International Journal of Food Microbiology 149 (2011) 152–158

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International Journal of Food Microbiology

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In vitro evaluation of gastrointestinal survival of Lactobacillus amylovorus DSM 16698alone and combined with galactooligosaccharides, milk and/or Bifidobacteriumanimalis subsp. lactis Bb-12

Rafael C.R. Martinez a,b, Azz-Eddine Aynaou c, Simone Albrecht d, Henk A. Schols d, Elaine C.P. De Martinis e,Erwin G. Zoetendal b, Koen Venema c, Susana M.I. Saad f,⁎, Hauke Smidt b

a Departamento de Alimentos e Nutrição Experimental, Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, Av. Prof. Lineu Prestes 580, 05508-000, São Paulo, SP, Brazilb Laboratory of Microbiology, Wageningen University, Dreijenplein 10, 6703 HB, Wageningen, The Netherlandsc TNO Healthy Living, Utrechtseweg 48, 3700 AJ, Zeist, The Netherlandsd Laboratory of Food Chemistry, Wageningen University, Bomenweg 2, 6703 HD, Wageningen, The Netherlandse Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Av. do Café s/n, 14040-903,Ribeirão Preto, SP, Brazilf Departamento de Tecnologia Bioquímico-Farmacêutica, Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, Av. Prof. Lineu Prestes 580, 05508-000, São Paulo, SP, Brazil

⁎ Corresponding author. Tel.: +55 11 30912378; fax:E-mail address: susaad@usp.br (S.M.I. Saad).

0168-1605/$ – see front matter © 2011 Elsevier B.V. Adoi:10.1016/j.ijfoodmicro.2011.06.010

a b s t r a c t

a r t i c l e i n f o

Article history:Received 14 January 2011Received in revised form 19 April 2011Accepted 18 June 2011Available online 25 June 2011

Keywords:LactobacillusProbioticGalactooligosaccharidesIn vitro survivalBifidobacteriumMilk

Probiotic properties of Lactobacillus amylovorus DSM 16698 were previously demonstrated in piglets. Here, itspotential as a human probiotic was studied in vitro, using the TIM-1 system, which is fully validated tosimulate the human upper gastrointestinal tract. To evaluate the effect of the food matrix composition on thesurvival of L. amylovorus DSM 16698 in TIM-1, the microorganism was inoculated alone or with prebioticgalactooligosaccharides (GOS), partially skimmed milk (PSM) and/or commercial probiotic Bifidobacteriumanimalis subsp. lactis Bb-12 (Bb-12). Samples were collected from TIM-1 for six hours, at one-hour intervalsand L. amylovorus populations were enumerated on MRS agar plates with confirmation of identity of selectedisolates by randomly amplified polymorphic DNA (RAPD) fingerprinting. The cumulative survival for L.amylovorus alone (control) was 30% at the end of the experiment (t=6 h). Co-administration of L. amylovoruswith GOS, PSM and/or Bb-12 increased its survival in comparison with the control significantly from the 4thhour after ingestion onwards (Pb0.05). Furthermore, by the use of High Performance Anion ExchangeChromatography, both L. amylovorus and Bb-12 were observed to promptly degrade GOS compounds insamples collected from TIM-1, as assessed at t=2 h. Hence, food matrix composition interfered with survivaland growth of L. amylovorus during passage through TIM-1, providing leads towards optimization of probioticproperties in vivo.

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1. Introduction

Probiotics are defined as live microorganisms which whenadministered in adequate amounts confer a health benefit on thehost (FAO/WHO, 2001). Among the most well-known probioticmicroorganisms are strains belonging to the Lactobacillus andBifidobacterium genera (Saxelin et al., 2005). The search for potentialprobiotic strains and validation of their positive effects are of greatinterest for the functional foods industry. There is growing evidence ofhealth benefits derived from consumption of probiotic lactobacilli andbifidobacteria, including inhibition of pathogenic microorganisms,

improvement of lactose digestion, reduction of serum cholesterollevels, lowering of risk of cancer and enhancement of the host'simmune system (Ehrmann et al., 2002; Guarner and Malagelada,2003; Gobbetti et al., 2010; Vitali et al., 2010).

L. amylovorus DSM 16698 is a probiotic candidate, the potential ofwhich was already demonstrated in weaned piglets (Konstantinovet al., 2008). It was isolated from the feces of an unweaned piglet andis an abundant species in the intestine of healthy animals (Konstantinovet al., 2006a, 2006b).

Besides probiotic consumption, it has been suggested that themodulation of gastrointestinal tract (GIT)microbiotamay be achievedby the use of prebiotics, defined as selective fermentable ingredientsthat allow specific modifications in the composition and/or activity ofthe GIT microbiota (Gibson et al., 2004; Roberfroid, 2007; Wells et al.,2008). In fact, indigenous populations of L. amylovoruswere shown tobe stimulated in vivo in the ileum and colon of weaned piglets afteradministration of a mix of prebiotic carbohydrates, including wheat

153R.C.R. Martinez et al. / International Journal of Food Microbiology 149 (2011) 152–158

starch, sugar beet pulp, inulin and fructooligosaccharides (FOS)(Konstantinov et al., 2004). There are three fundamental criteriaused to characterize a prebiotic compound: i) it must not be degradedby human enzymes; ii) it should be fermented in the GIT and iii) itshould selectively stimulate beneficial members and/or the metabolicactivity of the GIT microbiota (Van Loo, 2004). According toMacfarlane et al. (2006), the vast majority of studies on prebioticshave focused on inulin, FOS, and galactooligosaccharides (GOS).

GOS are produced through elongation of the dimer lactose by theuse of the enzyme β-galactosidase (Mahoney, 1998). It has beendemonstrated that GOS is able to stimulate the multiplication ofbifidobacteria and lactobacilli in the host (Rabiu et al., 2001; Smiricky-Tjardes et al., 2003; Macfarlane et al., 2008; Davis et al., 2010).

In the development of probiotic foods, research is required toselect adequate vehicles for the delivery of probiotics to ensure thatthey overcome the physical and chemical barriers found in the GIT(Piano et al., 2006;Wang et al., 2009). To achieve higher survival ratesfor probiotics in the GIT, different strategies have been exploited,including the co-administration with prebiotics, other probiotics and/or use of milk as a food matrix (Bezkorovainy, 2001; Su et al., 2007;Sanders and Marco, 2010).

The validated in vitro model TIM-1 is very useful to study thesurvival of probiotics, as it closely simulates the physiologicalconditions found in the stomach and small intestine. TIM-1 is avalidated system for the evaluation of survival of probiotics (Marteauet al., 1997). It consists of four successive compartments that simulatethe stomach, duodenum, jejunum, and ileum. In that system, theexperiments are relatively easy to perform under controlled condi-tions, it displays reproducible results and it is not limited by ethicalconstraints (Minekus et al., 1995; Marteau et al., 1997).

The use of the TIM-1 system for evaluating L. amylovorus DSM16698 survival under simulated human upper GIT conditions maycontribute to the validation of probiotic activity of the strain and addfurther knowledge on the effect of foodmatrixes in the delivery of livemicroorganisms to the GIT. In this sense, survival of L. amylovorusDSM16698 was evaluated when it was administered in TIM-1 alone and inthe presence of the commercial probiotic Bifidobacterium animalissubsp. lactis Bb-12, partially skimmed milk and/or GOS.

Fig. 1. Schematic view of the TIM-1 system for water soluble compounds. A: gastric compartcompartment; F: peristaltic valve; G: ileum compartment; H: ileo-cecal valve; I: stomach secand bicarbonate; K: secretion of bicarbonate to control the intestinal pH; L: pre-filter systemabsorption system; O: dialysate; P: pH electrodes; Q: level sensor; R: temperature sensor;

2. Material and methods

2.1. Bacterial strains

L. amylovorus DSM 16698 (from the culture collection of theLaboratory of Microbiology of Wageningen University, TheNetherlands) and B. animalis subsp. lactis Bb-12 (from the culturecollection of TNO-Zeist, The Netherlands) were used in this study.L. amylovorus DSM 16698 was maintained in de Man, Rogosa andSharpe broth (MRS-Difco-Sparks, MD, EUA), supplemented with20% (v/v) of glycerol (Sigma) at −80 °C, whereas Bb-12 was keptin MRS broth containing 0.05% (w/v) of L-cysteine hydrochloridehydrate (Acros Organics, Geel, Belgium) and also stored at −80 °C.

2.2. Experimental design

2.2.1. Dynamic gastrointestinal model and conditions testedThe TIM-1 system (Minekus et al., 1995; Fig. 1) was used to

estimate the survival of L. amylovorus DSM 16698 after passagethrough the GIT. For every experiment, one cleaned and dried unit ofTIM-1 system was used. The cleaning procedure was done by sinkingglass and plastic parts with soap composed of 40 g of NaOH plus 200 gof cleansing agent (RBS® phosphate-free, Carl Roth) per litre ofdistilled water. Parts were brushed for more efficient removal ofdirties and rinsed with tap water. The system was immersed for ca.30 min in a sodium hypochlorite solution (2.5%, w/v) and subse-quently rinsed with tap water, 0.5 M HCl solution, distilled water andfinally with 70% (v/v) ethanol for cleaning. Before attaching all thecaps, sensors and electrodes to the unit, these parts were rinsed with70% ethanol and allowed to dry.

The gastric and duodenal survival of L. amylovorus was determinedunder six different conditions: (C) L. amylovorus DSM 16698 alone(control); (G) L. amylovorusDSM16698plusGOS; (M) L. amylovorusDSM16698plus partially skimmedmilk (PSM) (FriescheVlag, Veenendaal, TheNetherlands); (B) L. amylovorus DSM 16698 with B. animalis subsp. lactisBb-12(Bb-12); (B+G) L. amylovorusDSM16698plusGOSandBb-12, and(B+M) L. amylovorus DSM 16698 plus PSM and Bb-12.

ment; B: pyloric sphincter; C: duodenum compartment; D: peristaltic valve; E: jejunumretion of water, enzymes and acid; J: duodenum secretion bottles with bile, pancreatin; M: semi-permeable membrane system (hollow fibers); N: closed dialyzing and waterS: pressure sensor (Minekus et al., 1995 with modifications).

Fig. 2. Gastric pH (■), and ileal (▲) and gastric emptying (◊) values expressed in termsof percentage (%) of intake, simulated in the TIM-1 system in the present study in thesix different conditions tested.

154 R.C.R. Martinez et al. / International Journal of Food Microbiology 149 (2011) 152–158

Prior to tests in the TIM-1 system, for conditions C, M, B, and B+M,L. amylovorus DSM 16698 was grown and sub-cultivated byinoculation at 4% (v/v) in MRS broth and incubation at 37 °C inanaerobic jars (BBL, Sparks, MD, USA) containing Anaerocult, for thegeneration of an anaerobic atmosphere (Merck, Darmstadt, Germany).The culture was centrifuged at 3000 g for 5 min at 4 °C (Allegra X-22R,BeckmanCoulter, Germany) and the pellet obtainedwas re-suspended in10 ml of sterile phosphate buffer solution (PBS, composed of 8.0 g NaCl,0.2 g KCl, 1.44 g Na2HPO4, and 0.27 g KH2PO4). This suspensionwas usedfor TIM-1 experiments. For conditions G and B+G L. amylovorus DSM16698 was grown and sub-cultivated in “modified MRS broth”formulated by replacing dextrose (2%, w/v) by Vivinal GOS® 1% (w/v).Vivinal GOS® (FrieslandCampina Domo, Zwolle, The Netherlands) had adry matter content of 75%, of which 59% corresponded to GOS, 21.0%lactose, 19.0% glucose, and 1.0% galactose, according to the supplier.Incubation was carried out overnight at 37 °C under anaerobic atmo-sphere. The culturewas centrifuged and the pellet was re-suspended andused for TIM-1 experiments as described above.

For conditions B and B+M, Bb-12 was grown and sub-cultivatedanaerobically by inoculation at 4% (v/v) and incubation overnight inMRS broth containing 0.05% (w/v) of L-cysteine hydrochloridehydrate. For condition B+G, Bb-12 was grown and sub-cultivatedtwice (4.0%, v/v) overnight at 37 °C, but using “modified MRS broth”containing 0.05% (w/v) of L-cysteine·HCl hydrate and Vivinal GOS®1% (w/v) under anaerobic atmosphere. Cultures of each conditionwere treated as described above for L. amylovorus and subsequentlyused for tests in TIM-1.

The complete compositionsof the “meals”used for feeding TIM-1 areshown in Table 1, and they simulated in vivo gastric conditions throughthe addition of salivary juice and enzymes. ThepHof the suspensionwasadjusted to 5.5 (with 1 M HCl) and 10 ml of the liquid were taken forenumeration of the initial populations (t=0) of L. amylovorus,whereasthe remaining content (ca. 300 g) was introduced into the gastriccompartment. Each survival experiment lasted for six hours.

The simulated secretion in the gastric compartment comprised of agastric electrolyte solution (NaCl [6.2 g/l, Merck], KCl [2.2 g/l, Sigma]and CaCl2 [0.30 g/l, Merck]) (Table 1) with 600 U per ml of pepsin(Sigma Chemical Co., St. Louis, MO, USA) and 37.5 U per ml of lipase(Amano Pharmaceutical, Japan) at a flow rate of 0.25 ml/min. The pHcurve in the stomach was computer controlled, with an initial pHvalue of 5.0, but dynamically dropping to pH 1.7 during 120 min andremaining steady after that (Fig. 2). Secretion of gastric acid (1 MHCl)occurred at a flow rate of 0.25 ml/min when it was necessary to adjustthe pH. The residence half time in the gastric compartment was30 min (Fig. 2).

Duodenal secretion, which comprised the electrolyte solution(Table 1), 1 M NaHCO3 (Sigma), 7% Pancreatin® (Pancrex V, Paines &Birne, Greenford, England), and bile (porcine bile extract, which iscomparable with human bile, Sigma, Saint Louis, MO, USA) was set ata concentration of 4% during the first hour and 2% from the secondhour until the end of the run to simulate the dynamic changes in bilesecretions in vivo. All the solutions were secreted at a rate of

Table 1Composition of the “meal” fed in TIM-1 for the six different conditions tested in this study.

Condition Bifidobacteriuma

(log CFU/ml)PSMb

(g)Vivinal GOS®(g)

L. amylovorus DSM 16698(log CFU/ml)

Wat(g)

C – – – 7.0 215G – – 5 6.8 210M – 215 – 7.1 –

B 7.5 – – 7.0 205B+G 7.3 – 5 6.8 200B+M 7.5 205 – 7.3 –

a Bifidobacterium: B. animalis subsp. lactis Bb-12.b PSM: Partially skimmed milk.

0.25 ml/min and the pH of the duodenal compartment wasmaintained at 6.5.

The volume in the duodenum was 55 ml, and the volume in thejejunal and ileal compartments was 115 ml. The jejunal and ilealdialysis flow rate was 10 ml/min. The jejunal absorption wasautomatically regulated and pH was set at 6.8. Fixed ileal absorptionrate was set at 0.4 ml/min and the pH of the ileal compartment wasmaintained at 7.2.

The residential half-time in the small intestinal compartment was160 min (Fig. 2), corresponding to ca. 10 min, 60 min, and 90 min forduodenum, jejunum, and ileum, respectively.

Jejunal dialysis fluid contained 5.43 g/l of NaCl, 0.65 g/l of KCl,0.37 g/l of CaCl2, and 1.55% bile extract. Ileum dialysis fluid wassimilar, except for the absence of bile salts. The flow rates through thehollow fibers were adjusted to 10 ml/min.

Furthermore, in every TIM-1 run, 30 g of citrate buffer (C6H5Na3-O7·2H2O 29.4 g/l pH 4.5, Sigma) and ca. 5 g of a start residue solution(composed of 5 g from a prepared gastric electrolyte solutioncontaining the enzymes Lipase [37.5 mg; Rhizopus F-AP15 fromAmano Pharmaceuticals], Pepsin [42.0 mg, Sigma P7012] and acetatebuffer pH 5.0 [prepared by addition of 21.6 g acetic acid to 87.1 g/lsodium salt trihydrate, Sigma]) were added to the model.

Two replicates of every condition tested were included in theexperiments.

2.2.2. Determination of bacterial survivalViable numbers of L. amylovorus DSM 16698 were determined in

the initial “meal” (t=0) and in the ileum efflux of TIM-1 for up to sixhours with sampling every hour (t=1, 2, 3, 4, 5, and 6 h). All samplescollected were serially ten-fold diluted in sterile PBS and 10 μl of each

er Citrate buffer(g)

Start residue(g)

Final volume(ml)

Gastric electrolyte solution(g)

30 5 310 5030 5 310 5030 5 310 5030 5 310 5030 5 310 5030 5 310 50

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dilution (100 to 10−7) were seeded on the surface of plates containingMRS agar, as previously described by Herigstad et al. (2001). Platesprepared from samples inoculated only with L. amylovorus wereincubated in anaerobic condition (Anaerobic System Anaerogen,Oxoid, Basingstoke, UK) for 48 h at 37 °C. However, when L. amylovoruswas introduced in the model together with Bb-12, the MRS plates forenumeration of lactobacilli were incubated at aerobic atmosphere for48 h at 37 °C, since the presence of oxygen inhibits the growth ofBifidobacterium spp., whereas L. amylovorus is aerotolerant (Vinderolaand Reinheimer, 1999; Jakava-Viljanen et al., 2008).

Viable counts were determined by counting agar plates obtainedfrom dilutions that yielded between 3 and 30 Colony Forming Units(CFU), according to Cunningham et al. (2008). The differentiationbetween bacterial isolates at strain level was carried out by randomlyamplified polymorphic DNA (RAPD) analysis (Collado and Hernández,2007). For that purpose, the number of CFU corresponding to thesquare root of the total number of microbial colonies obtained in theappropriate dilution were randomly picked and re-cultivated in MRSbroth for 48 h at 37 °C under anaerobic atmosphere (AnaerobicSystem Anaerogen, Oxoid). The DNA of these cultures was extractedby using the Wizard® Genomic DNA Purification Kit (Promega,Madison, WI, USA), according to instructions of the manufacturer. Theamount of DNA extracted was determined with a NanoDrop® (ND-1000, Spectrophotometer V.3.1.0, Wilmington, Delaware, USA). Theextracted DNA was used as template for RAPD analysis, and thereactions were performed in a 50 μl-reaction volume composed of200 μM deoxynucleoside triphosphate (Promega, USA), 0.2 μM ofboth primers 5′ AGTCAGCCAC 3′ (Collado et al., 2006) and 5′CCGCAGCCAA 3′ (Torriani et al., 1999), 5× Colorless GoTaq® reactionbuffer (Promega, USA), MgCl2 at a final concentration of 4 mM, 2.5 Uof GoTaq® DNA Polymerase (Promega, USA), and 5 μl of the template(DNA set at a concentration of 20 ng/μl). The temperature profile inthe thermocycler (Biometra®, Model T-1 Thermoblock, Göttingen,Germany) consisted of an initial denaturation step at 94 °C for 5 min,followed by 35 cycles of 94 °C for 1 min, 32 °C for 2 min, and 72 °C for2 min, and a final extension performed at 72 °C for 5 min. Theamplified products were analyzed electrophoretically in 1.0% (w/v)agarose gels containing ethidium bromide (0.5 μg/ml) using 0.5 TAEbuffer for approximately 35 min at 100 V, and visualized under UVlight (Transilluminator Bio Rad, Gel Doc 2000, Hercules, CA, USA).RAPDprofiles obtained fromthe colonies testedwere compared visuallywith the positive control obtained from a pure culture of L. amylovorusDSM 16698.

2.2.3. Evaluation of GOS degradation by L. amylovorus and Bb-12The degradation profile of GOS by L. amylovorus DSM 16698 and

Bb-12 in the samples collected from the TIM-1 system (conditions Gand B+G) at time-intervals t=0, 2, 4, and 6 h was obtained by HighPerformance Anion Exchange Chromatography (HPAEC; Dionex ISC3000; Dionex, Sunnyvale, CA, EUA), equipped with a Dionex CarboPacPA-1 column (2×250 mm) in combination with a Carbopac PA-1guard column (2×50 mm) (Albrecht et al., 2009). Briefly, in order toobtain an approximate concentration of 0.05 mg GOS/ml, sampleswere diluted accordingly, assuming that GOS was not degraded in thesystem. Next, the samples were heated at 100 °C for 10 min forinactivation of digestive enzymes and 20 μl were injected by means ofa Dionex ISC3000 autosampler. The oligosaccharides were eluted(0.3 ml/min) by a gradient of 0–400 mM sodium acetate in 100 mMNaOH during 40 min. Each elution was followed by a washing step(5 min, 1 M NaOAc in 100 mM NaOH) and an equilibration step(20 min, 100 mM NaOH). The detection was carried out using aDionex ED40 detector in pulsed amperometric detection mode.Chromatographic profile of non-fermented Vivinal GOS® was usedfor comparison of the results on GOS utilization from samples of TIM-1 experiments and the Chromeleon software-Version 6.70 (Dionex

Corporation) was used for the integration and evaluation of thechromatograms obtained.

2.2.4. Calculations and statistical analysisThe survival of L. amylovorus during passage through the TIM-1

systemwas expressed in terms of cumulative delivery of viable cells fromthe ileal compartment as a percentage of intake, and the values wereshown as mean percentages±range. The survival of L. amylovorus DSM16698 was calculated taking into account the corresponding volume ofchyme in each fraction. To compare the obtained survival of L. amylovorusDSM 16698 for the six tested conditions, one-way Repeated MeasuresANOVA with a significance level set at Pb0.05 was used. Wheneverstatistically significant differences among treatments were found, thePost-Hoc Dunnet test was applied, with a significance level set at Pb0.05.Statistical analyses were performed using the software package Statistic7.0 (StatSoft, USA).

3. Results and discussion

Studies on the survival of probiotic bacteria are important for theselection and development of new probiotic products, of oral vaccines,and also for a better understanding of the possible mechanismsinvolved in the beneficial effects of these microorganisms (Marteauet al., 1997; Kailasapathy, 2006). Many efforts have been donetowards increasing survival of lactic acid bacteria (LAB) in the GIT,including the selection of strains adapted to stressing conditions,buffering of yogurts by mixing with whey proteins, use of microen-capsulation techniques, and co-administration of probiotic strainswith milk, milk proteins or prebiotics (Conway et al., 1987; Charteriset al., 1998; Chou and Weimer, 1999; Sultana et al., 2000; Toppinget al., 2003; Kim et al., 2007). In the present study, to improve survivalof the potential probiotic strain L. amylovorusDSM 16698, six differentstrategies were tested in a validated in vitro model that simulates theupper GIT: the strain tested alone (control—C), the addition ofprebiotic GOS (G), the co-administration with PSM (M), the additionof Bb-12 (B), the co-introduction with GOS plus Bb-12 (B+G), andthe addition of PSM plus Bb-12 (B+M).

A total of 344 bacterial colonies were recovered from samplesobtained from TIM-1 system at different time-intervals and evaluatedby RAPD analysis aiming to confirm there was no contaminationduring the experiments in the model system. When incubation wasperformed under aerobiosis, to exclude the growth of Bifidobacteriumspp., all colonies tested were confirmed as L. amylovorus.

The cumulative survival (survival plus growth) of L. amylovorusDSM 16698, expressed as cumulative deliveries from the ilealcompartment observed is shown in Fig. 3. When the microorganismwas administered alone in the TIM-1 system, the cumulative survivalafter 6 h was 30%. In comparison with this control, conditions G, M, B,B+G, and B+M favored the survival of L. amylovorus from the 4thhour after intake onwards (Pb0.05).

No significant statistical difference was detected at 6 h amongconditions G, M, B, B+G, and B+M (PN0.05), although descriptiveanalysis indicated that probiotic strain Bb-12 combinedwith prebioticGOS (v) yielded a survival rate of ca. 120% (Fig. 3). This could bepartially attributed to the composition of the product Vivinal GOS®used. Based on dry matter content (75%), only 59% corresponds togalactooligossacharides, whereas the other 41% include lactose,glucose, and galactose. As it has been shown previously, L. amylovorusis able to use these compounds as growth substrates (Ben et al., 2008;Konstantinov et al., 2008). Furthermore, it was already demonstratedthat Bifidobacterium can utilize and grow on GOS (Barboza et al.,2009).

Although the positive effect of GOS on the growth of lactobacilliand bifidobacteria in the colon is well-known (Ben et al., 2008), theother substances present in Vivinal GOS® formulation may haveoffered an important extra source of energy that is readily available

0 1 2 3 4 5 60

20

40

60

80

100

120

140

L. amylovorus L. amylovorus + GOS L. amylovorus + PSM L. amylovorus + Bb-12 L. amylovorus + Bb-12 + GOS L. amylovorus + Bb-12 + PSM

Cum

ulat

ive

deliv

ery

(% o

f in

gest

ion)

Time-intervals (h)

Fig. 3. Survival of L. amylovorus DSM 16698 (expressed as cumulative delivery from theileal compartment) in the samples collected from the in vitromodel at 1 h, 2 h, 3 h, 4 h, 5 hand 6 h, assessed under six different conditions. In all experiments L. amylovorus wasadded at ca. 107 CFU/ml and the different conditions comprised: basal simulated secretiononly (control; ♦), GOS (galactooligosaccharides; ), PSM (partially skimmed milk; ▲),Bb-Ś12 (B. animalis subsp. lactis Bb-12; ●), Bb-12+GOS (★), and Bb-12+PSM (■).

156 R.C.R. Martinez et al. / International Journal of Food Microbiology 149 (2011) 152–158

for L. amylovorus during its passage through TIM-1 and this could be acompetitive advantage in vivo towards the background intestinalmicrobiota.

The HPAEC profile of the reference GOS mixture and the profilesobtained from the samples taken at t=0, 2, 4, and 6 h for conditions Gand B+G were similar and Fig. 4 illustrates results obtained forcondition G. The GOS reference and a sample taken at t=0 were

Fig. 4. Degradation profile of Vivinal GOS®, comprising monomers, dimers, trimers, tetrametin samples obtained at different time-intervals from TIM-1 system for both conditions G (L. amB. animalis subsp. lactis Bb-12 and GOS). nC (Nanocoulomb). Vivinal GOS® was used as ref

similar and indicated the high abundance of monomers and lactose inthe GOS mixture. Microbial degradation of GOS was evident at 2 h, asindicated by the presence of two high peaks corresponding tomonomers and lactose, while only trace amounts of GOS wereretrieved. However, no selectivity in structural degradation wasobserved. The monomers and lactose retrieved after t=2 h wereassumed to result from the degradation of GOS and decrease in peakareas for monomers and lactose was observed between t=2 h andt=6 h, indicating further microbial metabolization. Due to the non-molar response of the HPAEC detector, no quantitative conclusions onthe rate of degradation of GOS and the consumption of mono- anddimers can be drawn.

Similarly, Buriti et al. (2010) also observed that, under in vitrosimulated gastrointestinal conditions, the survival of a Lactobacillusstrain was increased in the presence of a prebiotic ingredient. Theauthors evaluated the resistance to simulated gastrointestinal fluids ofL. acidophilus La-5 present in synbiotic guava mousses kept underrefrigeration and freezing conditions and observed that the additionof inulin in refrigerated mousses enhanced the probiotic survivalduring the first week of storage. Furthermore, for frozen mousses, theauthors also observed that inulin improved the probiotic survivalwhen milk fat was also present in the product and was only partiallysubstituted by inulin.

The high survival found for L. amylovorus in the present studywhen the microorganism was co-administered with milk (ca. 114%,assessed at t=6 h) (Fig. 3) is in agreement with the results obtainedby Charteris et al. (1998). In their study, using an in vitro model thatsimulated the upper GIT to determine the survival capacity ofpotential probiotics, the survival of lactobacilli and bifidobacteriawas improvedwhen theywere tested in the presence of milk proteins.The authors hypothesized that milk proteins could act as buffering

ers and pentamers, determined by High Performance Anion Exchange Chromatography,ylovorusDSM 16698 in the presence of GOS), and B+G (L. amylovorus DSM16698 plus

erence.

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agents and inhibitors of digestive proteases in vivo. Similar protectiveeffect of PSMcould be involved in the present studywith L. amylovorus,although pH-controlled conditions were used.

Furthermore, the high survival for L. amylovorus observed in thepresence of the probiotic strain Bb-12 (Fig. 3) suggests a synergisticeffect. This is in line with observations made by Gardiner et al. (2004),who conducted a trial in which five porcine-derived Lactobacillus orPediococcus isolates with probiotic potential were administered topigs (n=4), either singly or as a combination at 1010 CFU per day.These authors observed that, in the post-administration period, thehighest intestinal survival was obtained when the microorganismshad been co-administered to the animals. Although the mechanismsinvolved in the positive interaction between L. amylovorus and Bb-12observed in the present study remain to be investigated, it can behypothesized that in co-administration in TIM-1, the larger bacterialmass present in the device diluted acids and bile salts per bacterialcell, creating a less stressful environment. Also, some members of theLAB group, including lactobacilli and also bifidobacteria, are able todeconjugate bile salts (Begley et al., 2006; Kim and Lee, 2008; Joneset al., 2008; Burns et al., 2010), and this may have played a role in thehigher survival rate observed in the in vitro model tested.

The results of thepresent study also showed that in some conditions,L. amylovorus not only survived, but grew during passage through theTIM-1 system, as indicated by values of survival above 100% (Fig. 3). Thiscould be important for the microorganism to exert its probiotic activityin vivo in the small intestine, through the inhibition of pathogenadhesion as observed by Roselli et al. (2007). These authors used anintestinal porcine intestinal epithelial cell line (IPEC-1) as a model toinvestigate a possible protective activity of L. amylovorus againstintestinal injury induced by enterotoxigenic Escherichia coli (ETEC)K88 infection and the underlying mechanisms. The authors observedthat L. amylovorusDSM 16698 conferred protection against the harmfuleffects of ETEC by decreasing adhesion of E. coli K88 and maintainingmembrane barrier integrity through IL-10 regulation.

Probiotic bacteria should survive food processing and in the GITenvironment to be able to reach the small intestine and transitorilycolonize the colon (Pan et al., 2009). Bezkorovainy (2001) reviewedseveral studies described by other researchers that studied the survivalof different probiotic strains in vitro and in vivo during their passagethrough the upper GIT, and observed that values ranged from 20% to40%. In the current study, using TIM-1, similar results were obtained forL. amylovorus DSM 16698 (Fig. 3). Other Lactobacillus strains tested inTIM-1 showed a much lower survival (Marteau et al., 1997). Survival inTIM-1 of Lactobacillus strains (L. acidophilus and L. bulgaricus) was alsoconfirmed in vivo (Marteauet al., 1997). This indicates that L. amylovorusDSM 16998 presents a relatively high gastrointestinal viability withinthe genus Lactobacillus, showing that survival is largely species and evenstrain-dependent.

In conclusion, the development of food products containingL. amylovorus, milk, B. animalis subsp. lactis Bb-12 and/or GOScould be an interesting approach for the area of functionalfoods for human beings and animals, improving the survival ofL. amylovorus during its passage throughout the harsh condi-tions found in the GIT. Further studies, involving quantitativedetection of metabolites generated and even performance ofquorum sensing assays are required in order to elucidate theexact mechanisms involved in the protection of L. amylovorusDSM 16998 in the GIT environment conferred by the presenceof PSM, B. animalis subsp. lactis Bb-12 and/or GOS.

Acknowledgments

This work has been carried out as part of a collaborative project (#17/07) between CAPES (Coordination for the Improvement of HigherEducation Personnel, Brazil) and Wageningen University (TheNetherlands). R. C. R. Martinez is grateful to CAPES for granting a

scholarship to support his Post-Doctoral stage in The Netherlands(Individual project #4252/08-0). Additional support was obtainedfrom the INTERPLAY project, funded through the European Commu-nity's Seventh Framework Programme (FP7/2007-2013) under grantagreement #227549.

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