LWT - Food Science and Technology 56 (2014) 187e193

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LWT - Food Science and Technology

journal homepage: www.elsevier.com/locate/lwt

Development of a novel synbiotic dark chocolate enriched with

Bacillus indicus HU36, maltodextrin and lemon ber: Optimization by
response surface methodology
zlem Erdem a, Mine Gltekin-zgven a, Ijlal Berktas a, Sevcan Ersan a, H. Ezgi Tuna a,

a, Beraat zelik a, *, Grbz Gnes a, Simon M. Cutting b
Ayse Karadag
a
b

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

* Corresponding author. Tel.: 90 212 2856042; fax: 90 212 2857333.

E-mail addresses: ozcelik@itu.edu.tr, beraat.ozc@gmail.com (B. zelik).
0023-6438/$ e see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.lwt.2013.10.020

Bacillus species like Bacillus subtilis, Bacillus pumilus, Bacillus

coagulans, Bacillus cereus and Bacillus clausii have probiotic properties (Cutting, 2010; Hong, Duc, & Cutting, 2005). Bacillus indicus
HU36 is spore-forming bacteria with a high resistance to gastrointestinal environment and it has been characterized for its safety
as probiotic supplements and its high content of dietary carotenoids (Duc, Fraseer, Tam, & Cutting, 2006; Hong et al., 2008).
Dietary bers are carbohydrates of plant origins which are
indigestible in small intestine. They are claimed to possess many
health benets such as; lowering calorie intake, shortening bowel
transit time, increasing fecal bulk, delaying gastric emptying,
helping to reduce the risk of cancer and heart disease, slowing
glucose absorption, enhancing immune functions and lowering
serum cholesterol levels (Drehner, 2001). They are added to a variety of food products for nutritional and functional enhancement
purposes. Carboxymethylcellulose, locust bean gum, inulin and
polydextrose added high-ber bread (Angioloni & Collar, 2011); the
defatted rice bran hemicelluloses B and insoluble dietary ber
added bread (Hu, Huang, Cao, & Ma, 2009); inulin, guar gum and
pea ber added pasta (Tudorica, Kuri, & Brennan, 2002); frozen
pizzas containing high content dietary ber stabilized rice bran
our (deDelahaye, Jimenez, & Perez, 2005); cakes enriched with

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. Erdem et al. / LWT - Food Science and Technology 56 (2014) 187e193

apple pomace (Sudha, Baskaran, & Leelavathi, 2007); ice cream

enriched with oat ber, wheat ber, apple ber and inulin
(Soukoulis, Lebesi & Tzia, 2009); and yoghurts including apple ber,
wheat ber, bamboo and inulin (Staffolo, Bertola, Martino &
Bavilacqua, 2004) might be given as some examples to the products enriched with dietary bers.
Beards, Tuohy, and Gibson (2010) tested the effects of maltitol,
polydextrose and resistant starch addition to chocolate. Forty volunteers consumed reformulated chocolate samples for over a six
week period. Their results showed that, consumption of samples
containing polydextroseemaltitol blend increased the level of
Lactobacilli and Bidobacteria levels in faeces after 6 weeks. Increase in the levels of short chain fatty acids i.e. propionate and
butyrate were observed. Formula development of chocolate provided prebiotic effects to consumers in addition to the decrease in
energy values.
Although chocolate is basically a uniform blend of cocoa, cocoa
butter and sugar, its production process is very unique and
complicated. It consists of 6 main stages as mixing, rening, conching, tempering, molding and packaging. Tempering is a stage of
controlling cocoa butter crystallization, helps to stabilize the
polymorphic transitions of cocoa butter crystals during storage and
provides the smooth and shiny appearance of chocolate, as
well (Beckett, 2008, 2009; Windhab, 2009).
The aim of this study is to develop the optimum formulation of a
novel potentially probiotic and synbiotic dark chocolates enriched
with novel probiotic strains B. indicus HU36; to investigate the
survival of B. indicus HU36 in formulations; to evaluate the addition
of dietary ber on color and sensorial properties of the product.
Formula optimization for synbiotic products was done by using the
Response Surface Methodology (RSM) method.
2. Materials and methods
2.1. Inoculum preparation
B. indicus HU36 was supplied from EU 7th Framework acronymed Project COLORSPORE (Project number: 207948). B. indicus
HU36 spores were obtained by exhaustion method described by
Nicholson and Setlow (1990). According to the method, Difco
Sporulation Medium (DSM) was streaked with colony of B. indicus
HU 36 and incubated overnight at 37  C. Then, a fresh colony was
inoculated into Luria Bertoni (LB) Broth and incubated at 30  C on a
rolling drum overnight. DSM agar and LB broth were supplied from
Oxoid (Basingstoke, UK). Afterward, the culture was grown in an
orbital shaking water bath at 37  C until OD600 of the culture was
reached approximately 1.00. Then culture was spread plated on
DSM agar and incubated at 30  C for 24 h. Afterward, the level of
visible sporulation of the culture was checked frequently. When
more than 90% of visible sporulation was detected, spores were
harvested with water after removal of vegetative cells by washing
with distilled water. Finally, harvested spores were lyophilized by
using Christ Alpha 1-2 LD Plus Freeze Dryer (Martin Christ GMBH,
Osterede am Harz, Germany).
2.2. Chocolate production
Dark chocolate couvertures containing 50 g cocoa/100 g were
donated from Nestle Turkey Gida A.S. Dietary bers (maltodextrin,
carboxymethylcellulose, inulin, lemon ber, apple ber, wheat ber) were all obtained from various local companies in Turkey in
dried and powdered from, no further process had been applied. All
samples were produced in laboratory conditions. After couvertures
were melted at 45  C in water bath, lyophilized B. indicus HU36
spores were added to have 6.08 log cfu/g of chocolate. Dietary

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).

. Erdem et al. / LWT - Food Science and Technology 56 (2014) 187e193

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.

Commercial bitter chocolate (Nestle Dark) was used as reference

sample for comparison.

Whiteness indexWI 100 

2  2  2 i0:5
h
100  L* a* b*
(1)

2.7. Sensory proling of samples

The main principles of Quantitative Descriptive Analysis (QDA)
(Stone, Sidel, Oliver, Woolsey, & Singleton, 1974) were used to
evaluate the sensory attributes of chocolates containing maltodextrin and lemon ber at different concentrations [1.5, 3.5, 5.5 (g/
100 g)].

Focus group studies were conducted by 8 panelists (5 females &

3 males, between 25 and 40 years), chosen from both graduate
students and faculty members of Istanbul Technical University of
Food Engineering Department. Panelists were trained in 3 different
sessions, 2 h long each. The glossary of terms and their denitions
and scores for references were determined and scaled in consensus
(Table 3). Afterward, each panelist was asked to score the samples
individually in a 7-point scale. Each panel consisted of 3 consecutive sessions where panelists were asked to score 4, 4 and 3 samples in each session. All panels were completed in 3 days. Panels
were performed in duplicates. Judges scored the samples according
to the glossary and reference scores as shown in Table 3. Chocolate
formulations produced for descriptive analysis did not contain
bacteria.

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

Factor 2:LFa (g/100 g)

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

MD Maltodextrin, LF Lemon ber.

The values represent the average of duplicated measurements for viable bacteria counts in log cfu/g.
The values represent the average of triplicate measurements for color analysis.
The values represent the average of duplicated measurements for sensory analysis.
Responses are listed in order, following the results of microbiological, color and sensory analysis, consecutively.

190

. Erdem et al. / LWT - Food Science and Technology 56 (2014) 187e193

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

Smooth appearance of product surface without lumps of grits

Intensity of light reection in the product, opposite of opaque
Brown color intensity; from light brown to dark brown
White-gray layer of visible lipid/sugar crystals on
the surface
The aroma of cocoa powder (from none > very)
Unpleasant and unwanted aroma in the product
The intensity of the taste of cocoa in the product
Taste quality most often associated with sucrose
(none > very)
Taste quality associated with caffeine as in caffeine
and quinine
(none > very)
Residual bitter taste intensity in mouth after swallowing
Residual cocoa taste intensity in mouth after swallowing
The avor of aqueous quinine solution (none > very)
The avor of cocoa (none > very)
Chocolate avor intensity, characteristic of chocolate
The force required to cut using central incisor teeth
The breakage level of the piece of chocolate in rst bite
Force required to compress sample between tongue
and palate
Level of even and consistent continuity of the product
in mouth
Time required to melt half of the sample while chewing
(slow > fast)
Level of stickiness to molar teeth
Level of covering the surface of the mouth
The after-feel lm which covers mouth surface

dark chocolate 7, halva 0

cocoa powder 0, dark chocolate 4, pudding 7
milk chocolate 0, dark chocolate 5, bitter chocolate 7
bitter chocolate 0, under-tempered laboratory made sample
stored for 1 month 7
white chocolate 0, dark chocolate 4, bitter chocolate 7
none 0, intermediate 4, extreme 7
white chocolate 0, dark chocolate 5, bitter chocolate 7
water 0, dark chocolate 2, milk chocolate 5, honey 7
white chocolate 0, quinine solution 7

white chocolate 0, quinine solution 7

white chocolate 0, bitter chocolate 7
white chocolate 0, quinine solution 7
white chocolate 0 , bitter chocolate 7
white chocolate 0, dark chocolate 7
marshmallow 0, bitter chocolate 4, carrot 7
halva 0, carrot 7
mufn 0, milk chocolate 4, bitter chocolate 7
halva 0, pudding 7
dark chocolate 2, milk chocolate 5, white chocolate 7
pudding 0, Nutella 5, honey 7
dark chocolate 3, Nutella 7
pudding 0, Nutella 7

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.

2.8. Statistical analysis

Experimental data was analyzed by multiple regressions to t
the second order polynomial equation to all independent factors.
The goodness of t of the model was evaluated by the coefcient
determination (R2) and the analysis of variance (ANOVA). To visualize the relationships between the responses and the independent
factors, surface response and contour plots of the tted polynomial
regression equations, and the optimal conditions for the targeted
responses were generated by Design Expert 8.0.4 (Stat-Ease, Inc.,
MN, USA) Software.
3. Results and discussions

control. In terms of overall acceptability scores, those bers were

distinctively separated from the samples containing carboxymethylcellulose (CMC), inulin (IN), wheat ber (WF) and apple ber
(AF) samples. Addition of CMC, IN, WF and AF showed negative
effect (p < 0.01) on taste, mouthfeel, texture and overall acceptability. Based on the results obtained, maltodextrin and lemon ber
were chosen as the most suitable dietary ber types for further
studies.

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

3.1. Preliminary sensory analysis for dietary ber determination

Hedonic analysis showed that (Table 1), samples containing
maltodextrin (MD) or lemon ber (LF) had the highest scores after
Lack of Fit
Firmness

Table 4
Results of viability of Bacillus indicus HU36 and yield %.
Sample code

MDa (g/100 g)

LFa (g/100 g)

Final count (log cfu/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.

. Erdem et al. / LWT - Food Science and Technology 56 (2014) 187e193

3.2. Viable bacteria count

Results of viable cell counts showed that in spite of the
observed reduction (w<1 log cfu/gr) of B. indicus HU36 concentration compare to the initial count, all samples contained more
than 5 log cfu/g bacteria survival (Table 4). Yield values indicate the
resistance of bacteria to our production process, and the range of
88e91% in all samples showed that B. indicus HU36 was highly
resistant. Maragkoudakis et al. (2006) considered probiotic load
between 5 and 8 log cfu/g acceptable for dairy products. Since
studies showed that chocolate is a better sample for probiotics

191

than dairy products (Possemiers et al., 2010), it can be accepted

that all our samples (S1eS9) are adequate as probiotic products
(Table 4).
3.3. Color analysis
The color analysis of top and bottom surface of samples was
done to identify L*, a*, b* values and WI values calculated afterward
(Table 2). Darkness in color is a desired feature in chocolate; it also
indicates the lack of white lines, which can be associated with
bloom formation. In terms of L* values, all samples showed higher

Fig. 1. Contour plots of rmness (1), adherence (2) and sweetness (3) as a function of maltodextrin (MD) and lemon ber (LF) conc.

192

. Erdem et al. / LWT - Food Science and Technology 56 (2014) 187e193

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.

3.4. Model tting (RSM)

The results of color, sensory analysis, and bacterial counts (responses) of the chocolate formulations all the experiments are
shown in Table 2. The experimental data was used to calculate the
coefcients of the quadratic polynomial equations, which were

used to predict the responses. Analysis of variance (ANOVA) in

Table 5 indicated that quadratic polynomial models were adequate
for the prediction of only sweetness, rmness and adherence features of samples. The models showed no lack of t for sweetness,
rmness and adherence because p values of were higher than
p > 0.05 (0.759, 0.068 and 0.713) and coefcients of multiple determinations, R2, being 0.985, 0.946, 0.965, all indicate that models
t the experimental data points. The model equations for the responses can be written as follow where MD and LF is maltodextrin
and lemon ber concentration (g/100 g):

Sweetness 0:841 0:955MD 0:819LF 4:934:103 MD2

 0:0266LF 2
Firmness 1:037 0:887MD 1:293LF 6:578:103 MD2
 0:037LF 2
Adherence 0:596 0:143MD 0:549LF 0:023MD2
 8:223:103 LF 2
For any of the terms in the models, a small p-value would
indicate a more signicant effect on the respective responses. The
linear term of maltodextrin (MD) and lemon ber (LF) concentration had positive effects on sweetness, rmness and adherence
values.
3.5. Response surface plots
The impact of maltodextrin and lemon ber levels on sweetness,
rmness and adherence can be seen in Fig. 1. The increase in lemon
ber and maltodextrin concentration results in the increase of
sweetness. According to Table 3, sweetness scores of samples are
expected to be between 2 (dark chocolate) and 5 (milk chocolate)
and our results for all samples were in this range. Fig. 1a showed
that the more maltodextrin and lemon ber concentration, the
elevated rmness of the samples was observed. According to
Table 3, the rmness scores are expected to be around 4. In our
model rmness score reached to 4, when maltodextrin concentration was 4.5 (g/100 g) and lemon ber concentration was slightly
over 3.5 (g/100 g) Fig. 1b summarized the effect of maltodextrin and
lemon ber concentration on adherence. Mutual increase in the
concentration of bers showed the most increase on adherence
scores, this value is always aimed to be kept at minimum; however
Fig. 1c showed that even in minimum ber concentrations, adherence score was around 1.75 which showed that dietary ber
addition brought slight increase on adherence at any level in the
described range in the model.
The results of Quantitative Descriptive Analysis (QDA) took
part in RSM studies as responses. Flavor proling of samples are
summarized graphically in Fig. 2. Neither of the samples showed
bloom nor off-avor and all samples showed very high levels of
smoothness similar to control sample. In general, QDA results
showed that dietary ber addition has signicant impact on only
sweetness, rmness and adherence features of samples among all
features compared to control sample.
3.6. Validation of model

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.

Design Expert 8.0.4 software generated optimum formulations

for targeted responses (sweetness, rmness and adherence). Three
optimum formulations and one control sample were produced.
Sensory analysis was applied to all samples and responses were
scored according to the glossary and references set at Table 3.
Maltodextrin and lemon ber concentration of optimum

. Erdem et al. / LWT - Food Science and Technology 56 (2014) 187e193

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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