Food and Nutrition Sciences, 2013, 4, 1301-1306
Published Online December 2013 (
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: *
Received November 3rd, 2013; revised December 3rd, 2013; accepted December 10th, 2013
Copyright © 2013 Mimoza Basholli-Salihu et al. This is an open access article distributed under the Creative Commons Attribution
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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·g1 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
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
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
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,
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.
where μ-specific growth rate is calculated using Equation
(2) as follows:
 
ln lnCCtt
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
N—number of viable cells after 4 weeks of storage
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
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.
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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-
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-
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The Use of Cellobiose and Fructooligosaccharide on Growth and Stability of Bifidobacterium infantis in Fermented Milk
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 -
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.
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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-
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FOS: Fructooligosaccharide
RCM: Reinforced Clostridial Medium
RCA: Reinforced Clostridial Agar