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Department of Food Engineering, Faculty of Chemical and Metallurgical Engineering, Istanbul Technical University, Maslak, TR-34469 Istanbul, Turkey
School of Biological Sciences, Royal Holloway University of London, Egham, Surrey TW20 0EX, United Kingdom
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 16 June 2011
Received in revised form
7 December 2012
Accepted 11 October 2013
The aim of this study was to investigate the effects of probiotic Bacillus indicus HU36 and dietary bers
(maltodextrin and lemon ber) addition on color and organoleptic quality properties of dark chocolate.
The viability of B. indicus HU36 in dark chocolate was examined as well in the study. Three-level [1.5, 3.5,
5.5 (g/100 g)], two factorial (maltodextrin, lemon ber) Central Composite Design (CCD) was performed
for developing synbiotic chocolate formulation. According to our results, B. indicus HU36 showed survival
rate between 88 and 91% in samples. Descriptive sensory analysis (QDA) and color analysis were performed to examine the effects of factors and their levels on quality attributes and describe developed
chocolates in detail. While bacteria and dietary ber addition did not show any negative effects on
product sensory and color properties; dietary ber addition improved some sensorial features signicantly i.e. sweetness, rmness and adherence, The validation of the model had been accomplished by
applying the conditions generated by the RSM model. This study is the rst report on the use of B. indicus
HU36 in potentially probiotic chocolate production.
2013 Elsevier Ltd. All rights reserved.
Keywords:
Bacillus indicus HU36
Probiotic
Synbiotic chocolate
RSM
Sensory proling
1. Introduction
Chocolate is an internationally craved and highly consumed
product among confectionery products. Latest studies have shown
that chocolate was not only a simple blend of fat and sugar, but also
a rich source of avonoids and polyphenols which shows high
antioxidant activities (Pimentel, Nitzke, Klipel, & de Jong, 2010;
Schinella et al., 2010; Vanzani, Rosetto, De Arco, Rigo, & Scarpa,
2011). In addition to exhibiting antioxidant activity, chocolate
might serve as a better probiotic carrier than dairy products for
intestinal delivery. Possemiers, Marzorati, Verstraete, and Ven de
Wiele (2010) claimed that chocolate ensured probiotic survival up
to 4 times higher than milk-containing products. However, dairy
products are still the most dominant sources for probiotic products
in the market.
Although, probiotic sources are not limited with Lactobacillus
and Bidobacteria species, they are the most dominant species
studied in probiotic formulations. Recent studies showed that
188
bers, particle size 40 micron at most, were mixed with couvertures. Then, the mixture was tempered manually according to
the tabliering method (Brown, 2008; Wybauw, 2004). After cooling
to room temperature samples were wrapped in aluminum foil for
packaging, stored at 18 C until the corresponding analysis.
2.3. A pre-study on dietary ber determination
A 9-point scale hedonic sensory analyses was performed to
determine the most suitable dietary ber type. To conduct the
sensory analysis, chocolate samples were manufactured by using 6
different types of dietary ber (maltodextrin, carboxymethylcellulose, inulin, lemon ber, apple ber, wheat ber), each containing
5 (g/100 g) dietary ber and one control sample without ber was
produced, as well. None of the samples contained B. indicus HU36.
10 voluntary panelists (6 females & 4 males, between 25 and 40
years) were chosen from both graduate students and faculty
members of Istanbul Technical University of Food Engineering
Department. Panelists were selected according to their interests in
chocolate consumption and willingness to participate. They were
given information about basic sensory attributes of chocolate
(appearance, aroma, taste, mouthfeel, texture). Scoring was performed by using a 9-point scale, in which points represented the
expressions from 1 to 9 (dislike extremely to like extremely). The
panels were conducted in two sessions, 4 samples were given to
each panelist in each session.
2.4. Experimental design by using RSM
RSM was employed to investigate the effects of dietary ber
addition on probiotic, sensorial and color properties of product
formulation. Since the results of sensory analysis conducted on 6
dietary bers showed maltodextrin and lemon ber had the
highest acceptability scores, we decided to use those bers as independent factors in the following optimization studies. A two
factorial (dietary ber type: maltodextrin and lemon ber), three
level [ber concentration: 1.5, 3.5, 5.5 (g/100 g)], central composite
design (CCD) was applied. 11 sample formulations were generated
by the model, and the results of microbiological, color and
descriptive sensory analysis were used as responses. Sample formulations, factor levels and responses are summarized at Table 2,
formulations were randomly prepared.
2.5. Viable bacteria count
Viable bacteria count of samples packaged in aluminum foils
and stored at 18 C was performed on the following day of their
preparation. B. indicus HU36 colonies were determined on DSM
agar in accordance with spread plate method; the plates were
incubated at 37 C for 24 h. The microbiological analysis was conducted in duplicates.
2.6. Color analysis
Chroma Meter (Model CR-400 Konica Minolta Sensing Inc.,Osaka, Japan) was used for describing color properties of samples.
CIELAB color parameters (L*, a*, b*) were measured. L* value
denes luminance of the samples between 0 and 100 scale in which
0 denes black and 100 denes white color, a* value describes
color categorizing from green () to red (), while b* value describes color categorizing from yellow() to blue () (Briones &
Aguilera, 2005; Nopens et al., 2008). Bottom and top surface color
measurements of each sample was performed in triplicates.
Whiteness Index (WI) for each sample was calculated according to
the Equation (1) (Briones & Aguilera, 2005; Nopens et al., 2008).
189
Table 1
Hedonic analysis results for the determination of bers to be used in the formulations.
a
Appearance
Control
LF
MD
CMC
Inulin
WF
AF
8.83
8.50
8.67
8.17
7.50
7.00
7.33
Aroma
0.41
0.83
0.52
0.75
0.55
1.10
1.21
8.50
6.83
8.33
6.83
6.67
5.33
6.17
Taste
0.84
0.98
0.82
0.98
0.82
1.97
1.47
8.00
6.33
7.33
1.83
3.67
4.17
4.00
Mouthfeel
0.63
0.82
0.82
0.41
1.37
1.17
1.10
8.33
6.50
7.17
1.00
3.17
3.33
5.00
Texture
0.82
0.84
0.75
0.89
1.17
1.37
2.10
8.17
6.17
7.00
1.17
3.50
3.00
4.50
Overall Acceptability
0.75
0.98
0.89
0.98
1.05
1.10
2.17
8.50
6.83
7.83
1.17
3.33
3.67
4.58
0.55
0.75
0.98
0.98
1.03
1.21
1.11
The results represent the means std deviation of the scores performed by panelists in duplicate. Scoring was performed by using a 9-point scale, in which points represented
the expressions from 1 (dislike extremely) to 9 (like extremely).
a
LF lemon ber, MD maltodextrin, CMC carboxymethylcellulose, WF wheat ber, AF apple ber.
2 2 2 i0:5
h
100 L* a* b*
(1)
Table 2
Experimental design with factors and responses.
Factor 1: MDa (g/100 g)
1.5
5.5
5.5
3.5
1.5
3.5
3.5
1.5
3.5
3.5
5.5
3.5
1.5
5.5
3.5
1.5
3.5
3.5
5.5
1.5
5.5
3.5
Sample code
S1
S2
S3
S4
S5
S6
S7
S8
S9
S10
S11
5.38
33.13
32.62
6.26
5.36
32.52
32
6.65
5.12
7
5.5
5.5
0
3.25
3.5
3
1.75
1.25
3
2
3.75
4
5.5
3
3.25
5.5
3.5
2.5
3.5
2.75
5.40
33.43
32.96
6.42
4.66
31.03
30.45
6.21
6.49
7
5
5
0
3.5
4
5.25
0.5
0.5
3
0.5
3
5
4.75
3.75
4.75
6
3
2.75
4
3
5.51
33.29
32.89
6.43
3.32
30.54
30.12
6.19
4.47
7
5
5
0
3
3.25
6.5
1.25
0.5
2.5
1.5
3
4.75
5.25
4.25
6.25
6
3.25
4.25
3
3
5.50
33.36
32.99
6.15
3.48
34.68
34.3
6.51
2.51
7
4
4.75
0.75
3.75
2.5
4.5
1.25
1
2.25
1
2.5
3
5
4
5
5.75
4
3.25
4
3.25
5.47
33.29
32.9
6.26
3.6
32.93
32.52
6.44
3.67
7
4.5
4.75
0
4
3.25
1.5
2
1
3.5
2
3.5
4
5.5
3.5
2.25
5.5
3.25
1.75
4
3
5.53
34.47
32.02
6.46
4.11
31.77
31.12
7.52
5.26
7
5.5
4
1
4
4
4
2
1
2.75
2
3.75
5
5.25
4.25
4.75
5.5
3.5
2.75
3.5
3.25
5.44
32.58
31.89
7.59
5.99
29.33
28.5
7.58
7.43
7
5.75
5.5
0.5
3.25
2.75
4.5
1.25
0.25
2
1
3
4
5
3.5
5
6
3
3
3.75
3.25
5.42
33.45
32.6
8.14
6.9
32.65
31.79
7.74
7.48
7
5
5.75
0.5
3.25
3
3.75
1
0.75
2
1
3
4
5
4.25
5.5
5.25
3.5
3.5
3.5
2.75
5.35
34.21
33.49
6.9
6.88
31.93
31.27
6.93
6.43
7
5
5.25
0.25
4.25
2.75
3.5
1.5
1
2.75
1.25
3
4.25
5.5
4
2.75
5.75
3.5
2
3.25
2.75
5.54
35.97
35.52
5.63
5.12
33.96
33.39
6.33
5.9
7
4.5
4.5
0
3.75
3
5
1
0.75
2.5
1
2.25
4
5.25
4.5
5.75
6
3.5
4
4
2.75
5.52
32.96
32.48
6.94
4.05
32.06
31.54
6.23
5.68
7
4.25
5.25
0
3.5
3.5
5.75
1.75
0.25
2.25
1.25
2.5
4.75
5.25
4.5
5.5
6
3.75
3.75
4
3
Responsese
a
b
c
d
e
Bacteriab
L* bottomc
WI bottomc
a* bottomc
b* bottomc
L* topc
WI topc
a* topc
b* topc
Smoothnessd
Brightnessd
Brown colord
Bloomd
Cocoa aromad
Cocoa tasted
Sweetnessd
Bitternessd
Bitter aftertasted
Cocoa aftertasted
Bitter avord
Cocoa avord
Chocolate avord
Hardnessd
Breakaged
Firmnessd
Smootnessd
Melting rated
Adherenced
Spreadinessd
Mouthcoatingd
190
Table 3
The glossary of terms, references and their scores dened the quality attributes used in sensory analysis.
Quality
Appearance
Smoothness
Brightness
Brown color
Bloom
Aroma
Cocoa aroma
Off-avor
Cocoa taste
Sweetness
Taste
Bitterness
Bitter aftertaste
Cocoa aftertaste
Bitter avor
Cocoa avor
Chocolate avor
Hardness
Breakage
Firmness
Flavor
Texture
(rst bite)
Texture
(mastication)
Smoothness
Melting rate
Adherence
Spreadiness
Mouthcoating
Denition
Referencesa
a
Dark chocolate denes a commercial product that includes %50 cocoa in formulation. Halva is a commercial product of a national brand. Bitter chocolate denes a
commercial product that includes %99 cocoa in formulation. A commercial chocolate avored pudding is used.
Table 5
Regression coefcients and p-values for signicant responses.
Responses
Factorsa
Coefcient
p-value
Model t
Sweetness
MD conc. (L)
MD conc. (Q)
LF conc. (L)
LF conc. (Q)
MD con. X LF conc.
Intercept
0.955
4.934.103
0.819
0.026
0.0625
0.842
<0.0001b
0.898
0.0003b
0.504
0.085
R2 0.985
F 68.79
p 0.0001
MD conc. (L)
MD conc. (Q)
LF conc. (L)
LF conc. (Q)
MD con. X LF conc.
Intercept
0.887
6.578.103
1.293
0.038
0.109
1.038
MD conc. (L)
MD conc. (Q)
LF conc. (L)
LF conc. (Q)
MD con. X LF conc.
Intercept
0.143
0.023
0.549
8.223.103
0.015
0.596
Table 4
Results of viability of Bacillus indicus HU36 and yield %.
Sample code
MDa (g/100 g)
LFa (g/100 g)
S1
S2
S3
S4
S5
S6
S7
S8
S9
S10
S11
1.5
5.5
5.5
3.5
1.5
3.5
3.5
1.5
3.5
3.5
5.5
3.5
1.5
5.5
5.5
1.5
3.5
3.5
5.5
1.5
5.5
3.5
5.38
5.40
5.51
5.50
5.47
5.53
5.44
5.42
5.35
5.54
5.52
0.13
0.14
0.20
0.23
0.12
0.17
0.16
0.22
0.12
0.18
0.21
Yield (%)
88.44
88.85
90.69
90.46
89.42
90.99
89.49
89.18
88.09
91.14
90.77
Initial count of the bacteria added to all samples was 6.08 log cfu/g. The values
represent the mean std deviation of duplicated measurements for viable bacteria
counts. Yield values calculated as the ratio of nal bacterial count to initial count.
a
MD Maltodextrin, LF Lemon ber.
Lack of Fit
Adherence
Lack of Fit
0.759
0.0033b
0.925
0.0007b
0.596
0.095
0.068
0.0021b
0.517
0.0002b
0.813
0.578
R2 0.946
F 17.50
p 0.0035
R2 0.965
F 27.72
p 0.0012
0.713
L: Linear; Q: Quadratic.
a
MD conc. Maltodextrin concentration (g/100 g); LF conc. Lemon ber
concentration (g/100 g); MDXLF represent the interaction term of maltodextrin and
lemon ber concentrations.
b
Statistically signicant at p < 0.01.
191
Fig. 1. Contour plots of rmness (1), adherence (2) and sweetness (3) as a function of maltodextrin (MD) and lemon ber (LF) conc.
192
bottom and top surface L* values than the reference sample (30.52
and 29.30 for bottom and top surface). WI values showed similar
results to L* values; showing that the reference sample was darker
than all samples we produced.
Fig. 2. Spider diagram of quality attributes (terms and scores were dened in Table 3)
of samples (S1eS11) depending on the results of QDA. MD Maltodextrin conc.;
LF Lemon ber conc.
193
Table 6
Optimum formulations, predicted and experimental results used for model validation.
Samples
Opt 1
Opt 2
Opt 3
Control
MD (g/100 g)
3.91
3.71
3.20
0
LF (g/100 g)
1.5
1.5
1.5
0
Predicted scores
Experimental scores
Sweetness
Firmness
Adherence
Sweetness
3.77
3.59
3.06
3.54
3.41
3.06
2.22
2.16
2.02
3.70
3.38
3.00
2.25
0.27
0.13
0.00
0.17
Firmness
3.62
3.38
3.14
2.45
0.12
0.12
0.13
0.11
Adherence
2.50
2.35
2.10
2.00
0.14
0.13
0.18
0.30
Opt 1, 2 and 3 stands for the optimum formulations designed by the model. MD Maltodextrin, LF Lemon ber.
Experimental scores are the average of responses of 8 panelists performed in duplicate.
formulations, predicted scores and experimental results for responses are summarized in Table 6. Optimization study showed
that, lemon ber level was kept constant at 1.5 (g/100 g), while
maltodextrin levels changed between 3.20 and 3.91 (g/100 g) in
formulations. Based on Table 3, sweetness scores fell in the range
between bitter and milk chocolate reference scores. On the other
hand, the sweetness score of control chocolate was the closest one
to bitter chocolate (Tables 3 and 6). All samples showed similar
rmness and adherence scores to control as bitter chocolate
reference. Table 6 also showed that sweetness, rmness and
adherence scores were decreased as the level of maltodextrin
concentration decreased. The experimental scores were satisfactorily close to the values predicted by the model (R2 was 0.95, 0.93
and 0.99 for sweetness, rmness and adherence). These results
conrm the validation of the model generated by Design Expert
8.0.4 Software.
4. Conclusion
In conclusion, this study shows B. indicus HU36 can be used
efciently for probiotic bitter chocolate production. Microbiological
analysis proved the B. indicus HU36 had a high survival rate in dark
chocolate; and all inoculated samples showed desired probiotic
bacteria load (over 5 log cfu/g product). Descriptive sensory analysis showed that dietary ber addition didnt show negative effects,
such as off-flavor, unwanted aroma or taste, on color and organoleptic properties of samples. Among bers, maltodextrin and lemon
ber addition had positive effects on the sensory characteristics.
Optimum formulations were generated by the RSM model and the
model was validated successfully. According to the results of optimization, the lemon ber concentration should be kept constant at
1.5 (g/100 g) while maltodextrin concentration should be kept
between 3.20 and 3.91 (g/100 g) in order to obtain the best
organoleptic properties of the product.
Acknowledgments
The authors acknowledge the nancial support of EU 7th
Framework Programme acronymed Project COLORSPORE (Project
number: 207948).
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