Research Article

Received: 7 April 2015 Revised: 25 May 2015 Accepted article published: 29 May 2015 Published online in Wiley Online Library: 22 June 2015

(wileyonlinelibrary.com) DOI 10.1002/jsfa.7278

Effect of immobilisation materials on viability

and fermentation activity of dairy starter
culture in whey-based substrate
Tanja Ž Krunić,a* Maja LJ Bulatović,b Nataša S Obradović,a
Maja S Vukašinović-Sekulićb and Marica B Rakinb

Abstract
BACKGROUND: The main objectives of the paper were to study influence of immobilisation of dairy starter culture ‘Lactoferm
ABY 6’ on fermentation and probiotic potential of fermented whey-based substrate.

RESULTS: Fermentation with free cells takes 1.5 h less than fermentation with encapsulated cells, but samples with encapsulated
cells have better characteristics after 28 days of storage. Chitosan coating provides additional protection of cells in bile salt
solution (95.86% of viable cells compared to the initial number) and simulated gastric juice (37.8% for pH 2.5) compared to the
alginate beads (94.54% in bile salt solution and 36.18% in simulated gastric juice for pH 2.5). Free cells had a drastic reduction
in the number of viable cells (83.0% in bile salt solution and no viable cells in simulated gastric juice for pH 2.5).

CONCLUSION: Samples with alginate beads and chitosan-coated alginate beads have significantly (P < 0.05) higher viable cell
count than samples with free cells, during 4 h monitoring survival at pH 2.5, pH 3.0 and 0.3% bovine bile solution. These beads
can be used to improve survival of probiotic cells in fermented whey-based beverage during storage and consummation, which
improves the quality of the product.
© 2015 Society of Chemical Industry

Keywords: whey; fermentation; immobilisation; probiotic

INTRODUCTION The use of immobilised lactic acid bacteria of starter cultures in

Whey represents a very good substrate for use in a various biotech-
nological processes. In recent years, the bioconversion of whey
has become an interesting process that achieves the reduction of
environmental pollution with the production of microbial biomass
and metabolites. Fermentation of whey by probiotic starter cul-
tures designed for yoghurt production could be an alternative for
increasing the amount of exploited whey and incorporation of this
by product in human nutrition. Since whey is not a natural habi-
tat for lactic acid bacteria (LAB), they have significantly less activity
in whey in comparison to milk. Reduced activity is explained by
the fact that whey is composed of a significantly lower content of
nutrients necessary for the growth of LAB which demands a highly
nutritionally rich medium.1
In the recent decade there has been an explosion of probiotic
health-based products. Probiotics have been consumed in foods
such as yoghurt for perhaps thousands of years. Members of the
genera Lactobacillus and Bifidobacterium have a long and safe
history in the manufacture of dairy products.2 After applications of
probiotics in food such as milk and dairy products, new application
of probiotics has been proposed: chocolate, bread, fermented
vegetables. The self life of probiotics should be controlled in
order to manufacture products with satisfactory number of live
bacteria (at least 107 CFU g−1 ) to obtain health benefits of probiotic
cultures.3 Unfortunately, many studies indicated that there is poor
the conventional and especially non-conventional dairy products
is one of the applications for the next decade. The effect of pro-
tection by immobilisation varies depending on the conditions of
the study, such as natural resistance of the microorganism, differ-
ent pH values and different wall materials.4 Cell immobilisation
confers protection to sensitive probiotic lactic acid bacteria from
oxygen,5 freezing6 and acidic conditions during manufacture
and storage7 and gastrointestinal transit.8 In the dairy industry,
immobilisation has been applied to improve survival and deliv-
ery of bacterial cultures.9 The most widely used encapsulating
material is alginate. Sodium alginate may be ionically cross-linked
with multivalent cations to form gels. The use of alginate is lim-
ited due to its low stability and fast degradation in the presence
of chelating agents. Polycations such as chitosan form strong
complexes with alginates. Chitosan is biodegradable and biocom-
patible. Due the possibility of a negative impact in the viability
∗ Correspondence to: Tanja Ž Krunić, Innovation Center Faculty of Technology
and Metallurgy, University of Belgrade, Karnegijeva 4, 11000 Belgrade, Serbia.
E-mail: tkrunic@tmf.bg.ac.rs
a Innovation Center Faculty of Technology and Metallurgy, University of Bel-
grade, 11000 Belgrade, Karnegijeva 4, Serbia
b Faculty of Technology and Metallurgy, University of Belgrade, 11000 Belgrade,
1723

survival of probiotic bacteria in these products. Karnegijeva 4, Serbia

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 www.soci.org
of bacteria, because of antibacterial activity of this polymer and
because chitosan has a very good film-forming ability, usually
chitosan is used as external shell in capsules made with anionic
polymers as alginate.9 Coating alginate beads with chitosan can
improve their chemical and mechanical stability, survival of the
probiotic bacteria during storage and in the gastrointestinal
tract.10
This work is a continuation of the study about the application
of whey as functional fermented beverage. The present study
examined fermentative activity, viability and survival in simulated
gastrointestinal conditions and viability during storage by free
and immobilised probiotic starter culture ABY-6 in whey substrate
enriched with milk. Therefore, the aim of this work was to com-
pare the studied parameters for free and immobilised cells and
influence of wall materials of beads (alginate and chitosan-coated
alginate beads) on viability of immobilised probiotic cells, with
the objective of improving the quality and durability of the
whey-based product.
MATERIALS AND METHODS
Culture and media
Commercial lyophilised dairy starter culture (Lactoferm ABY 6;
Biochem s.r.l. Monterotondo, Rome, Italy) used in this work is
mixture of Streptococcus salivarius subsp. thermophilus (80%),
Lactobacillus acidophilus (13%), Bifidobacterium bifidum (6%),
Lactobacillus delbrueckii subsp. bulgaricus (1%). This starter culture
is currently used in the dairy industry. The culture was maintained
according to the manufacturer’s instructions at −18 ∘ C until use
(no longer than 20 months).
Whey and sterile skim milk was obtained from domes-
tic dairy plant Imlek a.d. (Belgrade, Serbia). After collection,
whey was stored at −18 ∘ C until use. The chemical compo-
sition of whey was: total solids 9.8 ± 0.03% (w/v); protein
2.6 ± 0.012% (w/v); fat 1.05 ± 0.08% (w/v) and lactose 5.6 ± 0.114%
(w/v). The chemical composition of milk was: fat 0.5% (w/v),
proteins 2.9% (w/v), carbohydrates 4.7% (w/v) and calcium
0.12% (w/v).
Immobilisation
Alginate beads were produced using an electrostatic extrusion
technique. Sodium alginate (Sigma–Aldrich, St Louis, MO, USA)
were dissolved in distilled water and pasteurised at 70 ∘ C for
30 min. A 1% (w/v) ABY-6 starter culture diluted in 100 mL of
whey was mixed with 150 mL of the sodium alginate solution.
Two hundred and fifty millilitres of the alginate–cell suspen-
sion containing 1.60% sodium alginate was added dropwise by
syringe pump (Racel; Scientific Instruments, Stamford, CT, USA)
to the solution of 2% (w/v) CaCl2 (Acros Organics, Thermo Fisher
Scientific, NJ, USA). Alginate drops solidified upon contact with
CaCl2 , formed beads and thus entrapped bacterial cells. The
beads were allowed to harden in gelling solution for 30 min.
The beads were washed with sterile physiological solution (0.85%
NaCl) to remove excess calcium ions and unimmobilised cells. The
beads were stored at 4 ± 1 ∘ C in yeast extract solution (0.2% w/v)
until use.
Coating drops
Coating procedures were performed using low-molecular-weight
chitosan (Acros Organics) and followed the methods reported by
1724
Zhou et al.10
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TŽ Krunić et al.
Fermentation
Experiments were conducted in 100-mL Erlenmeyer flasks
containing 50 mL medium. Medium contained 70% pasteurised
whey and 30% sterilised milk with 0.5% fat. Based on preliminary
experiments (data not shown) 30% milk was used for beverage
formulation as concentration that appropriate for appreciably sen-
sory quality improvement. Samples were inoculated differently in
three experiments.
In Experiment 1, the sample was inoculated by adding 6%
(w/v) ABY-6 commercial culture encapsulated in alginate beads.
In Experiment 2, the sample was inoculated by adding 6% (w/v)
ABY-6 commercial culture encapsulated in alginate beads, coated
with chitosan; and in Experiment 3, the sample was inoculated by
adding 6% (v/v) ABY-6 commercial culture, previously activated by
dissolving 1% (w/v) ABY-6 commercial culture in sterile milk with
0.5% fat, for 30 min at 42 ∘ C.
After inoculation all samples were incubated at 42 ∘ C. Dur-
ing incubation, samples were taken after 4 h, 5 h and 5 h
30 min for determination of the pH value. Fermentations were
stopped by quick cooling when a pH between 5.0 and 4.5 was
reached.
Determination of cell leakage
Cell leakage was determined by using the following equation:
cell release (%)
free cell number
= × 100
free cell number − cell number in beads
where the free cell number and cell number on beads are given as
colony forming units (CFU).
Cell viability during the storage
After the fermentation, beverages were refrigerated at 4 ± 1 ∘ C.
The fermented beverages were sampled at intervals of 7 days, for
a period of 28 days and analysed for viable cell number, pH value
and titratable acidity.
Acid and bile tolerance (probiotic properties)
To determine acid and bile tolerance of free and encapsulated
probiotic cells, different MRS broths (Sigma–Aldrich) were spe-
cially prepared. Experiments were conducted in 100-mL Erlen-
meyer flasks containing 50 mL MRS broth. For determination of
acid tolerance, the pH value of MRS broth was adjusted to 2.5 and
3.0 using 1 mol L−1 HCl. For determination of bile tolerance MRS
broth was supplemented with 0.3% (w/v) dehydrated bovine bile
(Torlak, Belgrade, Serbia). Usually, prepared MRS broth was used as
control sample. Prepared MRS broths were autoclaved for 30 min
at 120 ∘ C. Sterile MRS broths were inoculated by 2.0% (v/v) of free
cells or 2% (w/v) of beads collected after the fermentation. The
MRS broths were incubated under anaerobic conditions at 37 ∘ C.
Viable cell count was determined in regular intervals after 0, 60,
120, 180 and 240 min.
Analytical methods
Analysis of the bead size
The diameters of randomly selected beads of each treatment
were measured with an optical microscope (Carl Zeiss Microscopy
GmbH, Cambridge, UK) using magnification of ×5.
J Sci Food Agric 2016; 96: 1723–1729
Effect of immobilisation on dairy starter culture
Analysis of mechanical strength of beads
Compression tests of packed alginate beads were performed using
Universal Testing Machine, AG-Xplus (Shimadzu, Japan) equipped
with a 100 N force load cell. The mechanical stability of beads
was determined by measuring the force required to cause 30%
deformation of the samples. Each measurement was carried out at
room temperature on 6 g of packed beads using the compression
method between two flat surfaces. Pre-testing conditions such as
force, compression speed and percentage of a sample deforma-
tion, were pre-defined in the materials testing software TRAPEZI-
UMX 1.13 (Shimadzu Group, Kyoto, Japan).
Enumeration of viable cells
Cell number of S. salivarius subsp. thermophiles was determined by
the pour-plate counting method on M17 agar. Cell numbers of Lb.
delbrueckii subsp. bulgaricus, Lb. acidophilus and Bifidobacterium
bifidum were determined by the pour-plate counting method on
MRS agar.11 Cell number was expressed in log10 CFU mL−1 for free
cells and log10 CFU g−1 for beads.
Titratable acidity and pH
The pH value was measured using a pH meter (WTW 82362; Inolab,
Wellheim, Germany) at room temperature. Titratable acidity was
determined by the Soxhlet–Henkel method.12 Titratable acidity
was thus obtained in Soxhlet–Henkel degrees (∘ SH).
Statistical analysis
Experiments were performed in triplicate. All values are expressed
as mean ± standard deviation. Mean values were analysed using
one-way ANOVA. The Tukey post hoc test was performed for
means comparison [Origin Pro 8 (1991–2007) computer package;
Origin Lab Co., Northampton, MA, USA]. Data were considered
significantly different when P < 0.05.
RESULTS AND DISCUSSION
Number of cells entrapped, size and mechanical strength
The beads were globular in shape. The diameter of uncoated
beads was 0.82 ± 0.09 mm, which was significantly lower than the
diameter of coated beads (1.23 ± 0.10 mm). The shape of the beads
did not change when chitosan was added to alginate beads. The
shape and dimensions of the particles are shown in Fig. 1.
The number of viable cells in the particles was 7.65 ± 0.15 (log10
CFU g−1 beads) for alginate beads and 7.55 ± 0.14 (log10 CFU g−1
beads) for coated beads.
The results of mechanical test obtained at compression speed
of 0.25 mm s−1 min and mentioned sample deformation for algi-
nate and alginate–chitosan beads with immobilised probiotic cells
showed that the measured maximal forces were in the range of
2.174 ± 0.015 N for alginate and 2.316 ± 0.028 N for coated beads,
before fermentation. After fermentation the measured maximal
forces were in the range of 2.384 ± 0.024 N and 2.963 ± 0.027 N,
respectively. In general, the chitosan coating was found to increase
the mechanical strength of beads tested before and after the fer-
mentation process. During the fermentation, the probiotic culture
produces lactic acid, decreases the pH of the medium, which leads
to the creation of conditions that adversely affects the mechani-
cal strength of the particles. Despite this, the mechanical strength
of particles increased indicating that alginate builds bonds with
calcium ions from milk and whey that have a positive impact on
particles strength. The results are shown in Table 1.
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Fermentation and stability
Fermentation
Fermentation of whey by commercial cultures designed for
yoghurt production could be an interesting method of includ-
ing whey in human consumption.13 Encapsulating the bacterial
culture brings many benefits’ for example, cells can be isolated
from the original substrate and used again for the fermentation.
Also, cells are protected from external factors and they have a
greater ability to survive in the human intestinal tract. The abil-
ity to survive adverse environmental conditions and course of
fermentation were examined.
Fermentation with free cells takes 1.5 h less than fermenta-
tion with encapsulated cells. It is the result of difficulties in the
exchange of nutrients between medium and cells inside of algi-
nate and coated beads. Titratable acidity after fermentation is
lower and pH decrease during fermentation is less for encapsu-
lated cells. Growth of culture in these conditions is also slightly
lower but these differences can be overcome with a slightly
extended fermentation time.
Number of leaked cells in the fermentation medium was
3.43 ± 0.11% of the total bacterial population for sample with
Ca-alginate beads. Slightly lower cell leakage (2.48 ± 0.10% of the
total bacterial population) was observed for sample with calcium
alginate beads coated with chitosan (Table 1). Based on the results
it could be said that chitosan and alginate develop a complex that
reduces the porosity of alginate beads and decreases the leakage
of the encapsulated probiotic.
Stability
Titratable acidity of samples, after 5.5 h of fermentation, was
14.8∘ SH for alginate beads and 12.4∘ SH for coated beads. A bev-
erage is considered to be of good quality if it has a titratable
acidity of approximately 44∘ SH.13 On the other hand, unpleasant
acid tastes can be detected at titratable acidity values greater
than 53.0∘ SH.14,15 Since whey is a poor substrate, yoghurt culture
produces significantly less lactic acid, even if it is not encapsulated.
Titratable acidity of beverage with free cells was 23.2∘ SH, with
alginate beads 14.8∘ SH and for coated beads 12.4∘ SH (Fig. 2). This
could be explained by the presence of alginate and chitosan as
additional barrier to the exchange of nutrients. Titratable acidity
of samples, after 28 days of storage, was 31.2∘ SH for free cells,
19.6∘ SH for alginate beads and 16.4∘ SH for coated beads. Com-
paring samples fermented by free cells and samples fermented
by encapsulated cells, it was observed that immobilisation and
coating significantly (P < 0.05) affects titratable acidity during the
fermentation and storage time.
The pH is one of the most important factors for stability of
probiotic bacteria in fermented products.16 The optimum pH for
growth of Lb. acidophilus is 5.5–6.0.17 Bifidobacterium bifidum is
not as acid tolerant as Lb. acidophilus. Most strains of bifidobacteria
are sensitive to pH values below 4.6, and because of that the
pH value of the final product must be maintained above 4.6.18 A
gradual decrease of pH was observed in all samples during both
5.5 h fermentations and during 28 days of storage (Fig. 3). At the
end of fermentation, the pH was 4.70 for alginate and 4.90 for
coated beads. After 28 days of storage, the pH was 4.49 for alginate
and 4.69 for coated beads. Comparing Fig. 2 and Fig. 3 it was
observed that the reduction of pH is accompanied by an increased
titratable acidity. That occurs because probiotic cultures are active,
even during storage in the refrigerator. There is a decrease in pH
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for storage 28 days, and an increase in titratable acidity, in all
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Figure 1. Ca-alginate beads coated with chitosan (A), and uncoated Ca-alginate beads (B), viewed with an optical microscope (magnification, ×5).

Table 1. Mechanical properties of coated and uncoated Ca-alginate beads with yogurt culture, measured before and after the fermentation and cell
release after 5.5 h of fermentation

Maximal force (N)

Material Deformation (%) Compression speed (mm s−1 ) Before fermentation* After fermentation* Cell release (%)

Alginate beads 30 0.25 2.196 ± 0.051 2.384 ± 0.024 3.43 ± 0.11

Alginate beads coated with chitosan 30 0.25 2.873 ± 0.021 2.963 ± 0.027 2.48 ± 0.10
* Results are given as the mean values ± standard deviation.

Figure 2. Variation trend in titratable acidity during the fermentation and Figure 3. Variation trend in pH during the fermentation and 28 days
28 days of refrigerated storage for samples with free cells, alginate beads of refrigerated storage for samples with free cells, alginate beads and
and chitosan-coated alginate beads. Mean values ± standard deviation. chitosan-coated alginate beads. Mean values ± standard deviation.

samples. Decreasing pH and increasing titratable acidity is more
pronounced in the sample with alginate beads, because chitosan
further hinders the exchange of matter between the culture and
the substrate, so the change of pH and titratable acidity in such
situation is less pronounced. Comparing samples fermented by
free cells and samples fermented by encapsulated cells, it was
observed that immobilisation and also coating beads, significantly
(P < 0.05) affects the pH value during the fermentation and storage
time for the examined yoghurt culture.
The most critical factor of dairy products is viability of pro-
biotic cells until the time of consumption. One of the proper-
ties for a given microorganism to be considered probiotic is its
1726
have examined the survival of bacteria at different storage tem-
peratures. According to Mortazavian et al.19 food products with
probiotic should preferably be preserved at a temperature of
4–5 ∘ C. In this paper, the beverage was stored at 4 ± 1 ∘ C. Figure 4
shows the stability of free and encapsulated probiotic bacteria
during 4 weeks of storage in the refrigerator at 4 ± 1 ∘ C. What
can be seen from the Fig. 4 is that viability of encapsulated cells
is more stable then viability of free cells; there is no large vari-
ation in the number of cells during storage. After 28 days the
number of cells was approximately the same as the number of
cells after fermentation for encapsulated cells, which is impor-
tant for probiotic beverage. The rate of population reduction

capacity to survive storage as a formulated product. Varies studies was significantly (P < 0.05) slower in samples with encapsulated

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Effect of immobilisation on dairy starter culture
Figure 4. Variation trend in the viable cell count of yogurt starter culture
before and after the fermentation and during 28 days of refrigerated stor-
age period, at 7-day intervals for free cells (log CFU mL−1 ), encapsulated
cells in alginate beads (log CFU g−1 ), encapsulated cells in chitosan-coated
alginate beads (log CFU g−1 ). Mean values ± standard deviation.
cells (0.03 Δlog10 CFU mL−1 for alginate beads and 0.001 Δlog10
CFU mL−1 for chitosan coated beads), than in sample with free
cells (0.50 Δlog10 CFU mL−1 ) at the end of storage period. The
observed results suggest that using alginate encapsulation and
chitosan coating, probiotic cells from the examined yoghurt cul-
ture are protected from unfavourable impacts contained in the
dairy product.
Probiotic properties
Acid tolerance
Acid tolerance of probiotics is important not only for resistance
to gastric pH, but is also needed for their use as dietary adjuncts
in high acid carrier foods such as yoghurt.20 The gastric pH in
healthy humans is about 2.5. This causes the destruction of most
microorganisms.21 Lactic acid starter bacteria showed low resis-
tance to simulated gastric juice and low pH environment, Lb.
delbrueckii subsp. bulgaricus, Lb. acidophilus, and S. thermophiles
showed losses in cell viability ranging from 0.7 to more than 6.0
log.22 According to Sultana et al.23 aciduric members such as Lb.
acidophilus, generally could not survive in low pH environment,
because low pH inhibits metabolism activity of Lb. acidophilus,
thus reducing the viability of the probiotic. The survival of free
and encapsulated probiotic culture at pH 2.5 and 3.0 were com-
pared (Fig. 5). Free cells had a drastic reduction in the number of
viable cells in 2 h (50%) and in the third hour there were no living
cells at pH 2.5 (Fig. 5A). Figure 5B shows that samples with algi-
nate beads had 75.61% surviving cells in the second hour and after
4 h the number of viable cells was 36.18% compared to an initial
number. Samples with coated beads had 81.96% surviving cells
in the second hour and after 4 h the number of viable cells was
37.8% of the initial number (Fig. 5C). As shown in Fig. 5, samples
with alginate beads and coated beads had significantly (P < 0.05)
higher probiotic viable cell count than samples with free cells, dur-
ing 4 h monitoring, at pH 2.5. At pH 3.0, samples with free cells had
72% surviving cells and samples with alginate beads and coated
beads had more than 96% of cells survived after 4 h, what is also,
significant (P < 0.05) contribution to the survival. According to this
study encapsulation in alginate beads and coating with chitosan
J Sci Food Agric 2016; 96: 1723–1729 © 2015 Society of Chemical Industry
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provides the best protection for the examined yoghurt culture in
simulated gastric juice. Chavarri et al.24 also reported that chitosan
coating provides better protection to probiotic organisms com-
pared to uncoated microcapsules.
Bile tolerance
Bile tolerance is often used as a criterion for probiotic strain
selection. Bile secreted in the small intestine adversely affects to
bacterial cell membrane and reduces the survival of bacteria. In
order to exert a beneficial effect, probiotic culture must survive
passage through the stomach and small intestine.25 The relevant
physiological concentration of human bile is about 0.3%.26 Bile salt
solution was used here to determine whether encapsulation and
coating of the alginate beads would increase survival of cells in
this environment, which is similar to that of the digestive system.
The results of analysis of probiotic characteristic of beverages with
free yoghurt culture, with entrapped cells in alginate beads and
coated beads with chitosan are given in Fig. 5. Free cells had a
significant (P < 0.05) reduction in the number of viable cells in 2 h
there were 85.15% of viable cells compared to the initial number
and in the fourth hour the number of viable cells decreased
to 83.0% (Fig. 5A). As shown in Fig. 5, samples with alginate
beads (94.54% of viable cells compared to the initial number)
and coated beads (95.85% of viable cells compared to the initial
number) have significantly (P < 0.05) higher probiotic viable cell
count than samples with free cells (83.00% of viable cells compared
to the initial number), during 4 h monitoring. Krasaekoopt et al.27
reported that the rate of survival of Lb. acidophilus cells in both
coated and uncoated beads was significantly (P < 0.05) higher than
the rate of survival of free cells. Koo et al.,28 Yu et al.29 and Chavarri
et al.24 found that the survival rate of Bifidobacterium bifidum in
alginate beads containing chitosan was higher than that found in
alginate beads without chitosan. According to the observed results
the chitosan coated alginate beads provides additional protection
of cells in bile salt solution compared to the alginate beads. The
reason for that can be an ion-exchange reaction. An ion-exchange
reaction takes place when the beads absorb bile salt and an
insoluble complex between chitosan and bile salt forms in the
chitosan–alginate membrane,30 this can decrease the diffusion of
bile salt into the beads and consequently protect encapsulated
cells from interacting with the bile salt.
CONCLUSIONS
The results show that encapsulation is a possible way to sig-
nificantly improve the properties of the product. The study has
indicated that coating alginate beads with chitosan, reduces the
porosity of beads and decreases the leakage of the encapsulated
probiotic during the fermentation in comparison with not coated
alginate beads. During 28 days of storage, the number of encap-
sulated cells in the beverage has fluctuated less than the number
of cells in beverage with free cells. This means that, by consum-
ing the beverage with encapsulated cells, a consumer would take
approximately the same amount of probiotics at any time within
those 28 days. Also, this study has shown that encapsulation sig-
nificantly improves bacterial survival in simulated gastrointestinal
environment, especially if alginate beads are coated with chitosan.
These characteristics allow viable cells to reach a beneficial level as
probiotic. Consequently, chitosan-coated alginate beads could be
a good way to administer these beneficial microorganisms in the
1727
production of functional beverages.
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 www.soci.org
A This work was funded by the Serbian Ministry of Education, Science
B 7 Adhikari K, Mustapha A, Grün IU, and Fernando L, Viability of microen-
C with milk and a probiotic strain. RSC Advances 4:55503–55510
Figure 5. Number of surviving cells when free (A), or encapsulated in
alginate beads (B), or encapsulated in chitosan-coated alginate beads (C),
during 4 h anaerobic incubation at 37 ∘ C in MRS broth as control, MRS broth
with pH 2.5, MRS broth with pH 3.0 and MRS broth containing 0.3% bile salt.
Mean values ± standard deviation.
1728
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TŽ Krunić et al.
ACKNOWLEDGEMENT
and Technological development (TR 31017).
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