JFS M: Food Microbiology and Safety

Acid, Bile, and Heat Tolerance of Free

and Microencapsulated Probiotic Bacteria
W.K. DING AND N.P. SHAH

ABSTRACT: Eight strains of probiotic bacteria, including Lactobacillus rhamnosus, Bifidobacterium longum,
L. salivarius, L. plantarum, L. acidophilus, L. paracasei, B. lactis type Bl-O4, and B. lactis type Bi-07, were stud-
ied for their acid, bile, and heat tolerance. Microencapsulation in alginate matrix was used to enhance survival of
the bacteria in acid and bile as well as a brief exposure to heat. Free probiotic organisms were used as a control. The
acid tolerance of probiotic organisms was tested using HCl in MRS broth over a 2-h incubation period. Bile toler-
ance was tested using 2 types of bile salts, oxgall and taurocholic acid, over an 8-h incubation period. Heat tolerance
was tested by exposing the probiotic organisms to 65 ◦ C for up to 1 h. Results indicated microencapsulated probiotic
bacteria survived better (P < 0.05) than free probiotic bacteria in MRS containing HCl. When free probiotic bacteria
were exposed to oxgall, viability was reduced by 6.51-log CFU/mL, whereas only 3.36-log CFU/mL was lost in mi-
croencapsulated strains. At 30 min of heat treatment, microencapsulated probiotic bacteria survived with an aver-
age loss of only 4.17-log CFU/mL, compared to 6.74-log CFU/mL loss with free probiotic bacteria. However, after 1 h
of heating both free and microencapsulated probiotic strains showed similar losses in viability. Overall microen-
capsulation improved the survival of probiotic bacteria when exposed to acidic conditions, bile salts, and mild heat
treatment.
Keywords: acid tolerance, bile tolerance, microencapsulation, probiotics

Introduction verse environment, and mild heat treatment from food processing

I n order to exert health benefits, probiotic bacteria are expected

to be at the level of 107 CFU of live microorganisms per milliliter
of product at the time of consumption (Guarner and Schaafsma
1998; Ouwehand and Salminen 1998; Shah 2000). Since the via-
bility and activity of probiotics are needed at the lower digestive
tract, these organisms should withstand the adverse conditions en-
countered in the host’s upper gastrointestinal tract (GIT). How-
ever, many probiotic bacteria lack the ability to survive the harsh
acidity and bile concentration commonly encountered in the GIT
(Shah and Jelen 1990; Berrada and others 1991; Mituoka 1992; Shah
and Lankaputhra 1997; Gardiner and others 2000). All bile, depend-
ing upon its concentration, can inhibit the growth of bacteria. Bile
acids inhibit many Gram-positive probiotic organisms such as Bifi-
M: Food Microbiology & Safety
thus potentially reducing cell injury and death. The development
of a heat stable microcapsule may be suitable for heated functional
food products such as cakes and sausages (Arihara 2006). There is
a need for microencapsulation not only to help the probiotic bac-
teria to survive in the food product but also during the passage
though the human stomach, where the pH can be as low as 2.0. It
has been reported that microcapsules made of alginate polymers
increased the survival and viability of probiotic bacteria in acidic
food products during cold storage (Adhikari and others 2000; Picot
and Lacroix 2004). Alginate has been widely used as an encapsulant
material because it has the benefits of being nontoxic and it is an
accepted food additive (Prevost and Divies 1992). The reversibility
of microencapsulation, that is, solubilizing of alginate by sequester-

dobacterium and Lactobacillus genera; however, they have little ef-
fect against Gram-negative organisms such as Escherichia coli. This
information has been known for decades and is actually incorpo-
rated into the taxonomic identification methods (Stacey and Webb
1947; Floch and others 1970).
The immobilization of probiotics using microencapsulation may
improve the survival of these microorganisms in products, both
during processing and storage, and during digestion (Champagne
and others 1993; Cui and others 2000; Lee and Heo 2000; Sultana
and others 2000; Goderska and others 2003; Capela and others
2005).
Microencapsulation of probiotic bacteria is currently gaining at-
tention as a method to improve the stability of probiotic organ-
isms in functional food products (Kailasapathy 2002; Ouwehand
and others 2002; Godward and Kailasapathy 2003; Krasaekoopt and
others 2003). Microencapsulation protects the cells from the ad-
MS 20070502 Submitted 7/1/2007, Accepted 8/28/2007. Authors are with
School of Molecular Sciences, Victoria Univ., Werribee Campus, PO Box ing Bifidobacterium spp. was supplemented with filter sterilized
14428, Melbourne, Victoria 8001, Australia. Direct inquiries to author Shah 0.05% (w/v) L-cysteine hydrochloride (Sigma Chemical Co., Castel
(E-mail: nagendra.shah@vu.edu.au).
ing the calcium ions, and the possible release of entrapped cells in
the lower intestine is another advantage thus allowing the probiotic
bacteria to exert their health benefits for the consumer. The aim of
this work was to improve the survivability of probiotic organisms in
acidic conditions and exposure to bile using microencapsulaiton,
and to evaluate the survival of microencapsulated probiotic bacte-
ria after a brief exposure to heat stress at 65 ◦ C.
Materials and Methods
Probiotic bacteria
Eight strains of probiotic bacteria, including L. rhamnosus type
Lr-32, B. longum typeBl-05, L. salivarius type Ls-33, L. plan-
tarum Lpc-37, L. acidophilus NCFM, L. paracasei Lp-115, B. lactis
type Bl-04, and B. lactis type Bi-07, were obtained from Danisco
(Copenhagen, Denmark). The probiotic strains were maintained
individually and grown in MRS broths (Oxoid Ltd., Melbourne, Vic-
toria, Australia) at 37 ◦ C using a 1% inoculum. MRS broth contain-
Hill, Sydney, NSW, Australia) to create a more favorable anaerobic

M446 JOURNAL OF FOOD SCIENCE—Vol. 72, Nr. 9, 2007 

C 2007 Institute of Food Technologists
doi: 10.1111/j.1750-3841.2007.00565.x
Further reproduction without permission is prohibited
Acid, bile, and heat tolerance. . .

Table 1 --- Effect of low pH (2.0) on viability of 8 strains of free probiotic bacteria.
Viable cell count (log CFU/mL)
Strain 0 min 30 min 60 min 90 min 120 min
L. rhamnosus 10.01 ± 0.63a 8.54 ± 0.41a 6.43 ± 0.13a 5.32 ± 0.03bc 3.41 ± 0.09ab
B. longum 10.34 ± 0.46a 8.38 ± 0.61a 6.34 ± 0.84a 5.78 ± 0.31b 3.29 ± 0.81bc
L. salivarius 10.93 ± 0.45a 9.34 ± 0.31a 6.31 ± 0.41a 5.65 ± 0.84c 4.10 ± 0.32a
L. plantarum 10.59 ± 0.41a 8.98 ± 0.12a 7.38 ± 0.74a 5.34 ± 0.12a 3.98 ± 0.29ab
L. acidophilus 10.22 ± 0.70a 8.45 ± 0.64a 6.42 ± 0.13a 4.98 ± 0.03a 4.21 ± 0.56a
L. paracasei 10.69 ± 0.52a 8.42 ± 0.38a 7.35 ± 0.31a 5.23 ± 0.07a 3.78 ± 0.74b
B. lactis type Bl-O4 10.73 ± 0.03a 8.32 ± 0.42a 6.21 ± 0.36a 5.32 ± 0.13c 3.98 ± 0.34c
B. lactis type Bi-07 10.43 ± 0.71a 9.26 ± 0.10a 6.98 ± 0.07a 5.65 ± 0.19ab 3.42 ± 0.17c
Means in the same column followed by different lowercase letters are significantly different (P < 0.05).
abc
Results are expressed as mean + SEM; each data point is the average of 3 repeated measurements from 3 independently replicated experiments, n = 3.

Table 2 --- Effect of low pH (2.0) on viability of 8 strains of encapsulated probiotic bacteria.
Viable cell count (log CFU/mL)
Strain 0 min 30 min 60 min 90 min 120 min
L. rhamnosus 10.49 ± 0.13a 8.76 ± 0.23a 7.53 ± 0.52a 7.22 ± 0.46a 7.11 ± 0.41a
B. longum 10.63 ± 0.45a 8.45 ± 0.56a 7.42 ± 0.74a 6.93 ± 0.73b 6.45 ± 0.23bc
L. salivarius 10.86 ± 0.52a 9.13 ± 0.25a 7.34 ± 0.87a 6.24 ± 0.53ab 6.14 ± 0.93a
L. plantarum 10.45 ± 0.31a 8.88 ± 0.06a 7.25 ± 0.31a 6.93 ± 0.08c 6.64 ± 0.87c
L. acidophilus 10.75 ± 0.07a 9.15 ± 0.08a 7.93 ± 0.34a 7.41 ± 0.05a 7.43 ± 0.53ab
L. paracasei 10.34 ± 0.35a 8.56 ± 0.41a 8.10 ± 0.61ab 7.14 ± 0.46c 6.41 ± 0.76b
B. lactis type Bl-O4 10.46 ± 0.42a 8.58 ±.0.76a 6.72 ± 0.03ac 6.31 ± 0.34ab 6.27 ± 0.42b
B. lactis type Bi-07 10.88 ± 0.0.24a 9.24 ± 0.21ab 8.23 ± 0.35a 7.31 ± 0.42b 6.93 ± 0.04ab
Means in the same column followed by different lowercase letters are significantly different (P < 0.05).
abc
Results are expressed as mean + SEM; each data point is the average of 3 repeated measurements from 3 independently replicated experiments, n = 3.

A Figure 1 --- (A) Viability of

free probiotic bacteria in
12
L. rhamnosus the presence of 3% (w/v)
oxgall. (B) Viability of
B. longum encapsulated probiotic
Viable cells log (CFU/mL)

10
bacteria in the presence of
L. salivarius 3% (w/v) oxgall.
8
L. plantarum

6 L. acidophilus

L. paracasei
4
B. lactis type Bl-O4

M: Food Microbiology & Safety

2 B. lactis type Bi-07
0 min 4h 8h
Incubation time at 37ºC
B 12
L. rhamnosus

10 B. longum
Viable cells log (CFU/mL)

L. salivarius
8
L. plantarum

L. acidophilus
6
L. paracasei
4
B. lactis type Bl-O4

B. lactis type Bi-07

2
0 min 4h 8h
Incubation time at 37ºC

Vol. 72, Nr. 9, 2007—JOURNAL OF FOOD SCIENCE M447

 Acid, bile, and heat tolerance. . .
environment. Before use, the probiotic organisms were activated by
growing 3 times successively in MRS at 37 ◦ C for 18 h. The identity
of all probiotic bacteria was confirmed using biochemical methods
as described by Shah and Lankaputhra (1997).
Microencapsulation of probiotics
The microencapsulation of probioitc bacteria was performed us-
ing a modified method of Shah and Ravula (2000). Briefly, 100 mL
of 3% (w/v) sodium alginate (D3247 AJAX Chemicals Ltd., Mel-
bourne, Victoria, Australia) were prepared in a volumetric flask and
sterilized by autoclaving. The sterile alginate was mixed with 25
mL of washed and concentrated (1010 CFU/mL) probiotic organ-
isms. An initial emulsion was made by mixing the alginate-bacteria
mixture with 600 mL of vegetable oil and 1 mL of Tween 80 in a
glass beaker. To reduce the particle size, the emulsion was mixed
thoroughly using an M-110Y microfluidizer (Microfluidics, Newton,
Mich., U.S.A.). After several cycles in the microfluidizer, calcium
chloride (0.01 M) was gently added to the emulsion until the emul-
sion was broken. After 30 min, the calcium alginate beads were re-
moved from the aqueous phase and were refrigerated at 4 ◦ C for
10 h to allow the beads to fully harden.
Acid tolerance
Acid tolerance of microencapsulated probiotic organisms and a
control consisting of free probiotic organisms was studied accord-
ing to the method of Liong and Shah (2004). In brief, MRS broth was
adjusted to pH 2.0 with 5.0 M HCl. Approximately 1010 CFU/mL of
each probiotic strain was inoculated into the modified MRS broth
and were incubated at 37 ◦ C for up to 2 h and samples were taken
at 30 min intervals for enumeration. Probiotic bacteria can form
A 12
10
Viable cells log (cfu/ml)
8 taurocholic acid.
6
M: Food Microbiology & Safety
4 B. lactis type Bl-O4
2
0 min 4h
Incubation time at 37ºC
B 12
10
Viable cells log (cfu/ml)
8
6
4
2
Initial 4h
Incubation time at 37ºC
M448 JOURNAL OF FOOD SCIENCE—Vol. 72, Nr. 9, 2007
chains or clusters particularly in MRS broth, thus cultures were
sonicated for 5 s to disperse the cells before serial dilutions were
preformed. Subsequent serial dilutions were vortexed for 30 s in-
dividually before inoculation to MRS agar plates. Plates were in-
cubated at 37 ◦ C for 72 h in an anaerobic jar (Becton Dickinson
Microbiology Systems, Sparks, Md., U.S.A.) with an anaerobic gas
generating kit (Oxoid Ltd.). For the enumeration of microencap-
sulated probiotic organisms, the bacteria were released from the
capsules by sequestering calcium ions with phosphate buffer at pH
7.0. Acid tolerance was determined by comparing the final plate
count after 2 h with the initial plate count at 0 h. All acid toler-
ance tests were repeated 3 times to estimate average and standard
error.
Bile tolerance
Two types of bile salts were used to study the bile tolerance of
probiotic bacteria: oxgall and taurocholic acid (Sigma Chemical
Co.). Taurocholic acid was used as the conjugated bile, and oxgall
contained both conjugated and deconjugated bile. Bile tolerance
was studied according to the method of Gilliland and Walker (1990).
Briefly, MRS broth containing 3% (w/v) of oxgall or 3% (w/v) tauro-
cholic acid was adjusted to pH 5.8 using 5.0 M HCl. Approximately
1010 CFU/mL of each probiotic strain was inoculated into the MRS
broths with bile salts and incubated at 37 ◦ C for up to 8 h. Al-
though the concentration of bile acid and pH varies largely between
individuals, the levels used in our study are within the physiological
concentrations found in the human duodenum (Floch 2002). The
control comprised MRS broth without bile salts. Bacterial growth
was observed by monitoring viable cell counts at 0-, 4-, and 8-h
intervals on MRS agar supplemented with 0.05% (w/v) L-cysteine
Figure 2 --- (A) Viability of free
L. rhamnosus probiotic bacteria in the
presence of 3% (w/v)
B. longum taurocholic acid. (B) Viability of
encapsulated probiotic bacteria
L. salivarius in the presence of 3% (w/v)
L. plantarum
L. acidophilus
L. paracasei
B. lactis type Bi-07
8h
L. rhamnosus
B. longum
L. salivarius
L. plantarum
L. acidophilus
L. paracasei
B. lactis type Bl-O4
B. lactis type Bi-07
8h
Acid, bile, and heat tolerance. . .

hydrochloride (Sigma Chemical Co.) to maintain a more favorable Results and Discussion
anaerobic environment. The bile tolerance of each strain was de-
termined by comparing the plate count after 4 and 8 h of exposure Acid tolerance
to bile salts with the initial pate count at 0 h. All experiments were The effect of acidic conditions on the viability of free probi-
repeated 3 times to estimate average and standard error. otic organisms is shown in Table 1. All probiotic organisms tested
showed a steady loss in viability when exposed to acidic conditions.
Heat tolerance Initially there was an average of 10.49-log CFU/mL of viable pro-
Heat tolerance of free and microencapsulated probiotic bacte- biotic bacteria, but after 1 h of exposure the average viability of
ria was studied by exposing 1010 CFU/mL of organisms in pep- cells was reduced to 6.66-log CFU/mL and the viability was fur-
tone water (pH 6.4 ± 0.2) (Oxoid Ltd.) to 65 ◦ C for up to 1 h. For ther reduced to an average of 3.77-log CFU/mL after 2 h of expo-
each strain, 1 g of microcapsules was inoculated into 9 mL of pep- sure. Lctobacillus acidophilus and L. salivarius were the most acid-
tone water in a 15 mL Falcon tube (BD, Franklin Lakes, N.J., U.S.A.) tolerant strains, with > 104 CFU/mL surviving after 2 h incubation
and 1 mL of free cells was used as a control. After heat treatment, at pH 2.0. The Bifidobacterium strains, including B. longum, B. lac-
the tubes were cooled to 22 ◦ C and serial dilutions were made us- tis type Bl-O4, and B. lactis type Bi-07, were the most acid-sensitive
ing phosphate buffer at pH 7.0 that helped release the encapsu- strains.
lated probiotic bacteria. Heat tolerance was determined by com- The effect of low pH on the viability of microencapsulated pro-
paring the final plate count after 1 h of heat treatment with the biotic bacteria is shown in Table 2. There was a loss in viability of
initial plate count at 0 h. All heat tolerance tests were repeated all the 8 strains tested however, at 1 h of exposure most microen-
3 times. capsulated probiotic organisms survived at > 107 CFU/mL and at
2 h of exposure most microencapsulated probiotic organisms sur-
Statistical analysis vived at > 106 CFU/mL. This level of survival is needed to confer
Results from 3 individual experiments were used to calculate the health benefits to the consumer (Ouwehand and Salminen 1998).
mean of cell counts. A single factor analysis of variance (ANOVA) Microencapsulation of probiotics in alginate beads has previously
was used to calculate the SEM of the cell counts. Significant dif- been tested for improving viability of probiotic bacteria in simu-
ferences between the means of cell counts were determined using lated gastric conditions (Kebary and others 1998; Krasaekoopt and
Tukey’s HSD test. All statistical analysis was carried out using SPSS others 2003) and the results of this study concur with other sim-
15.0
for Windows XP. ilar studies where microencapsultaion in alginate beads has been

initial 30 min 60 min

Figure 3 --- (A) The
effect of exposure to
A 12
65 ◦ C on the viability of
free probiotic bacteria.
10 (B) The effect of
Viable cells log (CFU/mL)

exposure to 65 ◦ C on
the viability of
8
microencapsulated
probiotic bacteria.
6

M: Food Microbiology & Safety

0
L. rhamnosus B. longum L. salivarius L. plantarum L. acidophilus L. paracasei B. lactis type Bl- B. lactis type Bi-
O4 07
Probiotic bacteria

initial 30 min 60 min

B 12

10
Viable cells log (CFU/mL)

0
L. rhamnosus B. longum L. salivarius L. plantarum L. acidophilus L. paracasei B. lactis type Bl- B. lactis type Bi-
O4 07
Probiotic bacteria

Vol. 72, Nr. 9, 2007—JOURNAL OF FOOD SCIENCE M449

 Acid, bile, and heat tolerance. . .
found to greatly increase the survival of probiotic bacteria in acidic
conditions (Audet and others 1988; Jankowski and others 1997;
Krasaekoopt and others 2003; Doleyres and Lacroix 2004).
Bile tolerance of probiotic bacteria
The effect of the bile salt oxgall on the viability of the 8 free
probiotic bacteria is presented in Figure 1A. All strains showed a
loss of viability when exposed to 3% (w/v) oxgall. The initial vi-
able count of 10.52-log CFU/mL was reduced to 6.35-log CFU/mL
after 4 h of exposure and the average viable number was further
reduced to 4.01-log CFU/mL after 8 h. The strains most suscepti-
ble to oxgall were B. lactis type Bl-O4 and B. lactis type Bi-07 with
7.32- and 7.41-log CFU/mL reduction in viability at 8 h of incuba-
tion, respectively. The initial viable counts of the microencapsu-
lated probiotic bacteria exposed to oxgall are shown in Figure 1B.
The initial viability count of 10.55-log CFU/mL was reduced to 8.65-
log CFU/mL after 4 h and 7.19-log CFU/mL after 8 h of incubation.
The effect of 3% (w/v) taurocholic acid on the viability of the 8
free and microencapsulated probiotic bacteria is presented in Fig-
ure 2A and 2B. Taurocholic acid was slightly more lethal to free pro-
biotic bacteria than oxgall when used at the same concentrations.
The most susceptible bacteria to taurocholic acid were B. longum
and L. plantarum both with > 7-log CFU/mL loss in viability.
The effect of taurocholic acid on the viability of microencapsu-
lated probiotic bacteria was less severe, with an average of 8.47-
and 6.24-log CFU/mL of organisms surviving after 4 and 8 h of ex-
posure, respectively. Studies have found that most probiotic bac-
teria can grow in MRS supplemented with up to 0.5% conjugated
bile salts (Noriega and others 2006). Studies using concentrations of
bile salts of between 1% and 3% have shown that encapsulated pro-
biotic bacteria can survive better than free probiotic cells (Chan-
dramouli and others 2004; Kailasapathy 2005).
Heat tolerance
The heat tolerance of free and microencapsulated probiotic bac-
teria incubated at 65 ◦ C for up to 1 h is shown in Figure 3A and 3B,
respectively. A temperature of 65 ◦ C was found to be lethal to all free
probiotic strains tested. Microencapsulated probiotic bacteria sur-
vived well at 30 min with an average loss of only 4.17-log CFU/mL
compared to free probiotic bacteria with an average loss of 6.74-log
CFU/mL at the same time point. However, after 1 h of incubation
the survival of free and microencapsulated probiotic bacteria was
M: Food Microbiology & Safety
similar. Results indicate that after 1 h of heating the alginate ma-
trix offered little protection for the probiotic bacteria. These results
concur with similar results of improved heat tolerance gained by
microencapsulation (Mandal and others 2006).
Conclusions
M icroencapsulation with alginate can be applied to a many
different probiotic strains and results show better survival
than free cells at low pH of 2.0, high bile salt concentrations, and
moderate heat treatment of up to 65 ◦ C. Microencapsulation may
prove to be an important method of improving the viability of pro-
biotic bacteria in acidic food products and help deliver viable bac-
teria to the host’s gastrointestinal tract. Furthermore, microencap-
sulation appeared to be effective in protecting cells from mild heat
treatment and thus could stimulate research in functional food
products that receive a mild heat treatment.
Acknowledgment
We would like to thank DANISCO for providing the probiotic strains
that were used in the study.
M450 JOURNAL OF FOOD SCIENCE—Vol. 72, Nr. 9, 2007
References
Adhikari K, Mustapha A, Grun IU, Fernando L. 2000. Viability of microencapsu-
lated bifidobacteria in set yoghurt during refrigerated storage. J. Dairy Sci 83:1946–
51.
Arihara K. 2006. Strategies for designing novel functional meat products. Meat Sci
74:219–29.
Audet P, Paquin C, Lacroix C. 1988. Immobilized growing lactic acid bacteria with k-
carrageenan-locust bean gum gel. Appl Microbiol Biotechnol 29:11–8.
Berrada N, Lemeland JF, Laroche G, Thovenot P, Piaia M. 1991. Bifidobacterium from
fermented milks: survival during gastric transit. J Dairy Sci 74:409–13.
Capela P, Hay TKC, Shah NP. 2005. Effect of cryoprotectants, prebiotics and microen-
capsulation on survival of probiotic organisms in yoghurt and freeze-dried yoghurt.
Food Res Int 39:203–11.
Champagne CP, Girard F, Rodrigue N. 1993. Production of concentrated suspensions
of thermophilic lactic acid bacteria in calcium-alginate beads. Int Dairy J 3:257–
75.
Chandramouli V, Kailaspathy K, Peiris P, Jones M. 2004. An improved method of mi-
croencapsulation and its evaluation to protect Lactobacillus spp. in simulated gas-
tric conditions. J Microbiol Meth 56:27–35.
Cui J-H, Goh J-S, Dim P-H, Choi S-H, Lee B-J. 2000. Survival and stability of bi-
fidobacteria loaded in alginate poly-l-lysine microparticles. Int J Pharm 210:51–
9.
Doleyres Y, Lacroix C. 2004. Technologies with free and immobilosed cells for probi-
otic bifidobacteria production and protection. Int Dairy J 15:973–88.
Floch MH. 2002. Bile salts, intestinal microflora and enterohepatic circulation. Digest
Liver Dis 34:54–7.
Floch MH, Gershengoren W, Diamond S, Hersh T. 1970. Cholic acid inhibition of in-
testinal bacteria. Am J Clin Nutr 23:8–10.
Gardiner GE, O’Sullivan E, Kelly J, Auty MA, Fitzgerald GF, Collins JK, Ross RP, Stanton
C. 2000. Comparative survival rates of human derived probiotic Lactobacillus para-
casei and L. salivarius strains during heat treatment and spray drying. Appl Environ
Microbiol 66:2605–12.
Gilliland SE, Walker DK. 1990. Factors to consider when selecting a culture of L. aci-
dophilus as a dietary adjunct to produce a hypercholesterolemic effect in humans.
J. Dairy Sci 73:905–9.
Goderska K, Zybals M, Czarnecki Z. 2003. Characterization of microencapsulated Lac-
tobacillus rhamnosus LR7 strain. Pol J Food Nutr Sci 12/53:21–4.
Godward SE, Kailasapathy K. 2003. Viability and survival of free, encapsulated and
co-encapsulated probiotic bacteria in yoghurt. Milk Sci Int 58:396–9.
Guarner F, Schaafsma GJ. 1998. Probiotics. Int J Food Microbiol 39:237–8.
Jankowski T, Zielinska M, Wysakowska A. 1997. Encapsulation of lactic acid bacteria
with alginate/starch capsules. Biotechnol Tech 11:31–4.
Kailasapathy K. 2002. Microencapsulation of probiotic bacteria: technology and po-
tential applications. Curr Iss Intest Microbiol 3:39–48.
Kailasapathy K. 2005. Survival of free and encapsulated probiotic bacteria and effect
on the sensory properties of yoghurt. Food Sci Technol 1:1–2.
Kebary KMK, Hussein SA, Badawi RM. 1998. Improving viability of bifidobacteria and
their effect on frozen ice milk. Egypt J Dairy Sci 26:319–37.
Krasaekoopt W, Bhandari B, Deeth H. 2003. Review: evaluation of encapsulation tech-
niques of probiotics for yogurt. Int Dairy J 13:3–13.
Lee KY, Heo TR. 2000. Survival of Bifidobacterium longum immobilized in calcium
alginate beads in simulated gastric juices and bile salt solution. Appl Environ Mi-
crobiol 66:869–73.
Liong MT, Shah NP. 2004. Acid and bile tolerance and cholesterol removal ability of
Lactobacilli strains. J Dairy Sci 88:55–6.
Mandal S, Puniya AK, Singh K. 2006. Effect of alginate concentrations on sur-
vival of microencapsulated Lactobacillus casei NCDC-298. Int Dairy J 16:1190–
5.
Mituoka T. 1992. The human gastrointestinal tract. In: Wood, B.J.B., editor. The lactic
acid bacteria. The lactic acid bacteria in health and disease, Vol. 1. London: Elsevier.
p 69–144.
Noriega L, Cuevas I, Margolles A, De los R-G, Clara G. 2006. Deconjugation and bile
salts hydrolase activity by Bifidobacterium strains with acquired resistance to bile.
Int Dairy J 16:850–5.
Ouwehand AC, Salminen SJ. 1998. The health effects of cultured milk products with
viable and non-viable bacteria. Int Dairy J 8:749–58.
Ouwehand AC, Salminen S, Isolauri E. 2002. Probiotics: an overview of beneficial ef-
fects. Antonie Van Leeuwenhoek 82:279–89.
Picot A, Lacroix C. 2004. Encapsulation of Bifidobacteria in whey protein-based mi-
crocapsules and survival in simulated gastrointestinal conditions and in yoghurt.
Int Dairy J 14:505–15.
Prevost H, Divies C. 1992. Cream fermentation by mixed culture of lactococci en-
trapped in two-layer calcium alginate gel beads. Biotechnol Lett 14:583–8.
Shah NP. 2000. Probiotic bacteria: selective enumeration and survival in dairy foods.
J Dairy Sci 83:894–907.
Shah NP, Jelen P. 1990. Survival of lactic acid bacteria and their lactases under acidic
conditions. J Food Sci. 55:506–9.
Shah NP, Lankaputhra WEV. 1997. Improving viability of Lactobacillus acidophilus
and Bifidobacterium spp. in yogurt. Int Dairy J 7:349–56.
Shah NP, Ravula RI. 2000. Microencapsulation of probiotic bacteria and their
survival in frozen fermented dairy desserts. Aust J Dairy Technol 55:41–
5.
Sultana K, Godward G, Reynolds N, Arumugaswamy R, Peiris P, Kailasapathy K. 2000.
Encapsulation of probiotic bacteria with alginate-starch and evaluation of sur-
vival in simulated gastro-intestinal conditions and in yoghurt. Int J Food Microbiol
62:47–55.
Stacey M, Webb M. 1947. Studies on the antibacterial properties of the bile
acids and some compounds derived from cholanic acid. Proc Roy Soc 134:523–
37.