Bull Vet Inst Pulawy 48, 437-441, 2004
ROLE OF THE PROPORTION OF YOGHURT BACTERIAL
STRAINS IN MILK SOURING AND THE FORMATION
OF CURD QUALITATIVE CHARACTERISTICS
DOROTA CAIS-SOKOLIŃSKA1, MIROSŁAW M. MICHALSKI2 AND JAN PIKUL1
1
Dairy Technology Department, Food Technology Faculty,
August Cieszkowski Agricultural University, 60-624 Poznań, Poland,
2
Department of Hygiene of Food of Animal Origin,
National Veterinary Research Institute, 24-100 Puławy, Poland
e-mail:[email protected]
Received for publication May 12, 2004.
Abstract
The objective of the undertaken investigations was to
evaluate the course of the milk souring process using
Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus
thermophilus bacteria at 1:1 and 3:2 ratios. The concentration
of lactic acid and titratable acidity in the developed curd were
determined. In addition, its active acidity, dynamic viscosity
and sensitivity to syneresis were also measured. It was found
that the acidity of the obtained yoghurt curd was raising along
with the increase in the number of milk lactic acid bacteria in
the applied inoculum. At the same time, the examined yoghurt
showed higher viscosity. As the storage duration of the
yoghurt curd increased, a further increase in the curd acidity
and its sensitivity to syneresis were observed.
Key words: yoghurt, acidity, quality, shelf
life.
The quality of the yoghurt curd, similarly to
other products obtained as the result of the milk
fermentation processes, depends on the quality and
composition of the applied bacterial cultures.
Appropriate proportions used in the bacterial culture
precondition their mutual development and, hence, the
proper course of the milk protein coagulation process
following the acidification of the environment resulting
in the formation of the casein gel of ordered network
structure (5). The microflora applied in the technology
of yoghurt production most commonly is made up of
strains of Lactobacillus delbrueckii subsp. bulgaricus
and Streptococcus thermophilus. These bacteria utilise
milk lactose in the course of the fermentation with
different speed and in different directions leading to
considerable differences in the acidity of the final
product (14). The environmental acidity, resulting from
the growth of microflora deliberately introduced during
the manufacturing process, determines not only the
degree of survival of individual strains in the course of
further yoghurt storage but, later on, leads to changes in
the yoghurt structure and viscosity as well as its
sensitivity to syneresis (12, 16).
The purpose of the performed investigations
was the determination of the effect of the mutual strain
proportions of L. delbrueckii subsp. bulgaricus and Str.
thermophilus bacteria on souring process of milk and the
quality of the developed yoghurt.
Material and Methods
The experimental material was milk curd
obtained using traditional yoghurt cultures and
manufactured in industrial conditions. The initial raw
material was selected crude milk of the highest quality
purchased by a dairy situated in the region of
Wielkopolska. The milk intended for further processing
contained: 12.65% dry matter, 3.26% protein and 4.40%
lactose. The content of fat in the milk used was
standardised to 2% and then milk was homogenised and
pasteurised. Milk cooled down to 43oC was inoculated
with one of the following two bacterial cultures:
A/ low-aromatising culture containing strains of
L. delbrueckii subsp. bulgaricus and Str.
thermophilus at 1:1 ratio,
B/ high-aromatising culture containing strains
of L. delbrueckii subsp. bulgaricus and Str.
thermophilus at 3:2 ratio.
The experimental cultures were added in a
freeze-dried Direct Vet Set (DVS) form in the amount of
50 units of activity to 250 dm3 of processed milk, which
corresponded to a 2% addition of activated working
starter. The inoculated milk was incubated at 43oC. The
incubation was terminated after 6 h by an instantaneous
reduction of the temperature to 6oC and, for the next 18
h, the manufactured yoghurt was allowed to mature at
this temperature. The obtained yoghurt curd was stored
at 8±1oC. The experiment was based on six production
batches. Four yoghurt samples were collected from each
batch (n=24).
438
The results of yoghurt titratable acidity
determinations were expressed in Soxhlet-Henkl degrees
(oSH) (7).
The measurement of the yoghurt active acidity
was carried out using a CP-315 type Elmetron pH-meter
equipped with a combined electrode of the ESAgP301W Eurosensor type consisting of a glass half-cell and
a saturated chlorosilver half-cell.
The content of lactic acid in the examined
yoghurts was determined employing the method
suggested by Lunder (9). The absorbance was estimated
at the wavelength of 400 nm using the Pharmacia
Biotech Novaspech II spectrophotometer.
Rheological properties were evaluated using a
rotational rheometre, type Brookfield RVT ll of the
Brookfield Engineering Lab. Inc. Stoughton utilising the
Couette flow between two coaxial cylinders.
Measurements were carried out at 10±1oC and at the
range of the shear rate γ from 2.2 to 111.4 s-1 (15).
Calculations of the corrected values of the shear rate and
the shear stress were performed using the Kilijański
method (8). Flow curves for yoghurts were drawn from
measured values of shear stresses and apparent dynamic
viscosity. For purposes of a comparative evaluation, the
value of dynamic viscosity was applied, which was
calculated for one-point shear rate equal to γ 10 s-1,
which corresponds to the shear rate in the mouth during
the food consumption (10).
The sensitivity of the curd to syneresis was
assessed on the basis of the released whey using the
method by Dannenberg and Kessler (3).
Results of measurements and assays, obtained
in the course of the performed experiments, were then
subjected to statistical analysis using the Excel
calculation sheet of the EAV program, ELSQ. The
obtained results were employed as the basis for the
determination of the standard deviation and verification
of hypotheses at the set level of significance P=0.05 (2).
Results
The performed statistical analysis of the
obtained results showed that the type of the bacterial
culture applied during milk inoculation had a significant
impact on the titratable acidity of the obtained curd
(Table 1). The titratable acidity of the yoghurt
containing the culture with the proportion of the applied
bacterial strains 1:1 was significantly lower than the
titratable acidity of yoghurts manufactured using the 3:2
proportion. The incubation time of the inoculated
processed milk had a significant impact on the titratable
acidity of the examined yoghurts, irrespective of the
type of the applied bacterial cultures. On average, the
acidity of the inoculated processed milk increased
during the incubation 5.5 times, although it did not
exceed the value of 40oSH. The increase in the titration
acidity was also demonstrated during three weeks of
storage. During the final period of yoghurt storage, the
titratable acidity was found to increase by 20%, in
comparison to their acidity determined directly after
production.
When analysing the obtained results, a
significant influence of the type of the applied cultures
on values of the curd active acidity, both during its
development as well as in the course of the further
storage was demonstrated (Table 2). However, higher
pH values were observed in yoghurts manufactured
using low-aromatising cultures (1:1) than in yoghurts
manufactured using high-aromatising (3:2) cultures.
These differences did not change during incubation,
maturing and storage of the yoghurts. Incubation time,
irrespective of the type of the applied bacterial culture,
was found to have a significant impact on active acidity
of the yoghurts. The pH values of milk under
processing, from the time it was inoculated with
bacterial cultures to the time the yoghurt was
manufactured, decreased from 6.70 to 4.34. It was
demonstrated that during the 21-day period of storage,
the pH values decreased to 4.11. The highest increases
concerned the active acidity of yoghurts manufactured
with the addition of traditional, high-aromatising
cultures.
On the basis of our experiments, it was found
that the quantity of lactic acid in the yoghurts depended
on the type of the applied bacterial cultures (Table 3).
The concentration of the lactic acid in the yoghurt
containing the traditional, low-aromatising culture made
up of the Lactobacillus and Streptococcus genera in the
proportion of 1:1, directly after its manufacture was by
approximately 10% lower than the concentration of
lactic acid determined in the yoghurt with the culture
containing the same bacterial strains but at the 3:2 ratio.
Following the performed incubation of the processed
milk, irrespective of the type of the applied bacterial
cultures, over a five-fold increase was observed in the
contents of lactic acid. The storage of the yoghurts for
21 d was found to lead to a further increase in the
contents of lactic acid by other 27%. At the termination
of storage, the concentration of lactic acid in the
examined yoghurts ranged from 1.08% (in the yoghurts
with the bacterial culture of 1:1 ratio) to 1.11% (in the
yoghurts the ratio of strains was 3:2).
Statistically significant differences in the
dynamics were found between the curd obtained from
milk inoculated with the same bacterial culture but of
different proportion of bacterial strains (Table 4). The
curd manufactured using the high-aromatising culture
was characterised by higher viscosity than that obtained
with the addition of the low-aromatising one. This
difference did not change throughout the period of
storage. During the storage period, the apparent
viscosity of the obtained milk curds decreased in
relation to the curd viscosity directly after its
manufacture. However, the observed decrease was
stronger (by 48%) in the yoghurt containing the
traditional, high-aromatising culture than in the one,
which was obtained with the addition of the lowaromatising culture (by 39%). It was found that changes
in the yoghurt apparent viscosity were not statistically
significant on days 14 and 21 of the storage.
439
The highest sensitivity to syneresis was
observed in the curd of the yoghurt manufactured from
the milk inoculated with the traditional, higharomatising culture after 21 d of storage (Table 5). The
sensitivity to syneresis
of this yoghurts was
significantly higher than in yoghurts containing lowaromatising cultures, irrespective of the storage time. In
the course of storage of the examined yoghurt, a
significant increase was observed in their susceptibility
to syneresis with the duration of storage, which was
associated with the aging process of the curd. It was
found that the sensitivity to syneresis increased over
four times at the end of yoghurt storage.
Table 1
Results of statistical analysis of the influence of the type of bacterial culture on titratable acidity
in yoghurts during their manufacture and storage (n = 24)
Analysed parameter
Incubation time
(h)
Mature time (h)
Storage time
(days)
0
2
4
6
18
7
14
21
1
2
3
4
5
6
7
8
Type of the applied bacterial cultures
(Lactobacillus : Streptococcus)
1:1
3:2
Titratable acidity (oSH)
x
SD
x
SD
A
B
6.58
0.588
6.58
0.588
11.12
0.599
12.55
0.569
28.12
0.560
29.03
0.263
36.50
0.264
39.50
0.478
36.35
0.478
39.92
0.597
40.10
0.452
45.70
0.463
43.80
0.158
47.40
0.365
44.27
0.599
48.30
0.379
1 A, B – lack of difference
2, 3, 4, 5, 6, 7, 8 A < B
A, B 1 < 2; 2 < 3; 3 < 4, 5; 4, 5 < 6; 6 < 7; 7 < 8
Table 2
Results of statistical analysis of the influence of the type of bacterial culture on active acidity (pH) in yoghurts during
their manufacture and storage (n = 24)
Analysed parameter
Incubation time
(h)
Mature time (h)
Storage time
(days)
0
2
4
6
18
7
14
21
1
2
3
4
5
6
7
8
Type of the applied bacterial cultures
(Lactobacillus : Streptococcus)
1:1
3:2
Acidity (pH)
X
SD
x
SD
A
B
6.70
0.563
6.70
0.563
6.18
0.359
6.17
0.265
5.15
0.347
5.04
0.493
4.34
0.748
4.29
0.480
4.34
0.630
4.29
0.149
4.22
0.124
4.11
0.507
4.16
0.540
4.03
0.249
4.11
0.201
3.97
0.077
1, 2 A, B – lack of difference
3, 4, 5, 6, 7, 8 A < B
A, B 1 < 2; 2 < 3; 3 < 4, 5; 4, 5 < 6; 6 < 7; 7 < 8
440
Table 3
Results of statistical analysis of the influence of the type of bacterial culture on lactic acid content in yoghurts during
their manufacture and storage (n = 24)
Analysed parameter
Incubation time
(h)
Mature time (h)
Storage time
(days)
0
2
4
6
18
7
14
21
1
2
3
4
5
6
7
8
Type of the applied bacterial cultures
(Lactobacillus : Streptococcus)
1:1
3:2
Lactic acid (%)
X
SD
x
SD
A
B
0.16
0.089
0.16
0.089
0.23
0.025
0.25
0.047
0.53
0.068
0.66
0.125
0.82
0.024
0.89
0.255
0.82
0.048
0.90
0.589
0.90
0.123
1.07
0.563
1.05
0.145
1.09
0.176
1.08
0.147
1.11
0.441
1, 2 A, B – lack of difference
3, 4, 5, 6, 7, 8 A < B
A, B 1,2 < 3; 3 < 4, 5; 4, 5 < 6, 7, 8
Table 4
Results of statistical analysis of the influence of the type of bacterial culture
on dynamic viscosity in yoghurts during their storage (n = 24)
Analysed parameter
Storage time
(days)
0
7
14
21
1
2
3
4
Type of the applied bacterial cultures
(Lactobacillus : Streptococcus)
1:1
3:2
Apparent viscosity (m Pas)
x
SD
x
SD
A
B
31.74
0.006
39.74
0.003
30.29
0.009
37.29
0.019
20.36
0.073
22.36
0.004
19.44
0.046
20.44
0.053
1, 2, 3, 4 A < B
A, B 1 > 2, 3, 4; 2 > 3, 4
Table 5
Results of statistical analysis of the influence of the type of bacterial culture
on sensitivity to syneresis in yoghurts during their storage (n = 24)
Analysed parameter
Storage time
(days)
0
7
14
21
1
2
3
4
Type of the applied bacterial cultures
(Lactobacillus : Streptococcus)
1:1
3:2
Syneresis (%)
x
SD
x
A
B
4.8
0.01
7.3
6.1
0.01
16.4
12.3
0.01
24.2
19.2
0.05
33.1
1, 2, 3, 4 A < B
A, B 1 < 2, 3, 4; 2 < 3, 4; 3 < 4
SD
0.01
0.18
0.04
0.07
441
Discussion
A faster reduction of the yoghurt pH value
during storage was observed in cultures dominated with
the L. delbrueckii subsp. bulgaricus. These results are
similar to those reported by Salji and Ismail (11), who
found a decline of the pH value from 1.3 to 9.6% of the
yoghurt stored for 3 weeks at the temperature of 7oC.
Results obtained in this study of lactic acid
assays are in keeping with the data reported by Tamime
and Robinson (13) who found over a five-fold increase
in the content of lactic acid found after 3 h and 30 min
of incubation of milk containing 0.15% lactic acid and
12% dry matter, i.e. a value similar to the dry matter
content in the experimental yoghurt.
The more increased potential acidity of the curd
manufactured with the L. delbrueckii subsp. bulgaricus
and Str. thermophilus genera at the 3:2 ratio than at the
1:1 ratio can be attributed to the increased souring
capacity of milk Lactobacillus in comparison to
Streptococcus (5).
According to Vlahopoulou and Bell (16),
yoghurts containing traditional cultures in which the L.
delbrueckii subsp. bulgaricus and Str. thermophilus
strains occur at 1:1 ratio are characterised by a lower
viscosity than those with the traditional, higharomatising culture with the domination of the L.
delbrueckii subsp. bulgaricus strain. The abovementioned researchers reported a 26.3% lower viscosity
of yoghurts manufactured from milk inoculated with the
traditional, low-aromatising culture in comparison with
the viscosity of yoghurts manufactured form milk
inoculated with the traditional high-aromatising culture.
The investigations comprised yoghurts of a 16% dry
matter content, which were inoculated with these
cultures at the amount equivalent to 2% working starter
and incubated at the temperature of 42oC. In addition, L.
delbrueckii subsp. bulgaricus bacteria lead to the
increase in yoghurt viscosity by producing special
mucous substances of polysaccharide nature (1, 4).
These substances form, primarily with casein,
permanent aggregates of high resistance to mechanical
action and, hence, of high resistance to syneresis (6).
Dannenberg and Kessler (3), when carrying out
investigations on natural yoghurts of 4.6 pH value
containing
traditional,
high-aromatic
cultures,
demonstrated their sensitivity to syneresis ranging from
38 to 58%, depending on the extent of the denaturation
of β-lactoglobulin in the processed milk. Similar
interrelations were demonstrated in studies conducted by
Savello and Dargan (12).
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