CHAPTER 4

RESULTS AND DISCUSSION

This chapter deals with the results of the experiments carried out during the course of present
study. Physico-chemical properties of carrot juice and skim milk powder were studied. The
effect of different process parameters such as skim milk powder, carrot juice and inoculum
level, on the physico-chemical, microbial, overall acceptability and storage properties were
observed. The optimization of carrot fortified probiotic yoghurt was carried out according to
central composite rotatable design (CCRD). Twenty different experimental runs were carried
out using Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus as
basic yoghurt cultures and Lactobacillus acidophilus and Bifidobacterium bifidum as
adjuvant cultures for the preparation of carrot fortified probiotic yoghurt. The results were
analyzed for different physico-chemical (syneresis, pH, and water holding capacity),
microbial counts (Lactobacillus acidophilus and Bifidobacterium bifidum counts), overall
acceptability and storage study which are discussed further.
4.1 Some Physico-chemical Properties of Skim Milk Powder and Carrot Juice
The purpose of determination of chemical composition was to get idea about nutritional
quality of ingredients and its suitability to prepare good quality of final product. Various
physico-chemical attributes of Skim Milk Powder and Carrot juice are shown in Table 4.1-
4.2. Total Solids (TS) of carrot juice (by oven drying) were found 8.30 % (w/w) .The results
were in accordance with findings of Salwa et al. reported 7.15 % (w/w) Total solids in carrot
juice [10]. Carrot juice pH was found to be 4.98. Salwa et al. reported that carrot juice pH
falls about 5.85 [10]. The variation in data may be due to various geographical and climatic
conditions such as soil condition, water level, temperature, etc.
Table 4.1 Physio-chemical properties of Skim Milk Powder.
Sr. No. Parameters Quantity
1 Moisture 3.02% (w/w)
2 pH 6.40
3 Acidity 0.158
4 Colour Chalky-White
5 Reconstitutability Very-Good
Table 4.2 Physico-chemical properties of the Carrot juice obtained from the fresh
Carrot.
Sr. No. Parameters Quantity
1 Moisture content 91.7 % (w/w)
2 T.S ( Oven drying) 8.30 % (w/w)
4 pH 4.98
5 Acidity 2.58
6 Total phenolic content 76g(G.A.E)/ml
7 -carotene 30.29g/100g

4.2 Process Optimization by Response Surface Methodology (RSM)
Central composite rotatable design (CCRD) of response surface methodology (RSM) was
applied to optimize the amount of skim milk powder, carrot juice and inoculum level for
syneresis, pH, water holding capacity, overall acceptability, Lactobacillus acidophilus and
Bifidobacterium bifidum count. The experimental range of independent variables was
selected from the literature reviewed. The low and high levels of carrot juice, skim milk
powder and inoculum level were 10 and 20 % (v/v), 14 and 18 % (w/v), 2 to 4 % (v/v)
respectively. For the determination of optimal level of three variables, response surface
approach was applied by using a set of experimental design having three independent
variables at five levels each by using design-expert 6.0 (trial version). The total number of
experiments with three variables were 20 (= 2
k
+ 2k+8), where k is the number of factors.
Twenty experiments were augmented with six replications at the center points to evaluate the
error. The optimization of the carrot fortified probiotic yoghurt was aimed at finding the level
of independent variables that would give minimum syneresis, pH in range of 3.6 to 4
maximum water holding capacity, maximum overall acceptability, maximum Lactobacillus
acidophilus and Bifidobacterium bifidum count . The second order polynomial equation was
fitted to the experimental data of each dependent variable as given below,

n
i
i ii j i
n
i
n
i j
ij i
n
i
i i
x x x x Y
1
2
1
1 1 1
0


Where Yi = Response {Y
1
= Syneresis (%, v/w), Y
2
= pH, Y
3
= Water Holding Capacity
(%w/w), Y
4
= Lactobacillus acidophilus count (cfu/ml), Y
5
= Bifidobacterium bifidum count
(cfu/ml), Y6= Overall Acceptability}
x
i
= Independent variables (x
1
= Skim milk powder (%, w/v), x
2
= Carrot juice (%, v/v), x
3
=
Inoculum level (%, v/v), o is the value of fitted response at the central point of design i.e. i,
ij, ii are the linear, quadratic and cross product regression coefficients, respectively.
The statistical analysis of the experimental data was performed to observe the effect of above
given parameters on measured responses. The adequacy of quadratic models for all responses
was on basis of R
2
, F-value and p-value at 5 % level of significance. The relative effect of
each process parameter on individual response was compared from the values
corresponding to that parameter. The magnitude of values allows comparing the relative
contribution of each independent variable in the prediction of the dependent variable. Higher
the positive value of of a parameter; higher would be the effect of that parameter and vice
versa. The negative value of indicates the negative effect of that parameter on measured
response. The response surface and contour plots were generated for different interaction of
the any two variables, while holding the value of remaining variables at the central level.
Such three dimensional surfaces could give accurate geometrical representation and provide
useful information about the behavior of the system within the experimental design. The
experimental design along with the values of various responses for the above parameters is
given in Table 4.3. The experiments were conducted randomly to minimize the effects of
unexplained variability in the observed responses because of external factors.

4.2.1 Diagnostic Checking of Fitted Model and Surface Plots for Syneresis
The results of second order response model in the form of Analysis of variance (ANOVA) for
syneresis of yoghurt are given in Table 4.4.
The ANOVA results indicated that quadratic regression to produce the second order model
was significant (F-value = 51.94). The value of R
2
= 97.91 % indicates that only 2.09 % of
total variation was not explained by the model. The value of adjusted determination
coefficient (Adjusted R
2
= 96.02 %) was high to advocate a high significance of the model
(Myers and Montegomery) [42]. This suggested that model accurately represents the data in
the experimental region. This also indicated that second order terms were sufficient and
higher order terms were not necessary.

Table 4.4 Regression summery and ANOVA table for syneresis for coded values of
process variables
Source Symbol
-
coefficient
Sum of
Squares
df
Mean
Square
F-
Value
p-Value
Model 40.84 498.29 9 55.37 51.94 <0.0001*
A x
1
-2.99 122.00 1 122.00 114.45 <0.0001*
B x
2
3.99 217.49 1 217.49 204.03 <0.0001*
C x
3
-0.81 8.93 1 8.93 8.38 0.0160*
A*A x
1
2
2.83 115.31 1 115.31 108.18 <0.0001*
B * B x
2
2
-0.53 4.05 1 4.05 3.80 0.0799
C*C x
3
2
-0.53 4.05 1 4.05 3.80 0.0799
A*B x
1
x
2
-1.00 8.00 1 8.00 7.50 0.0209*
A*C x
1
x
3
0.75 4.50 1 4.50 4.22 0.0670
B*C x
2
x
3
0.75 4.50 1 4.50 4.22 0.0670
R
2
0.9791
Adjusted
R
2

0.9602
*Significant at 5 % level.
The magnitude of p-value from Table 4.4 indicates that all linear terms of the variables had
significant effect on syneresis of yoghurt at 5 % level of significance (p < 0.05). The
quadratic terms of skim milk powder, interactive terms of skim milk powder and carrot
juice, also had significant effect on syneresis at 5 % level of significance (p < 0.05).
The quadratic model obtained from the regression analysis for syneresis in terms of coded
levels of the variables after eliminating the non-significant terms is as follows:

Syneresis (%, v/w) = 40.84-2.99x
1
+3.99x
2
-0.81x
3
+2.83x
1
2
-1.00x
1
x
2
(4.1)

The magnitude of value from the Table 4.4 revealed that skim milk powder ( = -2.99) has
highest negative linear effect followed by inoculum level (= -0.81) on the syneresis of
yoghurt at 5 % level of significance (p < 0.05); whereas carrot juice ( = +3.99) has highest
positive linear effect on the syneresis of yoghurt at 5 % level of significance (p < 0.05). This
indicates that with increase in amount of skim milk powder and percent inoculum, there will
be decrease in the syneresis of yoghurt. Decrease in the syneresis of yoghurt with increase in
amount of skim milk powder is due to reduction in free water in more concentrated yoghurt
[43]. The decrease in syneresis of yoghurt with increase in inoculum level is may be due to
the reason that faster acidification rate inhibits the rearrangement in protein network. With
increase in inoculum level, there will be a decrease in electrostatic repulsion between casein
particles which might be a cause of inhibition of rearrangements in protein network [31]. The
values from the Table 4.4 indicate that the increase carrot juice concentration, will increase
the syneresis of yoghurt. Increase in carrot juice concentration, an increase in syneresis of
yoghurt has been observed, which might be due to sudden decrease in pH. The sudden
decrease in pH with increasing concentration of carrot juice may have resulted in very less
solublisation of casein micelles from colloidal calcium phosphate. Less solublisation of
casein micelles causes less availability of casein to form three dimensional network resulting
in increased syneresis of yoghurt. The increase in syneresis of yoghurt with increase in
strawberry juice which is also acidic in nature has been reported by Vahedi et al. in
strawberry yoghurt [44]. Quadratic terms of skim milk powder had shown a significant
positive effect (p < 0.05) on syneresis produced. The interaction terms of skim milk powder
and carrot juice ( = -1.00), had a significant positive effect on syneresis at 5 % level of
significance (p < 0.05).

Fig 4.1 indicates a decrease in syneresis of yoghurt with the increase in skim milk powder
concentration due to reduction in free water in more concentrated yoghurt and increase in
syneresis of yoghurt with increase in carrot juice.

Fig.4.1 Effect of skim milk powder and carrot juice on syneresis (at 3%, v/v inoculum
level).

4.2.2 Diagnostic Checking of Fitted Model and Surface Plots for pH
The results of second order response model in the form of Analysis of variance (ANOVA) for
pH are given in Table 4.5.
The ANOVA results indicated that quadratic regression to produce the second order model
was significant (F-value = 19.56). The value of R
2
= 94.62 % indicates that only 5.38 % of
total variation was not explained by the model. The value of adjusted determination
coefficient (Adjusted R
2
= 89.79 %) was high to advocate a high significance of the model
(Myers and Montegomery) [42]. This suggested that model accurately represents the data in
the experimental region. This also indicated that second order terms were sufficient and
higher order terms were not necessary.
The magnitude of p-value from Table 4.5 indicates that only linear terms of the inoculum
level had significant effect on pH at 5 % level of significance (p < 0.05). The quadratic terms
of skim milk powder, carrot juice and inoculum level, interactive teams skim milk powder
and carrot juice, and skim milk powder and inoculum level, also had significant effect on
pH at 5 % level of significance (p < 0.05).The quadratic model obtained from the regression
analysis for pH in terms of coded levels of the variables after eliminating the non-significant
terms is as follows:

pH = 3.73-0.018x
3
-0.029x
1
2
-0.016 x
2
2
-0.053 x
3
2
-0.022x
1
x
2
-0.022 x
1
x
3
(4.2)

The magnitude of value (Table 4.5) revealed that skim milk powder ( = -5.325 x10
-4
) has a
highest negative linear effect followed by carrot juice(2.230 x10
-3
), inoculum level(-0.018) on
the pH of yoghurt at 5 % level of significance (p < 0.05). The magnitude of values suggest
that with the increase in inoculum level, amount of skim milk powder and carrot juice
concentration, pH decreased. With increase in inoculum level of yoghurt, the population of
microorganisms will increases [42] conversion of lactose to lactic acid resulting in overall
decrease in pH of yoghurt [14]. With increase in skim milk powder a decrease in pH has been
observed, may be due to more availability of lactose to produce lactic acid [14]. A decrease in
pH of yoghurt has been observed with increase in carrot juice concentration, might be due to
acidic nature of carrot juice.Quadratic terms of skim milk powder,carrot juice and inoculum
level had shown a significant (p < 0.05) effect on pH of yoghurt. The interaction term of
skim milk powder and carrot juice ( = -0.022) and skim milk powder and inoculum
level ( = -0.022), had a significant negative effect on pH at 5 % level of significance
respectively.

Table 4.5 Regression summery and ANOVA table for pH for coded values of process
variables
Source Symbol
-
coefficient
Sum of
Squares
df
Mean
Square
F-
Value
p-Value
Model 3.73 0.064 9 7.057 x10
-3
19.56 <0.0001*
A x
1
-5.325 x10
-4
3.872 x10
-6
1 3.872 x10
-6
0.011 0.9195
B x
2
-2.230 x10
-3
6.791 x10
-5
1 6.791 x10
-5
0.19 0.6736
C x
3
-0.018 4.477 x10
-3
1 4.477 x10
-3
12.41 0.0055*
A*A x
1
2
-0.029 0.012 1 0.012 33.01 0.0002*
B * B x
2
2
-0.016 3.864 x10
-3
1 3.864 x10
-3
10.71 0.0084*
C*C x
3
2
-0.053 0.041 1 0.041 114.30 <0.0001*
A*B x
1
x
2
-0.022 4.050 x10
-3
1 4.050 x10
-3
11.22 0.0074*
A*C x
1
x
3
-0.022 4.050 x10
-3
1 4.050 x10
-4
11.22 0.0074*
B*C x
2
x
3
7.500 x10
-3
4.500 x10
-4
1 4.500 x10
-4
1.25 0.2902
R
2
0.9462
Adjusted
R
2

0.8979
*Significant at 5 % level.
Fig 4.2 indicates maxima towards the centre and at 14% skim milk pH increased with the
increasing of carrot juice and the pH is decreased at 18% skim milk with the increasing of
carrot juice.
Fig 4.3 indicates that pH increased with the increasing of inoculum level upto 3.50%
inoculum level beyond this slightly decreased at low concentration of skim milk whereas at
18% skim milk pH increased firstly after sometime it decreased abruptly.


Fig. 4.2 Effect of skim milk powder and carrot juice on pH (at 3%, v/v inoculum level)


Fig. 4.3 Effect of skim milk powder and inoculum level on pH (at 15% v/v carrot
juice)
4.2.3 Diagnostic Checking of Fitted Model and Surface Plots for Water Holding
Capacity.
The results of second order response model in the form of Analysis of variance (ANOVA) for
Water Holding Capacity are given in Table 4.6.
The ANOVA results indicated that quadratic regression to produce the second order model
was significant (F-value = 182.25). The value of R
2
= 97.65 % indicates that only 2.35 % of
total variation was not explained by the model. The value of adjusted determination
coefficient (Adjusted R
2
= 95.54 %) was high to advocate a high significance of the model
(Myers and Montegomery) [42].

This suggested that model accurately represents the data in
the experimental region. This also indicated that second order terms were sufficient and
higher order terms were not necessary.

Table 4.6 Regression summery and ANOVA table for Water Holding Capacity for
coded values of process variables
Source Symbol -coefficient
Sum of
Squares
df
Mean
Square
F- Value p-Value
Model 40.76 220.84 9 24.54 46.20 <0.0001*
A x
1
1.30 22.97 1 22.97 43.24 <0.0001*
B x
2
1.92 50.22 1 50.22 94.56 <0.0001*
C x
3
1.77 42.59 1 42.59 80.19 <0.0001*
A*A x
1
2
0.58 4.78 1 4.78 9.00 <0.0133*
B * B x
2
2
-1.76 44.87 1 44.87 84.48 <0.0001*
C*C x
3
2
-0.83 9.83 1 9.83 18.51 <0.0016*
A*B x
1
x
2
-0.23 0.41 1 0.41 0.76 <0.4030
A*C x
1
x
3
0.22 0.38 1 0.38 0.71 0.4183
B*C x
2
x
3
-2.34 43.62 1 43.62 82.12 <0.0001*
R
2
0.9765
Adjusted
R
2

0.9554
*Significant at 5 % level.
The magnitude of p-value from Table 4.5 indicates that all linear terms of the variables had
significant effect on WHC at 5 % level of significance (p < 0.05). The quadratic terms of
skim milk powder, carrot juice and inoculum level, interactive terms carrot juice and
inoculum level, also had significant effect on WHC at 5 % level of significance (p <
0.05).The quadratic model obtained from the regression analysis for WHC in terms of coded
levels of the variables after eliminating the non-significant terms was as follows:

WHC= 40.76+1.30x
1
+1.92x
2
+1.77x
3
+0.58x
1
2
-1.76x
2
2
-0.83 x
3
2
-2.34x
2
x
3
(4.3)

The magnitude of value (Table 4.6) revealed that carrot juice ( = 1.92) has a highest
positive linear effect followed by inoculum level ( = 1.77 ),skim milk (=1.30) on the WHC
at 5 % level of significance (p < 0.05).This indicates that with the increase in amount of
inoculum level, skim milk powder and carrot juice concentration, there will be increase in
WHC. Fig 4.4 indicates that WHC increased with the increase in the concentration of
inoculum when carrot juice at 10 % whereas slightly decreased at 20% concentration of
carrot juice.


Fig. 4.4 Effect of carrot juice and inoculum level on WHC (at 16% w/v skim milk)





4.2.4 Diagnostic Checking of Fitted Model and Surface Plots for Lactobacillus
acidophilus Counts
The results of second order response model in the form of Analysis of variance (ANOVA) for
Lactobacillus acidophilus counts are given in Table 4.7.
The ANOVA results indicated that quadratic regression to produce the second order model
was significant (F-value = 2826.89). The value of R
2
= 99.96 % indicates that only 0.04 % of
total variation was not explained by the model. The value of adjusted determination
coefficient (Adjusted R
2
= 99.93 %) was high to advocate a high significance of the model
(Myers and Montegomery) [42].

This suggested that model accurately represents the data in
the experimental region. This also indicated that second order terms were sufficient and
higher order terms were not necessary.

Table 4.7 Regression summery and ANOVA table for Lactobacillus acidophilus for
coded values of process variables
Source Symbol
-
coefficient
Sum of
Squares
df
Mean
Square
F- Value p-Value
Model 28.27 6402.38 9 711.38 2826.89 <0.0001*
A x
1
8.63 1018.17 1 1018.17 4046.06 <0.0001*
B x
2
3.96 214.59 1 214.59 852.73 <0.0001*
C x
3
16.54 3737.62 1 3737.62 14852.70 <0.0001*
A*A x
1
2
3.63 190.15 1 190.15 755.64 <0.0001*
B * B x
2
2
-0.27 1.08 1 1.08 4.31 0.0647
C*C x
3
2
8.81 1118.61 1 1118.61 4445.18 <0.0001*
A*B x
1
x
2
0.18 0.25 1 0.25 0.97 0.3471
A*C x
1
x
3
-0.35 0.98 1 0.98 3.89 0.0767
B*C x
2
x
3
-4.65 172.79 1 172.79 686.66 <0.0001*
R
2
0.9996
Adjusted
R
2

0.9993
*Significant at 5 % level.
The magnitude of p-value from Table 4.7 indicates that all linear terms of the variables had
significant effect on Lactobacillus acidophilus counts at 5 % level of significance (p < 0.05).
The quadratic terms of skim milk powder and inoculum level, interactive terms carrot juice
and inoculum level also had significant effect on Lactobacillus acidophilus counts at 5 %
level of significance (p < 0.05).The quadratic model obtained from the regression analysis for
Lactobacillus acidophilus counts in terms of coded levels of the variables after eliminating
the non-significant terms was as follows:

Lactobacillus acidophilus count = 28.27+8.63x
1
+3.96x
2
+16.54x
3
+3.63x
1
2
+8.81x
3
2
-4.65x
2
x
3
(4.4)

The magnitude of value (Table 4.7) revealed that inoculum level ( = 16.54) has a highest
positive linear effect followed by skim milk powder ( = 8.63 ), carrot juice (=3.96) on the
Lactobacillus acidophilus count at 5 % level of significance (p < 0.05).This indicates that
with the increase in amount of inoculum level, skim milk powder and carrot juice
concentration, there will be increase in Lactobacillus acidophilus count. With increase in
skim milk powder, Lactobacillus acidophilus may get more availability of nutrients and
better protection from lowering of pH, resulting in increased Lactobacillus acidophilus count
[5]. There will be an increase in Lactobacillus acidophilus count with increase in inoculum
level. This might be due to high initial numbers of microorganisms [42].

Fig 4.5 Effect of carrot juice and inoculum level on Lactobacillus acidophilus count (at
16 %, w/v skim milk powder).

Fig 4.5 An increase in Lactobacillus acidophilus count has been observed with increase in
carrot juice concentration and inoculum level because the acidic nature of carrot juice will
favour the growth and multiplication of acid loving Lactobacillus acidophilus. Quadratic
terms skim milk powder and inoculum level had shown a significant (p < 0.05) effect on
Lactobacillus acidophilus count. The interaction term of carrot juice and inoculum level (
= -4.05), had a significant negative effect on Lactobacillus acidophilus count at 5 % level of
significance respectively.

4.2.5 Diagnostic Checking of Fitted Model and Surface Plots for Bifidobacterium
bifidumCounts
The results of second order response model in the form of Analysis of variance (ANOVA) for
Bifidobacterium bifidum count are given in Table 4.8.
The ANOVA results indicated that quadratic regression to produce the second order model
was significant (F-value = 193.15). The value of R
2
= 99.43 % indicates that only 0.57 % of
total variation was not explained by the model. The value of adjusted determination
coefficient (Adjusted R
2
= 98.91 %) was high to advocate a high significance of the model
(Myers and Montegomery) [42].

This suggested that model accurately represents the data in
the experimental region. This also indicated that second order terms were sufficient and
higher order terms were not necessary.
The magnitude of p-value from Table 4.8 indicates that all linear terms of the variables had
significant effect on Bifidobacterium bifidum count at 5 % level of significance (p < 0.05).
All quadratic term, interactive terms skim milk powder and inoculum level, carrot juice and
inoculum level, also had significant effect on Bifidobacterium bifidum count at 5 % level of
significance (p < 0.05). The quadratic model obtained from the regression analysis for
Bifidobacterium bifidum count in terms of coded levels of the variables after eliminating the
non-significant terms is as follows:

Bifidobacterium bifidum count = 17.92+3.25x
1
-1.84x
2
+5.08x
3
-3.27x
1
2
-0.801.24x
2
2
-2.21x
3
2
-
+0.84x
1
x
3
-1.20x
2
x
3
(4.5)

The magnitude of value from the Table 4.8 revealed that inoculum level ( = 5.08) has a
highest positive linear effect followed by skim milk powder ( = 3.25) on the
Bifidobacterium bifidum count whereas carrot juice has linear negative effect ( = -1.84) on
the Bifidobacterium bifidum count at 5 % level of significance (p < 0.05).
Table 4.8 Regression summery and ANOVA table for Bifidobacterium bifidumcount for
coded values of process variables
Source Symbol
-
coefficient
Sum of
Squares
df
Mean
Square
F-
Value
p-Value
Model 17.92 768.09 9 85.34 193.15 <0.0001*
A x
1
3.25 144.60 1 144.60 327.28 <0.0001*
B x
2
-1.84 46.01 1 46.01 104.14 <0.0001*
C x
3
5.08 352.80 1 352.80 798.49 <0.0001*
A*A x
1
2
-3.27 154.01 1 154.01 348.56 <0.0001*
B * B x
2
2
-0.80 9.29 1 9.29 21.03 <0.0010*
C*C x
3
2
-2.21 70.17 1 70.17 158.81 <0.0001*
A*B x
1
x
2
-0.27 0.56 1 0.56 1.27 0.2858
A*C x
1
x
3
0.84 5.68 1 5.68 12.85 0.0050*
B*C x
2
x
3
-1.20 11.47 1 11.47 25.96
<
0.0005*
R
2
0.9943
Adjusted
R
2

0.9891
*Significant at 5 % level.
This indicates that with the increase in inoculum level, skim milk powder there will be an
increase in Bifidobacterium bifidum count. An increase in Bifidobacterium bifidum count has
been observed with increase in inoculum level might be due to high initial number of
microorganisms [42].
With increase in amount of skim milk powder, Bifidobacterium bifidum get more availability
of nutrients and better protection from lowering of pH, resulting in increased count [5]. Fig
4.6 also indicates increase in Bifidobacterium bifidum count with increase in inoculum level
and there was an increase in Bifidobacterium bifidum count of yoghurt with increasing of
skim milk. Fig 4.7 indicates decrease in Bifidobacterium bifidum count with increase in
carrot juice might be due to higher number of Lactobacillus acidophilus which may have
suppressed growth of Bifidobacterium bifidum.

Fig. 4.6 Effect of skim milk powder and inoculum level on Bifidobacterium bifidum
count (at 15%, v/v carrot juice)


Fig. 4.7 Effect of carrot juice and inoculum level on Bifidobacterium bifidum count (at
16% w/v skim milk)
4.2.6 Diagnostic Checking of Fitted Model and Surface Plots for Overall acceptability
The results of second order response model in the form of Analysis of variance (ANOVA) for
Overall acceptability are given in Table 4.9
The ANOVA results indicated that quadratic regression to produce the second order model
was significant (F-value = 95.54). The value of R
2
= 98.85 % indicates that only 1.15 % of
total variation was not explained by the model. The value of adjusted determination
coefficient (Adjusted R
2
= 97.82 %) was high to advocate a high significance of the model
(Myers and Montegomery) [42].

This suggested that model accurately represents the data in
the experimental region. This also indicated that second order terms were sufficient and
higher order terms were not necessary.
The magnitude of p-value from Table 4.9 indicates that linear term of the skim milk and
inoculum had significant effect on overall acceptability at 5 % level of significance (p <
0.05). The quadratic terms of all the variables, also had significant effect on overall
acceptability at 5 % level of significance (p < 0.05).The quadratic model obtained from the
regression analysis for overall acceptability in terms of coded levels of the variables after
eliminating the non-significant terms was as follows:

Overall acceptability = 5.21+0.86x
1
+0.75x
3
-0.11x
1
2
-0.43x
2
2

-0.11 x
3
2
+0.14x
1
x
3
(4.6)

Table 4.9 Regression summery and ANOVA table for overall acceptability for coded
values of process variables
Source Symbol
-
coefficient
Sum of
Squares
df
Mean
Square
F-
Value
p-Value
Model 5.21 20.76 9 2.31 95.54 <0.0001*
A x
1
0.86 10.05 1 10.05 416.05 <0.0001*
B x
2
-0.054 0.040 1 0.040 1.66 0.2260
C x
3
0.75 7.68 1 7.68 318.01 <0.0001*
A*A x
1
2
-0.11 0.18 1 0.18 7.65 <0.0200*
B * B x
2
2
-0.43 2.68 1 2.68 111.06 <0.0001*
C*C x
3
2
-0.11 0.18 1 0.18 7.65 <0.0200*
A*B x
1
x
2
-0.013 1.250 x10
-3
1 1.250 x10
-3
0.052 0.8246
A*C x
1
x
3
0.14 0.15 1 0.15 6.26 <0.0313*
B*C x
2
x
3
-0.063 0.031 1 0.031 1.29 0.2818
R
2
0.9885
Adjusted
R
2

0.9782
*Significant at 5 % level.
The magnitude of value (Table 4.9) revealed that skim milk ( = 0.86) has a highest
positive linear effect followed by inoculum level ( = 0.75) on the Sensory at 5 % level of
significance (p < 0.05).Fig indicates that with the increase in amount of skim milk and
inoculum level, there will be increase in overall acceptability of yogurt.

Fig. 4.8 Effect of Skim milk powder and inoculum level on overall acceptability (at 15%
v/v carrot juice)

4.3 Optimization of Process Variables
A numerical multi-response optimization technique was used to find out the optimum levels
of carrot juice, skim milk powder and inoculum level that would give minimum syneresis,
maximum pH, WHC, Lactobacillus acidophilus, Bifidobacterium bifidum count and Sensory
for probiotic yoghurt. The optimum conditions obtained were 17.59 % (w/v) skim milk
powder,12.13 % (v/v) carrot juice , 3.76 % (v/v) inoculum level. Corresponding to these
optimum levels, the predicted value of syneresis, pH, WHC , Lactobacillus acidophilus
Bifidobacterium bifidum count and Overall acceptability were found to be 37.45 %, 3.65,
42.6 %, 54.34 x 10
9
cfu/ml , 22.96 x 10
9
cfu/ml and 6.33 respectively. The total phenolic
content and -carotene of optimized carrot fortified yogurt were found to be 35g/ml
and13g/100g.

Unal et al. [34] also used response surface methodology to describe the combined effect of
storage time, locust bean gum and dry matter of milk on the physical properties of low-fat set
yoghurt and found ideal concentrations of dry matter and locust bean gum as 14 and 0.02 gm
per 100 gm, respectively.


4.4 Storage Study of Optimized Sample
The tabular data for the effect of storage on physico-chemical and microbiological
characteristics of the carrot fortified probiotic yoghurt are shown in Table 4.10. The initial
values of percent syneresis for fresh yoghurts were found to be 37.45% which on storage of
28 days found to be 42.1 %. The results are in conformation with the research of Fox et al.
[45], wherein they stated that rate of syneresis is directly related to the acidity and therefore is
inversely related to pH.
During storage, the pH in fresh sample decreased from 3.65 to 3.43. Fig. 4.11 shows that pH
decreased constantly throughout the storage period. This decrease might be attributed to the
utilization of residual carbohydrates by viable microorganisms and production of lactic acid,
small amounts of CO
2
and formic acid from lactose. Decrease in pH may also be due to
microorganisms activity [44].
The water holding capacity of yoghurt decreased from 42.6% to 34.12%. Fig 4.12 shows that
water holding capacity decreased constantly throughout the storage period. Decrease in water
holding capacity; it might be depend on the more syneresis and non stable structure.
The changes in the viable counts of probiotic bacteria from manufacturing to storage during
four weeks of the carrot fortified probiotic yoghurt were monitored during manufacture and
storage of yoghurt for 28 days at 5C. It was observed that the strains of Lactobacillus
acidophilus and Bifidobacterium bifidum shown good viability in combination with the basic
yoghurt cultures. However, the counts after 28 days remained more than suggested level

of
>10
7
cfu gm
-1
[46]. The results are in close conformation with Martin who found that
probiotic were acid tolerant and can survived in sufficiently higher numbers to remain viable
in cultured dairy products even during storage. This point could be beneficial to manufacturer
in using these strains on industrial scale, to produce functional products with better viability
of beneficial microorganisms [47].




Table 4.10 Effect of storage on syneresis, pH, Lactobacillus acidophilus count and
Bifidobacterium bifidumcount
Response Fresh 1 day 7 days
14
days
21
days
28
days
Syneresis (%, v/w) 37.45 37.90 38.7 39.8 40.6 42.1
pH 3.65 3.64 3.59 3.54 3.49 3.43
WHC(%, w/w) 42.6 41.01 39.45 37.23 36.35 34.12
Lactobacillus acidophilus count
(x10
9
cfu/ml)
52.3 53.3 51.1 50.2 48.5 45.5
Bifidobacterium bifidum count
(x10
9
cfu/ml)
22.8 23.8 21.3 19.8 15.8 10.3

Lactobacillus acidophilus had shown sharp decrease after 21
st
day and shown good viability
for 21 days. Bifidobacterium bifidum count shown sharp decrease throughout the storage
period but is more than suggested value of 10
7
per gm. This dramatic loss termed acidophilus
death may be attributed to hydrogen peroxide produced by the starter lactobacilli. Mahmoud
et al. [48], stated decrease in Lactobacillus acidophilus and Bifidobacterium bifidum due to
antagonist relationship between yoghurt bacteria and probiotic strains and also stated that
dissolved oxygen content directly affected survival of L. acidophilus during storage.
According to Champagne et al. [49], oxygen affects the probiotic cultures in two ways. The
first is a direct toxicity to cells. Certain probiotic cultures are very sensitive to oxygen and die
in its presence, presumably due to the intracellular production of hydrogen peroxide. The
second way the oxygen effects the probiotic cultures is indirect, when oxygen is in the
medium certain cultures, particularly L. delbrueckii, excrete peroxide in the medium and a
synergistic inhibition of bifidobacteria by acid and peroxide has been demonstrated [49].
Shah [38], states that antagonism among the bacteria used in starter cultures caused by
antimicrobial substances such as bacteriocins may decrease the numbers of any sensitive
organisms that may be present in a product or starter culture.



Fig 4.9 Effect of storage on syneresis of carrot fortified probiotic yoghurt


Fig 4.10 Effect of storage on pH of carrot fortified probiotic yoghurt

Fig 4.11 Effect of storage on WHC of carrot fortified probiotic yoghurt


Fig 4.12 Effect of storage on Lactobacillus acidophilus count (cfu/ml) of carrot fortified
probiotic yoghurt

Fig 4.13 Effect of storage on Bifidobacterium bifidumcount (cfu/ml) of carrot
fortified probiotic yoghurt