International Journal of Food Microbiology 145 (2011) 2227

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International Journal of Food Microbiology

j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / i j f o o d m i c r o

Use of lactulose as prebiotic and its inuence on the growth, acidication prole and viable counts of different probiotics in fermented skim milk
Ricardo Pinheiro De Souza Oliveira a,b,, Ana Carolina Rodrigues Florence a, Patrizia Perego b, Maric Nogueira De Oliveira a, Attilio Converti b
a b

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

a r t i c l e

i n f o

a b s t r a c t
Lactulose can be considered as a prebiotic, which is able to stimulate healthy intestinal microora. In the present work, the use of this ingredient in fermented milk improved quality of skim milk fermented by Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus bulgaricus and Bidobacterium lactis in co-culture with Streptococcus thermophilus. Compared to control fermentations without lactulose, the addition of such a prebiotic in skim milk increased the counts of all probiotics, with particular concern to B. lactis (bidogenic effect), the acidication rate and the lactic acid acidity, and concurrently reduced the time to complete fermentation (tpH4.5) and the pH at the end of cold storage for 1 to 35 days. 2010 Elsevier B.V. All rights reserved.

Article history: Received 25 August 2010 Received in revised form 8 October 2010 Accepted 7 November 2010 Keywords: Probiotic Prebiotic Milk Yoghurt Lactulose Co-cultures

1. Introduction Signicant part of the world population suffers gastrointestinal diseases caused by pathogenic bacteria that invade the human intestine. A few days after the birth, the human intestine is colonized mainly by bidobacteria which play a very important role in the maintenance of a good health (Olguin et al., 2005). So, in order to solve this health problem, food industry and in particular dairy technology has developed dairy functional products enriched with probiotics like lactobacilli (Lactobacillus acidophilus, Lactobacillus casei, etc.) and bidobacteria (Donkor et al., 2007). The word probiotic was initially used as an antonym of the word antibiotic. It is derived from the Greek words and and translated as for life (Hamilton-Miller et al., 2003). In the past, different denitions of probiotics were given, but, following the recent recommendations of a working group on the evaluation of probiotics in food of FAO/ WHO (2002), probiotics are considered as live microorganisms that, when administered in adequate amounts, confer health benets on the host. Consequently, a wide variety of species and genera could be considered potential probiotics (Holzapfel et al., 1998); commercially, however, the most important strains are lactic acid bacteria (LAB).

Corresponding author. Biochemical and Pharmaceutical Technology Department, Faculty of Pharmaceutical Sciences, So Paulo University, Av Prof Lineu Prestes, 580, Bl 16, 05508-900, So Paulo, Brazil. Tel.: +39 0 10 3532593; fax: +39 0 10 3532586. E-mail address: rpsolive@usp.br (R.P. De Souza Oliveira). 0168-1605/$ see front matter 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2010.11.011

Lactic acid bacteria (LAB) are widely used in the production of fermented foods. Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus are traditionally used as starters for milk fermentation in yoghurt production. In addition to L. delbrueckii subsp. bulgaricus, which contributes to accelerate lactic acid development in yoghurt as well as to improve avor and textural properties (Curry and Crow, 2003), some other probiotics are used for this purpose, among which are Lactobacillus rhamnosus, L. acidophilus, Lactobacillus johnsonii and Bidobacterium lactis, because of their ability to grow in milk and to confer functional properties and benets to the health (Vasiljevic and Shah, 2008). The importance of certain technological and physiological characteristics of probiotic strains was recognized long time ago (Gordon et al., 1957). To achieve successful outcome of the lactobacilli therapy, the culture must have certain requirements: it should be a normal, nonpathogenic inhabitant of the intestine, capable of efcient gut colonization and present in substantially high concentrations in the product (107109 CFU/mL) (Mattila-Sandholm et al., 2002; Ouwehand, et al., 1999). The dairy industry has been quickly revitalized by the introduction of products characterized not only by their high nutritional value and pleasant taste, but also by their ability to exert positive effects on the consumer's health (Casiraghi et al., 2007). In this context, some nondigestible ingredients, called prebiotics, have received considerable attention, mainly because of their ability to selectively stimulate the growth and/or activity of probiotic bacteria in the colon (Gibson and Roberfroid, 1995; Huebner et al., 2007). However, the ability of

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probiotic microorganisms to use the prebiotics is strain and substrate specic (Shah, 2001). To be effective, prebiotics must escape digestion in the upper gastrointestinal tract and be used by a limited number of microorganisms comprising the colonic microora, mainly lactobacilli and bidobacteria (Gibson and Roberfroid, 1995; Isolauri, 2004); thus, in the later case they are referred to as bidogenic factors (Berg, 1998; Macfarlane and Cummings, 1999; Roberfroid, 2000). More rarely, they are reported to mitigate the virulence of pathogenic bacteria like Listeria monocytogenes (Park and Kroll, 1993). This family of compounds 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 and de Halleux, 2007), mainly as an effective drug against diseases like acute and chronic constipation (Tamura et al., 1993). Nevertheless, some promising applications are reported also in the nutraceuticals and food industries because of its benecial effects on human health (Donkor et al., 2007). It is a synthetic disaccharide obtained by isomerization of lactose, which is present in milk and whey in relatively high content, approximately 4.5% as an average (Zokaee et al., 2002), and contains fructose instead of glucose (Strohmaier, 1998). Since it is not absorbed in the small intestine, it has the potential to function as a prebiotic (Kontula et al., 1999). Moreover, several studies showed the effectiveness of lactulose to stimulate the growth of bidobacteria (Olano and Corzo, 2009; Sako et al., 1999; Shin et al., 2000). Taking into account all these considerations, lactulose appears as an important food ingredient that might be additionally explored for the production of functional foods, and one can expect its future large scale production for food and nutraceuticals purposes. To this purpose, the present study explores the effects of this ingredient on the acidication kinetics, post-acidication, growth and metabolism of binary co-cultures of L. bulgaricus, L. acidophilus, L. rhamnosus and B. lactis with S. thermophilus, as well on their survival during skim milk fermentation. 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) (yoghurt microorganisms); L. acidophilus LAC4 (La), L. rhamnosus LBA (Lr) and Bidobacterium 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 the presence (SM) or absence (M) of 4% (w/w) lactulose (trade name: Lactulose 61360) (Sigma Aldrich, Italy). The above solid content of milk corresponds to the average value reported by Restle et al. (2003) for integral cow milk, while the selected lactulose concentration was in the range (36% w/w) admitted by Brazilian legislation in yoghurts (ANVISA, 2002). Both milks were then thermally treated at 90 C for 5 min in water bath, model Grant Y6 (Cambridge, England). The heat treated milks were transferred to 1.0 L sterile asks, cooled in ice bath, distributed into 250-mL sterile Shott asks inside laminar ow chamber, 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 by dissolving 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 of milk. Bacterial counts in these pre-cultures ranged from 6.0 to 6.6 log CFU/mL. 2.4. Fermentations After inoculation, ask samples were transferred to a water bath, and batch fermentations were performed in triplicate at 42 C up to pH 4.5, which were selected as the conditions to stop the fermentation. Fermentations were monitored by pH determinations. 2.5. Post-acidication Once the fermentation of milk had been complete, post-acidication was determined after a) 1 day (D1), b) 7 days (D7) and c) 35 days of cold storage at 4 C (D35) by pH measurement using a pH meter, model pH 210 Microprocessor (Hanna instruments, Padova, Italy). 2.6. Counts of probiotic bacteria Bacterial counts were carried out in triplicate either after D1, D7 or D35. One mL of sample was diluted with 9 mL of 1 g/L sterile peptonated water. Afterwards, eight serial dilutions were done, and each bacterium was counted in the three most appropriate dilutions, applying the pour plate technique (Kodaka et al., 2005). Counts were nally presented as mean values. All media were obtained from Merck (Darmstadt, Germany). St colonies were counted in M17 agar (Oxoid) by aerobic incubation at 37 C for 48 h. Lb, La and Lr counts were carried out in MRS agar medium after pH adjustment to 5.4 by acetic acid addition and aerobic incubation at 37 C for 48, 72 and 72 h, respectively. Bl was counted in MRS Agar medium containing 50 g/L cysteine without any pH adjustment (IDF, 1996, 1997, 2003). 2.7. Kinetic parameters From the pH data collected during fermentation, the acidication rate (Vmax) was calculated as the time variation of pH (dpH/dt) and expressed as 103 pH units/min. During the incubation period, the following kinetic parameters were also calculated: (i) tmax (h), time at which 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 manually interrupted by aseptically agitating the clot by means of a stainless steel rod with perforated disks; the rod was gently moved upwards and downwards for 80 s. The fermented product was quickly cooled in an ice bath, and the fermented products were stored at 4 C. 2.8. Analytical methods A high-performance liquid chromatography, model 1100 (Hewlett Packard, Palo Alto, CA), was used for analysis of lactic acid (after D1, D7 and D35) and lactulose (every 1.5 h during the fermentation). The system consisted of a HP-1050 Intelligent Auto Sampler, a HP-1047A Refractive Index Detector (for organic acids), a HP-1050 UV Detector (for sugars) and a HP-1050 Pump. Separation was achieved using a Supelcogel H59304-U column (Sigma Aldrich, Bellefonte, PA) at 50 C with 0.01 M sulfuric acid as eluent at 0.4 mL/min. 2.9. Statistical analysis The experimental data of bacterial counts, post-acidication and lactic acid concentration, either after D1, D7 or D35, as well as those of lactulose concentration along the runs were presented as mean values. Variations with respect to the mean values were presented as standard deviations. Mean values of these parameters were submitted to analysis of variance (ANOVA) by the Statistica Software 6.0. They

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R.P. De Souza Oliveira et al. / International Journal of Food Microbiology 145 (2011) 2227 Table 1 Comparison of the acidication kinetic parameters in milk (M) and supplemented milk (SM) fermented by Lactobacillus bulgaricus (StLb), Lactobacillus acidophilus (StLa), Lactobacillus rhamnosus (StLr) and Bidobacterium lactis (StBl) in co-culture with Streptococcus thermophilus. Milk M M M M SM SM SM SM Co-culture StLb StLa StLr StBl StLb StLa StLr StBl Vmax (103 pH units/min) 21.79 0.27 20.35 0.24c 18.48 0.32b 15.66 0.48a 22.14 0.34d 24.03 0.13e 19.74 0.43c 17.51 0.53b
d

were compared using the Tukey test at signicance level (P) b 0.05, and different letters were used to label values with statistically signicant differences among them. 3. Results and discussion 3.1. Acidication kinetics Table 1 lists the acidication kinetics of milk, either in the presence (SM) or the absence (M) of 4% (w/w) lactulose, fermented by Lactobacillus bulgaricus (StLb), L. acidophilus (StLa), L. rhamnosus (StLr) and B. lactis (StBl) in binary co-cultures with S. thermophilus. Vmax ranged from 15.66 103 upH/min (StBl in M) to 24.03 103 upH/min (StLa in SM). In the absence of lactulose, the average Vmax value obtained with all the co-cultures (19.07 103 upH/min) was 15.6% higher than that previously obtained in completely skimmed milk (Oliveira et al., 2009a), likely due to the presence of 0.5% fat in the milk used in this work. It was also 1755% higher than in milk whey using StLa and StLr, respectively (Almeida et al., 2009). It should be noticed that the addition of 4% (w/w) lactulose increased the maximum rate of acidication in practically all cocultures studied in this work, with the exception of the StLb coculture for which no statistically signicant variation was observed. Taken as an average, Vmax was 8.5% higher in the presence of lactulose. Comparing this effect with those recently observed with other prebiotics (oligofructose, polydextrose and maltodextrin) and the same binary co-cultures (Oliveira et al., 2009a), one can realize that this disaccharide accelerated the acidication 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 stimulating effect of inulin, another important fructose-based prebiotic, which was

tmax (h) 2.28 0.23 4.14 0.06bc 4.50 0.32 cd 5.17 0.07e 2.03 0.05a 3.74 0.14a 4.57 0.37cde 4.91 0.34de
a

tpH4.5 (h) 4.27 0.21a 10.15 0.06d 10.68 0.24e 7.90 0.13b 4.15 0.12a 8.65 0.07c 8.92 0.11c 7.47 0.19b

Abbreviations: Vmax = maximum acidication 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 are signicantly different (P b 0.05).

shown to increase Vmax by 6.3% with respect to the same skimmed milk without any supplement (Oliveira et al., 2009b). As regards the effect of lactulose on the acidication rate of the different co-cultures, the largest increase in this parameter compared to the control (18%) was observed for StLa, with suggests that L. acidophilus could have metabolized the fructose moiety of lactulose more effectively than the other microorganisms, consistent with its proved ability to ferment fructooligosaccharides (Barrangou et al., 2003). Another possibility is that some compound released by L. acidophilus could have stimulated the metabolism of S. thermophilus. Consistent with these ndings, the addition of lactulose in the milk signicantly reduced (by 10.7%) the time to reach Vmax (tmax) only in StLa, while that to complete the fermentation (tpH4.5) was signicantly reduced either in this co-culture (by 17.3%) or in the StLr one

Fig. 1. Post-acidication (pH) of skim milk fermented by co-cultures of L. bulgaricus, L. acidophilus, L. rhamnosus or B. lactis with S. thermophilus, after 1 day (D1), 7 days (D7) or = Milk supplemented with 4% (w/w) of lactulose. Error bars are standard deviations with respect to the mean 35 days of storage at 4 C (D35). = Milk without lactulose; values of three different dilutions of triplicate fermentations.

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

(by 19.7%). Taking into consideration this result, the generalized slow fermentation ability of Lr (Oliveira et al., 2009a,b) and the 6.8% increase in Vmax of StLr induced by lactulose, it is evident the interest of additionally exploring the potential of the Lr-lactulose combination in yoghurt preparations. Taken as an average, tmax, which ranged from 2.03 h (StLb in SM) to 5.17 h (StBl in M), was about 8.5% shorter in the presence of lactulose, and an even higher reduction (11%) occurred in the mean time to complete the fermentations (tpH4.5). In summary, all these three parameters depended not only on the addition of lactulose, but also on the co-culture. Apparently, apart from StBl, there was a direct relationship between tmax and tpH4.5, either in the presence or the absence of lactulose. Moreover, as expected, the shortest fermentation time (tpH4.5 =4.154.27 h) was obtained in the presence of the typical yoghurt bacteria (StLb), with negligible inuence of the presence of lactulose. Following the same reasoning as for Vmax, one can see that the value 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 and maltodextrin, respectively, and 15 and 17% longer than with oligofructose and polydextrose, respectively. These results on the whole demonstrate that, although lactulose guaranteed the slowest acidication rate among the tested prebiotics, it exerted an intermediate effect on the whole duration of the fermentations. 3.2. Lactic acid acidity and post-acidication The lactic acid acidity (acidity expressed as mg/g of lactic acid) and the post-acidication (pH after a given storage time) are the best criteria to express the acidity of yoghurts (Souza, 1991). The same author reported that the acidity of commercial yoghurts is largely variable,

ranging from 7.0 to 12.5 mg/g of lactic acid or pH 3.74.6. Nevertheless, to avoid insipidness or excess acidity to the taste, optimal values should be in the ranges 7.09.0 mg/g and 4.04.4, respectively. The results of post-acidication and lactic acid acidity of milk fermented by the selected bacteria in co-culture with S. thermophilus are 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 was complete. As expected, these parameters are complementary. Moreover, comparing these values with the optimum ranges earlier suggested for post-acidication and lactic acid acidity, one can realize that the optimum storage period at 4 C should be around one week. The overall effects were evaluated making reference to average values gathering the results of all the co-cultures. An increase in the cold-storage time from 1 day to 35 days led to a statistically signicant increase in post-acidication, which passed, as an average, from 4.46 (D1) to 4.31 (D7) and 4.16 (D35), corresponding to additional acidication of 0.15 and 0.30 pH units, respectively. These results are consistent with the acidity due to lactic acid that progressively increased from 8.81 to 9.60 and 9.89 mg/g passing from D1 to D7 and D35. The addition of lactulose as a prebiotic signicantly inuenced the post-acidication only in 42% of the separate co-cultures; therefore, it was once more evaluated taking all the co-cultures as a whole. On this basis, it was found that this parameter stayed practically unvaried after D1 and decreased by only 4% after D7 and D35 compared to the situation without lactulose. On the contrary, the corresponding variations in lactic acid acidity were always statistically signicant for all the co-cultures. These results as well as those of post-acidication can be interpreted on the basis of the metabolic behavior of the selected microorganisms. Contrary to the well-known fact that heterofermentative bacteria produce less lactic acid than the homofermentative ones, the most signicant effects on lactic acid acidity (+3.44.1% and + 3.33.8%) were observed with the co-cultures (StLr and StBl) containing

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The values of pH after D1 were not so far from those reported by Moreira et al. (2000) (3.764.39), who analyzed samples of milk fermented by different strains of L. bulgaricus and S. thermophilus. Besides, Damin et al. (2006) detected average lactic acid concentration in commercial yoghurts from full fat milk (10.1 mg/g) practically coincident with the average value obtained in this work after D35 in the presence of lactulose. This comparison puts into evidence the potential of such a sugar as an ingredient for the preparation of functional foods. 3.3. Counts of viable cells Fig. 4 shows the counts in fermented milks of the above lactic bacteria in co-cultures with S. thermophilus. After D1, the counts of S. thermophilus were, on average, about 9.1 log CFU/mL with no statistically signicant 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 gure, almost the same occurred with the StLb co-culture. These results taken as a whole agree with those of Ozer et al. (2005), who did not observe any signicant effect of lactulose on the growth of yoghurt starter bacteria. The same decreasing trend could be observed for the counts of the other bacteria, but in this case the addition of lactulose stimulated signicantly their growth. The most important effect dealt with Bl, whose counts increased by almost three orders of magnitude (from 7.41 log CFU/mL in M to 10.3 log CFU/mL in SM after D1). This dramatic bidogenic effect of lactulose, which stayed almost the same also at longer storage times, was such that, even after D35, the counts of all the microorganisms were beyond the minimum value (7 log CFU/mL) recommended to exert probiotic effect (Mattila-Sandholm

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

), L. rhamnosus (

bacteria of the former metabolic type (Bongaerts et al., 2005; Jyoti et al., 2004), almost irrespective of the storage time. This behavior was likely 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, it did in fact behave as an additional slowly-metabolizable carbon source in this work, being partly consumed by all the tested cocultures (by 23.9, 27.2, 20.3 and 40.6% after 6 h of fermentation by St Lb, StLa, StLr and StBl, respectively).

Fig. 4. Effect of lactulose addition to skim milk on the counts of L. bulgaricus, L. acidophilus, L. rhamnosus or B. lactis in co-cultures with S. thermophilus, after 1 day (D1), 7 days (D7) or = Milk supplemented with 4% (w/w) of lactulose. Error bars are standard deviations with respect to the mean 35 days of storage at 4 C (D35). = Milk without lactulose; values of three different dilutions of triplicate fermentations.

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et al., 2002; Ouwehand et al., 1999). This result, already highlighted for the growth of B. bidum BB-02 (Ozer et al., 2005), is an additional conrmation of the potential of lactulose as a prebiotic. After D7, the stimulating effect of lactulose on the growth of probiotic bacteria was almost completely kept and, interestingly, the counts of all of them did not decrease signicantly compared to D1. Saarela et al. (2003) observed that lactulose improved the cold-storage stability of Lactobacillus salivarius at 4 C for 22 days, probably due to splitting of cell-chains. An enhancement by lactulose of -glucosidase and -galactosidase activities on intestinal microbiota was also reported (Juskiewicz and Zdunczyk, 2002; Pham and Shah, 2008). In particular, stimulation of the latter activity could have been responsible for quicker hydrolysis of lactulose to galactose and fructose, and then for its own metabolization in the present work. 4. Conclusions The lactulose, as a prebiotic, improves the quality of fermented skim milk by typical co-cultures of the yoghurt and probiotics cultures of L. acidophilus, L. rhamnosus and B. lactis in combination with S. thermophilus. The acidication rate (Vmax) was enhanced by lactulose addition to the skim milk, and decreased tmax and tpH4,5. All the probiotic bacteria exhibited better growth associated with lactulose metabolization. The addition of lactulose also favored the lactic acid acidity and post-acidication by all the tested co-cultures. After D7, the counts of all probiotics did not decrease signicantly. The viable counts of Bl in StBl co-culture with lactulose were signicantly higher (P b 0.05) than those of the others microorganisms and a bidogenic effect was observed. Acknowledgements The authors acknowledge the nancial support of FAPESP (Fundao de Amparo Pesquisa do Estado de So Paulo) and CAPES (Coordenao de Aperfeioamento de Pessoal de Nvel Superior), Brazil, for the PhD fellowships of R.P.S. Oliveira. References
Agncia Nacional de Vigilncia Sanitria - ANVISA. Legislao. VisaLegis. Resoluo RDC n.2, de 7 de janeiro de 2002. Aprova o regulamento tcnico de substncias bioativas e probiticos isolados com alegao de propriedades funcional e ou de sade. Diario Ocial da Unio, Brasilia, January 9th and July 17th, 2002. Aider, M., de Halleux, D., 2007. Isomerization of lactose and lactulose production: review. Trends in Food Science & Technology 18, 356364. Almeida, K.E., Tamime, A.Y., Oliveira, M.N., 2009. Inuence of total solids contents of milk whey on the acidifying prole and viability of various lactic acid bacteria. LWT - Food Science and Technology 42, 672678. Barrangou, R., Altermann, E., Hutkins, R., Cano, R., Klaenhammer, T.R., 2003. Functional and comparative genome analyses of an operon involved in fructooligosaccharide utilization by Lactobacillus acidophilus. Proceedings of the National Academy of Sciences of the United States of America 100, 89578962. Berg, R.D., 1998. Probiotics, prebiotics or conbiotics. Trends in Microbiology 6, 8992. Bongaerts, G., Severijnenb, R., Timmerman, H., 2005. Effect of antibiotics, prebiotics and probiotics in treatment for hepatic encephalopathy. Medical Hypotheses 64, 6468. Casiraghi, M.C., Canzi, E., Zanchi, R., Donati, E., Villa, L., 2007. Effects of a synbiotic milk product on human intestinal ecosystem. Journal of Applied Microbiology 103, 499506. Curry, B., Crow, V., 2003. Lactobacillus spp general characteristics. In: Roginski, H., Fuquay, J., Fox, P. (Eds.), Encyclopedia of dairy science. Academic Press Elsevier Science, Cornwall, UK, pp. 14791511. Damin, M.R., Almeida, K.E., Minowa, E., Oliveira, M.N., 2006. Propriedades fsico-qumicas e viabilidade de Streptococcus salivarius subsp. termophillus e Lactobacillus bulgaricus em diferentes marcas de iogurtes comerciais no perodo nal da vida-de-prateleira. Revista Leite e Derivados 94, 2030. Donkor, O.N., Nilmini, S.L.I., Stolic, P., Vasiljevic, T., Shah, N.P., 2007. Survival and activity of selected probiotic organisms in set type yoghurt during cold storage. International Dairy Journal 17, 657665. Fric, P., 2007. Probiotics and prebiotics renaissance of a therapeutic principle. Central European Journal of Medicine 2, 237270. Gibson, G.R., Roberfroid, M.B., 1995. Dietary modulation of the human colonic microbiota: introducing the concept of prebiotics. Journal of Nutrition 125, 14011412. Gordon, D., Macrae, J., Wheater, D.M., 1957. A Lactobacillus preparation for use with antibiotics. The Lancet 269, 899901.

Hamilton-Miller, J.M.T., Gibson, G.R., Bruck, W., 2003. Some insight into the derivation and early uses of the word probiotic. British Journal of Nutrition 90, 845. Holzapfel, W.H., Haberer, P., Snel, J., Schillinger, U., Huisin't Veld, J.H.J., 1998. Overview of gut ora and probiotics. International Journal of Food Microbiology 41, 85101. Huebner, J., Wehling, R.L., Hutkins, R.W., 2007. Functional activity of commercial prebiotics. International Dairy Journal 17, 770775. IDF, 1996. Preparation of samples and dilutions for microbiological examination, Standard N. 122C. Brussels: International Dairy Federation. IDF, 1997. Dairy starter cultures of lactic acid bacteria (LAB), Standard of Identity, Standard N. 149A. Brussels: International Dairy Federation. IDF, 2003. Yoghurt/Enumeration of Characteristic Microorganisms, Colony Count Technique at 37C, Standard N. 117. Brussels: International Dairy Federation. Isolauri, E., 2004. The role of probiotics in paediatrics. Current Paediatrics 14, 104109. FAO/WHO, Guidelines for the evaluation of probiotics in food, Joint FAO/WHO working group report on drafting guidelines for the evaluation of probiotics in food, London, ON, Canada, April 30th and May 1st, 2002, HYPERLINK "http://ftp://ftp.fao.org/es/ esn/food/wgreport2.pdf" ftp://ftp.fao.org/es/esn/food/wgreport2.pdf, accessed November 22nd, 2010. Juskiewicz, J., Zdunczyk, Z., 2002. Lactulose-induced diarrhoea in rats: effects on caecal development and activities of microbial enzymes. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology 133, 411417. Jyoti, B.D., Suresh, A.K., Venkatesh, K.V., 2004. Effect of preculturing conditions on growth of Lactobacillus rhamnosus on medium containing glucose and citrate. Microbiological Research 159, 3542. Kodaka, H., Mizuochi, S., Teramura, H., Nirazuka, T., 2005. Comparison of the compact dry TC method with the standard pour plate method (AOAC Ofcial Method 966.23) for determining aerobic colony counts in food samples: performancetested methodSM. Journal of AOAC International 88, 17021713. Kontula, P., Suihko, M.L., von Wright, A., Mattila-Sandholm, T., 1999. The effect of lactose derivatives on intestinal lactic acid bacteria. Journal of Dairy Science 82, 249256. Macfarlane, G.T., Cummings, J.H., 1999. Probiotics and prebiotics: can regulating the activities of intestinal bacteria benet health. British Medical Journal 318, 9991003. Mattila-Sandholm, T., Myllrinen, P., Crittenden, R., Fondn, R., Saarela, M., 2002. Technological challenges for future probiotic foods. International Dairy Journal 12, 173182. Moreira, M., Abraham, A., Antoni, G., 2000. Technological properties of milks fermented with thermophilic lactic acid bacteria at suboptimal temperature. Journal of Dairy Science 83, 395400. Olano, A., Corzo, N., 2009. Lactulose as a food ingredient. Journal of the Science of Food and Agriculture 89, 19871990. Olguin, F., Araya, M., Hirsch, S., Brunser, O., Ayala, V., Rivera, R., Gotteland, M., 2005. Prebiotic ingestion does not improve gastrointestinal barrier function in burn patients. Burns 31, 482488. Oliveira, R.P.S., Florence, A.C.R., Silva, R.C., Perego, P., Converti, A., Gioielli, L.A., Oliveira, M.N., 2009a. Effect of different prebiotics on the fermentation kinetics, probiotic survival and fatty acids proles in nonfat symbiotic fermented milk. International Journal of Food Microbiology 128, 467472. Oliveira, R.P.S., Perego, P., Converti, A., Oliveira, M.N., 2009b. Effect of inulin on growth and acidication performance of different probiotic bacteria in co-cultures and mixed culture with Streptococcus thermophilus. Journal of Food Engineering 91, 133139. Ouwehand, A.C., Kirjavainen, P.V., Shortt, C., Salminen, S., 1999. Probiotics: mechanisms and established effects. International Dairy Journal 9, 4352. Ozer, D., Akn, M.S., Ozer, B., 2005. Effect of inulin and lactulose on survival of Lactobacillus acidophilus LA-5 and Bidobacterium bidum BB-02 in acidophilus-bidus yoghurt. Food Science and Technology International 11, 1924. Park, S.F., Kroll, R.G., 1993. Expression of lysteriolysin and phosphatidylinositol-specic phospholipase C is repressed by the plant-derived molecule cellobiose in Listeria monocytogenes. Molecular Microbiology 8, 653661. Pham, T.T., Shah, N.P., 2008. Effects of lactulose supplementation on the growth of bidobacteria and biotransformation of isoavone glycosides to isoavone aglycones in soymilk. Journal of Agricultural and Food Chemistry 56, 47034709. Restle, J., Pacheco, P.S., Moletta, J.L., 2003. Genetic group and postpartum nutritional level on the milk yield and composition of beef cows. Revista Brasileira de Zootecnia 32, 585597. Roberfroid, M.B., 2000. Prebiotics and probiotics: are they functional foods. American Journal of Clinical Nutrition 71, 16821687. Saarela, M., Hallamaa, K., Mattila-Sandholm, T., Mtt, J., 2003. The effect of lactose derivatives lactulose, lactitol and lactobionic acid on the functional and technological properties of potentially probiotic Lactobacillus strains. International Dairy Journal 13, 291302. Sako, T., Matsumoto, K., Tanaka, R., 1999. Recent progress on research and applications of non-digestible galacto-oligosaccharides. International Dairy Journal 9, 6980. Shah, N.P., 2001. Functional foods, probiotics and prebiotics. Food Technology 55, 4653. Shin, H.S., Lee, J.H., Pestka, J.J., Ustunol, Z., 2000. Growth and viability of commercial Bidobacterium spp. in skim milk containing oligosaccharides and inulin. Journal of Food Science 65, 885887. Souza, G., 1991. Fatores de Qualidade do Iogurte. Coletnea do Instituto de Tecnologia de Alimentos 21, 2027. Strohmaier, W., 1998. Lactulose: status of health-related applications. International Dairy Federation 262271 Bulletin no. 9804. Tamura, Y., Mizota, T., Shimamura, S., Tomita, M., 1993. Lactulose and its application to the food and pharmaceutical industries. International Dairy Federation Bulletin 289, 4353. Vasiljevic, T., Shah, N.P., 2008. Probioticsfrom Metchnikoff to bioactives. International Dairy Journal 18, 714728. Zokaee, F., Kaghazchi, T., Zare, A., Soleimani, M., 2002. Isomerization of lactose to lactulosestudy and comparison of three catalytic systems. Process Biochemistry 37, 629635.