Stress Effect of Food Matrices on Viability of Probiotic Cells during Model Digestion

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 published maps and ins i u ional a il- ia ions. Copy igh : © 2021 by he au ho s. Licensee MDPI, Basel, Swi ze land. This a icle is an open access a icle dis ibu ed unde he e ms and condi ions o he C ea i e Commons A ibu ion (CC BY) license (h ps:// c ea i ecommons.o g/licenses/by/ 4.0/). 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. 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