International Dairy Journal

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1 Interntionl Diry Journl 21 (2011) 247e253 Contents lists ville t ScienceDirect Interntionl Diry Journl journl homepge: Microencpsultion of proiotic cteri using ph-induced geltion of sodium cseinte nd gelln gum Arup Ng, Kyoung-Sik Hn, Hrjinder Singh * Riddet Institute, Mssey University, Privte Bg , Plmerston North, New Zelnd rticle info strct Article history: Received 23 Mrch 2010 Received in revised form 19 August 2010 Accepted 11 Novemer 2010 A sodium cseinte nd gelln gum mixture ws gelled y grdully decresing ph with glucono-dlctone (GDL). Lctocillus csei cells were successfully entrpped into this gel mtrix y wter-in-oil emulsion. The optimum ingredient comintion, sed on elstic modulus nd reltive geltion time to ttin dequte gel strength, ws 10% (w/w) sodium cseinte, 0.25% (w/w) gelln gum nd 2.5% (w/w) GDL. A very fine, uniform cpsule prticle size distriution resulted. The surfce-weighted nd volumeweighted men cpsule dimeters were out 287 nd 399 mm, respectively. The rtio of the core cteri to the wll ingredients ws optimized to chieve high encpsultion yield of w89.5%. The survivl of encpsulted cells fter 30 min of incution in simulted gstric fluid ws significntly (P < 0.001) greter thn tht of free cells, oth with nd without the ddition of pepsin. The cpsules lso provided significnt protection for L. csei ginst detrimentl ile slts. Ó 2010 Elsevier Ltd. All rights reserved. 1. Introduction Microencpsultion hs een defined s the incorportion of food ingredients, enzymes, oils, cteril cells or other nutrceuticls into smll cpsules tht cn relese their contents t controlled rtes under specific conditions nd tht protect their contents from degrdtion y the detrimentl fctors in their environment (Desi & Prk, 2005). The purpose of microencpsulting proiotic cteri is to stilize nd mintin viility during storge (O Riordn, Andrews, Buckle, & Conwy, 2001), to protect ginst hrsh gstro-intestinl environment (Muthukumrsmy, Alln-Wojts, & Holley, 2006) nd controlled relese in the colon (Reid et l., 2005). Among the common techniques of microencpsultion, the widely used methods for proiotic cteri re spry drying, extrusion, spry coting nd cocervtion (Kilspthy, 2002; Mortzvin, Rzvi, Ehsni, & Sohrvndi, 2007). The use of diry proteins in microencpsultion hs een studied with gret interest ecuse of their well-known functionl properties (Chen & Suirde, 2005; Rosenerg & Sheu, 1996). Diry proteins esily meet GRAS (generlly recognized s sfe) stndrds nd hve high nutritionl vlue nd excellent geltion, foming nd wter-inding cpcity, thus llowing them to e highly suitle for * Corresponding uthor. Tel.: þ ; fx: þ E-mil ddress: h.singh@mssey.c.nz (H. Singh). encpsulting proiotics or other nutrceuticl crrier mterils tht cn e dministered orlly (Chen & Suirde, 2005). Among diry proteins, sodium cseinte ppers to offer idel physicl nd functionl properties for microencpsultion ecuse of its mphiphilic chrcter nd emulsifying chrcteristics (Hogn, McNmee, O Riordn, & O Sullivn, 2001; Mdene, Jcquot, Scher, & Desory, 2006). The geltion of protein hs trditionlly een chieved y het tretment, during which the polypeptide chins unfold nd susequently self-ggregte to form three-dimensionl network. However, het tretment is unsuitle for the encpsultion of vrious het-sensitive mterils, such s proiotic cteri. The cold-induced geltion of food proteins hs een suggested s potentil solution to this prolem (Brut & Foegeding, 1993; Heidech, Forst, & Kulozik, 2009, 2009; Mltis, Remondetto, Gonzlez, & Suirde, 2005). Heidech et l. (2009) recently developed method in which Lctocillus prcsei nd Bifidocterium lctis strins were encpsulted y the enzymtic geltion of sodium cseinte through crosslinking with trnsglutminse enzyme. In other work, Heidech et l. (2009) took similr pproch to milk protein geltion y encpsulting proiotic cells using rennet s cogulting gent. Sodium cseinte cn lso e cogulted nd gelled y cidifiction with glucono-d-lctone (GDL) (Lucey, vn Vliet, Grolle, Geurts, & Wlstr, 1997) nd the rheologicl properties of the milk gel hve een studied extensively (Coos, Horne, & Muir, 1995; Lucey et l., 1997; vn Vliet & Keetels, 1995) /$ e see front mtter Ó 2010 Elsevier Ltd. All rights reserved. doi: /j.idiryj

2 248 A. Ng et l. / Interntionl Diry Journl 21 (2011) 247e253 In our preliminry experiments, we could not chieve GDLinduced gel mtrix with sufficient rrier strength when using sodium cseinte s the sole wll mteril. However, proteinepolyscchride mixture of sodium cseinte nd gelln gum ws found to give etter properties for the encpsultion of cteril cells. The most significnt functionlity of gelln gum is tht it cn hold smll prticles in suspension without the viscosity incresing significntly (Bird & Pettitt, 1991). As gelln gum is not esily degrded y the ction of enzymes (Bird & Pettitt, 1991; Lee, 1996) nd is resistnt to cidic environments (Sun & Griffiths, 2000), complex of gelln gum with sodium cseinte ws hypothesized to e n idel wll mteril for encpsulting proiotic cteri. In the present study, we encpsulted strin of the common proiotic cterium, Lctocillus csei, into mixture of sodium cseinte nd gelln gum using comintion of geltion nd emulsifiction techniques. The ide of GDL-induced geltion of sodium cseinte confined in wter-in-oil emulsion system to encpsulte proiotic cteri in low ph mtrix hs not een ttempted previously. A comintion of sodium cseinte with gelln gum to provide dditionl gel strength for the encpsulting mtrix under these conditions hs not een descried efore. We optimized the encpsultion process nd compred the in vitro survivl of encpsulted cells nd free cells in simulted gstric fluid nd ile slt solution. 2. Mterils nd methods 2.1. Ingredients nd chemicls Sodium cseinte contining out 90% (w/w) protein ws otined from Fonterr Co-opertive Group Ltd (Plmerston North, New Zelnd). Gelln gum nd GDL were purchsed from Hwkins Wtts (Aucklnd, New Zelnd). Cnol oil ws purchsed from locl supermrket. All chemicls used in this study were of nlyticl grde nd were otined from Sigm Aldrich (St. Louis, MO, USA) Bcteril strin nd cell suspension A commercil strin of L. csei 431 ws otined from Christin Hnsen (Hørsholm, Denmrk) nd ws grown in MRS roth (Difco Lortories, Frnklin Lkes, NJ, USA) t 37 C for 18 h under neroic conditions (GzPk EZ neroe continer system; Becton, Dickinson & Compny, Frnklin Lkes, NJ, USA). The cteri were sucultured t lest three times prior to eing used for the preprtion of cell suspensions. For ech cell suspension, 8 ml of fresh culture ws inoculted into 400 ml of MRS roth nd ws incuted for 18 h. The culture ws centrifuged (Thermo Fisher Scientific, MA, USA) t 4600 g for 20 min. The superntnt ws discrded nd the cell precipitte ws wshed thoroughly with sterilized 0.2% peptone wter (Difco Lortories). This wshing process ws repeted three times nd the finl cell slurry ws mde up to 10 ml y dding the required mount of peptone wter nd ws mixed thoroughly. For enumertion of cteri, the numer of colony forming units (cfu) ws determined on MRS gr using the plte count method t 37 C for 48 h under neroic conditions Geltion of mixture of sodium cseinte nd gelln gum Gel formtion ws crried out using different concentrtions of the encpsultion ingredients. The grdul development of gel strength long with the elpsed time ws importnt to understnd, therefore, the rheologicl nlysis of the gels ws performed with rheometer (Model AR-G2, TA Instruments, Crwley, UK). A time sweep test ws performed for durtion of 180 min to monitor the development of the elstic modulus (G ) during gel formtion in the presence of GDL. The procedure ws crried out in oscilltory mode with flt plte geometry t fixed strin of 0.02 nd frequency of 1 Hz. The temperture of the smples ws mintined t 30 C. Initilly, 10% (w/w) sodium cseinte solution ws cidified with vrious concentrtions of GDL (1.0, 1.5, 2.0 nd 2.5%, w/w). Then sodium cseinte nd gelln gum mixture solutions contining different concentrtions of gelln gum (0.10, 0.25 nd 0.50%, w/w) were exmined t the constnt concentrtion of GDL chosen from the initil experiment. The chnges in the ph of solutions were lso mesured during incution of 240 min t 20 C Microencpsultion The microencpsultion technique used in this study is descried y process flow digrm in Fig. 1. All glsswre ws utoclved t 121 C for 15 min prior to use. The sodium cseinte nd the gelln gum were rehydrted into the required quntity of reverse osmosis (RO) wter for 16 h t 4 C under slight gittion with mgnetic stirrer (Biol, Aucklnd, New Zelnd) nd were therefter completely dissolved t 60 C. The mixture ws then heted to 90 C for 30 min for steriliztion nd cooled to room temperture. GDL ws dded directly into the mixture with continuous stirring t 300 rpm nd the ph of the mixture ws mesured. After 5 min, 5 ml of L. csei cell suspension ws dded to the mixture, followed y continuous stirring for nother 5 min; 100 g of this mixture ws then dded slowly into n Erlenmeyer flsk contining 400 ml of pre-sterilized cnol oil under constnt stirring t 1000 rpm. After holding for 120 min, the prticles were llowed to settle out nd the oil ws decnted off. The microcpsules were wshed three times with sterilized RO wter to remove ny residul oil tht dhered to the prticle surfces. The microcpsules were then stored t 4 C for susequent nlysis Encpsultion efficiency Vrious proportions of cell suspension were dded into the sodium cseinte nd gelln gum mixture of the constnt concentrtions decided previously. The cell suspension (10 ml) prepred s descried previously ws divided into four prts (1, 2, 3 nd 4 ml), which were mde up to the finl volume of 5 ml y dding the required quntity of sterilized 0.2% peptone wter. Ech of these portions ws dded to 100 g of the sodium cseinte nd gelln gum mixture (Tle 1). The cteril counts were mesured efore nd fter microencpsultion. The encpsultion efficiency (EE) ws clculted using slightly modified version of the eqution devised y Heidech et l. (2009), s shown elow, where WCM is the wll mteril nd cell suspension mix. EEð%Þ ¼ Totl solids wcm cfuðg cpsule slurryþ 1 Totl solids slurry cfuðg WCMÞ (1) The dry mtter contents of the WCM nd the cpsule slurry were determined y weighing smples efore nd fter drying in n oven mintined t 105 C for 24 h nd then mesuring the difference in weight s result of the moisture loss. The vile cells of the cpsule suspension in queous medi were counted y reking the microcpsules (1 g cpsule þ 9 ml peptone wter) with Colworth 400 lortory stomcher (Model BA6021, A.J. Sewrd, London, UK) until homogenous mss ws otined. The homogeniztion process ws confirmed gin with compound

3 A. Ng et l. / Interntionl Diry Journl 21 (2011) 247e % (w/w) Sodium cseinte % (w/w) gelln gum Heting the mix to 90ºC for 30 min Freeze dried Lctocillus csei cells Su-culturing into 10 ml MRS roth for 3 consecutive dys. Ech time cells were hrvested nd re-inoculted in sttionry phse Cooling down to room temperture Adding 2.5% (w/w) GDL directly to the mix in solid form Finl culturing in 400 ml MRS roth nd seprting out the cell suspension t sttionry phse fter 17 h t 37 ºC Centrifugtion t 4600 g for 20 min Thorough mixing for 5 min using mgnetic stirrer Wshing of cell suspension 3 times with 0.2% peptone wter Mixing with pre-sterilized Cnol Oil (100 ml mixed with 400 ml oil) under stirring t 1000 rpm Finl volume mke-up to 10 ml with 0.2% peptone wter Holding for 2 h Adding different proportions of cell suspension to optimize encpsultion efficiency Microcpsule formtion through geltion of sodium cseinte nd gelln gum mix. Decnttion of oil phse (centrifugtion not done to void gglomertion of microcpsules nd lso no emulsifiers used to fcilitte esy seprtion of cpsules) Wshing 3 times with sterile wter to remove the oil from the surfce of the cpsules, ech time under gittion with mgnetic stirrer for 5 min Storge t 4 ºC for susequent nlyticl studies. Fig. 1. Optimized process flow digrm of the cell culturing nd microencpsultion of Lctocillus csei 431 cteri. microscopic exmintion nd the vile cells were enumerted using seril dilution nd pour plting on MRS gr Prticle size distriution The surfce-weighted nd volume-weighted men dimeters nd the cumultive size distriutions of the microcpsules were mesured y Mlvern Mstersizer 2000 Ver (Mlvern Instruments Ltd, Mlvern, UK) using lser diffrction technology. The stndrd operting procedure (SOP) ws performed with prticle refrctive index (RI) of 1.37 nd the smple concentrtion Tle 1 Effects of vrious concentrtions of free cell suspensions on cteril counts in cpsules nd the encpsultion efficiency. Encpsultion Smple Composition (ml) log 10 cfu Cell suspension Peptone wter (g cpsule) 1 efficiency (%) A B C D ws % (v/v). The dispersnt used ws RO wter with n RI of Smples were nlysed in duplicte Survivl of encpsulted nd free cells in simulted gstric fluid Simulted gstric fluid (SGF) ws prepred ccording to method USP31-NF26 of the US Phrmcopei (2008) with 0.2% NCl, nd the ph ws then djusted to 2.0 with HCl. Two different tests were performed with nd without the ddition of 0.32% pepsin (from 800 to 2500 units of pepsin per milligrm of protein) to oserve the effect of pepsin on the sodium cseinte nd gelln gum gel mtrix. One grm of microcpsules ws dded to four pirs of Kimx tues, ech contining 9 ml of pre-wrmed SGF, nd ws incuted in wter th mintined t 37 C under oritl gittion t 100 rpm sed on the methods of Guerin, Vuillemrd, nd Suirde (2003). After every 30 min of incution, one smple ws removed nd the ph of the smple ws immeditely rised to 7.0 with 0.1 M NOH to stop the enzymtic rection. The cpsules were then smshed with the stomcher to relese the entrpped cteril cells, followed y plte counting (s descried in Section 2.5 ove). Exctly the sme pproch ws tken for free cells; 1 ml of cell suspension ws

4 250 A. Ng et l. / Interntionl Diry Journl 21 (2011) 247e253 dded into 9 ml of SGF. In ech cse, negtive controls in peptone wter (ph 7.0) were nlysed to mesure the initil counts. Smples were plted in duplicte for ech dilution level nd ll experiments were replicted three times to otin men vlues Survivl of encpsulted nd free cells under simulted ile conditions A simulted environment contining ile slts ws prepred ccording to the method descried y Muthukumrsmy et l. (2006). Monosic potssium phosphte (KH 2 PO 4, 0.68%, w/w) nd porcine ile extrct (1.0%, w/w) were dded to deionized MilliQ wter (Millipore, Molsheim, Frnce) nd the ph ws djusted to 6.8 with 0.2 M NOH. One grm of microcpsules ws dded into four pirs of Kimx tues, ech contining 9 ml of ile slt solution, prewrmed nd incuted in wter th mintined t 37 C with oritl gittion t 100 rpm. The sme process ws followed for free cells. The vile cells in the smples were counted fter 2, 4, 6 nd 8 h. Smples were plted in duplicte for ech dilution level nd ll experiments were replicted three times to otin men vlues Sttisticl nlysis The results were expressed s men stndrd devition (SD). The dt for survivl in SGF nd under simulted ile conditions were nlysed using SAS/PROC ANOVA (GLM) nd TTEST respectively (SAS, version 9.1, SAS Institute Inc., Cry, NC, USA). Mens for the survivl in SGF t ech incution time were compred using Duncn s multiple rnge test. The sttisticl significnce for survivl under simulted ile conditions ws ccepted t P < Results 3.1. Geltion of sodium cseinte nd gelln gum mixture An increse in the concentrtion of GDL resulted in more rpid formtion of the sodium cseinte gel nd higher G vlues (Fig. 2). Gel formtion ws initited fter out 30 min of incution with 2.5% GDL; lower concentrtions of GDL led to slower gel formtion nd the ddition of 1.0 nd 1.5% GDL ws not sufficient to produce gel with dequte strength, even fter 180 min of incution. Three distinct phses of geltion were visile with 2.5% GDL (Fig. 2): the initition of gel formtion; rpid increse in gel strength; trend to plteu when the GDL hydrolysis ws possily complete nd no more gluconic cid ws ville for further reduction in ph. Bsed on the elstic modulus nd the reltive geltion time, GDL concentrtion of 2.5% ws chosen for susequent nlysis. Gelln gum t different concentrtions (0.10, 0.25 nd 0.50%) ws dded to the comintion of 10% sodium cseinte nd 2.5% GDL. The interction etween sodium cseinte nd gelln gum ws evident y mrked increse in the G of the mixture fter 180 min of incution (Fig. 3). As very high G of the wll mteril might crete difficulty in relesing the core mteril from the gel network, gelln gum concentrtion of 0.25% ws chosen. To understnd the contriution of gelln gum in the gel formtion ehvior of the composite mix, we conducted similr rheologicl nlysis of 0.25% gelln gum solution lone cidified with 2.5% GDL. Fig. 2 shows tht gelln gum formed gel with stedy increse in G vlues during the 180 min time sweep test with finl G vlue of 2053 P. Becuse the G vlues of cseinteegelln mix (8339 P) were higher thn tht of cseinte lone (6447 P) nd of gelln lone (2053 P), it is cler tht interctions etween cseinte nd gelln contriuted to incresed gel strength. The rte of decrese in ph y the ddition of 2.5% GDL ws similr in oth sodium cseinte solution nd the sodium cseinte nd gelln mixture solution (Fig. 4) Encpsultion efficiency The EE of vrious concentrtions of free cell suspensions ws clculted (Tle 1). The EE vried from 41.9 to 89.5% with n increse in the cell loding ut showed slight decrese on further increse in the cell concentrtion ove certin level (3 ml of cell suspension) Prticle size distriution The size distriution for the cpsules ws found to e uniform nd exhiited dimeter rnge from out 40 to 1100 mm. The surfce-weighted nd volume-weighted men dimeters of the cpsules were found to e out 287 nd 399 mm respectively Survivl of encpsulted nd free cells in SGF nd under simulted ile conditions The initil count of free cells ws djusted with suitle dilution to out 10.7 log cfu, to mtch with the initil cell popultion of the cpsules. After 30 min of incution in SGF without pepsin, the G(P) Time (min) Fig. 2. Chnges in the elstic modulus (G ) of 10% sodium cseinte solution mixed with 1.0% (B), 1.5% (C), 2.0% (,) nd 2.5% (-) glucono-d-lctone nd 0.25% gelln gum solution mixed with 2.5% glucono-d-lctone (>) Time (min) Fig. 3. Chnges in the elstic modulus (G ) fter the ddition of 0.10% (C), 0.25% (,) nd 0.50% (-) gelln gum into control smple (B) contining 10% sodium cseinte nd 2.5% glucono-d-lctone.

5 A. Ng et l. / Interntionl Diry Journl 21 (2011) 247e ph Log 10 cfu g -1 cpsule * * * viility of free cells declined to 9.4 log cfu nd finlly reched 4.9 log cfu fter 120 min of incution. In contrst, ddition of pepsin to the SGF provided protection to the free cells (Fig. 5) ecuse the vile count in SGF with pepsin decresed only to 5.9 log cfu fter 120 min of incution, which ws significntly higher thn for the free cells in SGF without pepsin. However, pepsin hd lmost no effect on the viility of encpsulted cells in SGF. With or without the presence of pepsin in SGF, the vile count of the encpsulted cells ws significntly higher thn tht of the free cells. The vile count of encpsulted cells in ile slt solution ws lso significntly higher thn tht of free cells fter 4 h of incution (Fig. 6); eyond 4 h, the detrimentl ction of ile slt on the free cells ws ccelerted. The free cell count hd decresed y out 2.6 log cfu t 6 h ut then remined lmost constnt etween 6 nd 8 h of incution. For the encpsulted cells, the vile count decresed y only out 0.5 log cfu during the first 4 h of incution, incresed slightly t 6 h nd then lmost reched the initil cell count level fter 8 h of incution. Log 10 cfu g -1 cpsule Time (min) Fig. 4. Chnges in the ph y the ction of 2.5% glucono-d-lctone on 10% sodium cseinte (C) nd 10% sodium cseinte mixed with 0.25% gelln gum (B) during incution for 240 min t 20 C Incution time (min) Fig. 5. Survivl of encpsulted cells (6, with pepsin; B, without pepsin) nd free cells (;, with pepsin; C, without pepsin) of Lctocillus csei in simulted gstric fluid incuted t ph 2.0 for 120 min. The error rs indicte stndrd devitions from the men vlues of three replicted experiments. c c c 6 4. Discussion Incution time (hr) Fig. 6. Survivl of encpsulted cells (B) nd free cells (C) oflctocillus csei under simulted ile conditions during 8 h of incution. The error rs indicte stndrd devitions from the men vlues of three replicted experiments. The rheologicl properties of sodium cseinte gels induced y cold cidifiction with GDL hve een studied extensively (Lucey et l., 1997). In the present study, incresing the concentrtion of GDL resulted in more rpid formtion of the sodium cseinte gel nd higher G vlues, which is in greement with the findings of Lucey et l. (1997) nd Menendez, Schwrzenolz, Rohm, nd Henle (2004). However, the strength of the sodium cseinte gel ppered to e indequte for forming microcpsules with sufficient rigidity. It ws importnt tht the soft gel prticles retined their shpe nd tht their size ws intct without ny colescence. Therefore, we designed proteinepolyscchride complex using sodium cseinte nd gelln gum. Another reson for including polyscchride ws the instility of sodium cseinte gels t higher ph. Gelln gum in comintion with sodium cseinte ws found to provide synergistic effect in terms of the gel strength nd the stility t higher ph vlues. It is considered tht gelln gum does not simply result into higher viscosity ut lso forms complex structure when dded into sodium cseinte solution. This ws evident from the higher G vlues otined thn the corresponding G" vlues for ny comintion of cseinteegelln solutions (dt not shown). Similr rheologicl nlysis of cseinteegelln mix y Sos-Herrer, Berli, nd Mrtinez-Pdill (2008) supports this structure formtion. They found some intermedite complex formtion in cseinteegelln mix t ph 5.4 where oth polymers were negtively chrged, so the possiility of cocervtion ws ruled out. It ws lso concluded tht the oserved phenomenon could e due to electrosttic interction s suggested y De Kruif nd Tuinier (2001) nd more recently y Ye, Flngn, nd Singh (2006) for sodium cseinte/gum ric mix or intermoleculr hydrogen onding (Dickinson, 2003). In the dynmic microencpsultion system used in our study, the droplets move round continuously nd tend to colesce, form lrger prticles nd flocculte to form ggregtes in the sence of ny emulsifier. McClements (1999) explined this phenomenon s electrosttic ttrctions etween the csein molecules ner the isoelectric point. To void this sitution, the entire microencpsultion process ws completed within pproximtely 2 h, when the ph of the mixture hd decresed to out 5.2. Although finl ph of round 4.6 ws needed to complete the cogultion nd to ttin

6 252 A. Ng et l. / Interntionl Diry Journl 21 (2011) 247e253 firmer gel structure, the finl ph decrese my hve continued fter the microcpsules were stored t 4 C in distilled wter suspension. Our process ws cple of preventing further gglomertion of the csein prticles. The high EE cn e ttriuted to the entire process not including ny detrimentl steps such s het tretment or high sher force. In ddition, gelln gum in comintion with sodium cseinte my improve the EE. The EE ppered to e in greement with the results of Heidech et l. (2009), who used similr process with sodium cseinte emulsion. As shown in Tle 1, the EE improved stedily with n increse in the cell loding ut there ws slight decrese fter certin optimum loding, ecuse the cells present on the droplet surfce were possily lost into the oil phse or drined with the wshing wter. Microprticle size distriutions over very wide rnge hve een reported y severl reserchers. Muthukumrsmy et l. (2006) encpsulted Lctocillus reuteri using vriety of gel mtrices nd concluded tht n extrusion process generlly produces eds of much lrger dimeter (verge 2e4 mm) thn n emulsifiction process (from 20 mm to 1 mm). Adhikri, Mustph, nd Grun (2003) used comintion of emulsifiction nd cocervtion to encpsulte Bifidocterium longum nd reported microcpsules in the rnge 22e350 mm. The optimum microcpsule size is compromise etween the effectiveness of encpsultion nd the sensory properties. In generl, corseness in the mouth occurs for prticle size ove 1000 mm ut is not detectle elow 3 mm (Singer & Dunn, 1990). A minimum dimeter of 100 mm hs een suggested to offer etter protection for Bifidocterium in gstric juice (Hnsen, Alln-Wojts, Jin, & Pulson, 2002) nd n optimum rnge of 100e200 mm hs een proposed (McMster, Kokott, & Sltter, 2005). The men dimeter of our cpsules ws slightly ove this suggested rnge. Although the dimeter rnge showed wider vrition in size (40e1100 mm), 82.5% of the prticles were etween 100 nd 630 mm. The ctul impct of this size distriution cn e mesured only y proper sensory nlysis fter incorporting the microcpsules in suitle food formultion. Moreover, this study hs een conducted s proof of principle nd the process optimiztion steps will e crried out in our future work to ddress this issue. The reduction in viility of free cells in SGF without pepsin of out 6.1 log cfu fter 120 min of incution cn e compred with the 5 log cfu reduction of L. prcsei oserved fter 90 min t ph 2.5 y Heidech et l. (2009). However, Song, Cho, nd Prk (2003) oserved etter resistnce of L. csei YIT 9018 nd only 4 log cfu reduction ws recorded fter 3 h of incution t ph 1.2. A more pronounced lethl effect ws reported y Ding nd Shh (2009); when nine strins of lctocilli nd ifidocteri were tested for cid resistnce t ph 2.0, ll strins were found to e dly ffected, with n verge log cfu reduction of 6.5e7.0 fter 120 min of incution. The ddition of pepsin, proteolytic enzyme tht is secreted in the stomch, to SGF ppered to hve protective effect on free cells (Fig. 5). In this study, fter 120 min of incution, the reduction in free cells ws only out 4.6 log cfu in SGF with pepsin compred with 6.1 log cfu in SGF without pepsin. This oservtion is similr to the findings of Srel et l. (2005), who explined tht it could e due to the presence of other unknown compounds in commercil pepsin extrcts otined from porcine gstric mucos nd lso to the strin-specific ction of pepsin. The viility of encpsulted cells in SGF oth with nd without pepsin ws reduced y only out 3.1 log cfu fter 120 min of incution. When compred with the reduction for free cells, this finding is importnt for our microencpsultion technique. Heidech et l. (2009) used sodium cseinte gelled with trnsglutminse enzyme for L. prcsei encpsultion nd similrly found out 3.0 log cfu reduction fter 90 min of incution t ph 2.5. They lso investigted different gelling technique using rennet ut the differences in survivl rte for encpsulted cells compred with free cells of L. prcsei nd B. lctis were only 0.8 nd 2.8 log cfu higher respectively (Heidech et l., 2009). The etter survivl rte in n encpsulted environment cn e ttriuted to the sence ny direct contct of the cells with the cidic medium, which is common for ny kind of encpsultion technique; dditionlly, the uffering nture of milk protein might provide some enhnced protection (Guerin et l., 2003; Kos, Suskovic, Goret, & Mtosic, 2000; Reid et l., 2005). In this study, the etter survivility of encpsulted cells in SGF might lso e explined y the synergistic effect of gelln gum s well s y the pre-dptility of cteril cells in low ph cseinte gels. The neutrl ph of ile extrct solution my cuse destiliztion of the gel network nd the properties of the ile slts could possily cuse emulsifiction of the entrpped or surfce oil to some extent, therey relesing the L. csei cells (Ding & Shh, 2009). In the present study, very high ile tolernce ws oserved for encpsulted cells when compred with free cells. As different reserchers hve used vrious concentrtions nd sources of ile slts, it is difficult to mke ny comprison with our finding. However, lethl ction of ile slts on proiotic cteri hs generlly een oserved (Ding & Shh, 2009; Guerin et l., 2003; Song et l., 2003). Guerin et l. (2003) nd Trindde nd Grosso (2000) oserved opposite results; free cells nd encpsulted ifidocteri cells showed higher viility fter 3 h of incution in the presence of ile slts. 5. Conclusions The novel encpsultion technique developed in the present study offers high density gel network with low viscosity. The system is esy to hndle, gives full control over prticle sizes nd provides dequte protection for proiotic cteri ginst hrsh cidic environments nd the detrimentl ction of ile slts. However, the study is not complete without further nlysis of the produced microcpsules in terms of sensory properties, shelf life of entrpped proiotics nd relese ehvior of the encpsulting mtrix in the gstro-intestinl trct. We shll e crrying out further reserch on these spects. It cn e concluded tht this system my e nother promising encpsultion technique tht cn e effectively utilized for the ppliction of proiotic cteri in vrious foods. References Adhikri, K., Mustph, A., & Grun, I. U. (2003). Survivl nd metolic ctivity of microencpsulted Bifidocterium longum in stirred yogurt. Journl of Food Science, 68, 275e280. Bird, J. K., & Pettitt, D. J. (1991). 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