The Use of Cellobiose and Fructooligosaccharide on Growth and Stability of <i>Bifidobacterium infantis</i> in Fermented Milk

doi:10.4236/fns.2013.412167

Paper Menu >>

Journal Menu >>

Food and Nutrition Sciences, 2013, 4, 1301-1306

Published Online December 2013 (http://www.scirp.org/journal/fns)

http://dx.doi.org/10.4236/fns.2013.412167

Open Access FNS

The Use of Cellobiose and Fructooligosaccharide on

Growth and Stability of Bifidobacterium infantis in

Fermented Milk

Mimoza Basholli-Salihu1,2*, Monika Mueller1, Frank M. Unger1, Helmut Viernstein1

1Department of Pharmaceutical Technology and Biopharmaceutics, University of Vienna, Vienna, Austria; 2Department of Pharmacy,

Faculty of Medicine, University of Prishtina, Prishtina, Kosova.

Email: *[email protected]

Received November 3rd, 2013; revised December 3rd, 2013; accepted December 10th, 2013

License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ABSTRACT

The effects of cellobiose, fructooligosaccharide and their combination on fermentation of skim milk by probiotic Bifi-

dobacterium infantis were evaluated using mean doubling time as a parameter for sustaining growth. The lowest mean

doubling time was observed for 2% cellobiose, followed by a combination of 2% fructooligosaccharide (FOS) with 2%

cellobiose, while during storage at 4˚C for 4 weeks of fermented milk, no significant differences were observed between

fermented milk supplemented with 2% cellobiose and 2% FOS. The highest viability retention during storage was ob-

served for the combination of the two prebiotics, cellobiose and FOS. The results indicate that, in milk supplemented

with cellobiose or a combination of cellobiose and FOS, Bifidobacterium infantis remain viable during 4 weeks of stor-

age, suggesting the usefulness of cellobiose as a prebiotic ingredient in fermented products involving bifidobacteria.

Keywords: Bifidobacteria; Cellobiose; Synbiotic; Fermented Milk; Viability

1. Introduction

Dairy functional products containing probiotics, espe-

cially fermented milk, have attracted increased interest

for prevention of gastrointestinal disorders [1]. Con-

sumption of probiotic dairy products has been shown to

exert benefits to human health [2,3]. Probiotics are de-

fined as “live microorganisms which when administered

in sufficient numbers, confer a health benefit to the host”

[4]. Thus, probiotics (Lactobacillus spp. and Bifidobacte-

rium spp.) are more and more frequently used for pro-

duction of fermented dairy products. To provide potential

therapeutic benefits, probiotics should remain viable at

concentrations ≥6 log CFU·g−1 before consumption [5].

During production and storage of fermented dairy prod-

ucts, the probiotics are exposed to several stresses such

as low pH, presence of oxygen, cold and osmotic stress

[6-8].

The improvement of growth and viability in fermented

foods may be achieved by the use of growth enhancers

such as prebiotics [9-11].

Prebiotics are defined as “non-digestable carbohy-

drates that beneficially affect the host by selectively

stimulating the growth and/or activity of colonic micro-

flora” [12]. Several groups of oligosaccharides have been

used for improving the survival of probiotics [13-15]. In

addition to their effect on viability retention of probiotics,

prebiotics have been shown to increase the growth of

lactobacilli and bifidobacteria in the caecum and large

intestine. Furthermore, the prevention of travelers’ diar-

rhea, alleviation of irritable bowel syndrome symptoms

and reduction of risk factors for colon cancer have been

documented to be related to prebiotics intake [16-18].

Fructooligosaccharides are widely used as prebiotics

to improve the growth and viability of bifidobacteria in

dairy foods [8,9,19]. Recently, interest in new potential

prebiotics such as cellooligosaccharides has increased

[20,21].

Bioavailability of cellobiose in humans has been eval-

uated using cellobiose tolerance tests and breath hydro-

gen excretion [22]; it has been observed that after inges-

*Corresponding author.

The Use of Cellobiose and Fructooligosaccharide on Growth and Stability of Bifidobacterium infantis in Fermented Milk

1302

tion, cellobiose can be fermented by gut microflora. Fur-

thermore, the ingested cellobiose could not be hydro-

lyzed by the enzymes in the small intestine, reaching the

colon undigested [23-25].

Few studies have appeared on the effect of cellobiose

on growth rates of Bifidobacterium spp. [20]. According

to these reports, cellobiose has a higher prebiotic index

than FOS [26].

To the best of our knowledge, no study has been re-

ported on the effect of cellobiose on growth and viability

of probiotics in fermented milk.

The aim of the present study was to investigate the ef-

fects of cellobiose as food ingredient on the growth and

fermentation profiles of Bifidobacterium infantis UV16PR

in comparison to fructooligosaccharide as widely used

prebiotic. In addition, the effect of prebiotics alone or in

combination on cell viability retention during storage

was investigated.

2. Materials and Methods

2.1. Bacterial Growth and Culture Preparation

Bifidobacterium infantis (UV16PR) was kindly by Medi-

pharm (Kågeröd, Sweden). The cells were activated and

grown in Reinforced Clostridial Medium (RCM, Oxoid

Ltd., Hampshire, UK) by incubation at 37˚C in an an-

aerobic jar using Anaerogen kits (Oxoid Ltd., Hampshire,

UK).

2.2. Growth of Bifidobacterium infantis in Skim

Milk in the Presence of Glucose or Prebiotics

Cellobiose and FOS were used as prebiotics. Glucose

was used as reference. The prebiotics were added to 12%

(w/v) skim milk, reconstituted according to the manu-

facturer’s instructions, to give a final concentration of

2% (w/v). The supplemented reconstituted skim milk

(RSM) was divided in sterile tubes and pasteurized at

70˚C for 15 min, according to the method described ear-

lier [19]. Control samples did not contain glucose or pre-

biotics. A 5% inoculation of cells was used to determine

the growth. The inoculated samples were incubated un-

der anaerobic conditions in an anaerobic chamber using

Anaerogen kits at 37˚C for 48 h. For determination of

growth, aliquots of 1 ml were withdrawn immediately

after inoculation (time 0 h, baseline) and after 12 h, 24 h,

and 48 h. Growth was measured by enumeration of cells

using pour plate count methods. Aliquots of 1 ml were

serially diluted and then spread onto Reinforced Clos-

tridial Agar (RCA) plates, incubated anaerobically at

37˚C for 48 h - 72 h. The mean doubling time (Td) was

calculated using Equation (1) according to Shin et al. [19]

with minor modification.

Tln2



 (1)

where μ-specific growth rate is calculated using Equation

(2) as follows:



 

212

ln lnCCtt







(2)

where

C2—colony forming units per milliliter (CFU/ml) de-

termined at time t2;

C1—colony forming units per milliliter (CFU/ml) de-

termined at time t1.

2.3. Stability of Cells during Storage

Bifidobacterium infantis cells were cultured in RCM

supplemented with 2% cellobiose, 2% FOS or a combi-

nation of 2% cellobiose and 2% FOS; after 24 h fermen-

tation in anaerobic conditions, the samples were stored at

4˚C for 4 weeks. Aliquots of 1 ml were serially diluted

using 9 ml of sterile phosphate buffer. Viable cells were

determined after 24 h fermentation (before storage) and

after 4 weeks of storage, using plate count methods.

The viability during storage was expressed as percent,

calculated using Equation (3) as follows:





Viability %100N N

(3)

N—number of viable cells after 4 weeks of storage

(CFU/ml);

N0—number of viable cells before storage (CFU/ml).

2.4. pH Measurement

pH was determined before and after 24 h fermentation

and after 4 weeks of storage at 4˚C, using a pH meter

(Hanna Instruments, Woonsocket, RI, USA), calibrated

with fresh pH 4.0 and 7.0 standard buffers.

2.5. Statistical Analysis

All experiments were performed in triplicate. Statistical

analysis was performed using GraphPad Prism (Graph-

Pad Software, San-Diego, USA) by one-way ANOVA,

and Tukey test cross-comparing all study groups. Values

of p < 0.05 were considered significant.

3. Results and Discussion

3.1. Growth of Bifidobacterium infantis in Skim

Milk Supplemented with Glucose or

Prebiotics

The effectiveness of prebiotics toward growth promotion

of Bifidobacterium infantis was evaluated by measuring

mean doubling time. The mean doubling times (ex-

pressed in minutes) of Bifidobacterium infantis in RSM

containing 2% (w/v) cellobiose, 2% FOS, or a combina-

tion of 2% (w/v) cellobiose and 2% (w/v) FOS are shown

in Figure 1.

Open Access FNS

The Use of Cellobiose and Fructooligosaccharide on Growth and Stability of Bifidobacterium infantis in Fermented Milk 1303

Figure 1. Mean doubling times (in minutes) of Bifidobacte-

rium infantis grown in skim milk containing prebiotic s (cel-

lobiose and fructooligosaccharides-FOS) or glucose. Con-

trol represents samples without addition of any carbon

source. Each bar represent means ± standard deviation, n =

3. Determined by Tukey test cross-comparing all study groups.

Values of p < 0.05 were considered significant.

This is the first study to show the effectiveness of cel-

lobiose-supplemented milk on the growth of probiotics.

The fructooligosaccharides have been shown to reduce

the mean doubling times of several Bifidobacterium spp.

and Lactobacillus spp. [9,19,27].

In the present study, it was observed that in the pres-

ence of cellobiose, or FOS, or their combination the

mean doubling time was significantly lower compared to

the control (p < 0.001).

The 2% supplementation with cellobiose was shown to

be the most effective for growth of B. infantis in milk,

with a mean doubling time of 157.2 min, followed by a

combination of cellobiose and FOS with 163.85 min and

FOS with a mean doubling time of 183.66 min.

Mean doubling time in cellobiose supplemented milk

was significantly lower compared to FOS (p < 0.05), but

no significant difference was observed compared to the

combination of cellobiose and FOS (p > 0.05).

The effect of FOS and cellobiose on the growth of Bi-

fidobacterium spp. in milk during 48 h of fermentation

was shown in Figure 2. To the best of our knowledge,

the effect of cellobiose on the growth of Bifidobacterium

spp. in milk has not been studied so far. The cellobiose

and cellodextrins have been shown to increase the

growth of Bifidobacterium spp. to different extents in

growth medium [20,28].

Figure 2. Growth of Bifidobacterium infantis in skim milk

containing glucose, cellobiose, fructooligosaccharide (FOS),

or a combination of cellobiose and FOS, during 48 h of in-

cubation.

Even though the mean doubling time with cellobiose

was significantly lower compared to FOS, the differences

decreased during storage. This can be attributed to the

higher substrate preferences for cellobiose compared to

FOS which is in accordance to our previous work (data

not shown).

Thus, fermented milk with Bifidobacterium infantis in

the presence of cellobiose can be used as a functional

food product.

Differences in carbohydrate utilization can be attrib-

uted to differences in enzyme activities, responsible for

metabolizing the specific carbohydrates [20,29].

Similar results were reported earlier [16], who found

that carbohydrates with short chains are metabolized

faster than longer chain oligosaccharides.

The differences observed on the growth-promoting

properties of different carbohydrates on various probiot-

ics can be attributed to differences in transport systems

available, to the bacterial cells and to the presence and

localization of enzymes responsible for fermentation of

certain carbohydrates [30]. Furthermore the type of the

linkage between monomers and the degree of polymeri-

zation of the carbohydrate influence the growth of the

probiotics [31].

3.2. Viability of Cells during Storage in

Fermented Milk

The viability of Bifidobacterium infantis was decreased

during storage in fermented milk (Figure 3).

As shown in the figure, the presence of prebiotics sig-

nificantly increased the viability of cells during storage.

These results are in accordance to those previous re-

Open Access FNS

The Use of Cellobiose and Fructooligosaccharide on Growth and Stability of Bifidobacterium infantis in Fermented Milk

1304

Figure 3. Viability of Bifidobacterium infantis grown in skim

milk supplemented with glucose, cellobiose, fructooligosac-

charide (FOS) or a combination of cellobiose and FOS, af-

ter storage for 4 weeks at 4˚C. Each bar represent means ±

standard deviation, n = 3. Determined by Tukey test cross-

comparing all study groups. Values of p < 0.05 were con-

sidered significant.

ported [9]. The loss of viability during storage can be

attributed the presence of oxygen, low pH, cold, and os-

motic stresses [6].

In skim milk without prebiotics, the viability of cells

was 50%, which is not in accordance to the results shown

by [32] who observed an 83% loss of viability during 4

weeks of storage of Bifidobacterium infantis. Further-

more, studies reported earlierobserved 29.66% viability

of cells during storage of Bifidobacterium infantis [9].

These differences may be attributed to the different sub-

species of Bifidobacterium infantis used. In our study,

the selected strain of Bifidobacterium infantis was shown

to exert high β-galactosidase activity that is important for

utilization of the lactose present in milk.

These results suggest that the appropriate prebiotic for

a single probiotic strain depends on the enzyme activity

toward certain prebiotics.

No significant differences were observed in viability

during storage in the presence of 2% FOS or 2% cello-

biose (p > 0.05), namely 79.9% and 77.4%, respectively,

while significantly higher viability was observed in the

presence of the combination cellobiose + FOS (p < 0.01),

namely 87.6%.

Cellobiose was shown to be significantly more effec-

tive compared to FOS on the growth of B. infantis during

fermentation of skim milk, while no significant differ-

ences in viability during storage were observed. In the

present study, FOS was shown to be effectively enhanc-

ing the growth of B. infantis, but the utilization is slower

compared to cellobiose so that its effect is more evident

during storage. The rate of utilization of carbohydrates is

influenced by the degree of polymerization of oligosac-

charides and the type of linkage between monomer units

in the sugar [31].

Some of the differences in enzyme activities response-

ble for metabolizing the specific carbohydrates, leading

to differences in carbohydrate utilization, have been de-

scribed [20,29].

The pH values of the milk were determined before

fermentation, after 24 h of fermentation and after 4

weeks of storage of fermented milk at 4˚C. The pH val-

ues are shown in Figure 4.

Before fermentation the pH values ranged from 6.25 -

6.27.

During fermentation a strong decrease of pH was ob-

served, with values ranging from 4.39 - 4.59 due to the

formation of acetic and lactic acids which are the prod-

ucts of sugar metabolization [9,19]. The lower decrease

during the storage can be attributed to lower metabolic

activity of cells under the storage conditions (low pH and

4˚C), resulting in values from 4.09 - 4.29. Similar results

were observed on previous reported studies [9,19].

4. Conclusion

The presence of cellobiose and FOS supports the growth

of Bifidobacterium infantis in skim milk. Supplementa-

tion with cellobiose led to significantly lower mean dou-

bling times compared to controls, sucrose and FOS, while

Figure 4. The changes of pH value after fermentation and

storage at 4˚C of skim milk (SM) fermented by B. infantis,

in the presence of different carbon sources.

Open Access FNS

The Use of Cellobiose and Fructooligosaccharide on Growth and Stability of Bifidobacterium infantis in Fermented Milk 1305

during storage of fermented milk their effectiveness on

viability was shown to be comparable. Thus cellobiose

alone or in combination with FOS is suitable for syn-

biotic combinations with Bifidobacterium infantis for en-

hancement of quality of fermented milk.

5. Acknowledgements

The authors gratefully thank the Austrian Ministry of

Science and Technology (BMWF-Bertha von Suttner

program) for providing scholarship to Mimoza Bas-

holli-Salihu.

REFERENCES

[1] O. N. Donkor, S. L. I. Nilmini, P. Stolic, T. Vasiljevic

and N. P. Shah, “Survival and Activity of Selected Probi-

otic Organisms in Set-Type Yoghurt during Cold Stor-

age,” International Dairy Journal, Vol. 17, No. 6, 2007,

pp. 652-657.

http://dx.doi.org/10.1016/j.idairyj.2006.08.006

[2] M. De Vrese and P. R. Marteau, “Probiotics and Prebiot-

ics: Effects on Diarrhea,” Journal of Nutrition, Vol. 137,

No. 3, 2007, pp. 803S-811S.

[3] M. de Vrese, A. Stegelmann, B. Richter, S. Fenselau, C.

Laue and J. Schresenmeir, “Probiotics-Compensation for

Lactase Insufficiency,” American of Journal and Clinical

Nutrition, Vol. 73, No. 2, 2001, pp. 421-429.

[4] FAO/WHO, “Health and Nutritional Properties of Probi-

otics in Food including Powder Milk with Live Lactic

Acid Bacteria,” Report of a Joint FAO/WHO Expert Con-

sultation on Evaluation of Health and Nutritional Proper-

ties of Probiotics in Food Including Powder Milk with

Live Lactic Acid Bacteria, Córdoba, 1-4 October 2001.

http://www.who.int/foodsafety/publications/fs_managem

ent/en/probiotics.pdf

[5] D. Roy, “Technological Aspects Related to the Use of

Bifidobacteria in Dairy Products,” Lait, Vol. 85, No. 1-2,

2005, pp. 39-56. http://dx.doi.org/10.1051/lait:2004026

[6] D. Granato, G. F. Branco, A. G. Cruz, J. A. F. Faria and

N. P. Shah, “Probiotic Dairy Products as Functional Foods,”

Comprehensive Review of Food Science and Food Safety,

Vol. 9, No. 5, 2010 pp. 455-470.

http://dx.doi.org/10.1111/j.1541-4337.2010.00120.x

[7] R. P. S. Oliveira, P. Perego, M. N. Oliveira and A. Con-

verti, “Effect of Inulin as Prebiotic and Synbiotic Interac-

tions between Probiotics to Improve Fermented Milk

Firmness,” Journal of Food Engineering, Vol. 107, No. 1,

2011, pp. 36-40.

http://dx.doi.org/10.1016/j.jfoodeng.2011.06.005

[8] L. C. Maganha, R. E. Rosim, C. H. Corassin, A. G. Cruz,

J. A. F. Faria and C. A. F. Oliveira, “Viability of Probi-

otic Bacteria in Fermented Skim Milk Produced with

Different Levels of Milk Powder and Sugar,” Interna-

tional Journal of Dairy Technology, 2013.

http://dx.doi.org/10.1111/1471-0307.12087

[9] F. A. Bruno, W. E. Lankaputhra and N. P. Shah, “Growth,

Viability and Activity of Bifidobacterium spp. in Con-

taining Prebiotics,” Journal of Food Science, Vol. 67, No.

7, 2002, pp. 2740-2744.

http://dx.doi.org/10.1111/j.1365-2621.2002.tb08807.x

[10] B. M. Corcoran, R. P. Ross, G. F. Fitzgerald and C. Stan-

ton, “Comparative Survival of Probiotic Lactobacilli

Spray-Dried in the Presence of Prebiotic Substances,”

Journal of Applied Microbioloy, Vol. 96, No. 5, 2004, pp.

1024-1039.

http://dx.doi.org/10.1111/j.1365-2672.2004.02219.x

[11] P. Capela, T. K. C. Hay and N. P. Shah, “Effect of Cryo-

protectants, Prebiotics and Microencapsulation on Sur-

vival of Probiotic Organisms in Yoghurt and Freeze-

Dried Yoghurt,” Food Research International, Vol. 39,

No. 2, 2006, pp. 203-211.

http://dx.doi.org/10.1016/j.foodres.2005.07.007

[12] T. S. Manning and G. R. Gibson, “Prebiotics,” Best Prac-

tice and Research Clinical Gastroenterology, Vol. 18, No.

2, 2004, pp. 287-298.

http://dx.doi.org/10.1016/j.bpg.2003.10.008

[13] G. R. Gibson, H. M. Probert, J. Van Loo, R. A. Rastall

and M. B. Roberfroid, “Dietary Modulation of the Human

Colonic Microbiota: Updating the Concept of Prebiotics,”

Nutrition Research Reviews, Vol. 17, No. 2, 2004, pp.

259-275. http://dx.doi.org/10.1079/NRR200479

[14] M. Roberfroid, “Prebiotics: The Concept Revisited,” Jour-

nal of Nutrition, Vol. 137, 2007, pp. 830S-837S.

[15] R. G. Crittenden, L. F. Morris, M. L. Harvey, L. T. Tran,

H. L. Mitchell and M. J. Playne, “Selection of a Bifido-

bacterium Strain to Complement Resistant Starch in a

Symbiotic Yoghurt,” Journal of Applied Microbiology,

Vol. 90, No. 2, 2001, pp. 268-278.

http://dx.doi.org/10.1046/j.1365-2672.2001.01240.x

[16] J. H. Cummings, S. Christi and T. J. Cole, “A Study of

Fructooligosaccharides in the Prevention of Travellers’

Diarrhea,” Alimentary Pharmacology and Therapeutics,

Vol. 15, No. 8, 2001, pp. 1139-1145.

http://dx.doi.org/10.1046/j.1365-2036.2001.01043.x

[17] E. M. Quigley, “Prebiotics and Probiotics; Modifying and

Mining the Microbiota,” Pharmacological Research, Vol.

61, No. 3, 2010, pp. 213-218.

http://dx.doi.org/10.1016/j.phrs.2010.01.004

[18] E. M. Quigley, “Prebiotics and Probiotics: Their Role in

the Management of Gastrointestinal Disorders in Adults,”

Nutrition in Clinical Practice, Vol. 27, No. 2, 2012, pp.

487-492. http://dx.doi.org/10.1177/0884533611423926

[19] H.-S. Shin, J.-H. Lee, J. J. Pestka and Z. Ustunol,

“Growth and Viability of Commercial Bifidobacterium

spp in Skim Milk Containing Oligosaccharides and Inu-

lin,” Journal of Food Microbiology and Safety, Vol. 65,

No. 5, 2000, pp. 884-887.

[20] K. Pokusaeva, M. O’Connell-Motherway, A. Zomer, J.

MacSharry, G. F. Fitzgerald and D. van Sinderen, “Cel-

lodextrin Utilization by Bifidobacterium breve UCC2003,”

Applied and Environmental Microbiology, Vol. 77, No. 5,

2011, pp. 1681-1690.

http://dx.doi.org/10.1128/AEM.01786-10

[21] S.-K. Yeo and M.-T. Liong, “Effect of Prebiotics on Vi-

ability and Growth Characteristics of Probiotics in Soy-

milk,” Journal of the Science of Food and Agriculture,

Open Access FNS

The Use of Cellobiose and Fructooligosaccharide on Growth and Stability of Bifidobacterium infantis in Fermented Milk

Open Access FNS

1306

Vol. 90, No. 2, 2011, pp. 267-275.

http://dx.doi.org/10.1002/jsfa.3808

[22] S. Nakamura, T. Oku and M. Ichinose, “Bioavailability of

Cellobiose by Tolerance Test and Breath Hydrogen Ex-

cretion in Humans,” Nutrition, Vol. 20, No. 11, 2004, pp.

979-983. http://dx.doi.org/10.1016/j.nut.2004.08.005

[23] M. Kim and D. F. Day, “Optimization of Oligosaccharide

Synthesis from Cellobiose by Dextransucrase,” Applied

Biochemistry and Biotechnology, Vol. 148, No. 1-3, 2008,

pp. 189-198.

http://dx.doi.org/10.1007/s12010-007-8042-x

[24] M. Satouchi, T. Watanabe, S. Wakabayashi, K. Ohokuma,

T. Koshijima and M. Kuwahara, “Digestibility, Absorp-

tivity and Physiological Effects of Cellooligosaccharides

in Human and Rat,” Nippon Eiyo Shokuryo Gakkaishi,

Vol. 49, No. 3, 1996, pp. 143-148.

http://dx.doi.org/10.4327/jsnfs.49.143

[25] T. Watanabe, “Development of Physiological Functions

of Cellooligosaccharides,” Cellulose Communications, Vol.

5, No. 2, 1998, pp. 91-97.

[26] M. L. Sanz, G. R. Gibson and R. A. Rastall, “Influence of

Disaccharide Structure on Prebiotic Selectivity in Vitro,”

Journal of Agricultural and Food Chemistry, Vol. 53, No.

13, 2005, pp. 5192-5199.

http://dx.doi.org/10.1021/jf050276w

[27] A. R. Desai, I. B. Powell and N. P. Shah, “Survival and

Activity of Probiotics Lactobacilli in Skim Milk Con-

taining Prebiotics,” Journal of Food Microbiology and

Safety, Vol. 69, No. 3, 2004, pp. 57-60.

[28] L. Roncaglia, A. Amaretti, S. Raimondi, A. Leonardi and

M. Rossi, “Role of Bifidobacteria in the Activation of the

Lignin Secoisolariciresinol Diglucoside,” Applied Micro-

biology and Biotechnology, Vol. 92, No. 1, 2011, pp.

159-168. http://dx.doi.org/10.1007/s00253-011-3338-8

[29] S.-K. Yeo and M.-T. Liong, “Effect of Prebiotics on Vi-

ability and Growth Characteristics of Probiotics in Soy-

milk,” Journal of the Science of Food and Agriculture,

Vol. 90, No. 2, 2010, pp. 267-275.

http://dx.doi.org/10.1002/jsfa.3808

[30] C. L. Vernazza, G. R. Gibson and R. A. Rastall, “Carbo-

hydrate Preference, Acid Tolerance and Bile Tolerance in

Five Strains of Bifidobacterium,” Journal of Applied Mi-

crobiology, Vol. 100, No. 4, 2006, pp. 846-853.

http://dx.doi.org/10.1111/j.1365-2672.2006.02832.x

[31] R. A. Rastall, G. R. Gibson, H. S. Gill, F. Guarner, T. R.

Klaenhammer, B. Pot, G. Reid, I. R. Rowland and M. E.

Sanders, “Modulation of the Microbial Ecology of the

Human Colon by Probiotics, Prebiotics and Synbiotics to

Enhance Human Health: An Overview of Enabling Sci-

ence and Potential Applications,” FEMS Microbiology

Ecology, Vol. 52, No. 2, 2005, pp. 145-152.

http://dx.doi.org/10.1016/j.femsec.2005.01.003

[32] W. E. V. Lankaputhra, N. P. Shah and M. Britz, “Survival

of Bifidobacteria during Refrigerated Storage in Presence

of Acid and Hydrogen Peroxide,” Milchwissenschaft, Vol.

17, 1996, pp. 65-70.

Abbreviations

FOS: Fructooligosaccharide

RCM: Reinforced Clostridial Medium

RCA: Reinforced Clostridial Agar