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International Dairy Journal 22 (2012) 31e43

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International Dairy Journal

journal homepage: www.elsevier .com/locate/ idairyj

Survival of entrapped Lactobacillus rhamnosus GG in whey protein micro-beadsduring simulated ex vivo gastro-intestinal transit

S.B. Dohertya,b, M.A. Autya, C. Stantona,c, R.P. Rossa,c, G.F. Fitzgeraldb,c, A. Brodkorba,*a Teagasc Food Research Centre Moorepark, Fermoy, Co. Cork, IrelandbDepartment of Microbiology, University College Cork, Cork, IrelandcAlimentary Pharmabiotic Centre, Cork, Ireland

a r t i c l e i n f o

Article history:Received 15 February 2011Received in revised form16 June 2011Accepted 17 June 2011

* Corresponding author. Tel.: þ353 25 42222; fax:E-mail address: andre.brodkorb@teagasc.ie (A. Bro

0958-6946/$ e see front matter � 2011 Elsevier Ltd.doi:10.1016/j.idairyj.2011.06.009

a b s t r a c t

Cell survival of Lactobacillus rhamnosus GG entrapped in gelled whey protein isolate (WPI) micro-beadswas elucidated relative to cells suspended in native WPI and free-cell controls during ex vivo porcinegastro-intestinal incubation. Probiotic gastric tolerance was investigated as a function of pH (2.0e3.4)and time with subsequent intestinal incubation (pH 7.2). Free cells showed no survival after 30 minex vivo stomach incubation (�pH 3.4), while native WPI enhanced survival by 5.7� 0.1, 5.1� 0.2 and2.2� 0.2 log10 cfumL�1 following 180 min incubation at pH 3.4, 2.4 and 2.0, respectively. Protein micro-beads augmented ex vivo probiotic acid resistance (8.9� 0.1 log10 cfumL�1) and demonstrated signifi-cant micro-bead adsorption capacity coupled with micro-bead digestion and controlled release of viable,functional probiotics within 30 min intestinal incubation. This technology potentially envisions wheyprotein micro-beads as efficacious entrapment matrices and binding vehicles for delivery of bioactiveingredients.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Cell encapsulation, immobilization and entrapment epitomizeelementary practices for proficient bioactive delivery and controlledrelease in pharmaceutical, food and flavour industries (de Vos, Faas,Spasojevic, & Sikkema, 2010). Many in vitro studies illustrate theaptitude of entrapment technologies for amplification of probioticsurvival during quasi-stomach conditions (Burgain, Gaiani, Linder, &Scher, 2011). However, the selection of entrapment proceduresvaries according to the research field since protection of cells andbioactive molecules is governed by different physico-chemical andmolecular requirements unique to individual bacterial species andcompounds. Alginate e the quintessential encapsulation material eis susceptible to disintegration in the presence of excess monovalentions and Ca2þ chelating agents (Smidsrod & Skjak-Braek, 1990);hence, stable cell entrapment in alginate remains an difficultchallenge.

According to common credence, research has failed to fabricatea single matrix with all the essential entrapment characteristics;meanwhile, dairy proteins are attracting industrial and academiccuriosity as potential alternatives to coated-alginate and composite

þ353 25 42340.dkorb).

All rights reserved.

entrapment matrices. Whey proteins, in particular, have a highbiological value (Smithers, 2008) partly due to the high content ofbranched-chain essential amino acids, which stimulate specificintracellular pathways. From a formulation perspective, wheyproteins have been exploited as operational scaffolds for drug andcell entrapment due to their aptitude to form emulsions (Beaulieu,Savoie, Paquin, & Subirade, 2002) and gastro-resistant hydro-gels(Ainsley Reid, Champagne, Gardner, Fustier, & Vuillemard, 2007).b-Lactoglobulin (b-Lg), the most abundant whey protein, is a smallglobular protein with specific affinity for a variety of hydrophobicand amphipathic compounds, including retinol (Kontopidis, Holt, &Sawyer, 2002), phospholipids (Lefevre & Subirade, 2001) andaromatic compounds (Collini, D’Alfonso, & Baldini, 2000). Hence,whey proteins are emblematic of versatile carriers of hydrophobicmolecules and probiotic bacteria in controlled release applications.

Probiotic bacteria, defined as ‘live micro-organisms, whichwhen administered in adequate amounts, confer a health benefiton the host’, are considered safe for human consumption andillustrate potential for immunomodulation, treatment andprevention of disorders and diseases (Mattila-Sandholm et al.,2002). However, commercially available probiotic products andsupplements often provide inadequate cell populations (<107

viable cells) due to harsh processing conditions encountered duringmanufacture of the carrier system.

S.B. Doherty et al. / International Dairy Journal 22 (2012) 31e4332

Probiotic research focus has recently shifted due to the reali-zation that probiotic effects are dependent upon the presence offunctional ligands or “effector molecules” in the probiotic cellenvelope (van Baarlen et al., 2009). These characteristic bioactivemolecules are adversely affected by enzymatic action of pepsin andlow pH of the stomach with further antagonism associated withantimicrobial activity of bile salts and protease-rich conditions ofthe intestine (Konstantinov et al., 2008). Hence, effector moleculefunctionality requires specific conservation requisites duringproduct manufacture and gastro-intestinal (GI) transit; a probioticfunctionality consideration more influential than cell survivalconcentration. In essence, validation of cell survival via plateenumeration will not necessarily demonstrate the preservation ofprobiotic functional benefits. This indispensable issue has recentlycatalyzed industrial consciousness for an auxiliary requirement ofprobiotic functionality. Probiotic bacteria would therefore profitfrom entrapment ascendancy to avert cell demise for targetedfunctional delivery to their absorption site.

This research focused on developing a cell-entrapment method(Doherty et al., 2011), which validated in vitro probiotic delivery inwhey protein micro-beads. Progressive investigations evaluatedwhey protein matrices as preservation edifices for intestinaldelivery of viable, functional cell populations of Lactobacillusrhamnosus GG during ex vivo gastro-intestinal studies. Analysis ofwhey protein micro-beads and innate cell-surface features wereauxiliary elements of this research endeavour for determination ofprotein matrix compatibility as cell adhesion and functionaldelivery devices.

2. Materials and methods

2.1. Biochemicals

BiPro, a commercial whey protein isolate (WPI) obtained fromDavisco Foods International Inc. (Minnesota, U.S.A.) contained 98%(w/w) protein. Native b-Lg and a-lactalbumin (a-La) content inWPIwere analyzed by reverse phase-HPLC and estimated at 82% and16%, respectively. Pepsin (P-7000), sodium acetate and fast greenfluorescent dye (product code F7252) were obtained from SigmaAldrich (Dublin, Ireland) and Tween� 20 and Aristar� Plus gradeacetic acid were obtained from VWR International Ltd., Dublin,Ireland. Chemical products acetonitrile (MeCN) and trifluoroaceticacid (TFA), both HPLC grade, were purchased from Fisher ScientificLtd. (Dublin, Ireland). Milli-Q water (Millipore, Cork, Ireland) wassterilized and utilized in all cases for dispersion of samples, culturemediums and buffer solutions.

2.2. Bacterial strain and culture conditions

The probiotic strain Lb. rhamnosus GG (ATCC 53103, LGG, ValioLtd., Helsinki, Finland), was sourced from the Moorepark culturecollection, under a restricted materials transfer agreement. Cellswere harvested and stored at �20 �C as stock solutions in de ManRogosa Sharpe (MRS) broth (Oxoid Ltd., Hampshire, U.K.) contain-ing 50% (v/v) aqueous glycerol (Ref. G5516; Sigma Aldrich). Due tothe porcine origin of GI digesta, a spontaneous rifampicin-resistantderivative (LGGRif) was required to facilitate selective enumerationof the administered strain during ex vivo studies. Since many lac-tobacilli demonstrate resistance to vancomycin (Klein et al., 2000),rifampicin-resistant variants were generated according to themethod outlined by Gardiner et al. (1999) whereby single colonieswere selected and stocked after anaerobic incubation at 37 �C for48 h. Randomly amplified polymorphic DNA-PCR (RAPD-PCR)(Coakley, Ross, & Donnelly, 1996), growth characteristics, heat andacid tolerance was evaluated for both strains to ensure homology

between parent and variant strains. Subcultures were routinelychecked for purity using pulse-field gel electrophoresis (PFGE)(Simpson, Stanton, Fitzgerald, & Ross, 2002) and all cell cultureswere propagated from 1% (v/v) inoculations for 19 h at 37 �C underanaerobic conditions, achieved using Anaerocult gas packs (MerckKGaA, Darmstadt, Germany). This study utilizes stationary phasecolony forming units (cfu) since the stress response is generallymore resistant to environmental factors (van de Guchte et al., 2002)and all cell cultures were propagated from 1% (v/v) inoculations for19 h at 37 �C under anaerobic conditions.

2.3. Micro-entrapment procedure

WPI micro-beads were prepared according to the method out-lined by Doherty et al. (2011) whereby a dispersion of WPI (11%, w/v) was adjusted to pH 7, heated (78 �C, 45 min) and the suspensionof reactive WPI aggregates was subsequently cooled and refriger-ated overnight. A proteineprobiotic blend containing 9% (w/v) WPIand 109 cfumL�1 Lb. rhamnosus GG was aseptically extrudedthrough a 150 mm nozzle into 250 mL of curing media (0.5 M

sodium acetate; 0.04% Tween� 20; pH 4.6) tempered to 35 �C usingan Encapsulator Model IE-50R from EncapBioSystem (Greifensee,Switzerland). Micro-bead batches containing 1.7�1010 cfu werepolymerized in curing buffer, recovered and subjected to imme-diate analysis.

2.4. Collection of specimens

All ex vivo studies were performed using extracted gastro-intestinal (GI) contents (gastric e lower stomach; intestine e

ileum and caecum) collected from six finisher pigs starved for 16 hprior to slaughter. Porcine slaughter was performed in compliancewith European Union Council Directive 91/630/EEC (outlinesminimum standards for the protection of pigs) and European UnionCouncil Directive 98/58/EC (concerns the protection of animalskept for farming purposes). Upon receipt of GI contents, gastric andintestinal digesta were filtered through glass wool, centrifuged(8600� g, 45 min) and clarified using Whatman paper no. 4(Whatman International Ltd., Kent, U.K.). All samples were checkedfor sterility using brain heart infusion agar (Merck KGaA) andincubated at 37 �C for 48 h, while total Lactobacillus counts weredetermined on Lactobacillus-selective agar (LBS; Becton Dickinson,Oxford, U.K.) following anaerobic incubation for 5 d at 37 �C. GIdigesta were also tested on MRSRif plates to detect the presence/absence of LGG. RAPD analysis was also performed to furtherinvestigate the resident microbiota of respective porcine GI regions.All GI contents were analyzed within 6 h of slaughter for (i) pHdetermination (Mettler Toledo MP220 pH meter; VWR Interna-tional Ltd.), (ii) protein content according to the Bradford assay(Bradford, 1976) and (iii) activity of respective GI enzymes (seeSection 2.5). Following this, stomach and intestinal digesta wereindividually pooled and stored at �20 �C.

2.5. Enzyme assays

2.5.1. Pepsin activityEnzymatic activity of pepsin (EC 3.4.23.2) in porcine gastric

contents was evaluated using denatured haemoglobin as a substrateand purified pepsin as the reference material. The reaction wasstarted by the addition of 5 mL gastric juice to an equal volume ofacid-denatured haemoglobin solution (20 mgmL�1) prepared in10 mM HCl and incubated at 37 �C. Sample reactions were stoppedafter 10 min by the addition of 10 mL 5% (w/v) trichloroacetic acid(TCA), followed by centrifugation (5000� g, 5 min, 20 �C), filtration(0.45 mm) and absorbance measurement at 280 nm using a Cary

Table 1Analysis of ex vivo stomach and intestinal contents based on protein content(mgmL�1), enzyme activity (IU units), total and individual free amino acidconcentrations (mgmL�1).a

Characterization assays Stomach Intestine

Protein content (mgmL�1) 3.9 7.8Pepsin activity (IU units) 44.5 NDChymotrypsin (IU units) ND 319.8Trypsin (IU units) ND 21.5Total free amino acids (mgmL�1) 28,926� 23 27,603� 34Asp 1705� 3 1728� 1Thr 2145� 2 2142� 2Ser 1362� 4 1439� 1Glu 3213� 5 3193� 2Gly 2204� 2 2167� 3Ala 1870� 1 1851� 2Cys 375� 1 434� 1Val 1892� 1 1881� 2Met 723� 3 686� 1.5Ile 1562� 2 1570� 1Leu 2743� 2 2721� 2Tyr 1311� 3 1050� 2Phe 1351� 2 1371� 1His 801� 1 770� 2Lys 2707� 2 2519� 1NH3 422� 2 264� 2Arg 2540� 1 1817� 2

a One enzyme unit (IU) is defined as the amount of enzyme that liberated 1 mmolof tyrosinemin�1mg protein�1 and all amino acid data represent the mean value offive independent tests conducted in triplicate; ND denotes no enzyme detection.

S.B. Doherty et al. / International Dairy Journal 22 (2012) 31e43 33

spectrophotometer (JVA Analytical, Dublin, Ireland). The pepsinconcentration in gastric contents was estimated from referencesamples of known pepsin concentration, which determined thepepsin activity using the following equation:

y ¼ ½A280nmðsampleÞ � A280nmðblankÞ� � 1000� VT � E � EC

(1)

where y¼ pepsin unitsmL�1 GI contents; V¼ filtrate volume (mL);T¼ time of assay (min); E¼ enzyme in the reaction mixture (mg);EC¼ extinction coefficient for tyrosine (280 nm) andA280nm¼ absorbance at 280 nm. One unit (IU) enzyme activity wasdefined as the amount of enzyme which liberated 1 mmol oftyrosine (min�1 mg protein�1). Optimum pepsin activity was ach-ieved at ratios of substrate to porcine gastric juice of 1 mg mL�1 andresults demonstrated 3.9 mg protein per mL gastric juice with44.5 IU pepsin activity.

2.5.2. Endopeptidase activity in intestinal juiceCasein was chosen as the substrate to evaluate the activity of

endo-proteases in porcine intestinal contents. Reference proteaseassays were initially performed for trypsin (EC 3.4.21.4) anda-chymotrypsin (EC 3.4.21.1) standard solutions using casein(prepared by the Hammarstein method; Dunn, 1949) as thesubstrate during TCA-precipitation reactions. At 30 s intervals aftermixing, terminated reactions were held for 1 h at room tempera-ture and the increase in TCA-soluble products was measured at280 nm. Absorbance values were plotted as a function of time andthe slope was converted into mmol tyrosine equivalents released(mg proteinmin�1)�1 for standard trypsin and a-chymotrypsinsolutions. Following this, sulphanilamide-azocasein (Megazyme�,Wicklow, Ireland) was utilized to determine specific trypsin anda-chymotrypsin activity in intestinal contents. Samples wereblended with equal volumes of substrate for 10 min at 40 �C.Following this, non-hydrolyzed Azocasein was precipitated by theaddition of 5% TCA, mixtures were centrifuged (1000� g, 10 min)and casein hydrolysates were assayed spectrophotometrically at440 nm against the relevant blanks. Analogous to pepsin activity,one unit of trypsin/a-chymotrypsin activity was defined as theamount of enzyme required to hydrolyze 1 mmol tyrosine equiv-alents (mg proteinmin�1)�1 from soluble casein under standardconditions (pH 7.0; 40 �C) using the following equation:

y ¼ milliUnits ðassayÞ�1�0:001� df (2)

where y¼ endopeptidase units mL�1 of intestinal contents; milli-Units (assay)�1, which referred to the standard curve for enzymeaction on casein and df¼ dilution factor applied to original intes-tinal samples. Intestinal assays aforementioned were performed intriplicate on five independent porcine GI samples. Data determinedenzyme activities of 21.5 and 318.8 IU for trypsin and chymotrypsin,respectively, in ex vivo intestinal juice. Table 1 illustrates aminoacid profiles of stomach and intestinal porcine digesta and revealscopious amounts of essential amino acids and protein precursorswith total concentrations of 47.6 and 45.4 mgmL�1, respectively.

2.6. Viability of encapsulated Lb. rhamnosus GG in ex vivo porcinegastric contents

The protective effect of micro-entrapment on LGGRif upon expo-sure to gastric juice at various natural pH values was assessed asfollows: entrapped, suspended and free LGGRif (approx 109 cfumL�1)were incubated in gastric contents (1:10 dilution) for 180 min at 37 �Cwith orbital agitation (150 rpm) in a temperature-controlled envi-ronment incubator. To determine probiotic viability, treatmentsamples from entrapped, suspended and free LGGRif were recovered

from gastric digesta at pre-determined time points, dispersed ina selective phosphate buffer (0.5 M; pH 7) and stored on ice for 5 minto terminate any residual enzymatic reactions. Samples werehomogenized according to a previously validated procedure (Dohertyet al., 2010b) as 10-fold dilutions (w/w) using an Ultra-Turrax� T10(IKA�Werke, GmbH & Co. KG, Staufen, Germany) to ensure completeliberation of bacteria from the protein matrix with no adverse effecton cell viability. Planktonic (free) cells were treated similarly tomaintain consistent treatment conditions. Homogenates were seriallydiluted in sterile maximum recovery diluent (MRD, Oxoid, Ltd.,) andappropriate dilutions were spread-plated on two media for enumer-ation of LGGRif and total Lactobacillus. Cell counts for LGGRif wereobtained using MRS agar containing rifampicin (Sigma Aldrich) asa selective agent and 50 UmL�1 of nystatin (Sigma Aldrich) to inhibityeasts andmoulds after anaerobic incubation for 2 d at 37 �C (MRSRif)and total Lactobacillus counts were detected on Lactobacillus-selectiveagar following anaerobic incubation for 5 d. RAPD-PCR was per-formed as described by Coakley et al. (1996), which validated thepresence of LGGRif on MRSRif plates and all tests were conducted intriplicate and mean log survivor counts were plotted as a function ofincubation time. pH values of recovered GI digesta were recorded asa function of incubation time in order to investigate buffer capacity ofthe micro-bead treatments.

2.7. Release of encapsulated Lb. rhamnosus GG as a functionof porcine gastro-intestinal section

Entrapped cells (108 cfumL�1) were anaerobically incubated formaximum of 12 h in porcine digesta from different sections of the GItract (stomach, ileum and caecum). Aliquots of GI digesta wereremoved at specific time intervals, resuspended in phosphate buffer(0.5 M; pH 7), stored on ice for 5 min and homogenized for subse-quent LGGRif and total lactobacilli enumeration as outlined above.

2.8. Live/dead discrimination by flow cytometry

In addition to plate counts, viability of encapsulated and freeLGGRif suspensions were assessed by flow cytometry (FACS) as

S.B. Doherty et al. / International Dairy Journal 22 (2012) 31e4334

outlined previously by Doherty et al. (2010b) using voltages andthreshold values calibrated for the enumeration of LGGRif. Briefly,samples from gastric and intestinal incubation studies werehomogenized to release cells from respective protein matrices,followed by dilution (107 cfu mL�1) with working solutions ofThiazole Orange (TO) and Propidium Iodide (PI) fluorescentstains.

2.9. Physico-chemical characterization of micro-bead degradation

2.9.1. Micro-bead digestionDegree of hydrolysis (DH) was also assayed as described by

Doherty et al. (2011) using the o-phthaldialdehyde (OPA) spectro-photometric assay. Proteolysis was analyzed in each GI sample foreach time point during five independent studies.

2.9.2. Protein analysisTo assess the degree of micro-bead degradation, the amount of

free protein after GI incubation was determined using the Bradfordassay (Bradford,1976) and reference samples were also prepared byblending micro-beads with 0.2% NaCl solution at neutral pH,instead of GI digesta solution. At specific time points, samples werecentrifuged (1000� g, 5 min), supernatant removed, filtered andanalyzed using the Bradford assay. Size exclusion chromatography(SEC) was also performed using an automated 2695 Waters� HPLCsystem (Waters, Dublin, Ireland) equipped with a TSK G2000 SWcolumn (600� 7.5 mm; Tosu Hass, Japan) according to the methodoutlined by Doherty et al. (2010a) using 30% acetonitrile containing0.1% (v/v) TFA. Samples were analyzed in triplicate for each timepoint for evaluation of pH and enzymatic synergism during micro-bead disintegration. Free amino acid analysis was also performed intriplicate on recovered GI media at specified time points asdemonstrated previously (Doherty et al., 2011) using a Jeol JLC-500/V amino acid analyser (Jeol (UK) Ltd., Herts, U.K.).

2.9.3. MicroscopyThe dimensions of the whey protein micro-bead were deter-

mined by bright-field light microscopy using a BX51 light micro-scope (Olympus, Essex, U.K.) and all micro-bead batches wereexamined following staining with fast green fluorescent dye.Additional microscopy work was performed using a Leica TCS SP5confocal scanning laser microscope (CSLM) (Leica Micro systems,Wetzlar, Germany) for visualization of micro-bead digestion duringgastric and intestinal incubation as a function of time. Probioticmicro-beadswere stainedwith BD Cell Viability kit (BD Biosciences,Oxford, U.K.) and imaged as previously described by Auty et al.(2001) while atomic force microscope (AFM) images were alsoobtained using MFP-3D-AFM instrumentation (Asylum ResearchUK Ltd., Oxford, U.K.) according to Doherty et al. (2010a).

2.9.4. ElectrophoresisThe average molecular weights (AMW) of peptides procured

during micro-bead digestion in intestinal media were estimated bySDS-PAGE under reducing conditions according to the methoddescribed by Laemmli (1970). Treated samples were loaded ontoa stacking gel of 4% (w/v) acrylamide (pH 6.8) and separated ona gel containing 15% (w/v) acrylamide (pH 8.8) whereby each gelcontained 0.1% SDS. The electrophoresis was performed ata constant voltage of 180 V in a mini Protean II system (Bio-RadAlpha Technologies, Dublin, Ireland) and gels were stained in a 0.5%Coomassie brilliant blue R-250, 25% iso-propanol, 10% acetic acidsolution. The AMW of the protein bands of electrophoreticallyseparated matrix components were estimated by comparison oftheir mobility to those of standard proteins (Precision PlusProtein� Standards, Bio-Rad Alpha Technologies).

2.10. Hydrophobicity

2.10.1. Micro-bead surface hydrophobicitySurface hydrophobicity (SH) of whey protein micro-beads were

determined using the SDS bindingmethod outlined by Kato,Matsuda,Matsudomi, and Kobayashi (1984) with particular adjustment forwhey protein profiles. Proteinmicro-bead batches were suspended insodiumdihydrogenphosphate dihydrate buffer (0.02 M; pH6.0),whileSDS reagent (40.37 mg L�1) and methylene blue (24.0 mg L�1) wereprepared separately in fresh buffer solutions. Individual micro-beadbatches (109 cfumL�1, 90 mgWPImL�1) were mixed with SDSreagent (1:2 ratio), incubated for 30 min at 20 �Cunder slight agitationand subsequently dialyzed against the phosphate buffer (ratio 1:25,v/v) for 24 h at 20 �C. Mixtures of 0.5 mL of dialysate, 2.5 mL ofmethylene blue, and 10 mL of chloroform were centrifuged at2500� g for 5 min. The extinction co-efficient ( 3) of the chloroformphase was assessed at a wavelength of l¼ 655 nm according to Hillerand Lorenzen (2008).Measurementswere performed in triplicate andSH of fresh micro-beads batches were assessed relative to batchesprocured as a function of ex vivo gastric incubation time. Native andheat-treated WPI represented positive and negative controls, respec-tively, and all treatments contained equivalent protein concentrations.

2.10.2. Probiotic strain hydrophobicityThe apparent hydrophobicity of Lb. rhamnosus GG and LGGRif

cell surfaces were evaluated as a function of microbial adhesionto hydrocarbons (MATH) according to a modified method ofRosenburg (1991). Bacterial cells were harvested by centrifugationand resuspended in potassium phosphate buffer (Doherty et al.,2010b), while MATH experiments were performed by spectropho-tometer analysis. Analogous to this, cells liberated from micro-beads following 3 h ex vivo intestinal incubation at 37 �C wererecovered and subject to similar hydrophobicity analysis.

2.11. Statistical analysis

All experimental measurements were conducted in triplicateduring five independent studies (unless stated otherwise). Averagevalues and the standard deviation (SD) were calculated and mean logsurvival counts were plotted as a function of incubation time. Studentt-tests were performed using Microsoft Excel, assuming two-taileddistribution and equal variance for all experimental data sets. Treat-mentmeanswere considered significantly different at p� 0.05 unlessstated otherwise (*p< 0.05; **p< 0.01; ***p< 0.001).

3. Results

3.1. Viability of entrapped Lb. rhamnosus GG in ex vivo porcinegastric contents

Fig. 1 illustrates the survival of LGGRif in porcine gastric juice at(A) pH 3.4, (B) pH 2.4 and (C) pH 2.0. LGGRif entrapment in wheyprotein micro-beads significantly (p< 0.001) increased gastric cellsurvival relative to free cell and native protein suspensions, both ofwhich demonstrated profound (p� 0.01) cell loss after 30 min at37 �C. Complementary FACS data further validated cell viabilitywithin micro-bead matrices since LGGRif populations were exclu-sively located in gate A3 of FACS dot plots, indicating the presenceof viable cells with intact, functional cell membranes after 180 mingastric incubation at pH 3.4 (Fig. 1A; top inset). In all cases (pH3.4e2.0), incubation of free LGGRif for 30 min gave complete cellmortality (8.9� 0.1 log10 cfumL�1) compared with maximum cellloss of approximately 2 log10 cycles in native protein suspensionsduring this time. After 60 min at pH 2.4 and 2.0, however, nativeprotein protective properties weakened significantly (p< 0.001)

Fig. 1. Survival of LGGRif entrapped in micro-beads (C), suspended in native WPI (:) and free-cell controls (-) in ex vivo porcine gastric contents at pH 3.4 (A), 2.4 (B) and 2.0 (C)at 37 �C for 3 h with agitation in an orbital shaker at 150 rpm. The insets represent flow cytometry (FACS) dot plots with a specified gating strategy distinguishing live (A3), injured(A2) and dead (A1) cells, while A4 represents debris from respective samples. Confocal scanning laser microscopy (CSLM) images illustrate green and red cells, representing live anddead probiotic bacteria, respectively. Significant differences (*p< 0.05; **p< 0.01; ***p< 0.001) within each treatment are illustrated as a function of respective incubation timeperiods, i.e., 0e30, 30e60, 60e120 and 120e180 min. Standard deviation is indicated by the vertical bars. (For interpretation of the references to colour in this figure legend, thereader is referred to the web version of this article.)

S.B. Doherty et al. / International Dairy Journal 22 (2012) 31e43 35

S.B. Doherty et al. / International Dairy Journal 22 (2012) 31e4336

expressing 3.4� 0.3 and 5.2� 0.6 log10 cycle reductions, respec-tively. Plate enumeration was supported by FACS analysis thatillustrated injured and dead cell populations, and mixtures thereof,in native protein and free-cell treatments, respectively (Fig. 1A;insets). Overall, native WPI provided a moderate stabilizing façadefor probiotic bacteria during stomach incubation but significant(p< 0.001) cell loss (3.8� 0.4 log10 cfumL�1) and cell injury wereillustrated after 120 min incubation at pH 2.4, since FACS datarevealed the rapid transition of probiotic populations from func-tional to compromised cell conditions (Fig. 1B; middle inset). After180 min at pH 2.0, cell mortality (6.8� 0.7 log10 cfumL�1) demon-strated the fragile fascia of native WPI, while FACS revealed theemergence of a comprehensively injured cell population (Fig. 1C;middle inset). Hence, FACS analysis revealed acid and pepticsensitivity of LGGRif; an attribute that cannot be fully compensatedby the presence of native WPI. Conversely, entrapped treatmentsrevealed a pronounced protective sheath during initial 30 minincubation, which persisted with no significant difference asa function of pH and incubation time. After 180 min at pH 2.4 and2.0, plate enumeration and FACS demonstrated a distinct amelio-ration of cell viability in entrapped WPI lattices relative to free-cellcontrols (8.7� 0.1 and 8.4� 0.2 log10 cfumL�1, respectively).

Confocal microscopy (CSLM) in Fig. 1B, C revealed transversesections of micro-beads with an apparent abundance of live LGGRif

evenly distributedwithin denaturedWPI lattices following 180 mingastric incubation at pH 2.4 and 2.0, respectively. To maintaintreatment homology, however, native protein treatments were alsoextruded through the entrapment system for an objectivecomparison with micro-bead treatments; hence, all treatmentswere subject to identical shear forces experienced during the

Fig. 2. Viability of LGGRif within micro-beads ( ) and native protein suspensions ( ) beviability was investigated after (i) preparation of cell-protein amalgams, (ii) micro-bead extrConfocal scanning laser microscopy (CSLM) revealed high entrapment efficiency of live LGGafter 180 min gastric incubation (C). Native protein suspensions illustrated acceptable LGsurvival was significantly reduced after 180 min gastric incubation (D). Measurement bars foCSLM analysis incorporated Thiazole Orange (TA) and Propidium Iodide (PI) staining to ildifference (p< 0.001) within respective treatments relative to the cell concentration in orig

extrusion process. It is noteworthy that extrusion through a 150 mmnozzle exhibited no significant (p> 0.01) effect on entrapmentefficiency of LGGRif in denatured micro-bead matrices (Fig. 2A; livecell indicated by green rods) and after 180 min gastric incubation(Fig. 2C). Meanwhile, native protein suspensions illustrated anapparent entrapment competence during an identical extrusionprocess (Fig. 2B); however, gastric exposure generated injured cells(Fig. 2D; dead cells indicated by red rods). Probiotic viability waspreserved at the micro-bead periphery after 180 min gastricexposure (Fig. 2C), which suggests the favourable absence of pHgradients during acid conditions. The graph in Fig. 2 illustrates cellviability at each step and endorses the acid-susceptible limitationof native proteins relative to denatured protein micro-beads(Fig. 2C, D). Plate enumeration and CSLM corroborated with FACSanalysis, which demonstrated a distinct acid-resistant element ofmicro-bead functionality. (For interpretation of the references tocolour in the text, the reader is referred to the web version of thisarticle.)

Gastric contents were characterized (Table 1) prior to treatmentanalysis. Interestingly, 180 min incubation of probiotic-loadedmicro-beads in gastric digesta reduced free amino acid concen-trations in gastric contents by 308.9� 25.7 mmolmL�1 (Fig. 3A).The plethora of amino acid residues in gastric digesta expressedsimilar magnitudes of reduction after 180 min incubation; anexclusive attribute recognized within micro-bead treatments.Hence, amino acid entrapment at the micro-bead periphery orpotential penetration to the core may be possible; a hypothesisattributable to electrostatic and/or hydrophobic interactions.Furthermore, chromatography analysis characterized gastriccontents during micro-bead incubation (Fig. 3B) and witnessed the

fore and after micro-bead extrusion and during subsequent gastric studies. Probioticusion and following (iii) ex vivo porcine gastric incubation for 180 min (pH 2.0, 37 �C).Rif following micro-bead extrusion (A), with subsequent retention of probiotic viabilityGRif entrapment potential (B) during an identical extrusion procedure; however, cellr images A and B represent 50 and 10 mm, respectively, while C and D show 25 mm bars.lustrate the presence of live and dead probiotics. Asterisks (***) indicate a significantinal mixtures.

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1-s)tnetnoclanitsetnifo

(

Fig. 3. Total amino acid concentration is shown (A) for gastric juice (columns) and intestinal digesta (solid line) as a function of incubation time at 37 �C; (B) shows the percentpeptide distribution, as measured by size exclusion HPLC, in ex vivo porcine stomach digesta during micro-bead incubation. Peptide fragments were evaluated within the followingmass range: >10 kDa ( ), 10e2 kDa ( ) and 2e1 kDa ( ) and <1 kDa ( ). Each peptide mass range is expressed in percent as a function of time with errors bars indicatingstandard deviation of five independent tests performed in triplicate. Significant differences (*p< 0.05; ***p< 0.001) are illustrated relative to the amino/peptide fraction in theoriginal ex vivo digesta extract (t¼ 0).

S.B. Doherty et al. / International Dairy Journal 22 (2012) 31e43 37

production of a broad size range of peptide fragments; possibleremnants of the porcine diet prior to starvation, slaughter anddissection. As gastric incubation proceeded, total peptides> 2 kDademonstrated a pronounced decline of approx 25% with frag-ments< 1 kDa expressing 10% average reduction after 180 min.

3.1.1. Atomic force microscopyAtomic force microscopy (AFM) revealed a variety of potential

proteineprobiotic cohesive mechanisms in operation withindenatured whey protein matrices (Fig. 4). Fabrication of anentrapment network at pH 4.6 with probiotic-protective capacityappeared to be characterized by two distinct features: (i) pro-biotic camouflage via whey protein layers (Fig. 4A) and (ii)progressive engulfment of Lb. rhamnosus GG by aggregates ofglobular proteins (Fig. 4B) resulting in comprehensive probioticentrapment by whey protein particles (Fig. 4C; arrow indicatingone probiotic cell). These differing probioticeprotein amalgamswere visualized at various locations throughout the proteinlattice, which revealed diverse and random protein orientations;an attribute possibly associated with the rapid gelation impetusgoverning matrix generation (Doherty et al., 2011). These findingscorrelated with zeta potential analysis, which was performedaccording to the method outlined by Doherty et al. (2011).In brief, micro-beads were homogenized (Ultra-Turrax� T10,

IKA� Werke, Germany) for 5 min at ambient temperature andzeta potential was derived from the velocity of the proteinsuspension under an applied electric field of 150 V. Zetameasurements supported the hypothesis relating to electrostaticinteractions within entrapment matrices since the net charge ofencapsulation matrices (17.5� 0.5 mV) revealed no significant(p< 0.001) change after 28 days storage at room temperature.This finding potentially supports the maintenance of probioticeprotein alliance during extended storage. Hence, probioticeprotein interactions visualized by AFM appear to be validcomponents of micro-bead matrices as a function of time andenvironmental conditions.

3.2. Liberation of entrapped Lb. rhamnosus GG in ex vivo porcineGI contents

3.2.1. Cell enumeration and identificationThe pH-sensitive release of entrapped LGGRif was evaluated

during gastric (Fig. 5A) and intestinal (Fig. 5B) incubation at 37 �C for3 h. No probiotic bacteria were released from micro-beads duringgastric incubation at pH 3.4; however, trivial concentrations wereliberated after 3 h at pH 2.4 and 2.0 (0.2 and 0.5� 0.1 log10 cfumL�1,respectively). Moreover, entrapment preserved cell viability after180 min gastric incubation at pH 2.4 and 2.0 to achieve maximum

Fig. 4. Visualization of the mechanism of interaction operating within the micro-bead system during probiotic entrapment using atomic force microscopy (AFM). Proteineprobioticmatrices illustrated a range of interactions using 2 mm magnification range. AFM analysis suggested the envelopment of LGGRif by whey protein strata or layers (A), while partial (B)and comprehensive (C) entrapment by whey protein aggregates appeared to be permanent features of proteineprobiotic systems.

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LGGRif liberation of 8.9� 0.1 log10 cfumL�1 in intestinal contents.Intestinal incubation liberated 6.2� 0.1 log10 cfumL�1 after 5 min,while complete cell liberation was achieved following 30 minincubation (Fig. 5B). Random intestinal isolates from MRSRif platesgenerated the RAPD-PCR profile (Fig. 5C) confirming the selectiveenumeration of the administered probiotic strain during ex vivostudies. Random colonies from MRSRif plates unveiled nine macrorestriction patterns, representing the predicted brand pattern of Lb.rhamnosus GG. Following intestinal liberation, probiotic cellsremained vulnerable to the adverse effects of bile and variousindigenous intestinal components. However, FACS analysis validatedthe retention of intact, functional cell membranes after 180 min(Fig. 5D; gate A3) since this methodology was capable of differen-tiating Lb. rhamnosus GG and LGGRif from background intestinalmicrobiota. Interestingly, micro-beads incubated in phosphatebuffer saline (PBS) (pH 7) under identical conditions failed to

illustrate any signs of degradation after 180 min incubation. Hence,micro-bead degradation responded synergistically to neutral pHand intestinal enzymatic action, which reflects true in vivoscenarios.

3.2.2. Cell-surface hydrophobicityIn the selection of probiotic strains with beneficial health effects,

adhesion to intestinal mucus is a fundamental criterion for newprobiotic strains (Vinderola, Matar, & Perdigon, 2005). Attachment ofcells to the intestinal wall is dependent on various phenotypic cell-surface properties including hydrophobicity, which is commonlyassessed by microbial adhesion to hydrocarbons (MATH). Hence, theadhesion capacity of fresh Lb. rhamnosus GG and LGGRif to organicsolvents elucidated the maintenance of cell-surface hydrophobicityfollowing (i) the generation of a rifampicin-resistant derivative,LGGRif, (ii) micro-bead extrusion and (iii) ex vivo gastro-intestinal

Fig. 5. Release of viable LGGRif from micro-beads during (A) ex vivo gastric incubation at pH 3.4 (C), pH 2.4 (,) and pH 2.0 (:) and (B) ex vivo intestinal incubation at pH 6.6 (A)for up to 180 min at 37 �C. RAPD-PCR analysis (C) further confirmed the presence of LGGRif in random gastric, intestinal and caecum isolates from MRSRif plates of micro-beadtreatments. Lanes 1 and 15 contain a molecular ladder (100e1527 bp); Lane 2¼ LGG, Lane 3¼ LGGRif, Lanes 4e7¼ isolates from gastric incubation (180 min); Lanes8e11¼ isolates from intestinal incubation; Lanes 12e14¼ caecum isolates. FACS analysis (D) confirmed the maintenance of Lb. rhamnosus GG viability and functionality (gate A3)following stomach incubation (180 min, pH 2.0, 37 �C) and subsequent intestinal digestion (180 min, pH 6.6, 37 �C).

S.B. Doherty et al. / International Dairy Journal 22 (2012) 31e43 39

delivery. Relative to fresh stationary phase cultures, entrappedLb. rhamnosus GG and LGGRif retained identical and specific adhesioncharacteristics following cell release from micro-beads (Fig. 6A).Entrapped probiotic populations expressed no significant differencerelative to fresh cells, for any hydrocarbon tested, which demon-strated the persistence of hydrophilic, basic cell-surface attributes ofLGG following gastric resistance and intestinal liberation. Averagehydrophobicity values for fresh and encapsulated LGGRif demon-strated a robust attraction for chloroform (approximately 22%; Fig. 6Ai) andweak interactionwith n-hexadecane (approximately 1%; Fig. 6Aii) with standard deviations remaining below 1%. This hydrophiliccharacter maybe affiliated with the presence of exopolysaccharide(EPS) produced by Lb. rhamnosus GG since Landersjo, Yang, Huttunen,and Widmalm (2002) identified an EPS containing galactose, rham-nose and N-acetylglucosamine in a molar ratio of 4:1:1.

3.2.3. Micro-bead surface hydrophobicityThe surface hydrophobicity (SH) of whey protein micro-beads

was determined according to Kato et al. (1984) with slight modi-fications applied for micro-bead matrices. Anionic SDS moleculeswere bound to solvent exposed, hydrophobic amino acid residuesand quantified spectrophotometrically. The resulting SH values ofwhey protein micro-beads, heat-treated and native WPI solutionsare illustrated in Fig. 6B. In agreement with previous findings(Hiller & Lorenzen, 2008), low SH values were observed for nativeWPI solutions; which was subsequently maintained during 3 hex vivo stomach incubation. On the contrary, heat-treated WPIillustrated high SH values, both in solution and in micro-beadmatrices, values which declined as a function of ex vivo gastricincubation time. Relative to heat-treated solutions, micro-beadmatrices accelerated the reduction of SH by 7.6 SDS units after3 h in stomach conditions. These results correlate with Hiller andLorenzen (2008), who demonstrated that heat-treatmentincreases SH of whey protein isolate. Moreover, these dataendorsed the potential existence of hydrophobic amino acid resi-dues on the micro-bead surface, which accelerated interfacialadsorption behaviour of micro-beads during stomach incubation.

3.2.4. Confocal microscopyFollowing gastric incubation ranging from pH 3.4 to 2.0, micro-

beads were subsequently washed and resuspended in intestinal

contents (pH 7.2) at 37 �C and CSLM analysis (Fig. 7A) visualizedmicro-bead integrity and degradation as a function of GI incubationtime. Protein matrices remained intact following 180 min gastricincubation (Fig. 7A i), with concomitant maintenance of cellviability and functionality, as determined by plate enumeration andFACS analysis, respectively. Matrix biodegradation was initiatedafter 5 min intestinal exposure (Fig. 7A ii) with probiotic cellsprogressively discharged from the protein milieu. Protein frag-ments and aggregates were the initial products of matrix digestion;however, significant cell concentrations remained lodged withinthe aggregate core. After 15 min, micro-bead digestion progressedfrom the micro-bead periphery to the core with concomitantliberation of live LGGRif after 30 min (Fig. 7A iii), demonstrating thetransition of micro-beads to malleable protein suspensions. Fig. 7Asupports plate enumeration and FACS analysis, which confirmedcomplete intestinal release of viable functional LGGRif populationsafter 30 min.

3.2.5. ChromatographySEC during intestinal incubation illustrated the acceleration and

timely release of proteins, aggregates and peptides (Fig. 7B) during180 min intestinal incubation. Micro-bead disintegration viaproteolytic digestion lead to the release of protein aggregateswithin the size range 6e67 kDa during initial 60 min incubation(Fig. 7B; black line). Following this, peptides predom-inantly< 1 kDa, were generated after 180 min incubation (Fig. 7B;blue line) with concomitant disappearance of b-Lg, the principlewhey protein. Furthermore, SEC revealed the indigenous aminoacid profile of intestinal digesta (Fig. 7B; red line), corresponding tothe baseline level following initiation of the intestinal assay.Interestingly, no peptides were released during micro-bead incu-bation in PBS.

4. Discussion

The maintenance of GI homeostasis is considered critical forprevention and development of immune-mediated and metabolic-related diseases. The positive influence of probiotics on guthomeostasis is achieved by cell interaction with the host, bacterialantagonism and immunomodulation, which may help the healthyhost to maintain a ‘physiological state’ of control over inflammatory,

Fig. 6. Adhesion (A) of LGGRif to hydrocarbons as fresh stationary phase cultures ( ) relative to cell adhesion following ex vivo intestinal incubation ( ) using chloroform, n-hexadecane, hexane and diethyl ether. Cell ‘clearing’ was visualized in sample supernatant as a result of (i) LGGRif adhesion to chloroform compared with (ii) cell retention insupernatant in the presence of n-hexadecane. Surface hydrophobicity (SH) of micro-bead batches (B) ( ), heat-treated whey protein ( ) and native ( ) whey protein solutions aseffected by ex vivo gastric incubation (pH 2.0, 3 h, 37 �C). Vertical bars represent standard deviations from three independent studies performed in triplicate with SH units definedas mg SDS (500 mgWPI)�1.

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infectious and immunological reactions (Lebeer, Vanderleyden, & DeKeersmaecker, 2010). Next to health-promoting characteristics,functional aspects of probiotic bacteria also involve their capacity toreach the colon in a metabolically active state. However, given therecent concerns regarding probiotic delivery and viability in theintestine, this study elucidates probiotic entrapment in wheyprotein micro-beads as a delivery mechanism capable of trans-ferring functional cell consignments to their target site. Hence,entrapment stability of rifampicin-resistant derivative of Lb. rham-nosus GG (LGGRif) was assessed using ex vivo GI contents to reflectthe range of obstacles encountered during in vivo situations.

Design of micro-beads for intestinal cell delivery was achievedusing ionotropic gelation, while peptic resistance and intestinaldelivery of LGGRif in micro-beads, native protein, and free-celltreatments were screened in GI digesta procured from finisherpigs. This approach differentiated probiotic viability pertaining tothe presence of whey proteins alone or micro-bead structuraleffects. Gastric contents varied from pH 3.4 to 2.0, possibly due to(i) the buffering affect of the animal diet or (ii) the adverse effectof pepsin upon stomach cells at acidic pH. Although gastric lumen

has a pH of approx 2.0, the effect of proton release and neutrali-zation by glycoproteins collectively influence the pH gradient instomach digesta (Campos & Sancho, 2003). Hence, ex vivo studiesprovide a suitable environment for evaluation of probioticencapsulation stability during conditions that resemble thestomach environment. Cell survival was assessed via selectiveplate enumeration, RAPD-PCR, FACS, CSLM and AFM analysis. Asa general tendency, free cells demonstrated high susceptibility tolow pH and pepsin activity with no apparent ability to surviveex vivo conditions at/below pH 3.4 in the presence of 44.5 IU unitsof pepsin activity. In contrast to the immediate mortality-motiveof free LGGRif, entrapment demonstrated a pronounced augmen-tation of probiotic acid resistance and continuously supported themetabolic activity of stationary phase cultures during gastricincubation.

Research has demonstrated that cellular or culture compo-nents of dead probiotic bacteria potentially mediate beneficialeffects on the host, in addition to viable metabolizing cells(Ouwehand & Salminen, 1998). In this regard, a validated FACSmethodology (Doherty et al., 2010b) was utilized in collaboration

Fig. 7. Gastric tolerance and progressive dissolution of micro-beads (A) during ex vivo incubation. Micro-bead integrity was maintained following 180 min stomach incubation atpH 2.0 (i), while micro-bead disintegration was initiated after 5 min intestinal (pH 6.6) incubation (ii) with complete cell liberation visualized after 30 min (iii); bar represents250 mm. Protein and peptide release from whey protein micro-beads is shown in (B), as measured by size exclusion HPLC, after 60 min (black line) and 180 min (blue line) ex vivointestinal incubation at 37 �C. Trace amounts of peptides were identified in the extracted intestinal digesta prior to micro-bead addition and thus represent the baseline reference(red line). The standard curve of protein/peptide standards (A) indicated the molecular masses of eluting ex vivo samples. (For interpretation of the references to colour in thisfigure legend, the reader is referred to the web version of this article.)

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with plate count techniques to evaluate the retention of cellfunctionality and viability following ex vivo incubation. Regard-less of protein structural diversity between treatment groups,FACS analysis complemented plate enumeration via differentia-tion of cell populations into live, injured and dead sub-groups,providing a real-time assessment of the progressive cell death,injury and viability in free, native protein and entrapped treat-ments, respectively. Meanwhile, CSLM provided an additionalvisual aid, which confirmed the maintenance of micro-beadmorphology, in addition to the absence of pH gradients duringgastric incubation. Micro-bead acid resistance did not appear tobe adversely affected by their cratered, porous surface features.In relation to alginate matrices (Krasaekoopt, Bhandari, & Deeth,2003), whey protein micro-beads represent a favourable matrixmaterial due to their peptic resistance and rigidity duringstomach incubation. Moreover, AFM analysis revealed probioticcell membranes camouflaged by whey protein strata in additionto partial and/comprehensive LGGRif entrapment by globular

protein aggregates. This cohesive attraction between matrixcomponents (Fig. 4) appeared to be a permanent resident withinmicro-beads potentially free of pH-gradient formation due to thehomogenous retention of cell viability throughout the matrixduring gastric incubation (CSLM images in Fig. 1). During thisstudy, CSLM validated cell viability and micro-bead integrityfollowing micro-bead extrusion with subsequent gastric incu-bation (Fig. 2C). Plate enumeration, CSLM and FACS analysissynergistically demonstrated the maintenance of probioticviability and functionality following gastric incubation, whichamplifies the proficiency of the entrapment procedure duringchallenging conditions.

During micro-bead incubation, the surrounding ex vivo gastricmedia illustrated a time-dependent reduction in free amino acidconcentrations, with all amino acid residues demonstratingsimilar reductions. Since GI extracts expressed a plethora of aminoacids with a broad profile of pI values, electrostatic and hydro-phobic interactions are both considered interactive mechanisms

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associated with potential amino acid binding to micro-beads. b-Lgpresent in micro-beads, is known to be a carrier of hydrophobicmolecules, particularly retinol (Fox & McSweeney, 2003) andresveratrol from grapes (Liang, Tajmir-Riahi, & Subirade, 2008).Surface hydrophobicity (SH) illustrated a dynamic approach forthe investigation of physico-chemical characteristics of micro-beads that are sensitive to polarity and hydrophobic entities inthe surrounding environment. Hence, binding of hydrophobiccompounds to b-Lg can be illustrated via changes in the surfacehydrophobicity of micro-beads. SH is defined based on amino acidcomposition (Bigelow, 1967); however it disregards secondary,tertiary and quaternary structure. Thus, effective hydrophobicityof exposed amino acids residues on the micro-bead peripherymay potentially mediate their interfacial adsorption behaviour,particularly during gastric incubation. High SH of heat-treatedwhey proteins deciphers a strong micro-bead binding capacity;a characteristic associated with heat denaturation of b-Lg(Shpigelman, Israeli, & Livney, 2009). This finding is in agreementwith previous work (Moro, Gatti, & Delorenzi, 2001) thatdemonstrated high SH values for bovine serum albumin and b-Lg,which corresponds to their biological function of transportingsurface-bound hydrophobic molecules. The weak SH of native WPIwas forecast since the compact globular structure of nativeprotein is frequently associated with low SH (Wagner & Gueguen,1999). Heat-treated protein suspensions and micro-bead matricesexhibit pronounced SH differentiation, which may reflect strongerhydrophobic interactions between free amino acid residues e

originating from gastric media e and unfolded, open micro-beadmatrices. Thus, SH reveals potential evidence to reconcile micro-bead binding capacity and the camouflage of amino acid resi-dues during gastric incubation.

Whey protein micro-beads illustrated a dynamic synergismlinking pH and enzymatic action, which created optimum micro-bead functionality via stomach integrity with concomitantencouragement of intestinal proteolytic action. As previouslydemonstrated by protein systems (Remondetto, Beyssac, &Subirade, 2004), probiotic release from whey protein micro-beadsis biphasic, with an initial relatively fast release rate (5 min) rep-resenting the proportion of cells entrapped on the micro-beadperiphery, followed by slower sustained liberation (after 30 min)associated with retarded liberation of LGGRif enclosed within themicro-bead core. Moreover, characterization of Lb. rhamnosusGG asa presumptive probiotic is based on its ability to persist in theintestinal lumen and epithelium via retention of cell-adhesivecapacity post-gastric incubation. Histology was not within thescope of this study; however, FACS analysis and hydrophobicitycharacterization e a phenotype related to cell-adhesive capacity e

both validated the retention of viable, functional and hydrophiliccharacteristics typical of stationary phase Lb. rhamnosus GG andLGGRif. Taking into consideration that colonization of the GI tractwith lactobacilli interferes with colonization of enteropathogenicmicro-organisms, the observed retention of probiotic functionalityadvocates the maintenance and expression of health benefitsthrough competition with Gram-negative pathogens for adhesionsites (Vinderola et al., 2005).

To facilitate comparison of enzymatic reactions during ex vivostudies, it was important to consider enzyme activity in the stomach(pepsin) and intestine (trypsin and chymotrypsin). Following micro-bead transit from stomach to intestinal media, enzyme penetrationof the matrix structure occurred within 30 min with subsequentrelease of the quasi-totality of encapsulated LGGRif. It is noteworthythat bile salts are toxic for many living cells since they can disorganizethe structure of the cell membrane and bile salt tolerance is consid-ered another essential property required for probiotic survival in theintestine (Succi et al., 2005); however, extension of intestinal

incubation from 3 to 12 h validated the retention of LGGRif viabilityand functionality in the presence of bile, generating a FACS resultidentical to Fig. 5D. Hence, specific probiotic delivery to the intestinein gelled whey protein micro-beads is significantly more effectivethan cell suspensions in native WPI.

Sequential gastric and intestinal ex vivo incubation demon-strated excellent sentinel properties for protein micro-beads withquasi-survival rates of previous in vitro studies (Doherty et al.,2011). Moreover, the release of essential amino acids from wheyproteins micro-beads during intestinal proteolysis may enhancethe nutritional quality of micro-beads as cell delivery vehicles.These attributes may be useful for site-specific controlledbiomolecule delivery with auxiliary promotion of their intestinalabsorption. This strategy is widely used in the pharmaceuticalfield and could find a broad spectrum of applications in thedevelopment of innovative bioactive foods. Cell entrapment inwhey protein micro-beads epitomizes an interesting alternative tospray-drying due to the high biological value of whey proteins(Smithers, 2008). In contrast to cold-renneting of skim milk-concentrates (Heidebach, Först, & Kulozik, 2009), acetate-induced cross-links generated protein networks with vulnerablehydrophobic patches buried within the globular whey matrix.Hence, micro-beads were fabricated with high resistance todissolution in enzyme-active environments. Other studies havedemonstrated the protective properties associated with spray-drying (Bielecka & Majkowska, 2000) and encapsulation in milkproteins (Ainsley Reid et al., 2007; Beaulieu et al., 2002); however,the present study illustrates micro-beads with sufficient strengthto defy ex vivo challenges with concomitant exploitation of thenutritional and functional properties of whey protein for expan-sion of the bioactive and cell delivery market. A few studies havebeen performed on cell growth and survival during entrapment inalginate and more rarely in whey protein and no detailed infor-mation was specified relating to proteolytic activity, cell func-tionality or amino acid release during cell liberation. Hence, thisstudy gained specific insights into the structural arrangementpost-extrusion, gastric and intestinal incubation using chroma-tography and image analysis to complement traditional culturetechniques.

5. Conclusion

Encapsulation matrices were created from denatured wheyproteins using an entrapment process devoid of high temperatures,shear forces and cell loss. This dense whey protein gel lattice wascapable of offering a micro-milieu for favourable cell entrapmentduring gastric incubation, with auxiliary fragmentation withinintestinal environments. The unique functional properties of wheyproteins alleviate the problems associated with capsule size, whichis highly important regarding the sensory impact of matrices on thefinal food products. Hence, it can be concluded that the use ofgelled protein matrices is a promising strategy to render milkproteins as valuable entrapment materials for targeted delivery ofbioactive compounds in food, beverage and pharmaceuticalapplications.

Acknowledgements

The support provided by the National Food Imaging Centre andthe advise of John O’Callaghan is gratefully acknowledged. Thework was funded by the Irish Dairy Research Trust, the NationalDevelopment Plan 2007e2013 and Science Foundation Ireland(SFI). S. B. Doherty was funded by the Irish Dairy Research Trustunder the Teagasc Walsh Fellowship Scheme.

S.B. Doherty et al. / International Dairy Journal 22 (2012) 31e43 43

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