Int.

Dairy Journal 7 (1997) 349-356

0 1997 Elsevier Science Ltd
All rights reserved. Printed in Great Britain
PII: SO958-6946(97)00023-X 0958-6946/97 $17.00 + 0.00
ELSEVIER

Improving Viability of Lactobacillus acidophilus

and Bijidobacterium spp. in Yogurt

Nagendra P. Shah”* and Warnakulsuriya E. V. Lankaputhrab

nDepartment of Biological and Food Sciences, Victoria University of Technology, Werribee Campus, PO Box 14428, MCMC,
Melbourne, Victoria 8001, Australia
bCentre ,for Bioprocessing and Food Technology, Victoria University of Technology, Werribee Campus, PO Box 14428, MCMC,
Melbourne, Victoria 8001, Australia

(Received 2 November 1996; accepted 26 April 1997)

ABSTRACT

Viability of probiotic bacteria was assessed in yogurt prepared using ruptured or whole cells of yogurt bacteria (Lactobacillus
delbrueckii ssp. bulgaricus 2515 and Streptococcus thermophilus 2010), and whole cells of probiotic bacteria (Lactobacillus
acidophilus 2409 and one species of Bzjidobacterium; B. longum 1941, B. pseudolongum 20099, B. infantis 1912, B. bifidum 1900 or B.
bifidum 1901). Yogurt bacterial cells were ruptured to release their intracellular /&galactosidase (/?-gal) and reduce their viable
counts to improve the viability of probiotic bacteria. Viable counts of probiotic bacteria after fermentation were 2 log cycles higher
in yogurt made with ruptured yogurt bacteria and whole cells of probiotic bacteria and their viability after 6 weeks storage
remained above the recommended level of 106cfygp1, possibly due to the higher level of P-gal released as a result of rupture of
yogurt bacterial cells. Yogurt made with ruptured cells of yogurt bacteria contained less hydrogen peroxide during fermentation.
Ruptured or whole cells of yogurt bacteria produced similar levels of acetaldehyde. Thus, with the approach outlined in this study,
viability of probiotic bacteria could be improved without compromising the flavour of the product. 0 1997 Elsevier Science Ltd.
All rights reserved

Keywords: viability; yogurt bacteria; probiotic bacteria; cell rupture; hydrogen peroxide; acetaldehyde

INTRODUCTION Shah et al., 1995) and in Europe (Iwana et al., 1993)

have shown poor viability of probiotic bacteria, and
Live cultures of probiotic bacteria such as L. especially bilidobacteria, in yogurt preparations.
acidophilus and Bifidobacterium spp. in the diet are Several factors have been claimed to affect the viability
claimed to provide several therapeutic benefits of probiotic bacteria in yogurt, including acid and
including prevention of cancer, reduction in the level of hydrogen peroxide produced by yogurt bacteria,
serum cholesterol, management of normal gut flora oxygen content in the product and oxygen permeation
and improvement in lactose utilization in lactose through the package (Gilliland and Speck, 1977;
malabsorbers (Modler, 1990; Hughes and Hoover, Schioppa et al., 1981; Hull et al., 1984; Ishibashi and
1991; Kurmann and Rasic, 1991; Mishra and Kuila, Shimamura, 1993; Lankaputhra and Shah, 1994,;
1991; Mital and Garg, 1992; Nakazawa and Hosono, Medina and Jordono, 1994; Lankaputhra et al.,
1992; Ishibashi and Shimamura, 1993). As a result, 1996b). Although L. acidophilus tolerates acidity, a
these organisms are increasingly incorporated into rapid decrease in their number has been observed
fermented dairy products such as yogurt. Yogurt under acidic conditions (Conway et al., 1987; Hood
bacteria (Lactobacillus delbrueckii ssp. bulgaricus and and Zottola, 1988; Shah and Jelen, 1990; Lankaputhra
Streptococcus thermophilus) are not indigenous to and Shah, 1995). Bifidobacteria are not as acid tolerant
humans and cannot colonize the intestine to promote as L. acidophilus; the growth of the latter organism
human health. Probiotic bacteria grow slowly in milk ceases below pH 4.0, while the growth of
and the usual practice is to add yogurt bacteria to Bifidobacterium spp. is retarded below pH 5.0 (Shah,
enhance the fermentation process for making probiotic 1997).
yogurt (Samona and Robinson, 1994). For therapeutic Among lactic acid bacteria, yogurt bacteria contain
benefits, the minimum level of probiotic bacteria in the highest lactase activity (Shah and Jelen, 1990).
yogurt has been suggested to be lo5 to lo6 viable cells Lactase or /3-D-galactosidase @-gal) is an intracellular
per mL or g of product (Robinson, 1987). and whole microbial cells exhibit very little
Despite the importance of viability of these beneficial extracellular lactase activity (Kilara and Shahani, 1976;
bacteria, surveys conducted in Australia (Anon, 1992; Shah and Jelen, 1991). Activity of the P-gal can be
increased several times by cell lysis induced by
*Corresponding author. sonication or Eaton press.

349
350 N. P. Shah et al.
B-Gal released after sonication could be used to
hydrolyse a portion of lactose in milk and the products
of lactose hydrolysis, glucose and galactose, could be
used by organisms such as L. acidophilus and
Btfidobacterium spp. Rupturing of yogurt bacteria
could also reduce their viable count and thus the
amount of hydrogen peroxide produced by these
bacteria. Our earlier study (Dave and Shah, 1997) has
shown that hydrogen peroxide produced by L.
delbrueckii ssp. bulgaricus affected the growth of
probiotic bacteria. The objective of this study was to
determine whether viability of probiotic bacteria could
be improved by rupturing yogurt bacterial cells in
order to release their intracellular b-gal and reduce
their viable counts.
MATERIALS AND METHODS
Bacterial strains
L. delbrueckii ssp. bulgaricus 2515, S. thermophilus
2010, L. acidophilus 2409, Bifidobacterium longum 1941,
B. pseudolongum 20099, B. infantis 1912 and B. bifidum
1900 and 1901 were obtained from the Dairy Research
Laboratory, Division of Food Science and Technology,
Commonwealth Scientific and Industrial Research
Organization (CSIRO), Highett, Victoria, Australia.
All strains carry CSCC (CSIRO Culture Collection)
numbers, except B. pseudolongum which carries a DSM
(Deutsche Sammlung von Mikroorganismen und
Zellkulteuren GmbH) number. L. delbrueckii ssp.
bulgaricus 2515 and S. thermophilus 2010 were selected
because of their high extracellular P-gal activity
(Lankaputhra and Shah, unpublished data) and L.
acidophilus 2409 was selected because of its acid and
bile tolerance as reported earlier (Lankaputhra and
Shah, 1995). Cultures were maintained and their
identity confirmed as described earlier (Lankaputhra
and Shah, 1995).
Rupturing and freeze-drying of yogurt bacteria
The two yogurt bacteria were grown separately in 1 L
of deMan; Rogosa and Sharpe (MRS) broth for 16 h at
37°C and the cells in their early log phase were
recovered by centrifuging at 15,344g for 15 min at 4°C
using a Beckman Model L-70 ultracentrifuge and JA-
14 rotor (Beckman Instruments, Palo Alto, CA). The
cells were washed by suspending in sterile phosphate-
buffered saline and re-centrifuged. The cell pellet was
suspended in 50mL (concentration factor 20) of sterile
saline solution, the suspension cooled to <4”C and
10 mL of glass beads of 0.1 mm size were added. The
cell suspension and glass beads were placed in a 70mL
sterile stainless steel adaptor and mechanical vibration
was applied using an MSK cell homogenizer (B. Braun
Melsungen AG, Melsungen, Germany) for 30, 60 or
90s in order to rupture the cells. Samples were taken
before and after cell rupture to enumerate viable
counts and to measure fl-galactosidase activity. The
ruptured cell suspension was centrifuged at 1380g for
1 min using the Beckman ultracentrifuge to remove the
glass beads. The cell suspension was mixed with 12%
(w/v) reconstituted non-fat dry milk (NDM) at a 1:1
ratio, frozen at -20°C and freeze-dried for 16 h at
-30°C using a Dynavac FD 300 freeze-drier (Dynavac
Engineering Pty Ltd Inc., Melbourne, Victoria,
Australia).
Harvesting and freeze-drying of probiotic cultures
L. acidophilus 2409 and five species of
Bifidobacterium (B. longum 1941, B. pseudolongum
20099, B. infantis 1912, B. bifi:dum 1900 and 1901) were
grown separately in MRS broth for 16 h and the cells
were harvested and washed as for yogurt bacteria. The
washed cells were suspended in NDM, frozen at -20°C
and freeze-dried as for yogurt bacterial cultures.
All freeze-dried starter cultures were packed in
MacCartney glass bottles with airtight seals and stored
at 4°C until use.
Preparation of yogurt
Homogenized and pasteurized milk supplemented
with 5% non-fat dry milk was heated to 85°C for
30 min, cooled to 42°C and 0.1% freeze-dried, ruptured
(60s) starter cultures of L. delbruckii ssp. bulgaricus
2515, S. thermophilus 2010 and L. acidophilus 2409 and
Btfidobacterium spp. were added. Five different types
of yogurt were prepared each containing L. delbrueckii
ssp. bulgaricus 2515, S. thermophilus 2010, L.
acidophilus 2409 and one species of BiJidobacterium (B.
longum 1941, B. pseudolongum 20099, B. infantis 1912,
B. bfidum 1900 or B. bfidum 1901). The yogurt mix
was incubated at 42°C and samples were taken during
fermentation at hourly intervals until the pH reached
4.5 for measurement of pH and titratable acidity,
enumeration of yogurt and probiotic bacteria and for
determination of B-D-galactosidase activity, hydrogen
peroxide and acetaldehyde. The yogurt was then stored
for 6 weeks at 4°C and viable counts of probiotic
bacteria were determined at weekly intervals.
Enumeration of bacteria and chemical analyses
Enumeration of yogurt and probiotic bacteria
L. delbrueckii ssp. bulgaricus was enumerated
according to the method of Dave and Shah (1996)
using MRS agar (Oxoid, W. Heidelberg, Australia)
adjusted to pH 5.2 and anaerobic incubation at 43°C
for 72 h. Streptococcus thermophilus agar and aerobic
incubation at 37°C were used for selective enumeration
of S. thermophilus as per the method of Dave and Shah
(1996). L. acidophilus was enumerated according to the
method of Lankaputhra and Shah (1996) using
modified MRS-salicin agar and bilidobacteria were
enumerated according to the method of Lankaputhra
et al. (1996a) using MRS-NNLP (nalidixic acid,
neomycin sulphate, lithium chloride and paromomycin
sulphate) agar.
Measurement of enzyme activity
/?-D-Galactosidase activity of the freeze-dried cell
preparations and that of the yogurt mix during
fermentation was determined according to the method
of Shah and Jelen (1990) using o-nitrophenyl$-D-
galactopyranoside (ONPG) as substrate. The unit of
lactase activity was estimated according to the method
 Viabilityofprobiotic bacteria in yogurt 351

of Mahoney et al. (1975) as the amount of the enzyme
which liberated one pmole o-nitrophenol from ONPG.
Measurement of hydrogen peroxide and acetaldehyde.
Concentrations of hydrogen peroxide and
acetaldehyde were determined according to the
methods of Gilliland (1968) and Millies et al. (1989),
respectively..
All experiments and analyses were replicated three
times. The results presented are averages of all
replicates.
RESULTS AND DISCUSSION
The viable counts and /?-gal activity of L. delbrueckii
ssp. bulgaricus 2515 and S. thermophilus 2010 before
and after cell rupture are shown in Table 1. After 60s
cell-rupture treatment, the viable counts of L.
delbrueckii ssp. bulgaricus 2515 were reduced from
4.2x 10” to l.oxlo5cfug~-’ and those of S.
thermophilus 2010 from 3.6~10” to 4.2x106cfug-‘.
The cell rupture time of 60 s was used throughout the
study. The P-gal activity increased from 38 to 310 unit
per gram of cell suspension for L. delbrueckii ssp.
bulgaricus 2515 and from 25 to 286 for that of S.
thermophilus 2010 culture. Shah and Jelen (1990)
observed a 5-fold increase in lactase activity upon
sonication of an L. delbrueckii ssp. bulgaricus culture,
while an S. thermophilus culture produced more
enzyme activity per gram of dry cell weight in glucose
and lactose containing APT (All Purpose Tween)
broths than that of L. delbrueckii ssp. bulgaricus. This
could be due to differences in strain or due to variation
in growth medium.
Five different batches of yogurt were made. The
strains of L. delbrueckii ssp. bulgaricus, S. thermophilus
and L. acidophilus were kept the same in all five
batches; however, the species of Bifidobacterium varied
in each batch of yogurt. Several studies reported
varying viability of various Btfidobacterium spp.
(Anon, 1992; Shah et al., 1995; Dave and Shah, 1997).
B. longum 1941 and B. pseudolongum 20099 have been
found to survive well in the presence of acid and bile
salts (Lankaputhra and Shah, 1995).
Figure 1 shows the changes in the viable counts of
yogurt and probiotic bacteria during yogurt
fermentation using ruptured (9 h) or whole (7 h) cells of
yogurt bacteria with whole cells of probiotic bacteria.
As shown, the viable counts of L. delbrueckii ssp.
bulgaricus 2515 and S. thermophilus 2010 gradually
increased from lo’-lo3 to 105-106cfug-’ at the end of
9 h of fermentation (Fig. la) using ruptured yogurt
bacteria and whole cells of probiotic bacteria, while the
numbers of L. acidophilus 2409 and B. longum 1941
increased to 107-lo* from initial counts of IO’cfug-‘.
Similar trends were observed during fermentation with
yogurt bacteria, L. acidophilus 2409 and other species
of Btjidobacterium such as B. pseudolongum 20099, B.
infantis 2912, B. b$dum 1900 and B. b$dum (data not
included).
Viable counts of L. delbrueckii ssp. bulgaricus 2515
and S. thermophilus 2010 increased from 105-lo6 to
10’~10* cfu gg’ in 7 h of fermentation (Fig. lb) when
whole cells of the two yogurt bacteria were used. The
fermentation time was 2 h shorter and the final counts
of the two yogurt bacteria were approximately 2 log
cycles higher in yogurt made with whole cells as
compared with that made with ruptured cells. Counts
of L. acidophilus 2409 and B. longum 1941 were the
same at 0 h in yogurts made with ruptured or whole
cells; however, the final counts of L. acidophilus 2409
and B. longum 1941 after 9 h of fermentation with
ruptured cells were l-2 log cycles higher. Viable counts
of the culture bacteria using whole cells of L.
delbrueckii ssp. bulgaricus 2515, S. thermophilus 2010,
L. acidophilus 2409 and the other four species of
Bijidobacterium during 7 h of fermentation showed
similar trends (data not included).
Rupturing yogurt bacteria decreased the viable
counts by about 7 to 8 log cycles and increased b-gal
activity about 15-fold (Table 1). The initial counts of
yogurt bacteria at 0 h were approximately 3 log cycles
lower as a result of cell rupture. Reduced viable counts
of yogurt bacteria may have helped the probiotic
bacteria to build up their numbers. L. acidophilus and
Bifidobacterium spp. are easily dominated and
outgrown by other bacteria while growing together.
Higher counts of probiotic bacteria in yogurt prepared
using ruptured yogurt bacterial cells could also be due
to increased levels of P-gal produced by yogurt bacteria
as compared with yogurt prepared with whole cells of
both groups of organisms.
The final counts of the two yogurt bacteria, L.
acidophilus 2409 and Btyidobacterium spp. in five
batches of yogurt after 9 or 7 h of fermentation are
summarized in Table 2. In all five batches, bacterial
counts of yogurt bacteria were 2-3 log cycles higher in
yogurt made from whole cells as compared with that
made using ruptured cells, while the probiotic bacterial

Table 1. Viable Counts and /?-Galactosidase Activity of Lactobacillus delbrueckii ssp. bulgaricus 2515 and Streptococcus
thermophitus2010 Before and After Cell Rupture
Organisms Before cell rupture After cell rupture for
30s 60s 90s
Viable counts (cfug- ‘)
L. delbrueckii ssp. 4.2x 10” 3.2x lo7 1.0x lo5 1.2x102
bulgaricus 25 15
S. thermophilus2010 3.6x 10” 1.0x lox 4.2x lo6 1.8x104
p-galactosidase activity”
L. delbrueckii ssp. 38 202 310 512
bulgaricus 25 15
S. thermophitus20 10 25 242 286 371

“pmole o-nitrophenol min-’ gP’culture.

352 N. P. Shah et al.

9.
q L. bulgartcus
6 __ n S. thermophilur
0 L. actdophilus
Fl

c 6
=8 6
E
a 4
:
P 3
s

0 1 2 3 4 5 6 7 6 9

Time of incubation (h)

1 HL. bulaaricus

0 1 2 3 4 5 6 7

Time of incubation (h)

Fig. 1. Changes in the viable counts of two yogurt bacteria (Lactobacillusdelbrueckii ssp. bulgaricus 2515 and Streptococcus ther-
mophilus2010) and two probiotic bacteria (Lactobacillus acidophilus2409 and Btfidobacteriumlongum 1941) during fermentation
of milk with (a) ruptured cells of yogurt bacteria and whole cells of probiotic bacteria, and (b) whole cells of yogurt and probiotic
bacteria.

counts were about l-2 log cycles lower. Among the
Bifidobacterium spp., B. longum 1941 and B.
pseudolongum 20099 gave the highest counts in yogurt
made using either whole or ruptured cells and B.
bifidum 1901 the lowest. However, B. pseudolongum is
of animal origin and may not provide therapeutic
benefits and thus could not be considered as a good
candidate for incorporation into fermented dairy foods
such as yogurt. B. btjidum is commonly used by the
Australian yogurt manufacturers, but this organism
has been found to be poorly tolerant to acid, bile and
hydrogen peroxide (Lankaputhra and Shah, 1995;
Shah, 1997).
These results are consistent with previous findings.
Reuter (1990) conducted a survey of fermented milk
products containing bifidobacteria in Germany, France
and Japan and found that B. longum was widely used
in Germany. Clark et al. (1993) studied the survival of
B. infantis, B. adolescentis, B. longum and B. bifidum in
acidic conditions and reported that B. longum survived
the best. Clark and Martin (1994) reported that B.
longum tolerated bile concentration as high as 4%.
Thus, it appears that B. longum could be the best
candidate for use as dietary adjunct in fermented dairy
products such as yogurt.
Changes in the viable counts of probiotic bacteria in
yogurt made with ruptured or whole cells of yogurt
bacteria during 6 weeks of storage are shown in Fig.
2(a,b). In general, the viability of L. acidophilus 2409
and B. longum 1941 decreased during storage. Viability
of probiotic bacteria in yogurts made with the two
yogurt bacteria, L. acidophilus 2409 and four other
 Viability of prabiotic bacteria in yogurt 353

Table 2. Counts of Yogurt Bacteria (Lactobacillus delbrueckii ssp. bulgaricus 2515 and Streptococcus thermophilus 2010) and
Probiotic Bacteria (Lactobacillus acidophilus 2409 and Bifidobacterium spp.) in Five Batches of Yogurt Made Using Ruptured or
Whole Cells of Yogurt Bacteria and Whole Cells of Probiotic Bacteria (Counts are Taken After 9 or 7 h of Fermentation of Milk at
42°C when the pH Reached 4.5)

Organisms Batches of yogurt made with various types of yogurt and probiotic organisms
1 2 3 4 5

Viable counts (cfu gg’) after 9 h of fermentation of milk with ruptured yogurt bacteria and whole cells of probiotic bacteria
L. delbrueckii ssp. 4.2x lo5 4.7 X lo5 4.1x105 4.6x lo5 1.0x IO5
bulgaricus 2.515
S. thermophilus 2.6x IO6 3.1x106 2.6x lo6 7.2x 10’ 7.2x lo6
2010
L. acidophilus 2409 7.8x 10’ 7.4x IO7 5.8x 10’ 3.1 x 10’ 1.5x10’
B$dobacterium 1.2x lo8 1.6~10~ 4.7x lo7 8.5x 10’ 8.5x IO6
SPP.”
Viable counts (cfu g ‘) after 7 h of fermentation with whole cells of yogurt and probiotic bacteria
L. delbrueckii ssp. 9.8x 10’ 8.6x 10’ 8.6x 10’ 2.8x 10’ l.9x108
bulgaricus 25 15
S. thermophilus 8.2x lo8 9.2x 10’ 6.8x lox 1.6x10” l.lx109
2010
L. acidophilus 2409 1.4x lo6 1.6x lo6 1.2x 10h 4.1x106 1.9x106
Bifi’dobacterium 6.7x lo6 1.0x IO7 9.0x lo6 l.5x106 9.7x IO4
spp.”

UB. longum 1941, B. pseudolongum 20099, B. infantis 2912, B. bifidum 1900 and B. bifidum 1901 were used for manufacturing batches
of yogurts 1 to 5, respectively.

species of Btfidobacterium showed similar trends (data in the whole vs ruptured cell fermentations. However,
not included). The counts of L. acidophilus 2409 and B. the counts of probiotic bacteria remained above the
fongum 1941 in yogurt made from ruptured yogurt recommended level of 1 million viable cells gg’ in
bacteria and whole cells of probiotic bacteria decreased yogurt prepared with ruptured cells; the improved
by about 58- and 3.4-fold, respectively, whereas the viable counts of probiotic bacteria could be due to
decay of these bacteria in yogurt made from whole higher initial counts of these organisms.
cells of yogurt and probiotic bacteria was 11.6- and Table 3 shows changes in titratable acidity (TA),
8.7-fold, respectively, after 6 weeks of storage. In the /I-gal activity, hydrogen peroxide and acetaldehyde
case of B. longum, a 2.6-fold faster death rate was during yogurt manufacture containing L. acidophilus
observed as compared with O.Zfold for L. acidophilus 2409 and B. longum 1941. Changes in these

Table 3. Changes (MeanfSD) in Titratable Acidity, /?I-Galactosidase Activity, Hydrogen Peroxide and Acetaldehyde
Concentrations During Fermentation of Milk with Ruptured or Whole Cells of Yogurt Bacteria (Lactobacillus delbrueckii ssp.
bulgaricus 2515 and Streptococcus thermophilus 2010) and Probiotic Bacteria (Lactobacillus acidophilus 2409 and Bifidobacterium
longum 194 1)

Time of incubation (h) Titratable acidity (%) /?-Gal activity” Hydrogen peroxide Acetaldehyde (pg g-i)
(M g-‘)
Yogurt made with ruptured cells of yogurt bacteria
and whole cells of probiotic bacteria
0 0.32f0.02 1.81*0.13 ND ND
1 0.33f0.02 1.82f0.10 ND ND
2 0.40f0.03 1.84f0.14 ND ND
3 0.51f0.02 1.90f0.12 l.lf0.1 0.6fO. 1
4 0.75f0.02 2.01f0.11 2.2f0.2 1.2*0.2
5 0.8lf0.02 2.02fO. 12 2.7fO. 1 1.9ztO.l
6 0.90*0.01 1.76f0.08 3.2f0.2 2.6&O. 1
7 1.2lztO.02 1.681tO.09 2.8f0.2 3.350.2
8 1.30f0.01 1.53ztO.12 2.5fO. 1 3.3ztO.l
9 1.4oIto.03 1.3lf0.10 1.8ztO.l 3.3ztO.l
Yogurt made with whole cells of yogurt and probiotic bacteria
0 0.32f0.02 0.12f0.04 ND ND
1 0.33f0.02 0.12f0.03 1.3f0.2 0.5ItO.l
2 0.53f0.01 0.14f0.06 2.6&O. 1 1.OfO. 1
3 0.74f0.01 0.58f0.09 2.8fO. 1 1.6ztO.2
4 0.91f0.03 0.86f0.08 3.lztO.3 2.2Ito.2
5 1.08f0.03 1.3lf0.10 3.8f0.2 2.6&O. 1
6 1.27f0.01 1.30f0.08 3.6f0.3 3.lztO.l
I 1.40f0.02 1.30f0.07 3.2f0.2 3.9+0.2
a pmole o-nitrophenol mm’ g-’ mix.
ND = not detected.
354 N. P. Shah et al.

q L. acidoohilus

2 3 4 5 6

Time of storage (week)

•I L. acidophilus
W 6. bngum

1 2 3 4 5 6
Time of storage (week)

b
J

Fig. 2. Changes in the viable counts of two probiotic bacteria (Lactobacillus acidophilus 2409 and Bifidobacterium longum 1941)
during storage of yogurt made with (a) ruptured cells of yogurt bacteria and whole cells of probiotic bacteria, and (b) whole cells
of yogurt and probiotic bacteria.

parameters in other batches of yogurt made using
the other four species of Bifidobacterium are not
included in Table 3. The TA increased to 1.4% in
yogurt after 9 or 7 h of fermentation. There was a
slight increase in the TA of yogurt during 6 weeks
of storage (data not shown). As expected, p-gal
activity was higher in the yogurt mix containing
ruptured cells. The enzyme activity reached about
2.0 unit per gram of mix during fermentation, then
declined as the pH decreased. Acidification of
sonicated culture has been found to result in loss of
enzyme activity (Shah and Jelen, 1990). The enzyme
activity increased to 1.31 unit per gram of mix
during fermentation and the loss in activity was
minimal in yogurt made with whole cells of yogurt
and probiotic bacteria. The microbial cell
membrane, cell wall, or both may have aided in
protecting the /?-gal from acid denaturation in
yogurt made with whole cells of yogurt and
probiotic bacteria.
Production of hydrogen peroxide was higher in
yogurt made with whole cells of yogurt and probiotic
bacteria as compared with that made using ruptured
cells. This may be due to reduction in the initial viable
count of yogurt bacteria as a result of cell rupture. No
hydrogen peroxide was detected after one week of
storage of the product (data not shown).
Yogurt made with ruptured or whole cells of yogurt
bacteria showed almost the same level of acetaldehyde
after 9 or 7 h of fermentation, respectively. The levels
of acetaldehyde should be sufficient to produce desired
flavour as the threshold level of acetaldehyde for
development of characteristic yogurt flavour is about
0.4ppm. The production of acetaldehyde was slow in
yogurt made with ruptured yogurt bacteria, obviously
due to lower levels of live yogurt bacteria.
 Viability ofprobiotic bacteria in yogurt 355

SUMMARY AND CONCLUSIONS Hughes, D. B. and Hoover, D. G. (1991) Bifidobacteria -

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lower and of probiotic bacteria l-2 log cycles higher in
Lactobacillus acidophilus in yoghurt. Australian Journal of
yogurt made with ruptured yogurt bacteria cells and Dairy Technology 39, 164-166.
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of probiotic bacteria decreased during storage but were commercial yoghurts sold in Europe. Bifidobacteria Micro-
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ACKNOWLEDGEMENT Bifidobacterium spp. Milchwissenschaft 51, 446451.
Lankaputhra, W. E. V., Shah, N. P. and Britz, M. L. (1996a)
The authors thank the Australian Research Council Evaluation of media for selective enumeration of Lactoba-
cillus acidophilus and Bifidobacterium spp. Food Australia
and Plumrose (Australia) Pty Ltd (now Nestle Dairy
48, 113-118.
Products) for funding this project.
Lankaputhra, W. E. V., Shah, N. P. and Britz, M. L. (1996b)
Survival of bifidobacteria during refrigerated storage in the
presence of acid and hydrogen peroxide. Milchwissenschaft
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