Almond milk fermented with different potentially probiotic bacteria improves iron uptake by intestinal epithelial (Caco-2) cells

ISSN: 2182-1054 DOI : 10.7455/ij s/4.1.2015.a4 Copy igh No ice Au ho s who publish in he In e na ional Jou nal o Food S udies ag ee o he ollowing e ms:  Au ho s e ain copy igh and g an he jou nal igh o i s publica ion wi h he wo k simul aneously licensed unde a C ea i e Commons A ibu ion License ha allows o he s o sha e he wo k wi h an acknowledgemen o he wo k's au ho ship and ini ial publica ion in his jou nal.  Au ho s a e able o en e in o sepa a e, addi ional con ac ual a angemen s o he non-exclusi e dis ibu ion o he jou nal's published e sion o he wo k (e.g., pos i o an ins i u ional eposi o y o publish i in a book), wi h an acknowledgemen o i s ini ial publica ion in his jou nal.  Au ho s a e pe mi ed and encou aged o pos hei wo k online (e.g., in ins i u ional eposi o ies o on hei websi e) p io o and du ing he submission p ocess, as i can lead o p oduc i e exchanges, as well as ea lie and g ea e ci a ion o published wo k. Almond milk e men ed wi h di e en po en ially p obio ic bac e ia imp o es i on up ake by in es inal epi helial (Caco-2) cells In e na ional Jou nal o Food S udies OFFICIAL JOURNAL OF THE ISEKI_FOOD ASSOCIATION In e na ional Jou nal o Food S udies IJFS Ap il 2015 Volume 4 pages 49–60 Almond milk e men ed wi h di e en po en ially p obio ic bac e ia imp o es i on up ake by in es inal epi helial (Caco-2) cells Neus Be na a*, Mai e Ch´ a e a, Ampa o Chi al a, Jos´ e Mois´ es Lapa ab, and Chelo Gonz´ alez-Ma ´ ıneza aIns i u o de Ingenie ´ıa de Alimen os pa a el Desa ollo. Uni e si a Poli `ecnica de Val`encia, Camino de Ve a s/n, 46022 Valencia, Spain bMic obial Ecophysiology and Nu i ion Labo a o y. Ins i u o de Ag oqu´ımica y Tecnolog´ıa de Alimen os (CSIC). A . Agus ´ın Esca dino, 7. 46980 Pa e na, Valencia, Spain *Co esponding au ho neube p[email p o ec ed] .es Tel: +34 963 877 056 Fax: +34 963 877 956 Recei ed: 6 Ma s 2014; Published online: 18 Ap il 2015 Abs ac New e men ed almond milks we e de eloped, using di e en po en ially p obio ic bac e ia, in o de o mee he cu en demand o heal hy, e sa ile non-dai y p oduc s. An in i o diges ion/Caco- 2 cell model was used o e alua e he e ec o bo h non- e men ed and e men ed almond milks on he mi ochond ial enzyma ic ac i i ies o en e ocy es. Mo eo e , mac ophages we e challenged wi h he in- i o diges ed samples and he p oduc ion o p o-in lamma o y bioma ke s TNF-αand IL-6 was quan i ied. Enzyma ic ac i i ies o cell cul u es seemed o be s imula ed by he exposu e o bo h e men ed and non- e men ed almond milks. Bo h bioma ke s dec eased (p<0.05) in e men ed almond milks wi h ei he B. bi idum o B. longum. Resul s showed ha e men ed almond p oduc s a o ed he ene ge ic me abolism o en e ocy es and had a lowe in lamma o y esponse han non- e men ed almond milk, sugges ing i s bene i s o he managemen o alle gies/in ole ances. Mo eo e , he e men a ion p ocess enhanced he up ake o i on by Caco-2 cells, especially when using L. hamnosus and ei he B. bi idum o B. longum as s a e s, hus imp o ing he p oduc bioac i i y. The e o e, new non- dai y e men ed p oduc s wi h unc ional p ope ies we e de eloped, which migh be posi ioned as al e na i es o cow-milk p oduc s o sensi ized g oups o popula ion (alle gic and/o in ole an o cow milk o anemic popula ion, among o he s). Keywo ds: Almond milk; Fe men a ion; P obio ics; I on a ailabili y; In lamma ion 1 In oduc ion The cu en ala ming inc ease in he incidence o alle gic diseases in bo h child en and adul s in de eloped coun ies has been a ibu ed o he so-called “hygiene hypo hesis”; his heo y sugges s ha he inc eased le el o hygiene in bo h he en i onmen and he ood supply leads o a educed exposu e o a a ie y o mic obes, which, ea ly in li e, is a c ucial ac o in he de elopmen o alle gies (Bj¨o ks ´en, 2009). In- deed, se e al p ospec i e ollow-up s udies ha e ound ha al e a ions in gu mic obio a p ecede alle gy de elopmen (Kalliom¨aki, 2010). Hence, ecen clinical alle gy esea ches ha e p incipally ocused hei a en ion on he manipula ion o Copy igh © 2015 ISEKI-Food Associa ion (IFA) 10.7455/ij s/4.1.2015.a4 50 Be na e al. gu mic obio a composi ion (Kalliom¨aki e al., 2010). Nowadays, p obio ics, p ebio ics and synbio ics (combina ion o p e- and p obio ics) a e conside ed as good ools wi h which o elici changes in he gu biomass composi ion, since hey can imp o e and s imula e bene icial gu mic o lo a, among o he e ec s ha a e bene icial o he heal h. Since he la e 1990s, o e 30 andomized clinical ials ha e been published, in which p obio ics ha e been used ei he in he ea men o p e en ion o alle gies (mainly a opic diseases) (Kalliom¨aki e al., 2010). Hence, conside ing he high p e alence o a opic disease in childhood in he indus ialized coun ies (Anandan, Nu ma o , an Schayck, & Sheikh, 2010), he use o yoghu - ype oods as ca ie s o p obio ics and/o synbio ics would be help ul as a means o a aining he ei he p e en i e o p ophylac ic ea men in his a ge ed popula ion. Mo eo e , despi e he li le known and un us wo hy concep o unc ional ood, consume s a e amilia wi h yoghu - ype p oduc s and conside hem as heal hy (Annunzia a & Vecchio, 2011), which would acili a e he inclusion o such unc ional e men ed p oduc s in hei die s. The immunomodula o y e ec s o e men ed dai y-milks, casein hyd olysa es and soy- be e age caused by lac ic acid bac e ia ha e been widely epo ed (Ba oja, Ki ja ainen, Hekma , & Reid, 2007; Su as e al., 1996; Waga , Champagne, Buckley, Raymond, & G een-Johnson, 2009). Howe e , in addi ion o he alle genic p o eins, bo h ma ices migh p o oke i on de iciencies in in an s and oddle s. On he one hand, he calcium oge he wi h he casein p o ided by cow-milk a e seen o inhibi he abso p ion o die a y non-heme i on, in addi ion o he in es inal blood loss obse ed in app oxima ely 40% o in an s du ing eeding wi h cow-milk and/o i s de i a i es (Agos oni & Tu ck, 2011). On he o he hand, soya-based p oduc s con ain phy a es, which nega i ely in e e e in he abso p ion o i on, among o he mine als (A azcoz, 2007). By con as , almond milk has no been epo ed o in e e e nega i ely in i on abso p ion. In- deed, almonds ha e high an i-oxidan ac i i y owing o he α- ocophe ol and polyphenolic cons i uen s (Chen, Lapsley, & Blumbe g, 2006), which migh imp o e he bioa ailabili y o die a y i on. As i has been e iewed, al hough non-heme i on is easily oxidized (no bioa ailable) due o he ise o pH in he lumen, he p esence o educ an s om ood can main ain his mic onu ien in i s educed o m and, he e o e, posi i ely imp o e i s abso p ion (Mi e , Simpson, & McKie, 2003). Mo eo e , almond milk is conside ed an app op ia e al e - na i e o cow-milk, since, besides he heal hy lipid p o ile, i has a low a io o Na/K and a balanced a io o Ca/P (Luengo, 2009). Ne e heless, wi h ega ds o he in lamma o y esponse, he e is lack o da a conce ning nu be e ages, and leas when i comes o de i a i e e men ed p oduc s. Only ecen ly has da a appea ed on almonds (Raja am, Connell, & Saba e, 2010), in which he au ho s s udied whe he , in addi ion o he lowe ing o blood lipids, monounsa u a ed a - ich almonds in lu- enced o he co ona y hea disease isk ac o s, such as in lamma ion. The s udy concluded ha inco po a ing abou 68 g almonds in a 2000 kcal choles e ol-lowe ing die dec eased se um E-selec in (molecules which a e indica i e o endo helial dys unc ion) and C- eac i e p o ein (sensi i e ma ke o in lamma ion ha , in high concen a ions, is s ongly linked wi h co ona y e en s, s oke and pe iphe al ascula disease) in heal hy men and women. Al hough a ound 50% o almond composi ion is a , in akes o 7g pe day o his nu ha e been shown o educe low-densi y lipop o ein choles- e ol concen a ion by 1% (Saba ´e, Haddad, Tanzman, Jambazian, & Raja am, 2003) and up o 84 g pe day can be consumed wi hou weigh gain (Chen e al., 2006). In addi ion, hese nu s ha e a low glycemic index ( hey do no ad e sely impac insulin sensi i i y) (Chen e al., 2006) and ha e been ound o possess p ebio ic e ec s, since hey s imula ed he g ow h o gu bi i- dobac e ia and Eubac e ium ec ale (Mandala i, Nueno-Palop, Bisignano, Wickham, & Na bad, 2008). Hence, aking in o accoun he heal h bene i s o almond in ake, almond milk migh be conside ed as a good ood ma ix wi h which o ob ain heal hy e men ed p oduc s. Mo eo e , i he e men a ion p ocess is ca ied ou by po en ially p obio ic bac e ia, he de eloped e men ed p oduc could be use ul as a means IJFS Ap il 2015 Volume 4 pages 49–60 Fe men ed almond milks imp o e bioa ailabili y o i on 51 o p e en ing some immunomodula o y diseases, such as alle gies. The Caco-2 cell line, commonly used in con- junc ion wi h in i o diges ion echniques, is a use ul model o s udying in es inal human i on up ake, which i allows o occu simul a- neously wi h ood diges ion, and is gene ally ega ded as he bes a ailable in es inal cell model o s udying he mechanisms associa ed wi h ec o ial i on anspo (Glahn, Lee, Yeung, Goldman, & Mille , 1998). In addi ion, he mac ophage-de i ed RAW 264.7 cell line exp esses key genes and p o eins o p incipal pa hways o he p oduc ion o egula o y cy okines (No ak, Babcock, Jho, Hel on, & Espa , 2003) and cons i u es a cell model used wo ldwide and a use ul ool wi h which o s udy he in lamma o y esponse(s) and me abolic ac i i y p omo ed by ood-de i ed componen s while s ill main aining a apid and inexpensi e sys em (Deepika, Ras all, & Cha alampopoulos, 2011; Kabee doss e al., 2011). The aim o his s udy, he e o e, was o e alua e whe he almond milk, e men ed wi h di e en po en ial p obio ic bac e ia, a ec s bo h he ene ge ic me abolism in in es inal cells and he p oduc ion o p o-in lamma o y bioma ke s in o de o gain insigh s in o he po en ial bene i s o he designed p oduc s o he consume ’s gu heal h. 2 Ma e ials and Me hods 2.1 P epa a ion and e men a ion o almond milk Almond milk was p oduced by soaking and g inding almonds (P unus amygdalus L. c . dul- cis) supplied by F u os Secos 3G S.L. (Valen- cia, Spain). The ex ac ion was ca ied ou in he Sojama ic ® 1.5 (Ba celona, Spain), equip- men speci ically designed o he p oduc ion o ege able milks, using a nu :wa e a io o 8:100. The milky liquid ob ained was hen mi- c o luidized in a high p essu e homogenize (M- 110P model; Mic o luidics In e na ional Co po- a ion, Wes wood, MA, USA) by applying 172 MPa, s e ilized (121 ◦C/15 min) and subse- quen ly cooled down o 37 ◦C ( e men a ion em- pe a u e). Lac obacillus hamnosus CECT 278, Lac obacil- lus plan a um 3O9, Bi idobac e ium bi idum CECT 870, Bi idobac e ium longum CECT 4551, S ep ococcus he mophilus CECT 986, Lac o- bacillus delb ueckii subs. Bulga icus CECT 4005 we e used as s a e s, pu e o mixed, as shown in Table 2. All he bac e ia we e pu chased om CECT (Pa e na, Spain), wi h he excep ion o he L. plan a um 3O9 s ain, which was isola ed om Gui a sheep milk and selec ed as a p obi- o ic in p e ious s udies (Amo ocho C uz, 2011). Fo he p epa a ion o s a e s inoculum, he s ains we e independen ly incuba ed o 24 h in hei selec i e b o hs and hen cen i uged a 100 g (Medig ige -BL-S, JP-Selec a; Ba celona, Spain) o 10 min a 4 ◦C o e-suspend he pel- le in PBS-1x bu e (10 mmol/L phospha e, 137 mmol/L NaCl, 2.7 mmol/L KCl, pH 7.4) un il eaching s ain concen a ions o 108c u/mL. Fo each o mula ion, 1% ( / ) o s a e sus- pension was added o he almond milk and sub- sequen ly incuba ed a 37 ◦C un il pH alues o 4.4-4.6 we e eached, which was con olled by us- ing a GLP 21+ pH-me e (C ison Ins umen s S.A.; Ba celona, Spain). The e men ed samples we e ozen and s o ed a -22 ◦C p io o anal- ysis. A non- e men ed almond sample was used as a con ol (C). 2.2 Simula ed gas oin es inal diges ion The human gas oin es inal diges ion p ocess was simula ed by using po cine pepsin (800- 2, 500 uni s/mg p o ein), panc ea in (ac i i y, 4USP speci ica ions) and bile, as p e iously de- sc ibed by Lapa a, Ba be a, Aleg ia, Glahn, and Mille (2009). All eagen s we e pu chased om Sigma-Ald ich Co. (S Louis, MO, USA). P io o he in i o diges ion, 1.5 mL aliquo o each assayed sample was dilu ed in 5mL o a saline solu ion (140 mmol/L NaCl, 5 mmol/L KCl ad- jus ed o pH 3). Fo gas ic diges ion (pepsin in 0.1 mol/L HCl adjus ed o pH 3; 1 h), samples we e placed on a ocking pla o m shake in an incuba o (37 ◦C; 5% CO2; 95% ela i e humid- i y). The in es inal diges ion (panc ea in-bile ex- ac in 0.1 mol/L NaHCO3adjus ed o pH 6.9-7; IJFS Ap il 2015 Volume 4 pages 49–60 52 Be na e al. 2 h) was ca ied ou in he uppe chambe o a wo-chambe sys em in 6-well pla es. The uppe chambe was o med by i ing he bo om o an app op ia ely sized T answell in- se ing (Co ning B.V. Li e Sciences, Ams e - dam, The Ne he lands) wi h a 15,000 molecu- la mass cu -o dialysis memb ane (Spec a/Po 2.1, Spec um Medical, Ga dena, CA, USA). Aliquo s (1.5 mL) o he gas oin es inal diges we e loaded in o he uppe chambe s and incu- ba ed o 2 h. A e wa ds, he inse s we e e- mo ed and he dialysa es we e dilu ed (1:4, / ) wi h cul u e media and incuba ed wi h in es inal epi helial (Caco-2) o mac ophage (RAW 264.7) cells. 2.3 Fe i in analysis in in es inal epi helial cell monolaye Caco-2 cells we e ob ained om he Ame ican Type Cul u e Collec ion (Rock ille, MD, USA) a passage 17 and used in expe imen s a pas- sages 33 o 38. Cells we e main ained wi h Dulbecco’s modi ied Eagle’s medium (DMEM) (Gibco ® , Mad id, Spain) unde condi ions p e- iously desc ibed by Glahn e al. (1998). Fo he assays, Caco-2 cells we e seeded a 50,000 cell/cm2in collagen- ea ed 6-well cul u e pla es (Cos a , Camb idge, MA, USA), and we e g own wi h DMEM o 12 days. On he day p io o he expe imen s, he DMEM medium was e- placed by 2 mL o minimum essen ial medium (MEM) (Gibco ® , Mad id, Spain) and hen he cells we e e u ned o he incuba o . 50 µmol/L o FeCl3we e added o he diges ed almond milk samples and he e i in o ma ion by Caco-2 cells o e a 24 h pe iod was p opo ional o he cell i on up ake. A la ex-enhanced u bidime - ic immunoassay (Fe i in- u bila ex; Spin eac , Gi ona, Spain) was used o measu e he Caco-2 cell e i in con en , as Glahn e al. (1998) de- sc ibed. The concen a ions o e i in we e no - malized by he de e mina ion o he o al p o ein con en in cell cul u es by using a mic oLow y ki (TP0200) (Sigma-Ald ich, S . Louis, MO, USA). The con ol cells (basal), exposed o in i o diges ions o con ol solu ions con aining diges i e enzymes bu no sample, we e moni- o ed h oughou . Base-line cell e i in in cul- u es g own in MEM a e aged 4.2 ng/mg cell p o ein. The samples we e analyzed in iplica e. 2.4 Mi ochond ia enzyme ac i i ies These ac i i ies we e e alua ed in Caco-2 cell cul u es by moni o ing MTT (3-(4,5- dime hyl hiazol-2-yl)-2,3-diphenyl e azolium b omide) con e sion on exposed cul u es a e an incuba ion pe iod o 3 h, ollowing he me hod desc ibed by Lapa a e al. (2009). This colo ime ic me hod is based on he educ ion o he e azolium ing o MTT by mi ochon- d ia dehyd ogenases yielding a blue o mazan p oduc , which can be measu ed spec opho- ome ically. Fo he assays, Caco-2 cells we e seeded a 50,000 cell/cm2in 24-well cul u e pla es (Cos a , Camb idge, MA, USA), and we e g own wi h DMEM o 12 days. Con ol cells (basal) exposed o diges s con aining enzymes bu no samples we e used h oughou each assay. Fou eplica es we e analyzed. 2.5 Analysis o p o-in lamma o y ma ke s The in lamma o y analyses we e ca ied ou ollowing he me hod desc ibed by No ak e al. (2003). Fo he assays, RAW 264.7 cells we e seeded a 50,000 cell/cm2and we e g own wi h Roswell Pa k Memo ial Ins i u e (RPMI) medium (Gibco ® , Mad id, Spain) o 24 hou s. Tumo Nec osis Fac o -α(TNF-α) and in e - leukin 6 (IL-6) (eBioscience L d., Ha ield, UK) we e de e mined by ELISA, ollowing he man- u ac u e ’s ins uc ions, on exposed RAW 264.7 cell cul u es a e an incuba ion pe iod o 3 h. Resul s we e exp essed as picog ams pe mL o media. Fou eplica es we e analyzed. 2.6 S a is ical analysis Each o he expe imen s was conduc ed on ou independen eplica es. A one-way analysis o a iance (ANOVA) and he Tukey pos hoc es we e applied. S a is ical signi icance was es ab- lished a a con idence le el o 95% o all he IJFS Ap il 2015 Volume 4 pages 49–60 Fe men ed almond milks imp o e bioa ailabili y o i on 53 compa isons. SPSS .15 so wa e (SPSS Inc., Chicago, IL, USA) was used o he s a is ical analysis. 3 Resul s and Discussion 3.1 Almond milk and hei e men ed-de i a i e p oduc s The use o high p essu e homogeniza ion (HPH) con ibu ed o a be e s abili y o he almond milk, since his eme gen mic o luidiza ion ech- nology is able o educe he size o a glob- ule pa icles, g ea ly delaying he loccula ion and coagula ion phenomena (Lujan Cap a e al., 2009; C uz e al., 2009). The chemical composi ion o he almond milk used o e men a ions, which is subsequen ly codi ied as con ol (C), is summa ized in Table 1. Taking in o accoun he nu :wa e a io used, hese alues a e simila o hose ound in he li - e a u e (Yada, Lapsley, & Huang, 2011). The ini ial pH o he almond milk (C) was 6.61 ±0.08. A signi ican decline in pH o 4.60 ±0.02 a e 20 h a 37 ◦C o incuba ion, ook place in he de eloped p oduc s along wi h a co espond- ing inc ease in acidi y caused by e men a ion. Table 1: Chemical composi ion o almond milks used in he s udy. Values (mean ±s anda d de- ia ion) a e exp essed as g ams pe 100 mL o be e age. Compound Concen a ion D y ma e 6.64 ±0.5 Lipids 3.96 ±0.2 P o eins 1.37 ±0.03 To al suga s 0.41 ±0.002 Ashes 0.325 ±0.012 Fibe 0.58* * Fibe concen a ion was ob ained by sub ac ing he d y ma e con en om he sum o he es o com- pounds shown. 3.2 Bac e ial e men a ion e ec s on TNFαand IL-6 p oduc ion Figu e 1shows he Tumo nec osis ac o (TNF)-αand in e leukin (IL)-6 p oduc ion in mac ophage (RAW 264.7 cells) cul u es exposed o diges s o e men ed milks. A non- e men ed almond milk was used as a con ol (C). Focusing on he cells ea ed wi h dialyzable ac- ion samples no e men ed by using bi idobac e- ia (F1 o F5) (Table 2), e men a ion wi h ei- he L. hamnosus (F2) o L. plan a um (F3) dec eased he TNF-αp oduc ion (p<0.05) by a simila amoun wi h espec o he con ol (C). When he s anda d yoghu bac e ia was used (F1), he TNF-αp oduc ion was simila o he con ol. Opposi e e ec s we e obse ed when he e men a ion was de eloped by combining hose lac obacilli wi h s anda d yoghu bac e ia (F4 and F5, espec i ely), since he TNF-αp oduc- ion inc eased (p<0.05) compa ed o he con- ol (Figu e 1). As ega ds he IL-6, and con- a y o ha obse ed in TNF-α, all samples had a posi i e e ec in he p oduc ion o his p o- in lamma o y ma ke wi h espec o he con ol, especially when he almond milk e men a ion was done by using mixed-cul u es (F4 and F5). These samples exhibi ed he lowes IL-6 concen- a ions (p<0.05), showing 55-70% o inhibi ion (p<0.05) o he ini ial IL-6 p oduc ion induced by non- e men ed almond milk (C) (Figu e 1). Wi h espec o he samples e men ed using he bi idobac e ia (F6 o F11), all he cells exposed o hose dialyzed samples exhibi ed a e y low TNF-αp oduc ion in compa ison o he con- ol (p<0.05). When conside ing IL-6 p oduc- ion, di e en pa e ns we e obse ed depending on he ype o bi idobac e ia p esen in s a e . As Figu e 1shows, he ac ions om e men ed samples wi h B. bi idum (F6, F8 and F10) in- duced cells o p oduce IL-6 amoun s simila o ha ob ained in e men ed samples wi h s an- da d yoghu bac e ia (F1). Howe e , wi h B. longum (F7, F9 and F11) lowe (p<0.05) IL-6 concen a ions we e quan i ied, especially when i was combined wi h ei he s anda d yoghu bac e ia (F7) o L. plan a um (F11). TNF-αis a p o-in lamma o y ac o p oduced by mac ophages ha exe s impo an e ec s on IJFS Ap il 2015 Volume 4 pages 49–60 54 Be na e al. Figu e 1: Tumo nec osis ac o (TNF)-αand in e leukin (IL)-6 p oduc ion in mac ophage (RAW 264.7 cells) cul u es exposed o diges s o e men ed milks. C: non- e men ed almond milk. Basal: cells no exposed o samples.*a-e Di e en supe sc ip le e s indica e signi ican di e ences be ween samples in TNF-α p oduc ion (p<0.05). * w-z Di e en supe sc ip le e s indica e signi ican di e ences be ween samples in IL-6 p oduc ion (p<0.05). Table 2: Mic obial s ains used o p oduce he di e en e men ed almond milks. Fo mula ion S a e s inoculum C - - - F1 - - S. he mophilus + L. delb ueckii F2 L. hamnosus - - F3 L. plan a um - - F4 L. hamnosus - S. he mophilus + L. delb ueckii F5 L. plan a um - S. he mophilus + L. delb ueckii F6 - B. bi idum S. he mophilus + L. delb ueckii F7 - B. longum S. he mophilus + L. delb ueckii F8 L. hamnosus B. bi idum - F9 L. hamnosus B. longum - F10 L. plan a um B. bi idum - F11 L. plan a um B. longum - L.: Lac obacillus B.: Bi idobac e ium S.: S ep ococcus IJFS Ap il 2015 Volume 4 pages 49–60 Fe men ed almond milks imp o e bioa ailabili y o i on 55 sys emic in lamma ion and induces he p oduc- ion o o he in lamma o y cy okines such as IL- 6 o IL-8 (Hu, Kobayashi, Zenda, & Shimamu a, 1997). The obse ed bac e ial e men a ion e - ec s could ha e impo an consequences on he in es inal ba ie unc ion because TNF-αplays a c ucial ole by inc easing pa acellula pe me- abili y and impai ing igh junc ion unc ionali y (Ma e al., 2004) and leukocy e in il a ion in he in es inal wall (Ho man, 2000). In addi ion, he educ ion in TNF-αp oduc ion migh also ha e impo an physiological consequences p e en ing alle gic in lamma o y p ocesses. Almonds a e known o ha e se e al nu i ional bene i s, including ha o lowe ing choles e ol and p o ec ion agains diabe es (Raja am e al., 2010; Saba ´e e al., 2003). Fu he mo e, sci- en i ic s udies ha e poin ed o hei po en ial p ebio ic and an i-in lamma o y ac i i ies (Ra- ja am e al., 2010; Mandala i e al., 2008). Al- mond lipids and ca bohyd a es a ailable o e - men a ion ha e been associa ed wi h inc easing bo h he numbe o bi idobac e ia s ains and he sho chain a y acids (SCFA) con en , es- pecially in bu y a e concen a ions (Mandala i e al., 2008). Bu y ic acid has he po en ial o bene i colonic heal h, since i educes hy- d ogen pe oxide in cells and main ains hei in- eg i y (Sco , Ma in, Duncan, & Flin , 2014). Al hough bi idobac e ia s ains a e no able o p oduce bu y a e, hey syn hesize o he SCFA such as ace a e which can be used by o he bac- e ia (i.e. lac obacilli) in hei me abolic ou e o syn hesize his cells’ p o ec i e acid (Falony, Vlachou, Ve b ugghe, & De Vuys , 2006). Mo e- o e , his bu y ic p oduc ion may be enhanced in he p esence o p ebio ics (i.e. almond ibe s) (Falony e al., 2006; Sco e al., 2014). These ac s a e cohe en and migh explain he ma ked posi i e e ec s obse ed in he in lamma o y e- sponses induced by dialysa es om samples in- ocula ed wi h bi idobac e ia. Ne e heless, he ac i e componen s abo e men ioned (SCFA and p ebio ics) ha e no been analysed since hey we e beyond he objec i e o his s udy. As he esul s sugges , he in ol emen o ei- he B. bi idum o B. longum in he de elop- men o e men ed almond-based p oduc s migh be o in e es in o de o educe in lamma o y in es inal p ocesses o a a ge ed popula ion. In- deed, p e ious in i o and in i o s udies ca ied ou by using hese bac e ia species also showed posi i e e ec s in immunomodula o y esponses (Lapa a, Oli a es, Gallina, & Sanz, 2012; La- pa a & Sanz, 2010). Ne e heless, despi e he esul s, he di e ences om di e en p obio ic s ains used a e ye o be in es iga ed in de ail. 3.3 Bac e ial e men a ion e ec s on ene ge ic me abolism o in es inal cells Resul s o he possible oxici y o e men ed sam- ples in in es inal epi helial (Caco-2) cells, which was quan i ied by moni o ing he mi ochond ial enzyme (MTT es ) ac i i ies, a e shown in Fig- u e 2. The in es inal epi helium cons i u es he i s physiological ba ie o exogenous com- pounds and nu ien abso p ion. Mi ochon- d ia and endo-lysosomal enzyme ac i i ies we e p o en o be sensi i e bioma ke s o changes in cellula me abolism in esponse o in e nalized ood-de i ed compounds (Lapa a e al., 2009; Wu e al., 2010). This MTT assay showed ha none o he dialysa es exposed o cell cul- u es seem o cause oxic e ec s, as concluded om he ac ha he MTT alues we e simila (p<0.05) o hose calcula ed o basal cul u e (cells no exposed o almond milk samples). As men ioned abo e, possible syne gism be ween al- mond cons i uen s (p ebio ics) and s a e bac e- ia migh ha e p oduced cell p o ec i e bioac i e compounds (i.e. bu y a e) (Falony e al., 2006; Sco e al., 2014) which p e en ed al e a ions o he Caco-2 my hocond ia and endo-lyososomal enzyma ic ac i i y. Pa icula ly, he MTT alues showed ha dialysa es om samples e men ed wi h L. plan- a um 3O9 (F3) (Table 2) s imula ed he en- e ge ic cell me abolism, simila o ha o non- e men ed almond milk (C) (p<0.05). How- e e , when hese bac e ia we e used in combina- ion wi h s anda d yoghu bac e ia, he esul ed dialysa es (F4 and F5) did no ha e his signi i- can s imula o y e ec . Bo h MTT esul s (Figu e 2) and he ones ob- ained in he p oduc ion o p o-in lamma o y cy- okines by immune cells (Figu e 1) indica ed ha almond e men ed p oduc s migh exe bene i- IJFS Ap il 2015 Volume 4 pages 49–60 56 Be na e al. Figu e 2: MTT con e sion pe cen ages in Caco-2 cell cul u es exposed o diges s o e men ed milks. (C: non- e men ed almond milk; Basal: cells no exposed o samples). *a,b Di e en supe sc ip le e s indica e signi ican di e ences(p<0.05) be ween samples. cial e ec s on human gu heal h, especially when using s anda d yoghu bac e ia wi h B. longum (F7) and L. hamnosus wi h B. longum (F9) as s a e s. 3.4 I on up ake in he p esence o e men ed almond milk Fe i in concen a ions in cell cul u es exposed o di e en dialyzed e men ed almond milks a e shown in Figu e 3. Appa en ly, he e men a- ion p ocess imp o ed he bioa ailabili y o i on, since he esul an Caco-2 i on up ake in e e y e men ed o mula ion was highe han ha ob- ained in he cells exposed o non- e men ed al- mond milk dialysa es (C) (p<0.05). In pa - icula , samples e men ed wi h L. hamnosus, wi h ei he B. bi idum (F8) o B. longum (F9), we e he ones which induced he mos i on up- ake (p<0.05). P e ious s udies ha e also shown he in i o enhancing e ec o p obio ic bac e- ia on i on up ake in e men ed ege able ma- ices, such as ca o juice (Be gq is , Andlid, & Sandbe g, 2006), maize (P oulx & Reddy, 2007) o beans (Lapa a, Tako, Glahn, & Mille , 2008). This s udy, hence, ex ends he bac e ial- media ed posi i e e ec s on i on up ake om e - men ed ege able p oduc s, pa icula ly hose in which he s a e cul u e con ains bi idobac e- ia s ains. In addi ion, i has been epo ed ha almond ex ac s exhibi ed excellen me al ion chela ion e icacies (abili y o main ain oli- goelemen s such as i on in he educed o m needed o be abso bed by he epi helial cells), owing o i s sou ce o bioac i e polyphenols wi h an ioxidan ac i i y (Ga ido, Monagas, Gomez- Co do es, & Ba olome, 2008; Wije a ne, Abou- Zaid, & Shahidi, 2006). These almond-de i ed componen s wi h unc ional cha ac e is ics may also be p esen in he e men ed samples and migh explain, a leas in pa , he e ec s ob- se ed. The e o e, as Figu e 3sugges ed, e men ed almond milk may p o ide heal h bene i s o IJFS Ap il 2015 Volume 4 pages 49–60