Resul s
3.1. Viabili y o P obio ics in Model Food Ma ices
The aim o he p esen s udy was o e alua e he iabili y o es ed p obio ic cul u es
in he p esence o selec ed model and eal oods and be e ages in model condi ions o he
diges i e ac . As a model ood, solu ions wi h a ious concen a ions o alcohol, suga ,
sal , p o ein and ace ic acid we e p epa ed. Real oods we e selec ed acco ding o he mos
common die a y p e e ences in he popula ion.
In all model oods excep acidic ood, he pH alue was se o pH 7, and he model acid
ood is o pH 3. In Table 3, pH alues in es ed eal be e ages and oods a e p esen ed. Wi h
he excep ion o juice (pH 3) and bee (pH 5.1), all es ed be e ages and oods exhibi ed
pH in he ange o 6.0–7.3. Thus, we canno expec some d ama ic in luence o pH o es ed
ood ma ixes on iabili y o p obio ic cells. Howe e , a mo e acidic pH (app oxima ely
5.5–6.5) will p obably be mo e sui able o p obio ic bac e ia g ow h.

Mic oo ganisms 2021,9, 1625 6 o 17
Table 3. pH alues in eal be e ages and oods.
Be e age/Liquid pH Food pH
Wa e 7.0 Pas a wi h c eam
sauce—ins an 7.1
Black ea 6.9 Chicken wi h ice—boiled 7.2
Black co ee 6.0 Po a o salad wi h ied po k 6.7
Bee (4.9% alcohol) 5.1 Hambu ge — as ood 7.1
Juice, o ange 3.2 Yoghu —chocola e 6.2
Milk (3.5% a ) 7.1 Pudding wi h whipped c eam 7.3
Coca-Cola 5.6 Chocola e sp ead 6.3
Bee b o h 6.2 Po idge 5.2
Vege able b o h 6.0 Pas y 7.0
Chicken b o h 6.3 Poppy seed cake 6.2
Bee b o h ins an 6.2 Chips—sal ed 7.1
Pea ins an soup wi h c ou ons
6.4 F ui s (apple + banana) 6.7
Ins an soup wi h li e
dumplings 6.5 Vege able mix 5.8
3.1.1. G ow h o Mixed P obio ic Cul u e in Model Foods Exposed o A i icial Diges ion
The in luence o basic model oods on concen a ion and iabili y o p obio ic cells
du ing a i icial diges ion was s udied (Figu e 1A–F). As he p obio ic cul u e, a mixed
comme cial p epa a i e was used. The measu emen was pe o med using low cy ome y,
and as model oods wa e (wa e -based ood), ace ic acid solu ion o pH 3 (acid ood),
glucose (swee ood), p o ein (p o ein-based ood), sodium chlo ide (sal ed ood) and
e hanol (alcohol-based ood) a di e en concen a ions (see Table 2) we e used. The
e alua ion o iabili y and CFU in indi idual model and eal oods is complica ed by
he con inuous g ow h o p obio ic cells in any en i onmen , hus, in some samples, an
inc ease o CFU a e model diges ion was obse ed. I is necessa y o no e ha he numbe
o CFU de e mined by low cy ome y is ela ed o 1
µ
L o sample because o he ex eme
sensi i i y o his me hod when compa ed wi h cul i a ion echniques.
Fi s , model diges ion using he p obio ic mix u e wi hou addi ion o ood ma ix
was measu ed. P obio ic capsule con en was e-hyd a ed in wa e and placed di ec ly in o
he s omach and hen o in es ine luids. This expe imen simula ed a model swallowing an
in ake o d y p obio ic capsule wi hou he addi ion o any ood o be e age. A signi ican
dec ease o he numbe o li ing cells was obse ed a e model diges ion wi hou he
p esence o some ood ma ix. A e d as ic pH changes, a d ama ic dec ease (app oxi-
ma ely 17.5
×
) o he numbe o o iginally p esen cells was eco ded a he beginning o
he in es inal s age o model diges ion (Figu e 1F). On he o he hand, a e model diges ion
o p obio ics in he p esence o wa e , an app oxima ely 12 imes inc ease o he o iginal
numbe o cells was eco ded. I is a model o swallowing he in ake o a p obio ic capsule
oge he wi h 300 mL o wa e . Subsequen ly, he e ec o p o ein (Figu e 1B) on cell
g ow h du ing diges ion was es ed. Two hou s a e he addi ion o in es inal juices, a
signi ican inc ease o cell concen a ion in sample wi h he addi ion o 10 g/L o bo ine
albumin was obse ed. In his sample, abou 3 imes mo e han he o iginal numbe o
cells was de e mined.
The e ec o sal en i onmen on cell g ow h du ing diges ion is in oduced in
Figu e 1C
. A e a mode a e inc ease o CFU in he en i onmen o in es inal luids,
wo hou s a e addi ion, a sligh dec ease o cell concen a ion was obse ed (excep
he lowes concen a ion used). Mo eo e , wi h inc easing sal concen a ion, he mo e
in ensi e dec ease o cell concen a ion was obse ed. Fo 0.4 g/L NaCl concen a ion,
he inal CFU alue a e wo hou s o model diges ion was abou 2 imes highe han he
o iginal CFU alue ound a he beginning o he exposi ion o a i icial diges ion.
Mic oo ganisms 2021,9, 1625 7 o 17
Mic oo ganisms 2020, 8, x FOR PEER REVIEW 7 o 17
(A) (B)
(C) (D)
(E) (F)
Figu e 1. Viabili y o mixed cul u e o lac ic acid bac e ia du ing he p ocess o diges ion o model oods con aining p o-
bio ics (comme cial p obio ic mix u e): (A) suga concen a ion, (B) p o ein concen a ion, (C) sal concen a ion, (D) al-
cohol concen a ion, (E) compa ison o model ac o s, pH 3 was se by ace ic acid, and (F) incuba ion o cell suspension
di ec ly in diges i e luid.
The e ec o sal en i onmen on cell g ow h du ing diges ion is in oduced in Figu e
1C. A e a mode a e inc ease o CFU in he en i onmen o in es inal luids, wo hou s
a e addi ion, a sligh dec ease o cell concen a ion was obse ed (excep he lowes con-
0.00E+00
5.00E+05
1.00E+06
1.50E+06
2.00E+06
2.50E+06
3.00E+06
CFU/uL
0,0002 g/ml glucose
0,002 g/ml glucose
0,01 g/ml glucose
0.00E+00
1.00E+06
2.00E+06
3.00E+06
4.00E+06
5.00E+06
6.00E+06
7.00E+06
8.00E+06
9.00E+06
1.00E+07
CFU/uL
0,005 g/ml p o ein
0,01 g/ml p o ein
0,02 g/ml p o ein
0.00E+00
2.00E+05
4.00E+05
6.00E+05
8.00E+05
1.00E+06
1.20E+06
1.40E+06
CFU/uL
0,0004 g/ml NaCl
0,0012 g/ml NaCl
0,004 g/ml NaCl
0,01 g/ml NaCl
0.00E+00
1.00E+06
2.00E+06
3.00E+06
4.00E+06
5.00E+06
6.00E+06
7.00E+06
8.00E+06
CFU/uL
5% e hanol
10% e hanol
20% e hanol
40% e hanol
0.00E+00
2.00E+06
4.00E+06
6.00E+06
8.00E+06
1.00E+07
1.20E+07
wa e pH3 0,002 g/ml
glucose
0,01 g/ml
p o ein
0,0012 g/ml
NaCl
20% e hanol
CFU/uL
15 min
incuba ion
s omach
beginning
0.00E+00
5.00E+03
1.00E+04
1.50E+04
2.00E+04
2.50E+04
3.00E+04
3.50E+04
4.00E+04
4.50E+04
5.00E+04
CFU/uL
Figu e 1.
Viabili y o mixed cul u e o lac ic acid bac e ia du ing he p ocess o diges ion o model oods con aining
p obio ics (comme cial p obio ic mix u e): (
A
) suga concen a ion, (
B
) p o ein concen a ion, (
C
) sal concen a ion, (
D
)
alcohol concen a ion, (
E
) compa ison o model ac o s, pH 3 was se by ace ic acid, and (
F
) incuba ion o cell suspension
di ec ly in diges i e luid.
Nex , he e ec o alcohol (Figu e 1E) on cell g ow h du ing diges ion was moni o ed.
A e he addi ion o in es inal juices, a mode a e inc ease o CFU was de ec ed, bu a e
Mic oo ganisms 2021,9, 1625 8 o 17
a longe diges ion, a dec ease o cell concen a ion was obse ed again. The dec ease in
cell numbe wi h inc eased alcohol concen a ion was obse ed as well. Ne e heless, a
low e hanol concen a ion (5–10%) exhibi ed a ela i ely good e ec on CFU (abou 3–4x
inc ease a e comple e model diges ion).
Fu he , he e ec o model ood wi h glucose on cell g ow h du ing diges ion was
moni o ed (Figu e 1A). The highes inc ease o cell numbe was eco ded in he sample
wi h 2 g/L o glucose. A e wo hou s o model diges ion, abou 5 imes highe CFU in
his sample was de e mined. Finally, he numbe o cells in acidic model ood was es ed
(Figu e 1E). A he beginning o he in es inal s age, a dec ease o he numbe o cells in
acidic ood was obse ed. Howe e , a e wo hou s o exposi ion o he in es inal juice,
he CFU numbe o acidic ood was al eady 2 imes highe .
Gene ally, acco ding o o al CFU numbe s in model oods, he mos sui able en i-
onmen s o p obio ic p epa a i e in ake we e ecognized as: (i) wa e , (ii) p o ein a he
concen a ion o 10 g/L, (iii) glucose a 2 g/L and low e hanol concen a ion (5–10%). The
mos nega i e e ec was exhibi ed p edominan ly by sal , which was p obably caused by
osmo ic s ess, simila o he e ec o highe glucose concen a ions. A nega i e e ec o
e hanol as a bac e ios a ic agen was obse ed a e long- e m exposi ion o highe concen-
a ions (20% and mo e), while lowe concen a ions o e hanol exhibi ed posi i e e ec s
on p obio ic iabili y. The eason could be a c oss- esponse o he ex e nal s ess e ec o
e hanol and p obably also an abili y o some p obio ics o use e hanol as an addi ional
ca bon sou ce.
3.1.2. Flow Cy ome y De e mina ion o Viabili y o P obio ics Exposed o Model
Diges ion in Liquid En i onmen s
In he ollowing sec ion, he in luence o model oods on he CFU numbe and ia-
bili y o p obio ic monocul u es o Lac obacillus acidophilus and Bi idobac e ium b e e a e
model diges ion was s udied (Figu e 2). Fo compa ison, a mixed p obio ic cul u e was
used. The scheme o he expe imen is desc ibed in Sec ion 2.5. A e a i icial diges ion
in s omach juice ollowed by incuba ion in in es inal juices, he CFU numbe was di ec ly
de e mined by low cy ome y. A he end o incuba ion, he cul u e o Lac obacillus aci-
dophilus exhibi ed a high inc ease o CFU p edominan ly in model ood con aining p o ein,
bu also sligh ly in o he model oods (Figu e 2A). Con e sely, he s ain Bi idobac e ium
b e e g ew in ensi ely, p edominan ly in model oods con aining saccha ide (Figu e 2B). In
he comme cial p obio ic mix u e, he highes inc ease o CFU numbe was ound a e he
model diges ion in he p esence o wa e a pH = 7 and in he p esence o acidic solu ion
(pH = 3). Fu he mo e, he CFU inc ease was de ec ed in oods con aining p o ein and 10%
alcohol as well (Figu e 2C).
Flow cy ome y was also used o de e mina ion o cell iabili y in he en i onmen
o clea (cen i uga ion 5000 pm, 10 min) mea and ege able b o h p epa ed acco ding o
Sec ion 2.1.2.
Resul s a e illus a ed in Figu e 3. When compa ed wi h model oods, monocul u es
as well as mixed p obio ics cul u e exhibi ed simila ends, co esponding o some o he
ypical model oods, such as p o ein (mea b o h) o suga ( ege able b o h). The bes
en i onmen o all es ed samples was chicken b o h, con aining high concen a ions o
p o eins. Excep o in his en i onmen , monocul u es g ew qui e slowly (app oxima ely
4–5x lowe biomass o ma ion) when compa ed wi h he mixed cul u e. G ow h o mixed
cul u e in mea ex ac was lowe han in he p esence o 10 g/L o p o ein.
The esis ance o he p obio ic mix u e agains acidic pH men ioned abo e is p obably
caused by he addi ion o some p o ec i e agen s o he p epa a i e, which could p e en
damage o cells by he acidic s omach en i onmen , as desc ibed by he p oduce . A simila
s udy desc ibed a neu al o sligh ly acidic pH (5–6) as a sui able ood en i onmen o
p o ec ion o lac ic acid bac e ia [
19
]. This pH was op imal o g ow h o p obio ics du ing
ood manu ac u ing and cell su i al du ing s o age and diges ion. Wa e ac i i y can
also imp o e cell p o ec ion du ing diges ion, as well as solids and gels. The p esence o
highly e men able suga s and ibe also p omo ed su i al o cells du ing ood s o age
Mic oo ganisms 2021,9, 1625 9 o 17
and diges ion. High a con en exhibi ed posi i e e ec s on cells, mainly due o good
bu e ing capaci y [19].
Mic oo ganisms 2020, 8, x FOR PEER REVIEW 9 o 17
(A) (B) (C)
Figu e 2. The in luence o model oods and he ype o p obio ic on he numbe o iable cells a e model
diges ion: (A) Lac obacillus acidophilus, (B) Bi idobac e ium b e e and (C) p obio ic mix u e.
Figu e 3. The in luence o he ype o ood ex ac (b o h) and he ype o p obio ic on he
numbe o iable cells a e model diges ion.
The esis ance o he p obio ic mix u e agains acidic pH men ioned abo e is p oba-
bly caused by he addi ion o some p o ec i e agen s o he p epa a i e, which could p e-
en damage o cells by he acidic s omach en i onmen , as desc ibed by he p oduce . A
simila s udy desc ibed a neu al o sligh ly acidic pH (5–6) as a sui able ood en i onmen
o p o ec ion o lac ic acid bac e ia [19]. This pH was op imal o g ow h o p obio ics
du ing ood manu ac u ing and cell su i al du ing s o age and diges ion. Wa e ac i i y
can also imp o e cell p o ec ion du ing diges ion, as well as solids and gels. The p esence
o highly e men able suga s and ibe also p omo ed su i al o cells du ing ood s o age
and diges ion. High a con en exhibi ed posi i e e ec s on cells, mainly due o good
bu e ing capaci y [19].
Flow cy ome y is a highly sensi i e me hod o cell iabili y de e mina ion, especially
when compa ed o he cul i a ion echniques (Supplemen a y Figu e S1). Abou a 1000
imes lowe amoun o sample was su icien o di ec e alua ion o cell iabili y (see
Supplemen a y Figu e S2). We can suppose ha clea be e ages (wa e , ea, mea b o h)
0.00E+00
1.00E+05
2.00E+05
3.00E+05
4.00E+05
5.00E+05
6.00E+05
7.00E+05
8.00E+05
9.00E+05
1.00E+06
CFU/uL
LA be o e
LA a e diges ion
0.00E+00
2.00E+05
4.00E+05
6.00E+05
8.00E+05
1.00E+06
1.20E+06
1.40E+06
BB be o e
BB a e diges ion
1.00E+04
5.01E+06
1.00E+07
1.50E+07
2.00E+07
2.50E+07
MIX be o e
MIX a e diges ion
0
500,000
1,000,000
1,500,000
2,000,000
2,500,000
ege able chicken bee
CFU/uL
ype o b o h
Lac obacillus acidophilus Bi idobac e ium bi idum MIX
Figu e 2.
The in luence o model oods and he ype o p obio ic on he numbe o iable cells a e model diges ion: (
A
)
Lac obacillus acidophilus, (B)Bi idobac e ium b e e and (C) p obio ic mix u e.
Mic oo ganisms 2020, 8, x FOR PEER REVIEW 9 o 17
(A) (B) (C)
Figu e 2. The in luence o model oods and he ype o p obio ic on he numbe o iable cells a e model
diges ion: (A) Lac obacillus acidophilus, (B) Bi idobac e ium b e e and (C) p obio ic mix u e.
Figu e 3. The in luence o he ype o ood ex ac (b o h) and he ype o p obio ic on he
numbe o iable cells a e model diges ion.
The esis ance o he p obio ic mix u e agains acidic pH men ioned abo e is p oba-
bly caused by he addi ion o some p o ec i e agen s o he p epa a i e, which could p e-
en damage o cells by he acidic s omach en i onmen , as desc ibed by he p oduce . A
simila s udy desc ibed a neu al o sligh ly acidic pH (5–6) as a sui able ood en i onmen
o p o ec ion o lac ic acid bac e ia [19]. This pH was op imal o g ow h o p obio ics
du ing ood manu ac u ing and cell su i al du ing s o age and diges ion. Wa e ac i i y
can also imp o e cell p o ec ion du ing diges ion, as well as solids and gels. The p esence
o highly e men able suga s and ibe also p omo ed su i al o cells du ing ood s o age
and diges ion. High a con en exhibi ed posi i e e ec s on cells, mainly due o good
bu e ing capaci y [19].
Flow cy ome y is a highly sensi i e me hod o cell iabili y de e mina ion, especially
when compa ed o he cul i a ion echniques (Supplemen a y Figu e S1). Abou a 1000
imes lowe amoun o sample was su icien o di ec e alua ion o cell iabili y (see
Supplemen a y Figu e S2). We can suppose ha clea be e ages (wa e , ea, mea b o h)
0.00E+00
1.00E+05
2.00E+05
3.00E+05
4.00E+05
5.00E+05
6.00E+05
7.00E+05
8.00E+05
9.00E+05
1.00E+06
CFU/uL
LA be o e
LA a e diges ion
0.00E+00
2.00E+05
4.00E+05
6.00E+05
8.00E+05
1.00E+06
1.20E+06
1.40E+06
BB be o e
BB a e diges ion
1.00E+04
5.01E+06
1.00E+07
1.50E+07
2.00E+07
2.50E+07
MIX be o e
MIX a e diges ion
0
500,000
1,000,000
1,500,000
2,000,000
2,500,000
ege able chicken bee
CFU/uL
ype o b o h
Lac obacillus acidophilus Bi idobac e ium bi idum MIX
Figu e 3.
The in luence o he ype o ood ex ac (b o h) and he ype o p obio ic on he numbe o iable cells a e model
diges ion.
Flow cy ome y is a highly sensi i e me hod o cell iabili y de e mina ion, espe-
cially when compa ed o he cul i a ion echniques (Supplemen a y Figu e S1). Abou a
1000 imes lowe amoun o sample was su icien o di ec e alua ion o cell iabili y (see
Supplemen a y Figu e S2). We can suppose ha clea be e ages (wa e , ea, mea b o h)
and model solu ions a e sui able o low cy ome y measu emen s o cell iabili y, in con-
as o colloid ma e ials (e.g., milk), ood homogena es and all en i onmen s con aining
pa icles. Thus, he compa ison o all es ed eal be e ages was pe o med by cul i a ion
echniques o elimina e he in e e ence o impu i ies.
Mic oo ganisms 2021,9, 1625 16 o 17
Rega ding eal oods and be e ages, he bes ood en i onmen s o inc easing p o-
bio ic iabili y we e complex meals wi h mea and ibe , milk p oduc s—po idge and
yoghu , chocola e sp ead and mixed ui s. Among be e ages, he bes op ion was milk,
bu Coca-Cola, co ee, bee and black ea we e also accep able o he iabili y o p obio ics.
We can conclude ha p obio ics a e mo e iable when consumed wi h meals compa ed
o he be e ages only. The iabili y o he mixed p obio ic cul u e was highe in all en-
i onmen s when compa ed wi h bo h monocul u es. The addi ion o p ebio ics u he
posi i ely inc eased he iabili y o p obio ics, e en in he p esence o p ocessed oods. In
gene al, he highes iabili y o p obio ics du ing a i icial diges ion was obse ed in he
mixed p obio ics cul u e in he p esence o p o ein, suga and a , o hei combina ion.
The inc ease o cell iabili y obse ed in such oods du ing model diges ion may u he
con ibu e o he posi i e e ec s o p obio ics on human heal h.
Supplemen a y Ma e ials:
The ollowing a e a ailable online a h ps://www.mdpi.com/a icle/10
.3390/mic oo ganisms9081625/s1, Figu e S1: Bac e ia Lac obacilus acidophilus,Bi idobac e ium b e e
and comme cial p obio ic mix u e cul i a ed o 48 h in MRS aga , Figu e S2: Viabili y o p obio ics
analyzed by low cy ome y— esul s o analysis o comme cial p obio ic mix u e incuba ed in
wa e medium.
Au ho Con ibu ions:
Concep ualiza ion, P.M., I.M. and P.R.; me hodology, P.M., I.M. and J.H.;
alida ion, P.M., J.H.; in es iga ion, P.M., I.M. and P.R.; w i ing—o iginal d a p epa a ion, P.M.
and I.M.; w i ing— e iew and edi ing, I.M.; isualiza ion, P.M.; supe ision, P.R. and I.M.; unding
acquisi ion, P.M. All au ho s ha e ead and ag eed o he published e sion o he manusc ip .
Funding:
This esea ch was unded by he p ojec N . FCH-S-21-7483 o he Facul y o Chemis y,
B no Uni e si y o Technology, B no, Czech Republic.
Ins i u ional Re iew Boa d S a emen : No applicable.
In o med Consen S a emen : No applicable.
Da a A ailabili y S a emen : The s udy did no epo any da a.
Acknowledgmen s: The au ho s would like o hank Kla a Vajglo a and Ma ina Poko na.
Con lic s o In e es : The au ho s decla e no con lic o in e es .
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