Journal of Food Biosciences and Technology,

Islamic Azad University, Science and Research Branch, Vol. 4, No. 1, 21-30, 2014

The Effect of Milk Supplementation on the Growth and Viability of

Starter and Probiotic Bacteria in Yogurt during Refrigerated Storage
M. H. Naji a*, Z. Hashemi b, M. Hoseini c
a

Ph. D. Student of Food Science and Technology, Academic Member of Zarindashat Branch, Islamic Azad
University, Fars, Iran.
b
Pediatrician Shiraz Medical Science University, Fars, Iran.
c
Ph. D. Student of Food Science and Technology, College of Agriculture, Mashhad University, Khorasane
Razavi, Iran.

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Received 8 October 2012; Accepted: 24 April 2013

ABSTRACT: In the present work, the effect of milk supplementation on viability of yogurt bacteria
(Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus) and probiotic bacteria
(Lactobacillus acidophilus and Bifidobacteria) was studied during yogurt manufacture and thirty three days
storage. Incubation time to reach pH value of 4.5 was greatly affected by the addition of casein fraction of milk
proteins. The viable counts of L. delbrueckii subsp. bulgaricus and Bifidobacteria were increased in yogurt
supplemented with tryptone and milk powder plus five fold starter culture. Addition of 500 mg L-1 of cysteine,
promoted the growth of L. acidophilus until three weeks from the date of production. Bifidobacteria counts
remained more than 105 cfu ml-1 in yogurt supplemented with 2% milk powder and inoculated with five fold
starter culture. Using a high level of inoculums promoted the viability of L. acidophilus and Bifidobacteria
significantly (p< 0.05) in the first week of storage.

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Keywords: Probiotic, Supplementation, Yogurt.

Introduction1
Probiotics are defined as viable
microorganisms that exhibit a beneficial
effect on the health of the host upon
ingestion by improving the properties of
indigenous microflora (Gomez & Malcata,
1999).
Probiotics enhance the population of
beneficial bacteria in the human gut,
suppress pathogens and build up resistance
against intestinal diseases. The modulation
of immunity, alleviation of lactose
intolerance, prevention of some forms of
cancers and the lowering of serum
cholesterol by these bacteria has also been
reported (Talwalker & Kialasapathy, 2003;
Prado et al., 2008).
Probiotic microorganisms can not affect

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Corresponding Author: [email protected]

the intestinal environment unless their

population reaches a certain minimum level.
There is no general agreement on the
minimum concentration for probiotic
bacteria, while some researchers suggest
concentration level of above 105-106 viable
cell per ml or gram of product (Dave &
Shah, 1997), other stipulate more than 107
and 108 as satisfactory level (De Vuys,
2000).
Several members of the lactic acid
bacteria have gained recognition as probiotic
bacteria, amongst them, Lactobacillus
acidophilus and Bifidobacteria are more
significant.
Yogurt has long been recognized as a
product with many desirable effects for
consumer. In recent years, there has been a
significant increase in the popularity of
yogurt as a food product (Lourens-Hattingh

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M. H. Naji et al.

& Viljoen, 2001).

Yogurt or yogurt-like products have been
used as the most popular vehicle for
incorporation of probiotic bacteria.
Commercially it is not feasible to ferment
milk using only probiotic organisms owing
to the longer time required to reduce the pH
of milk and also objectional taste imported
by some of the probiotic bacterial strains
(Dave & Shah, 1997; Lourens-Hattingh &
Viljoen, 2001; Tamime, 2005).
Most of the probiotic yogurts include live
strains of L. acidophilus and species of
Bifidobacterium in addition to the
conventional
yogurt
organisms,
Streptococcus
thermophilus
and
Lactobacillus delbrueckii subsp. Bulgaricus
(Tamime, 2000; Tamime, 2005).
Despite the importance of viability of
probiotic bacteria, recent market surveys
have revealed poor viability of these
microorganisms in commercial yogurt
preparations
(Shah
et
al.,
1995;
Nighswonger et al., 1996; Vinderola et al.,
2000).
Several works have been done to improve
the growth and viability of probiotic bacteria
by adding supplements to milk base. Milk
supplementation by adding dairy ingredients
(Dave & Shah, 1997; Oliviera et al., 2001;
Sodini et al., 2002), Oxygen scavengers
(Dave & Shah, 1997; Dave & Shah, 1998)
and carbohydrates (Chick et al., 2001) have
been reported.
In this regard, most of the efforts have
been done using ABT (L. acidophilus,

Bifidobacteria and S. thermophilus) starter

cultures that are devoid of L. delbrueckii
subsp. bulgaricus (Dave & Shah, 1998;
Oliviera et al., 2001).
L. delbrueckii subsp. bulgaricus produces
lactic acid during refrigerated storage. This
process is known in the industry as "postacidification" and, if it occurs, it causes a
loss in viability of the probiotic bacteria
(Tamime, 2005).
L. delbrueckii subsp. bulgaricus has a
critical role in lactic acid and flavor
production in yogurt and when excluded
from the starter culture, it profoundly affects
the texture, acidity and the aroma of the final
product.
Therefore, in recent years some yogurt
products have been made using AB-yogurt
cultures (live strains of L. acidophilus and
species of Bifidobacterium in addition to the
conventional
yogurt
organisms,
S.
thermophilus and L. delbrueckii subsp.
Bulgaricus (Lourens-Hattingh & Viljoen,
2001).
The effect of milk supplementation on
growth and viability of probiotic and yogurt
bacteria in such fermented products have not
been studied precisely.
Therefore the aim of this study was to
examine the effects of milk supplementation
on the growth and viability of yogurt and
probiotic
bacteria
using
AB-yogurt
commercial starter culture. These results
would be applicable to the development of
probioric containing fermented dairy
products.

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Table 1. Milk supplemented with various dairy and non-dairy products in different treatment groups
Treatment Groups
Control
TRY
WP
WPC
MP
YE
Suc
TC
TCS
MP-SC

Supplements
None
Tryptone, 0.2% W/V
Whey powder, 0.2% W/V
Whey powder concentrate, 0.2% W/V
Milk powder, 2% W/V
Yeast extract ,0.2% W/V
Sucrose, 0.2% W/V
Tryptone, 0.2% W/V + Cysteine, 500 mg /L
Tryptone, 0.2% W/V + Cysteine, 500 mg /L + Sucrose, 0.2% W/V
Milk powder 2% W/V + 5 fold starter culture

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J. FBT, IAU,Vol. 4, No. 1, 21-30, 2014

were taken at pre-determined intervals to

determine the pH and TA as well as the
viable counts of starter and probiotic
bacteria in yogurt samples.
The one day period represents the
analyses carried out after the samples pH
reached the value of 4.5 and periods of 6 to
33 days represent analyses of yogurt samples
after 6, 14, 23, 33 days of storage at 4C,
respectively.

Materials and Methods

- Starter culture and dairy ingredients
A commercial AB-yogurt starter culture
was used in this study. The starter culture
was in freeze dried DVS (Direct Vat
System) form.
The storage and maintenance of the
culture was carried out according to the
recommendation of the manufacturer.
Six ingredients and their combinations
have been tested (table 1).
- Yogurt production and storage
Pasteurized and homogenized milk
containing 2.5% fat and 10.4% total solid
non-fat, was tempered to 50C and fortified
with supplements as table 1. The mixtures
were heated to 85C for 30 min, cooled to
43C, and the starter culture was added (as
recommended by the supplier). The
inoculated fortified milk was dispensed in
100 ml polystyrene cups. The cups were
heat-sealed with aluminum foil (thickness.
80 m).
Incubation was carried out at 43 0.5C
and fermentation was terminated at pH of
4.5.
The time taken to reach the pH of 4.5 was
recorded for each group in order to study the
effects of added supplements.
When the fermentation was terminated,
yogurt cups were stored at 4C for 33 days.

- Microbiological analyses
Viable counts of S. thermophilus, L.
delbrueckii subsp. bulgaricus, L. acidophilus
and bifidobacteria were monitored during
manufacturing and storage period for 33
days at 4C.
One gram of each yogurt sample was
diluted with 9 ml of 0.09 sterile normal
saline and was mixed uniformly with a
vortex mixer.
Appropriate dilutions were made and
subsequently pour-plate method was applied
in duplicate order onto the selective media.
The counts of S. thermophilus were
enumerated on M17 agar (Merck,
Darmstadt, Germany) and by incubating the
plates aerobically at 37C for 24 3h (Dave
& Shah, 1998).
Differential enumeration of L. delbrueckii
subsp. bulgaricus was performed on MRS
agar (Merck, Darmstadt, Germany) adjusted
to the pH of 5.2 and anaerobic incubation at
43C for 72h (Dave & Shah, 1998).
MRS - clindomycin - ciprofloxacin agar
(MRS-CL/CIP Agar) was used for selective
enumeration of L. acidophilus by incubating
the plates anaerobically at 37C for 72h 3h
(ISO / DIS 20128 IDF 192).
Selective enumeration of Bifidobacteria
was performed on MRS agar supplemented
with 0.5 mg L-1 dichloxallin (Sigma
Chemical Co., St Louis, USA), 1 gr L-1
Lithium chloride (Merck, Darmstadt,
Germany) and 0.5 gr L-1 cysteine
hydrochloride
(Merck,
Darmstadt,
Germany). The anaerobic incubation at 37C

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- Chemical analysis (pH and titrable

acidity determination)
The pH values of the inoculated dairy
mixtures and yogurts were measured using a
pH electrode and meter (Hach pH meter,
Hach company, USA) after calibration.
The titrable acidity (TA) was determined
by the AOAC method and expressed as %
lactic acid (AOAC, 1984).
The pH and TA values of samples were
measured at 30 min intervals, 45-min after
incubation at 4C, until the pH value of 4.5
was reached.
During the refrigeration storage, samples
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M. H. Naji et al.

for 72h 3h was performed by modification

of the method described by Favaro-Trindade
& Grosso (2004).

Results and Discussion

- Effects of milk supplementation on pH
and titrable acidity
Changes in pH and TA during incubation
at 45C for the yogurt mixtures are shown in
table 2 and table 3 respectively.
The times taken to reach the pH value of
4.5 in the yogurt samples indicate the
significant effect of milk supplementation on
the incubation time. The shortest time (3.7
h) was observed in the milk supplemented
with yeast extract and the longest time (6.8
h) was observed in the milk without
supplementation (control group).

Statistical analyses
For
each
condition,
three
independentreplicates of the experiments
were carried out. Before statistical analysis,
the populations of bacteria were converted to
log CFU g-1. All data were analyzed using
the one way ANOVA procedure of the
SPSS, version 11.5 (SPSS, Chicago, Ill.).
Duncan's multiple range test was used to
determine if significant differences existed
among logs CFU g-1 of bacteria

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Table 2. Changes in pH during the fermentation of yogurt (to reach pH ~ 4.5)

Time (min)

Treatment
Groups

45

75

105

135

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6.18
0.16

5.98
0.25

4.99
0.10

4.76
0.04

4.62
0.08

5.37
0.10

5.05
0.04

Control

X
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6.48
0.02

6.41
0.06

6.36
0.07

6.35
0.05

6.32
0.02

TRY

X
S

6.50
0.00

6.41
0.05

6.21
0.05

6.00
0.06

5.60
0.05

WP

X
S

6.49
0.02

6.41
0.04

6.32
0.07

6.25
0.08

5.92
0.14

WPC

X
S

6.51
0.01

6.42
0.05

6.38
0.01

6.33
0.04

6.15
0.01

5.89
0.28

5.16
0.25

MP

X
S

6.37
0.04

6.20
0.11

5.88
0.25

5.36
0.40

4.98
0.21

4.68
0.07

4.54
0.01

YE

X
S

6.18
0.06

6.05
0.09

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255

285

315

345

375

405

5.76
0.32

5.32
0.14

5.05
0.11

4.90
0.14

4.75
0.17

4.57
0.09

4.77
0.01

4.66
0.04

4.54
0.01

4.85
0.13

4.71
0.05

4.60
0.10

4.53
0.04

5.82
0.21

5.54
0.20

5.25
0.13

5.00
0.00

4.78
0.04

4.68
0.08

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6.15
0.01

6.03
0.08

5.80
0.21

5.52
0.20

5.24
0.09

5.00
0.00

4.85
0.06

4.65
0.15

TC

X
S

6.38
0.04

6.29
0.00

6.23
0.01

6.20
0.01

5.82
0.47

5.46
0.66

5.22
0.57

4.86
0.29

TCS

X
S

6.38
0.01

6.30
0.01

6.18
0.04

5.77
0.34

5.30
0.21

4.90
0.00

4.72
0.01

5.06
0.67

MP-SC

X
S

6.41
0.01

6.34
0.01

6.23
0.01

5.91
0.03

5.29
0.04

4.92
0.03

4.72
0.02

4.58
0.02

4.62
0.12

* X and S are mean values and standard deviation respectively.

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J. FBT, IAU,Vol. 4, No. 1, 21-30, 2014

Table 3. Changes in titrable acidity during the fermentation of yogurt (to reach pH ~ 4.5)
Time (min)

Treatment
Groups

45

75

105

135

156

195

225

255

285

315

345

375

39.5
3.5

45.0
0.0

51.5
2.1

49.8
15.9

Control

X
S

14.8
0.8

16.1
1.4

16.6
0.8

16.8
1.1

17.3
0.7

20.1
2.0

27.8
9.6

31.3
11.0

TRY

X
S

16.1
0.1

17.8
1.0

20.5
1.4

24.8
3.9

34.1
1.5

51.3
1.8

58.0
2.8

62.0
0.0

WP

X
S

15.2
0.6

16.1
0.9

17.8
1.1

19.7
1.9

29.4
4.7

37.8
3.2

46.3
1.8

55.5
3.5

62.0
2.8

68.0
8.5

WPC

X
S

14.8
0.3

16.4
0.5

16.7
0.4

18.1
0.6

23.2
6.6

26.6
6.9

40.5
6.4

52.3
1.8

57.5
2.1

61.5
2.1

MP

X
S

16.9
0.6

20.5
3.5

25.9
7.3

39.5
14.4

52.5
13.4

58.5
9.2

64.2
5.9

YE

X
S

20.0
0.0

24.6
4.4

25.9
5.4

33.5
8.1

44.1
10.0

48.0
4.2

61.0
15.6

55.5
0.7

Suc

X
S

20.8
0.4

23.5
3.5

26.6
4.4

33.3
8.1

41.2
7.1

48.0
7.1

59.0
14.1

57.5
0.7

TC

X
S

15.1
0.1

16.4
0.9

17.8
2.5

21.0
5.4

28.3
13.8

33.5
16.3

49.0
33.)

50.6
15.0

TCS

X
S

17.2
1.2

18.9
2.3

21.5
4.2

30.4
10.1

44.0
10.6

57.5
3.5

66.6
3.7

72.0
2.8

MP-SC

X
S

18.5
0.7

19.5
0.7

22.5
0.7

27.5
0.7

44.7
0.5

59.5
0.7

65.0
1.4

71.0
1.4

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8.5

* X and S are mean values and standard deviation respectively.

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Based on these findings, the decrease in

the pH value was faster in yogurts that were
supplemented with casein fraction of milk
proteins than that observed in yogurt that
was supplemented only with whey proteins.
This is in agreement with the report of
Sodini et al. in 2002, who showed that milk
supplementation with casein hydrolysate and
milk protein concentrate decreases the
fermentation time required to reach the pH
value of 5 for milk when incubated with
single culture of S. thermophilus ST7, L.
delbrueckii subsp. bulgaricus LB12, L.
acidophilus LA5 and L. rhamnosus LR35.
The decrease in the pH was faster in
yogurt containing WP, WPC, acid
hydrolysate of casein (ACH), or tryptone

than that of the control group. An increase in

the concentration of cysteine from 50 mgl-1
to 500 mgl-1 caused a pronounced increase in
the time taken to reach the pH value of 4.5
(Dave & Shah, 1998).
- Effects of milk supplementation on
viability of starter and probiotic bacteria
Changes in viable counts of starter and
probiotic bacteria in yogurt supplemented
with various ingredients during 33 of
refrigerated storage are shown in table 4 to
table 7.
Statistical analysis showed, when the pH
reached the value of 4.5, counts of L.
delbrueckii subsp. bulgaricus were higher in
yogurt supplemented with tryptone, WP,

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M. H. Naji et al.

WPC and MP-SC5, than other groups.

During storage for 33 days, the counts of
L. delbrueckii subsp. bulgaricus declined
gradually in all yogurts, except those
supplemented with TC and yeast extract,
which showed the highest counts of this
bacterium at 23rd day and 14th day
respectively. At the 14th day, counts of L.
delbrueckii subsp. bulgaricus were highest
in yogurt supplemented with tryptone and
MP-SC5 while the counts of this bacterium,
didn't have meaningful different between
yogurt samples (p< 0.05) for other days.
The viability of L. delbrueckii subsp.
bulgaricus
Lb1466
was
enhanced
significantly (p< 0.05) in the presence of
probiotic organisms during storage at 4C for
28 day (Donkor et al., 2006).
The number of viable cells of S.
thermophilus remained high (>108 cfu ml-1)
until 33 days from the date of production in
all yogurt samples. This is in agreement with
the report of Dave & Shah, 1998, that the
counts of S. thermophilus were highest in
yogurt supplemented with tryptone.
The counts of S. thermophilus increased
slightly until the 14th day of storage and then
declined for the control, WP, WPC, yeast
and MP-SC5 groups. The incorporation of
cysteine at 250 or 500 mg L-1 adversely
affected the growth of S. thermophilus. In
contrast, cysteine at 50 mg L-1 was found to
promote the growth of S. thermophilus
(Dave & Shah, 1997; Dave & Shah, 1998).
Our results did not show significant effect
of milk supplementation with 500 mg L-1 of
cysteine on the viable counts of S.
thermophilus, while marked reduction (3-4
log) of counts related to S. thermophilus
occurred when fermented soymilk drinks
were held at 25C for 10 days. In contrast
the viable counts remained high in drinks
held at 4C (Wang et al., 2002).
The survival rate of S. thermophilus was
better as compared to the yogurt containing
probiotic bacteria.
Counts of L. acidophilus showed a

constant decline in all yogurt products

during refrigerated storage.
From 0 day until the 14th day, the counts
of L. acidophilus were significantly higher.
Addition of 500 mg L-1 of cysteine,
promoted the growth of L. acidophilus until
three weeks from the date of production.
This confirmed the findings of Dave & Shah
(1997 and 1998) who observed improved
viability of L. acidophilus in yogurt
supplemented with 250 or 500 mgl-1 of
cysteine.
They concluded that, these results could
be due to the adverse effect of cysteine on S.
thermophilus
that
caused
prolonged
fermentation time and perhaps favored the
multiplication of L. acidophilus in yogurt
supplemented with cysteine (Dave & Shah,
1997b).
In the present study, improved viability of
L. acidophilus was neither due to the adverse
effect of cysteine on S. thermophilus nor
longer fermentation time.
There were not significant (p< 0.05)
differences in the counts of S. thermophilus
between yogurts supplemented with cysteine
and other yogurt samples and the counts of
L. acidophilus yogurts with shorter
fermentation time.
All the products showed a decline in
viable counts of Bifidobacteria during
refrigerated storage except the yogurt
supplemented with milk powder and
inoculated with fivefold starter culture (MPSC5).
The counts of Bifidobacteria were higher
considerably between 15 to 33 day of
storage in yogurt supplemented with sucrose
and MP-SC5 than other yogurt samples.
Only, Bifidobacteria counts remained more
than 105 cfu ml-1 in yogurt supplemented
with MP-SC5 throughout the 33 days of
refrigerated storage.
When the time to reach the pH value of
4.5 was taken into consideration, the counts
of Bifidobacteria were significantly (p<0.05)
higher in the yogurt that was supplemented

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J. FBT, IAU,Vol. 4, No. 1, 21-30, 2014

inoculum caused significant (p< 0.05)

increase in the survival of Bifidobacteria
during storage period. This is in contrast
with Dave & Shah, (1997 b) findings who
showed that increased inoculum did not
improve the viability of Bifidobacteria in
yogurt made with ABT starter culture.

with tryptone.
The use of high level of inoculums, will
ensure a high cell count at the end of the
inoculation and survival of the probiotic
bacteria during storage until consumption
(Samona & Robinson, 1994).
In the present study, five fold increase in

Table 4. Changes in the number of Lactobacillus delbrueckii ssp. Bulgaricus during storage of yogurt
supplemented with various ingredients
Treatment
Groups
Control
TRY
WP
WPC
MP
YE
Suc
TC
TCS
MP-SC

0
6.45 (0.00)
7.89 (0.41)
7.43 (1.16)
6.77 (0.10)
6.37 (0.00)
6.36 (0.16)
6.56 (0.20)
6.27 (0.00)
6.55 (0.30)
8.95 (0.21)

7
6.40 (0.40)
6.28 (0.28)
6.50 (0.00)
6.39 (0.52)
6.09 (0.12)
6.22 (0.09)
6.20 (0.48)
6.50 (0.22)
6.17 (0.30)
6.00 (0.02)

Storage time (days)

14
6.38 (0.17)
6.73 (0.22)
6.49 (0.02)
6.30 (0.24)
6.23 (0.00)
6.52 (0.17)
6.32 (0.32)
6.65 (0.12)
6.40 (0.06)
6.86 (0.15)

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5.79 (0.24)
5.98 (0.41)
5.73 (0.58)
6.39 (0.60)
6.25 (0.18)
6.21 (0.03)
5.95 (0.04)
6.71 (0.02)
6.23 (0.06)
6.70 (0.08)

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5.98 (0.16)
5.25 (0.95)
6.19 (0.11)
5.95 (0.10)
5.82 (0.12)
5.95 (0.01)
5.37 (0.13)
5.93 (0.04)
6.07 (0.37)
5.06 (0.31)

Table 5. Changes in the number of Streptococcus thermophilus during storage of yogurt supplemented with
various ingredients
Treatment
Groups
Control
TRY
WP
WPC
MP
YE
Suc
TC
TCS
MP-SC

0
8.39 (0.10)
9.63 (0.15)
8.81 (0.31)
8.70 (0.74)
8.72 (0.46)
8.45 (0.08)
8.76 (0.12)
8.40 (0.45)
8.90 (0.40)
8.85 (0.40)

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Storage time (days)

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14
9.26 (0.31)
8.56 (0.15)
8.66 (0.29)
8.84 (0.06)
8.88 (0.24)
9.20 (0.45)
8.67 (0.37)
9.44 (0.55)
8.67 (0.45)
8.18 (0.67)
8.70 (0.44)
8.56 (0.40)
8.76 (0.52)
8.80 (0.05)
8.89 (0.29)
8.55 (0.03)
8.49 (0.54)
8.55 (0.02)
7.71 (0.40)
9.17 (0.05)

23
8.81 (0.13)
8.80 (0.10)
8.84 (0.25)
8.65 (0.19)
8.26 (0.19)
8.64 (0.34)
8.69 (0.08)
8.36 (0.11)
8.36 (0.07)
8.59 (0.20)

33
9.06 (0.15)
8.45 (0.28)
8.52 (0.07)
8.45 (0.10)
8.22 (0.22)
8.22 (0.05)
8.48 (0.16)
8.12 (0.16)
8.20 (0.12)
8.24 (0.12)

Table 6. Changes in the number of Lactobacillus acidophilus during storage of yogurt supplemented with
various ingredients
Treatment
Groups
Control
TRY
WP
WPC
MP
YE
Suc
TC
TCS
MP-SC

0
6.36 (0.03)
6.07 (0.07)
6.74 (0.05)
6.32 (0.32)
6.34 (0.06)
5.70 (0.28)
6.18 (0.00)
6.23 (0.35)
7.50 (0.14)
6.49 (0.06)

Storage time (days)

7
14
6.23 (0.06)
6.03 (0.01)
5.77 (0.17)
6.06 (0.04)
6.19 (0.19)
6.03 (0.04)
5.70 (0.36)
6.00 (0.03)
6.56 (0.13)
6.15 (0.03)
6.50 (0.27)
6.20 (0.03)
6.56 (0.38)
5.98 (0.20)
6.80 (0.08)
6.47 (0.04)
6.68 (0.15)
6.15 (0.25)
6.60 (0.15)
6.80 (0.02)

23
5.81 (0.11)
5.74 (0.20)
5.59 (0.24)
5.84 (0.13)
5.35 (0.59)
4.81 (0.02)
4.95 (0.04)
4.92 (0.02)
4.87 (0.02)
6.45 (0.08)

33
5.39 (0.55)
5.42 (0.54)
4.56 (0.68)
4.93 (0.17)
4.33 (0.28)
4.37 (0.17)
4.72 (0.13)
4.36 (0.11)
4.48 (0.00)
5.86 (0.17)

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M. H. Naji et al.
Table 7. Changes in the number of Bifidobacteria during storage of yogurt supplemented with various
ingredients
Treatment
Groups
Control
TRY
WP
WPC
MP
YE
Suc
TC
TCS
MP-SC

Storage time (days)

7
14
5.58 (0.01)
4.85 (0.02)
5.82 (0.34)
4.93 (0.19)
5.67 (0.12)
5.18 (0.07)
5.17 (0.05)
4.87 (0.17)
4.69 (0.08)
4.70 (0.01)
5.69 (0.15)
4.74 (0.06)
5.12 (0.25)
5.30 (0.38)
5.82 (0.07)
5.01 (0.24)
4.99 (0.14)
4.97 (0.20)
5.40 (0.08)
5.53 (0.24)

0
5.22 (0.02)
6.89 (0.01)
5.26 (0.20)
5.44 (0.03)
5.34 (0.39)
4.88 (0.41)
4.88 (0.41)
4.73 (0.73)
5.25 (0.21)
5.30 (0.17)

Conclusion
Milk supplementation by adding various
dairy and non-dairy ingredients showed
different patterns of decrease or increase in
pH or titrable acidity during manufacture
and refrigerated storage of probiotic yogurt.
The viable counts of S. thermophilus, L.
delbrueckii subsp. bulgaricus, L. acidophilus
and Bifidobacteria were considerably
affected by the added ingredients.
The time taken to reach the pH value of
4.5 decreased considerably by addition of 2
gr L-1 of yeast extract, tryptone, milk
powder and 500 mg L-1 cysteine, but the
incubation time increased in yogurt mixes
supplemented with 2 gr L-1 of whey protein
and whey protein concentrate.
The viability of L. delbrueckii subsp.
Bulgaricus was improved in yogurt
supplemented with tryptone, milk powder,
WP and WPC.
The addition of 500 mg L-1 of cysteine
promoted the viability of L. acidophilus up
to 15 days of refrigerated storage, while the
viability of L. acidophilus was adversely
affected on addition of cysteine from 21 to
33 days of storage.
The use of high level of inoculums
significantly increased viability of L.
acidophilus and Bifiodobacteria from 15 to
33 days of storage. Furthermore, the addition
of 2 gr L-1 tryptone increased the viable
count of Bifidobacteria for the first week of
the storage. While the addition of growth

23
4.43 (0.25)
4.73 (0.34)
4.44 (0.36)
4.80 (0.03)
4.65 (0.04)
4.33 (0.01)
5.37 (0.56)
4.71 (0.01)
4.73 (0.03)
5.04 (0.02)

33
4.65 (0.09)
4.81 (0.13)
4.80 (0.15)
5.20 (0.43)
4.15 (0.15)
4.60 (0.18)
4.64 (0.39)
4.48 (0.40)
4.23 (0.06)
5.40 (0.18)

D
I

promoting substances affected the growth

and viability of probiotic bacteria in yogurt
but based on related researches it is clear
that the proper selection and combination of
probiotic strains has a profound effect on the
growth and survival of probiotic bacteria in
fermented milks.

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