International Journal of Food Microbiology 145 (2011) 22–27

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International Journal of Food Microbiology

j ourna l homepage: www.e lsev ie r.com/ locate / i j foodmicro

Use of lactulose as prebiotic and its influence on the growth, acidification profile andviable counts of different probiotics in fermented skim milk

Ricardo Pinheiro De Souza Oliveira a,b,⁎, Ana Carolina Rodrigues Florence a, Patrizia Perego b,Maricê Nogueira De Oliveira a, Attilio Converti b

a Biochemical and Pharmaceutical Technology Department, Faculty of Pharmaceutical Sciences, São Paulo University, Av Prof Lineu Prestes, 580, Bl 16, 05508-900, São Paulo, Brazilb Department of Chemical and Process Engineering, Genoa University, Via Opera Pia 15, I-16145 Genova, Italy

⁎ Corresponding author. Biochemical and PharmaceuFaculty of Pharmaceutical Sciences, São Paulo University16, 05508-900, São Paulo, Brazil. Tel.: +39 0 10 353259

E-mail address: rpsolive@usp.br (R.P. De Souza Olive

0168-1605/$ – see front matter © 2010 Elsevier B.V. Adoi:10.1016/j.ijfoodmicro.2010.11.011

a b s t r a c t

a r t i c l e i n f o

Article history:Received 25 August 2010Received in revised form 8 October 2010Accepted 7 November 2010

Keywords:ProbioticPrebioticMilkYoghurtLactuloseCo-cultures

Lactulose can be considered as a prebiotic, which is able to stimulate healthy intestinal microflora. In the presentwork, the use of this ingredient in fermented milk improved quality of skim milk fermented by Lactobacillusacidophilus, Lactobacillus rhamnosus, Lactobacillus bulgaricus and Bifidobacterium lactis in co-culture withStreptococcus thermophilus. Compared to control fermentations without lactulose, the addition of such a prebioticin skim milk increased the counts of all probiotics, with particular concern to B. lactis (bifidogenic effect), theacidification rate and the lactic acid acidity, and concurrently reduced the time to complete fermentation (tpH4.5) andthe pH at the end of cold storage for 1 to 35 days.

tical Technology Department,, Av Prof Lineu Prestes, 580, Bl3; fax: +39 0 10 3532586.ira).

ll rights reserved.

© 2010 Elsevier B.V. All rights reserved.

1. Introduction

Significant part of the world population suffers gastrointestinaldiseases causedbypathogenic bacteria that invade the human intestine.A few days after the birth, the human intestine is colonized mainly bybifidobacteria which play a very important role in themaintenance of agood health (Olguin et al., 2005). So, in order to solve this healthproblem, food industry and inparticulardairy technologyhasdevelopeddairy functional products enriched with probiotics like lactobacilli(Lactobacillus acidophilus, Lactobacillus casei, etc.) and bifidobacteria(Donkor et al., 2007).

The word ‘probiotic’ was initially used as an antonym of the word‘antibiotic’. It is derived from the Greek words προ and βιοτοσ andtranslated as ‘for life’ (Hamilton-Miller et al., 2003). In the past, differentdefinitions of probiotics were given, but, following the recent recommen-dations of a working group on the evaluation of probiotics in food of FAO/WHO(2002), probiotics are consideredas livemicroorganisms that,whenadministered in adequate amounts, confer health benefits on the host.Consequently, a wide variety of species and genera could be consideredpotential probiotics (Holzapfel et al., 1998); commercially, however, themost important strains are lactic acid bacteria (LAB).

Lactic acid bacteria (LAB) are widely used in the production offermented foods. Lactobacillus delbrueckii subsp. bulgaricus andStreptococcus thermophilus are traditionally used as starters for milkfermentation in yoghurt production. In addition to L. delbrueckii subsp.bulgaricus, which contributes to accelerate lactic acid development inyoghurt as well as to improve flavor and textural properties (Curryand Crow, 2003), some other probiotics are used for this purpose,among which are Lactobacillus rhamnosus, L. acidophilus, Lactobacillusjohnsonii and Bifidobacterium lactis, because of their ability to grow inmilk and to confer functional properties and benefits to the health(Vasiljevic and Shah, 2008).

The importance of certain technological and physiological charac-teristics of probiotic strainswas recognized long time ago (Gordon et al.,1957). To achieve successful outcome of the lactobacilli therapy, theculture must have certain requirements: it should be a normal, non-pathogenic inhabitant of the intestine, capable of efficient gutcolonization and present in substantially high concentrations in theproduct (107–109 CFU/mL) (Mattila-Sandholm et al., 2002; Ouwehand,et al., 1999).

The dairy industry has been quickly revitalized by the introductionof products characterized not only by their high nutritional value andpleasant taste, but also by their ability to exert positive effects on theconsumer's health (Casiraghi et al., 2007). In this context, some non-digestible ingredients, called prebiotics, have received considerableattention, mainly because of their ability to selectively stimulate thegrowth and/or activity of probiotic bacteria in the colon (Gibson andRoberfroid, 1995; Huebner et al., 2007). However, the ability of

23R.P. De Souza Oliveira et al. / International Journal of Food Microbiology 145 (2011) 22–27

probiotic microorganisms to use the prebiotics is strain and substratespecific (Shah, 2001).

To be effective, prebiotics must escape digestion in the uppergastrointestinal tract and be used by a limited number of micro-organisms comprising the colonic microflora, mainly lactobacilli andbifidobacteria (Gibson and Roberfroid, 1995; Isolauri, 2004); thus, inthe later case they are referred to as bifidogenic factors (Berg, 1998;Macfarlane and Cummings, 1999; Roberfroid, 2000). More rarely, theyare reported to mitigate the virulence of pathogenic bacteria likeListeria monocytogenes (Park and Kroll, 1993). This family ofcompounds includes several oligosaccharides (namely fructo-,gluco-, galacto-, isomalto-, xylo-, and soyo-oligosaccharides), inulin,lactulose, lactosucrose, among others (Fric, 2007).

Lactulose is widely used in pharmaceutical industry (Aider andde Halleux, 2007), mainly as an effective drug against diseases likeacute and chronic constipation (Tamura et al., 1993). Nevertheless,some promising applications are reported also in the nutraceuticalsand food industries because of its beneficial effects on human health(Donkor et al., 2007). It is a synthetic disaccharide obtained byisomerization of lactose, which is present in milk and whey inrelatively high content, approximately 4.5% as an average (Zokaeeet al., 2002), and contains fructose instead of glucose (Strohmaier,1998). Since it is not absorbed in the small intestine, it has thepotential to function as a prebiotic (Kontula et al., 1999). Moreover,several studies showed the effectiveness of lactulose to stimulate thegrowth of bifidobacteria (Olano and Corzo, 2009; Sako et al., 1999;Shin et al., 2000).

Taking into account all these considerations, lactulose appears asan important food ingredient that might be additionally explored forthe production of functional foods, and one can expect its future largescale production for food and nutraceuticals purposes. To thispurpose, the present study explores the effects of this ingredient onthe acidification kinetics, post-acidification, growth and metabolismof binary co-cultures of L. bulgaricus, L. acidophilus, L. rhamnosus andB. lactiswith S. thermophilus, as well on their survival during skimmilkfermentation.

2. Materials and methods

2.1. Microbial cultures

Strains of pure starter freeze-dried cultures (Danisco, Sassenage,France)were used: S. thermophilus TA040 (St) and L. delbrueckii subsp.bulgaricus LB340 from here onwards called L. bulgaricus (Lb) (yoghurtmicroorganisms); L. acidophilus LAC4 (La), L. rhamnosus LBA (Lr) andBifidobacterium animalis subsp. lactis BL 04.

2.2. Milk preparation

Milk prepared by adding 13 g of skim powder milk (Castroni,Reggio Emilia, Italy) in 100 g of distilled water was used either in thepresence (SM) or absence (M) of 4% (w/w) lactulose (trade name:Lactulose 61360) (Sigma Aldrich, Italy). The above solid content ofmilk corresponds to the average value reported by Restle et al. (2003)for integral cow milk, while the selected lactulose concentration wasin the range (3–6% w/w) admitted by Brazilian legislation in yoghurts(ANVISA, 2002). Both milks were then thermally treated at 90 °C for5 min in water bath, model Grant Y6 (Cambridge, England). The heattreatedmilks were transferred to 1.0 L sterile flasks, cooled in ice bath,distributed into 250-mL sterile Shott flasks inside laminar flowchamber, and stored at 4 °C for 24 h before using.

2.3. Inoculum preparation

The La, Lb, Lr, Bl and St freeze-dried cultures were prepared bydissolving in 50 mL of milk 10% (w/w) of total solids; autoclaved at

121 °C for 20 min. After blending and activation at 42 °C for 30 min,1.0 mL of the pre-culture was inoculated into 250 mL ofmilk. Bacterialcounts in these pre-cultures ranged from 6.0 to 6.6 log CFU/mL.

2.4. Fermentations

After inoculation, flask samples were transferred to a water bath,and batch fermentations were performed in triplicate at 42 °C up topH 4.5, which were selected as the conditions to stop thefermentation. Fermentations were monitored by pH determinations.

2.5. Post-acidification

Once the fermentation ofmilk had been complete, post-acidificationwas determined after a) 1 day (D1), b) 7 days (D7) and c) 35 days ofcold storage at 4 °C (D35) by pHmeasurement using a pHmeter, modelpH 210 Microprocessor (Hanna instruments, Padova, Italy).

2.6. Counts of probiotic bacteria

Bacterial counts were carried out in triplicate either after D1, D7 orD35. One mL of sample was diluted with 9 mL of 1 g/L sterilepeptonated water. Afterwards, eight serial dilutions were done, andeach bacterium was counted in the three most appropriate dilutions,applying the pour plate technique (Kodaka et al., 2005). Counts werefinally presented asmean values. All media were obtained fromMerck(Darmstadt, Germany). St colonies were counted in M17 agar (Oxoid)by aerobic incubation at 37 °C for 48 h. Lb, La and Lr counts werecarried out in MRS agar medium after pH adjustment to 5.4 by aceticacid addition and aerobic incubation at 37 °C for 48, 72 and 72 h,respectively. Bl was counted in MRS Agar medium containing 50 g/Lcysteine without any pH adjustment (IDF, 1996, 1997, 2003).

2.7. Kinetic parameters

From the pH data collected during fermentation, the acidificationrate (Vmax) was calculated as the time variation of pH (dpH/dt) andexpressed as 10−3 pH units/min. During the incubation period, thefollowing kinetic parameters were also calculated: (i) tmax (h), time atwhich Vmax was reached; and (ii) tpH4.5 (h), time to reach pH 4.5 (i.e.,to complete the fermentation).

Once pH 4.5 had been reached, the fermentations were manuallyinterrupted by aseptically agitating the clot by means of a stainlesssteel rod with perforated disks; the rod was gently moved upwardsand downwards for 80 s. The fermented product was quickly cooled inan ice bath, and the fermented products were stored at 4 °C.

2.8. Analytical methods

A high-performance liquid chromatography, model 1100 (HewlettPackard, Palo Alto, CA), was used for analysis of lactic acid (after D1,D7 and D35) and lactulose (every 1.5 h during the fermentation). Thesystem consisted of a HP-1050 Intelligent Auto Sampler, a HP-1047ARefractive Index Detector (for organic acids), a HP-1050 UV Detector(for sugars) and a HP-1050 Pump. Separation was achieved using aSupelcogel H59304-U column (Sigma Aldrich, Bellefonte, PA) at 50 °Cwith 0.01 M sulfuric acid as eluent at 0.4 mL/min.

2.9. Statistical analysis

The experimental data of bacterial counts, post-acidification andlactic acid concentration, either after D1, D7 or D35, as well as those oflactulose concentration along the runs were presented as meanvalues. Variations with respect to the mean values were presented asstandard deviations. Mean values of these parameters were submittedto analysis of variance (ANOVA) by the Statistica Software 6.0. They

Table 1Comparison of the acidification kinetic parameters in milk (M) and supplemented milk(SM) fermented by Lactobacillus bulgaricus (St–Lb), Lactobacillus acidophilus (St–La),Lactobacillus rhamnosus (St–Lr) and Bifidobacterium lactis (St–Bl) in co-culture withStreptococcus thermophilus.

Milk Co-culture Vmax (10−3 pH units/min) tmax (h) tpH4.5 (h)

M St–Lb 21.79±0.27d 2.28±0.23a 4.27±0.21a

M St–La 20.35±0.24c 4.14±0.06bc 10.15±0.06d

M St–Lr 18.48±0.32b 4.50±0.32 cd 10.68±0.24e

M St–Bl 15.66±0.48a 5.17±0.07e 7.90±0.13b

SM St–Lb 22.14±0.34d 2.03±0.05a 4.15±0.12a

SM St–La 24.03±0.13e 3.74±0.14a 8.65±0.07c

SM St–Lr 19.74±0.43c 4.57±0.37cde 8.92±0.11c

SM St–Bl 17.51±0.53b 4.91±0.34de 7.47±0.19b

Abbreviations: Vmax =maximum acidification rate; tmax = time to reach Vmax; tpH4.5 =time to reach pH 4.5.Means (n=3)±standard deviation with different letters in the same line aresignificantly different (Pb0.05).

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were compared using the Tukey test at significance level (P)b0.05,and different letters were used to label values with statisticallysignificant differences among them.

3. Results and discussion

3.1. Acidification kinetics

Table 1 lists the acidification kinetics of milk, either in the presence(SM) or the absence (M) of 4% (w/w) lactulose, fermented byLactobacillus bulgaricus (St–Lb), L. acidophilus (St–La), L. rhamnosus(St–Lr) and B. lactis (St–Bl) in binary co-cultures with S. thermophilus.

Vmax ranged from 15.66·10−3 upH/min (St–Bl in M) to 24.03·10−3 upH/min (St–La in SM). In the absence of lactulose, the averageVmax value obtained with all the co-cultures (19.07·10−3 upH/min)was 15.6% higher than that previously obtained in completelyskimmed milk (Oliveira et al., 2009a), likely due to the presence of0.5% fat in the milk used in this work. It was also 17–55% higher thanin milk whey using St–La and St–Lr, respectively (Almeida et al.,2009). It should be noticed that the addition of 4% (w/w) lactuloseincreased the maximum rate of acidification in practically all co-cultures studied in this work, with the exception of the St–Lb co-culture for which no statistically significant variation was observed.

Taken as an average, Vmax was 8.5% higher in the presence oflactulose. Comparing this effectwith those recently observedwith otherprebiotics (oligofructose, polydextrose andmaltodextrin) and the samebinary co-cultures (Oliveira et al., 2009a), one can realize that thisdisaccharide accelerated the acidification less than oligofructose(+9.8%), but more either than polydextrose (+5.5%) or maltodextrin(+17.6%), which suggests some possible role exerted by fructose.Indeed, this suggestion is consistent with the well-known stimulatingeffect of inulin, another important fructose-based prebiotic, which was

Fig. 1. Post-acidification (pH) of skim milk fermented by co-cultures of L. bulgaricus, L. acid35 days of storage at 4 °C (D35). ■ = Milk without lactulose; = Milk supplemented wvalues of three different dilutions of triplicate fermentations.

shown to increase Vmax by 6.3% with respect to the same skimmedmilkwithout any supplement (Oliveira et al., 2009b).

As regards the effect of lactulose on the acidification rate of thedifferent co-cultures, the largest increase in this parameter comparedto the control (18%) was observed for St–La, with suggests thatL. acidophilus could have metabolized the fructose moiety of lactulosemore effectively than the other microorganisms, consistent with itsproved ability to ferment fructooligosaccharides (Barrangou et al.,2003). Another possibility is that some compound released byL. acidophilus could have stimulated themetabolism of S. thermophilus.

Consistent with these findings, the addition of lactulose in themilksignificantly reduced (by 10.7%) the time to reach Vmax (tmax) only inSt–La, while that to complete the fermentation (tpH4.5) was signifi-cantly reduced either in this co-culture (by 17.3%) or in the St–Lr one

ophilus, L. rhamnosus or B. lactis with S. thermophilus, after 1 day (D1), 7 days (D7) orith 4% (w/w) of lactulose. Error bars are standard deviations with respect to the mean

Fig. 2. Lactic acid acidity (mg/g of lactic acid) of skimmilk fermented by co-cultures of L. bulgaricus, L. acidophilus, L. rhamnosus or B. lactiswith S. thermophilus, after 1 day (D1), 7 days(D7) or 35 days of storage at 4 °C (D35).■= Milk without lactulose; = Milk supplemented with 4% (w/w) of lactulose. Error bars are standard deviations with respect to themean values of three different dilutions of triplicate fermentations.

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(by 19.7%). Taking into consideration this result, the generalized slowfermentation ability of Lr (Oliveira et al., 2009a,b) and the 6.8%increase in Vmax of St–Lr induced by lactulose, it is evident the interestof additionally exploring the potential of the Lr-lactulose combinationin yoghurt preparations.

Taken as an average, tmax, which ranged from 2.03 h (St–Lb in SM) to5.17 h (St–Bl in M), was about 8.5% shorter in the presence of lactulose,and an even higher reduction (11%) occurred in the mean time tocomplete the fermentations (tpH4.5). In summary, all these threeparameters depended not only on the addition of lactulose, but also onthe co-culture. Apparently, apart from St–Bl, there was a directrelationship between tmax and tpH4.5, either in the presence or the absenceof lactulose. Moreover, as expected, the shortest fermentation time(tpH4.5=4.15–4.27 h)was obtained in the presence of the typical yoghurtbacteria (St–Lb), with negligible influence of the presence of lactulose.

Following the same reasoning as for Vmax, one can see that thevalue of tmax in the presence of lactulose (3.81 h) was on average 23,21, 27 and 21% higher than in the presence of inulin, maltodextrin,oligofructose and polydextrose, respectively (Oliveira et al., 2009a).On the other hand, tpH4.5 was 10 and 5% shorter than with inulin andmaltodextrin, respectively, and 15 and 17% longer than witholigofructose and polydextrose, respectively. These results on thewhole demonstrate that, although lactulose guaranteed the slowestacidification rate among the tested prebiotics, it exerted an interme-diate effect on the whole duration of the fermentations.

3.2. Lactic acid acidity and post-acidification

The lactic acid acidity (acidity expressed as mg/g of lactic acid) andthe post-acidification (pHafter a given storage time) are thebest criteriato express the acidity of yoghurts (Souza, 1991). The same authorreported that the acidity of commercial yoghurts is largely variable,

ranging from 7.0 to 12.5 mg/g of lactic acid or pH 3.7–4.6. Nevertheless,to avoid insipidness or excess acidity to the taste, optimal values shouldbe in the ranges 7.0–9.0 mg/g and 4.0–4.4, respectively.

The results of post-acidification and lactic acid acidity of milkfermented by the selected bacteria in co-culture with S. thermophilusare shown in Figs. 1 and 2, respectively, either 1 day (D1), 7 days (D7)or 35 days (D35) of storage at 4 °C after the fermentation wascomplete. As expected, these parameters are complementary. More-over, comparing these values with the optimum ranges earliersuggested for post-acidification and lactic acid acidity, one can realizethat the optimum storage period at 4 °C should be around one week.

The overall effects were evaluated making reference to averagevalues gathering the results of all the co-cultures. An increase in thecold-storage time from 1 day to 35 days led to a statistically significantincrease in post-acidification, which passed, as an average, from 4.46(D1) to 4.31 (D7) and 4.16 (D35), corresponding to additionalacidification of 0.15 and 0.30 pH units, respectively. These results areconsistentwith the aciditydue to lactic acid thatprogressively increasedfrom 8.81 to 9.60 and 9.89 mg/g passing from D1 to D7 and D35.

The addition of lactulose as a prebiotic significantly influenced thepost-acidification only in 42% of the separate co-cultures; therefore, itwas once more evaluated taking all the co-cultures as a whole. On thisbasis, it was found that this parameter stayed practically unvaried afterD1anddecreased by only 4%afterD7 andD35 compared to the situationwithout lactulose. On the contrary, the correspondingvariations in lacticacid acidity were always statistically significant for all the co-cultures.These results aswell as those of post-acidification can be interpreted onthe basis of the metabolic behavior of the selected microorganisms.

Contrary to the well-known fact that heterofermentative bacteriaproduce less lactic acid than the homofermentative ones, the mostsignificant effects on lactic acid acidity (+3.4–4.1% and +3.3–3.8%)were observed with the co-cultures (St–Lr and St–Bl) containing

Fig. 3. Lactulose consumption by L. bulgaricus (■), L. acidophilus ( ), L. rhamnosus ( )and B. lactis ( ) in co-culture with S. thermophilus.

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bacteria of the former metabolic type (Bongaerts et al., 2005; Jyotiet al., 2004), almost irrespective of the storage time. This behavior waslikely due to a more effective consumption of lactose by Lr and Bl(results not shown) and, in the peculiar case of Bl, even of lactulose(Fig. 3). Although such a sugar is usually considered as a prebiotic, itdid in fact behave as an additional slowly-metabolizable carbonsource in this work, being partly consumed by all the tested co-cultures (by 23.9, 27.2, 20.3 and 40.6% after 6 h of fermentation by St–Lb, St–La, St–Lr and St–Bl, respectively).

Fig. 4. Effect of lactulose addition to skimmilk on the counts of L. bulgaricus, L. acidophilus, L. r35 days of storage at 4 °C (D35). ■ = Milk without lactulose; = Milk supplemented wvalues of three different dilutions of triplicate fermentations.

The values of pH after D1 were not so far from those reported byMoreira et al. (2000) (3.76–4.39), who analyzed samples of milkfermented by different strains of L. bulgaricus and S. thermophilus.Besides, Damin et al. (2006) detected average lactic acid concentra-tion in commercial yoghurts from full fat milk (10.1 mg/g) practicallycoincident with the average value obtained in this work after D35 inthe presence of lactulose. This comparison puts into evidence thepotential of such a sugar as an ingredient for the preparation offunctional foods.

3.3. Counts of viable cells

Fig. 4 shows the counts in fermented milks of the above lacticbacteria in co-cultures with S. thermophilus. After D1, the counts of S.thermophilus were, on average, about 9.1 log CFU/mL with nostatistically significant effect either of lactulose or the co-culture;afterwards, they progressively decreased to 8.2 (D7) and 7.2 log CFU/mL (D35) (results not shown). As shown in this figure, almost thesame occurred with the St–Lb co-culture. These results taken as awhole agree with those of Ozer et al. (2005), who did not observe anysignificant effect of lactulose on the growth of yoghurt starter bacteria.The same decreasing trend could be observed for the counts of theother bacteria, but in this case the addition of lactulose stimulatedsignificantly their growth. The most important effect dealt with Bl,whose counts increased by almost three orders of magnitude (from7.41 log CFU/mL in M to 10.3 log CFU/mL in SM after D1). Thisdramatic bifidogenic effect of lactulose, which stayed almost the samealso at longer storage times, was such that, even after D35, the countsof all the microorganisms were beyond the minimum value (7 logCFU/mL) recommended to exert probiotic effect (Mattila-Sandholm

hamnosus or B. lactis in co-cultures with S. thermophilus, after 1 day (D1), 7 days (D7) orith 4% (w/w) of lactulose. Error bars are standard deviations with respect to the mean

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et al., 2002; Ouwehand et al., 1999). This result, already highlightedfor the growth of B. bifidum BB-02 (Ozer et al., 2005), is an additionalconfirmation of the potential of lactulose as a prebiotic.

After D7, the stimulating effect of lactulose on the growth ofprobiotic bacteria was almost completely kept and, interestingly, thecounts of all of them did not decrease significantly compared to D1.Saarela et al. (2003) observed that lactulose improved the cold-storagestability of Lactobacillus salivarius at 4 °C for 22 days, probably due tosplitting of cell-chains. An enhancement by lactulose of β-glucosidaseandβ-galactosidase activities on intestinalmicrobiotawas also reported(Juskiewicz and Zdunczyk, 2002; Pham and Shah, 2008). In particular,stimulation of the latter activity could have been responsible for quickerhydrolysis of lactulose to galactose and fructose, and then for its ownmetabolization in the present work.

4. Conclusions

The lactulose, as a prebiotic, improves the quality of fermentedskim milk by typical co-cultures of the yoghurt and probioticscultures of L. acidophilus, L. rhamnosus and B. lactis in combinationwith S. thermophilus. The acidification rate (Vmax) was enhanced bylactulose addition to the skim milk, and decreased tmax and tpH4,5. Allthe probiotic bacteria exhibited better growth associated withlactulose metabolization. The addition of lactulose also favored thelactic acid acidity and post-acidification by all the tested co-cultures.After D7, the counts of all probiotics did not decrease significantly. Theviable counts of Bl in St–Bl co-culture with lactulose were significantlyhigher (Pb0.05) than those of the others microorganisms and abifidogenic effect was observed.

Acknowledgements

The authors acknowledge the financial support of FAPESP(Fundação de Amparo à Pesquisa do Estado de São Paulo) andCAPES (Coordenação de Aperfeiçoamento de Pessoal de NívelSuperior), Brazil, for the PhD fellowships of R.P.S. Oliveira.

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