sensors

Article
The Sensory Quality and Volatile Profile of Dark
Chocolate Enriched with Encapsulated Probiotic
Lactobacillus plantarum Bacteria
Milica Mirković 1, *, Sanja Seratlić 2 ID , Kieran Kilcawley 2 ID
, David Mannion 2 ,
Nemanja Mirković 3 and Zorica Radulović 1
1 Faculty of Agriculture, University of Belgrade, Nemanjina 6, Belgrade 11080, Serbia;
zradulovic@agrif.bg.ac.rs
2 Teagasc Food Research Centre, Moorepark, Fermoy, Co. Cork P61, Ireland; sanja.seratlic@teagasc.ie (S.S.);
kieran.kilcawley@teagasc.ie (K.K.); david.mannion@teagasc.ie (D.M.)
3 Institute for Molecular Genetics and Genetic Engineering, University of Belgrade, Vojvode Stepe 444,
Belgrade 11000, Serbia; nemanjamirkovic@imgge.bg.ac.rs
* Correspondence: petrusicm@agrif.bg.ac.rs; Tel.: +381-4413-201




Received: 2 July 2018; Accepted: 28 July 2018; Published: 6 August 2018


Abstract: Cocoa and dark chocolate have a wide variety of powerful antioxidants and other nutrients
that can positively affect human health. Probiotic dark chocolate has the potential to be a new
product in the growing number of functional foods. In this study, encapsulated potential probiotic
Lactobacillus plantarum 564 and commercial probiotic Lactobacillus plantarum 299v were added in
the production of dark chocolate. The results show very good survival of probiotic bacteria after
production and during storage, reaching 108 cfu/g in the first 60 days and over 106 cfu/g up to
180 days. No statistically significant difference (p > 0.05) in chemical composition and no major
differences in the volatile profiles between control and experimental chocolate samples were observed,
indicating no impact of probiotic bacteria on compositional and sensory characteristics of dark
chocolate. The sensory evaluation of control and both probiotic dark chocolate samples showed
excellent sensory quality after 60 and 180 days of storage, demonstrating that probiotics did not
affect aroma, texture and appearance of chocolate. Due to a high viability of bacterial cells and
acceptable sensory properties, it can be concluded that encapsulated probiotics Lb. plantarum 564 and
Lb. plantarum 299v could be successfully used in the production of probiotic dark chocolate.

Keywords: chocolate; probiotics; encapsulation; viability; sensory analysis; volatiles

1. Introduction
Specialized chocolate shops and supermarkets have a wide range of different chocolates but
probiotic chocolate remains a novel product. Probiotic bacteria are usually delivered within dairy
products, such as fermented milks and cheeses, where bacteria perform a major role in the development
of the final product characteristics. However, lactose intolerance, allergenic milk proteins [1,2] and
high fat content are the major drawbacks related to the intake of dairy products, especially for
health-conscious consumers. Additionally, required refrigeration and relatively short shelf-life of dairy
products represent also limitations in their use. In this context, the evolution of a new probiotic product
could be of significant importance. Cocoa and chocolate have been suggested as a good food medium
for functional healthy ingredients, because they are rich sources of catechins (flavan-3-ols, or flavanols),
epicatechin and procyanidins, which are polyphenols that have the ability to act as antioxidants [3–5],
showing cardio protective [6–8] and antidepressant effects [9]. Moreover, Possemiers et al. [10] revealed
that chocolate was a better probiotic carrier than dairy products for intestinal delivery, because bacterial

Sensors 2018, 18, 2570; doi:10.3390/s18082570 www.mdpi.com/journal/sensors

Sensors 2018, 18, 2570 2 of 16

survival rates through gastro-intestinal tract conditions were four times higher in chocolate than in
dairy products.
Dark chocolate is made from cocoa (in form of paste, powder or butter) and sugar (added
as an emulsifier and sometimes as a flavour) and it does not contain milk, while milk chocolate
contains whole-milk powder, including emulsifying agents and very often some flavourings [11,12].
Therefore, dark chocolate with 75% cacao was chosen as a probiotic carrier in this study. The use of a
dense food matrix which contains a higher content of protective ingredients has additional advantages
over typical dairy probiotic products.
Chocolate can be defined as a product made primarily of cocoa solids and cocoa fats, although
since 1999 up to 5% of its content can be comprised of vegetable fat alternatives, such as palm oil,
shea, sal, illipe or mango kernel butter [11]. Different chocolate flavours can be obtained by varying
the temperature and time when roasting the beans or adjusting the quantities of cocoa solids and/or
adding non-chocolate ingredients. The specific flavour of dark chocolate is primarily due to a very rich
volatile fraction which is composed of a mixture of hundreds of volatile and non-volatile compounds.
There are many descriptive studies that have identified more than 600 volatile compounds in chocolate
products and cocoa, mainly pyrazines, amines and amides, acids, esters and hydrocarbons [4,13,14].
Composition of dark chocolate depends on the genotype of the cocoa bean and the processes
used during chocolate production: fermentation, drying, roasting and conching [14]. Cocoa bean
fermentation is an essential step in the development of key volatile fractions (alcohols, esters and fatty
acids) and the formation of flavour precursors (amino acids and reducing sugars). Molecules generated
during cocoa bean fermentation, such as pyrazines, aldehydes (cocoa, nut aroma), esters (fruity
aroma) and polyphenols (astringent and bitter taste) are mainly responsible for cocoa and chocolate
flavour formation [15,16]. Important flavour-active compounds produced during fermentation
include 2-methylbutanoate, tetramethyl-pyrazine and other pyrazines. Bitter notes are evoked by
caffeine and threobromine, while other flavour precursor components derived from amino acids
during fermentation include phenyl-acetaldehyde, 3-methylbutanol, 2-methyl-3-(methyldithio)furan,
2-ethyl-3,5-dimethyl-methylpyrazine and 2,3-diethyl-5-methylpyrazine [17]. Flavour development
continues during drying (moisture decrease in cocoa bean from around 60% to around 7.5%), where the
characteristic brown colour is developed and the levels of acidity and astringency are reduced
by decreasing the volatile acids and total polyphenol content. The next crucial step for flavour
improvement is cocoa roasting and it involves Maillard reactions (chemical reactions between amino
acids and reducing sugars under heat treatment), which provide the desirable flavour and colour
formation. Roasting also affects the concentration of polyphenols and their ability to interact with
proteins, which causes a decrease in astringency [18,19]. Conching (agitating chocolate at >50 ◦ C) is a
final stage in flavour development during chocolate manufacture and is important in determining the
final flavour characteristics and texture formation [4,20].
Within the general quality of chocolate, sensory quality is the most important parameter
and it is therefore necessary that the sensory attributes should not be altered by the addition of
probiotic bacteria.
The aim of this study was to examine the viability of two encapsulated spray dried bacteria
Lactobacillus plantarum 564 (potential probiotic) and Lactobacillus plantarum 299v (commercial probiotic)
in dark chocolate after production and during 360 days of storage at room temperature and their
influence on volatile compounds and sensory characteristics of final product. To increase process
efficiency and improve survival of bacterial cells, an encapsulation process was applied using spray
drying. It is a low-cost process that achieves dried powder of small particle size with optimal moisture
content and fast production of large quantities of viable cells [21]. It is envisaged that this research will
provide a knowledge platform for the commercial development of probiotic dark chocolate.
Sensors 2018, 18, 2570 3 of 16

2. Materials and Methods

2.1. Strains and Culture Conditions

A potential probiotic strain Lb. plantarum 564, isolated from artisanal Serbian white brined
cheese [22] and a commercial probiotic strain Lb. plantarum 299v (DSM, Heerlen, The Netherlands)
were used in this study. Strain Lb. plantarum 564 belong to the strain collection of the Department for
Industrial Microbiology, Faculty of Agriculture, University of Belgrade, Serbia. This strain was selected
according to technological properties and probiotic potential which is tested by Radulović et al. [22].
Both strains were cultured in MRS broth (Merck, Darmstadt, Germany) at 37 ◦ C in anaerobic conditions.

2.2. Encapsulation Process of Lactobacillus plantarum

The spray drying encapsulation process was performed according to the method described by
Radulović et al. [23]. Overnight cultures (300 mL) were centrifuged (4500× g, 15 min, 15 ◦ C), the pellet
was washed twice in 50 mM K2 HPO4 (pH 6.5), re-suspended in 300 mL of sterilised reconstituted
skim milk (20% w/v) and spray-dried with a laboratory scale spray-dryer (Büchi mini spray dryer
model B-290, Flawil, Switzerland) using the constant inlet air temperature of 170 ◦ C and the outlet
temperature of 80 ◦ C.

2.3. Enumeration of Encapsulated Lactobacillus plantarum

Enumeration of spray dried probiotic cells was performed after reconstitution of 1 g of spray
dried powder in 9 mL of 2% (w/v) sodium citrate and appropriate decimal dilution was plated on
MRS agar (Merck, Darmstadt, Germany). The plates were incubated at 37 ◦ C for 48 h in anaerobic
conditions (Gas Pak, Merck, Darmstadt, Germany).

2.4. Chocolate Production

Production of dark chocolate was carried out in the semi-industrial scale in the factory of
confectionery products Soko Štark, Belgrade, Serbia. A control sample without and two experimental
samples containing spray dried probiotic strains (Lb. plantarum 564 and Lb. plantarum 299v) were
produced in three replicate trials according to Laličić-Petronijević et al. [24]. Powders of spray dried
probiotic bacteria were added to the chocolate masses after tempering when temperature was lower
than 40 ◦ C, by blender mixing for 5 min. Aiming to provide required number of probiotic cells in
chocolate samples (108 cfu/g), 10 g of powder of spray dried cells were added per kg of dark chocolate,
after which chocolate masses were moulded, cooled, removed from the form and packed in aluminium
foil and paper blanks and stored at 20 ◦ C for 360 days.

2.5. Viability of Probiotic Bacteria

Viable cell counts of both probiotic bacteria were determined by the standard plate method and
values expressed as colony forming units per gram (cfu/g) of chocolate. Ten grams of dark chocolate
was homogenized in 90 mL saline solution (0.9% NaCl) in a Stomacher apparatus (Lab Blender
Stomacher 400, Seward, West Sussex, UK). Serial dilutions were prepared and appropriate dilutions
were plated on MRS agar (Merck, Darmstadt, Germany). Total bacterial counts were determined
after 48 h of incubation at 37 ◦ C under anaerobic conditions (Gas Pak, Merck, Darmstadt, Germany).
Viability of both bacterial strains were analysed in triplicate immediately after production and after 60,
90, 180, 270 and 360 days of storage at 20 ◦ C.

2.6. Chemical Analysis of Chocolate

Chemical composition of dark chocolate samples (moisture, protein, fat, ash and carbohydrate
content) was determined according to the AOAC methods [25].
Sensors 2018, 18, 2570 4 of 16

2.7. Characterisation of Volatile Flavour Compounds by Head Space Gas Chromatography Mass Spectrometry
The volatile profile of the chocolate samples was analysed by static head space solid phase
microextraction (HS-SPME) gas chromatography mass spectrometry (GCMS).
Each sample (5 g) was weighed into a 20-mL headspace glass amber vial with a screw top and
a silicone/PTFE septum (Apex Scientific, Maynooth, Kildare, Ireland). Sample introduction was
accomplished using a CTC Analytics CombiPal autosampler. Samples were equilibrated at 40 ◦ C in
a controlled temperature agitator for 10 min at 500 rpm (5 s on/off) prior to exposure of the SPME
50/30 µm CarboxenTM /divinylbenzene/polydimethylsiloxane (CAR/DVB/PDMS) fibre. The fibre
was fully exposed to the headspace of the sample at a depth of 1 cm at 40 ◦ C at 350 rpm for 20 min.
The fibre was desorbed for 2 min at 250 ◦ C in split-less mode onto a Varian 450 gas chromatograph
with an 1177 injector (Aquilant Scientific Ltd., Dublin, Ireland) using a merlin microseal and a SPME
liner. The column was a DB-5ms (60 m × 0.25 mm × 0.25 µm) (Agilent Technologies, Cork, Ireland).
The extracted compounds were cyrofocussed using liquid carbon dioxide at −60 ◦ C directly onto the
column. The temperature of the column oven was initially held at −60 ◦ C for 2 min, increased to
20 ◦ C at 20 ◦ C/min, held for 20 min, then increased to 110 ◦ C at 10 ◦ C/min, followed by an increase
to 290 ◦ C at 15 ◦ C/min, yielding at total run time of 47.0 min. The carrier gas was helium, held at a
constant flow of 1 mL/min. The detector was a Varian 320 triple quad mass spectrometer (Aquilant
Scientific Ltd., Ireland), used in single quadrupole mode. Compounds were identified using mass
spectra comparisons to the NIST 2008 mass spectral library and from an internal data base of standards
created over time. An auto-tune of the GCMS was carried out prior to the analysis to ensure optimal
GCMS performance. A set of standards was also run at the start and end of the sample set to ensure
the MS was performing within specification. Each sample was analysed in triplicate after 180 days
of storage.

2.8. Sensory Analysis

The sensory quality of dark chocolate samples with probiotic bacteria during storage was analysed
using a modified scoring method for sensory evaluation of chocolate as described by Popov-Raljić
and Laličić-Petronijević [26]. Sensory evaluations included the following properties: appearance
(form, colour, gloss and surface), mechanical properties (structure, break, hardiness and chewiness),
geometrical properties (sandiness), surface properties (moisture and lubricity), aroma (odour, taste)
and other dynamic properties (solubility), as shown in Table 1. For each sensory property, the Weight
Coefficient was determined based on the influence of each attribute on overall sensory quality.
A scoring range from 1.00 to 5.00, with the possibility of assigning half and quarter points was
applied. Quality category was determined in scores spans; samples which were evaluated with less
than 2.5 points were considered as unsatisfactory, 2.5–3.5 good, 3.5–4.5 very good and 4.5–5 excellent
quality. Sensory evaluation of all three chocolate types was performed by seven experienced assessors
familiar with the product. All panellists met the criteria specified by the ISO standards for selection,
training and monitoring of assessors [27,28].
The sensory analysis of the probiotic chocolate samples was performed after 60 and 180 days of
storage at room temperature of 20 ◦ C.
Sensors 2018, 18, 2570 5 of 16

Table 1. Sensory evaluation of dark chocolate.

Weight
Basic Sensory Properties Score Description of the Evaluated Property
Coefficient
5 Appropriate form; spotless colour; smooth, glossy surface; clear print
Form
4 Insignificant deviation of form; spotless colour; smooth, glossy surface; less clear print
Colour
APPEARANCE 2.50 3 Deviations of form; lower quality colour; fingerprints on the surface; air bubbles; less clear print
Gloss
2 More pronounced form deviations; partially white or grey surface; presence of cracks
Surface
1 Distorted form; grey or white surface; higher damages; bad print
MEHANICAL 5 Straight break, homogenous, fragile; homogenous structure; appropriate chewiness
PROPERTIES 4 Uneven break; homogenous structure; appropriate hardness; very good chewiness
Structure 2.50 3 Uneven break, air bubbles; inappropriate hardness; fat bloom appearance on the break; average chewiness
Break 2 Uneven break; roughly-granular texture; fat bloom on the break; stickiness; chewiness satisfactory
Hardness
1 Crumbling; texture roughly granular; fat bloom; very bad chewiness and very strong stickiness
Chewiness
5 Without surface extracted water/fat
SURFACE
TEXTURE 4 Slight separation of water/fat on the surface
PROPERTIES
1.0 3 Separation of water/fat on the surface
Moisture
2 Clearly expressed separation of water/fat on the surface
Lubricity
1 Very pronounced separation of water/fat on the surface
5 Inherent gradual solubility
OTHER
4 Slight deviation from the characteristic, gradual dissolution
DYNAMIC
0.5 3 Deviation from the characteristic, gradual dissolution
PROPERTIES
2 Clearly pronounced deviation from the characteristic, gradual dissolution
Solubility
1 Very pronounced deviation from the characteristic, gradual dissolution—very slow or very fast solubility
5 Appropriate; round; aromatic
4 Appropriate; less round; aromatic
THE ODOUR 4.00 3 Appropriate; less round; poorly aromatic
2 Not appropriate; sour; staled
1 Foreign odour; sour; staled; mouldy
AROMA
5 Appropriate; round; aromatic
4 Appropriate; less round; aromatic
THE TASTE 7.00 3 Poorly round; poorly aromatic
2 Slightly sour; not round
1 Foreign taste; sour; bitter
Sensors 2018, 18, 2570 6 of 16

2.9. Statistical Analysis

Statistical significance was tested by means of ANOVA analysis and the differences between
individual mean values were tested using the Fisher’s least significant difference (LSD) test. Significant
differences were considered for p < 0.05. Calculations were made with STATISTICA 6.0 PL software
for Windows (StatSoft Inc, Tulsa, OK, USA).
The principal component analysis of sensory and volatile data was analysed using the
Unscrambler Software, version 9.7 (CAMO ASA, Trondheim, Norway).

3. Results and Discussion

3.1. Cell Count of Spray Dried Probiotic Bacteria

Bacterial count of spray dried cells of potential probiotic Lb. plantarum 564 was 10.20 log cfu mL−1 ,
while number of spray dried cells of Lb. plantarum 299v was 10.36 log cfu mL−1 . Obtained results
indicate that optimal conditions were achieved during spray drying process. Similar results were
obtained in our previous studies [23,29].

3.2. Viability of Probiotic Bacteria

According to the definition given by FAO/WHO [30] probiotics are “live microorganisms which,
when administrated in adequate amounts, confer a health benefits on the host”. To have a positive effect
in the intestinal tract, probiotics should be able to survive low pH, gastric acid and bile secretions and
should succeed in competing with the resident intestinal microbiota. The bacteria should also be able
to selectively stimulate the growth and/or activity of bacteria in the colon [31,32]. Probiotic bacteria
have been reported to have numerous health benefits such as balancing of intestinal microbiota,
stimulation of immune system, blood cholesterol reduction, enhancement of the digestibility of
protein and vitamin synthesis, anti-bacterial activities, treatment of lactose intolerance, food-related
allergies, amongst others [33–36]. Most of probiotic bacteria belong to the Lactobacillus genera and
Bifidobacterium strains [37]. However, variety of food grade lactic acid bacteria, have been evaluated for
their potential probiotic ability. Furthermore, some of autochthonous facultative heterofermentative
lactobacilli isolated from traditional dairy products, such as Lactobacillus casei, Lactobacillus plantarum,
Lactobacillus rhamnosus can also have probiotic properties [38].
Many destabilizing factors in food production could affect viability of probiotic bacteria.
In production of dark chocolate, the factors such as temperature, oxygen, osmotic pressure, water
activity and so forth could be critical for bacterial cell survival. Therefore, it is very important to
incorporate probiotic bacteria in dark chocolate mass at very low temperature during manufacturing
process, so their viability will not be impaired. In this experiment, probiotic bacteria were added
after the process of tempering when the temperature reached around 30 ◦ C. However, chocolate
composition, such as protein and fat content, sugar concentration, including high buffering capacity,
remains an important factor affecting the viability of probiotic bacteria [39], not only after production
but also during storage.
Based on the development of functional foods such as probiotic dark chocolate, one of the
main goals is achieving the desired amount of probiotic bacteria in dark chocolate during the
production and storage in order to provide a therapeutic effect on human health. To have a
beneficial effect on health, probiotics must be viable in the product at the time of consumption.
The threshold cell number should be above 106 cfu/g during manufacturing process and storage [40,41].
The viability of probiotic bacteria can be affected by many factors, particularly physicochemical
properties of dark chocolate (fat and protein content, type of sugars, etc.) and addition of certain
food ingredients (stabilizers, sweeteners, etc.) [42]. Different approaches that increase the viability
of probiotic bacteria have been adopted, including appropriate selection of acid- and bile-resistant
strains, use of oxygen impermeable containers, two-step fermentation, incorporation of micronutrients
and microencapsulation [43]. Microencapsulation by spray-drying is presumably the most effective
Sensors 2018, 18, 2570 7 of 16
Sensors 2018, 18, x FOR PEER REVIEW 7 of 15

economic
and economic method.
method. Also, spray
Also, drying
spray drying is is
suitable
suitablefor forlarge-scale,
large-scale, industrial
industrial applications [44].
applications [44].
Although high
Although high temperature
temperature of of hot
hot air
air can
canbebeaadisadvantage
disadvantageof ofspray-drying,
spray-drying, proper
properreadjustments
readjustments
of processing conditions can achieve better viability of probiotic bacteria
of processing conditions can achieve better viability of probiotic bacteria [45,46]. According to [45,46]. According to the
the
literature,an
literature, anairairoutlet
outlet temperature
temperature of 80 ◦
ofto8085toC85was °Coptimal
was optimal
for spray fordrying
spray todrying
preserveto preserve Lb.
Lb. paracasei
paracasei
and and Lb.strains
Lb. salivarius salivarius
withstrains
probioticwith probiotic
potential potential
[47,48]. [47,48]. studies
Our previous Our previous studies [29]
[29] demonstrated a
high viability and survival rate of spray dried Lb. plantarum 564 cells while using a constant inlet aira
demonstrated a high viability and survival rate of spray dried Lb. plantarum 564 cells while using
constant inletofair
temperature 170 ◦ C and outlet
temperature of 170 °C and outlet
temperature of 80 ◦ C, indicating
temperature of 80successful
°C, indicating
use ofsuccessful use of
applied spray
applied spray drying conditions as a method of bacterial
drying conditions as a method of bacterial preservation in the present research. preservation in the present research.
Accordingtotothe
According the results,
results, the the
sprayspray
drieddried Lb. plantarum
Lb. plantarum 564 and564 Lb. and Lb. plantarum
plantarum 299v cells 299v cells
remained
remained
viable for upviable for up tohaving
to 6 months, 6 months,
counts having
of overcounts6
10 cfu/gof over 10 cfu/g
(Figure 6 (Figure
1). The present1).study
The demonstrated
present study
demonstrated very good survival of both probiotic bacteria in dark chocolate
very good survival of both probiotic bacteria in dark chocolate after production and during storage after production and
during
at roomstorage at roomespecially
temperature, temperature, especially
in first 90 days.inDuring
first 90 the
days. During
first the first two
two months, months,
the cell the cell
numbers of
numbers
both of both
potential andpotential
commercial andspray
commercial spray dried
dried probiotic probiotic
bacteria were bacteria were
above 8 log above
units. 8 log
After 90 units.
days
After
of 90 days
storage, of storage,
there was a slight theredecrease
was a slight decreaseofinboth
in viability viability of both Lb.564
Lb. planatrum planatrum
and 299v 564(7.28
and 299v
and
(7.28log
7.49 andcfug −
7.49 log
1 cfug , respectively).
−1
, respectively). Afterwards, Afterwards, the cellof
the cell numbers numbers
probioticofbacteria
probiotic bacteriadecreased
gradually gradually
decreased
and after 360anddaysafter
the360 days the
numbers ofnumbers
probioticof probiotic
bacteria bacteria
in both in both experimental
experimental chocolates
chocolates were were
marginally
marginally above
above 5 log units. 5 log units.

Figure 1.1. Viability

Figure Viability of
ofspray
spraydried
driedLb.
Lb. planatrum
planatrum 564
564 and
and commercial
commercial probiotic
probiotic Lb.
Lb. plantarum
plantarum 299v
299v in
in
dark chocolate during storage.
dark chocolate during storage.

Nebesny et al. [49] reported that lyophilized lactic acid bacteria Lactobacillus casei and
Nebesny et al. [49] reported that lyophilized lactic acid bacteria Lactobacillus casei and Lactobacillus
Lactobacillus paracasei had very good survival rate in dark chocolate during storage at both 4 °C and
paracasei had very good survival rate in dark chocolate during storage at both 4 ◦ C and 18 ◦ C, where the
18°C, where the total number of live cells maintained at functional level of 7 log units even after 12
total number of live cells maintained at functional level of 7 log units even after 12 months of storage.
months of storage. Laličić-Petronijević et al. [24] also observed that the number of lyophilized cells of
Laličić-Petronijević et al. [24] also observed that the number of lyophilized cells of probiotic bacteria
probiotic bacteria Lactobacillus® acidophilus NCFM® remained at the level of 7 log units per gram of
Lactobacillus acidophilus NCFM remained at the level of 7 log units per gram of dark chocolate during
dark chocolate during 180 days of storage at 20 °C. Obtained results are also in accordance with
180 days of storage at 20 ◦ C. Obtained results are also in accordance with findings of Mandal et al. [39],
findings of Mandal et al. [39], who reported above 8 log cfu/g of lactobacilli in probiotic chocolate
who reported above 8 log cfu/g of lactobacilli in probiotic chocolate during 60 days of storage,
during 60 days of storage, while Aragon-Alegro et al. [50] detected over 7 log cfu/g of Lb. paracasei in
while Aragon-Alegro et al. [50] detected over 7 log cfu/g of Lb. paracasei in probiotic chocolate
probiotic chocolate mousse stored 28 days under refrigerated conditions. In the present study, 6.5
mousse stored 28 days under refrigerated conditions. In the present study, 6.5 log units of both strains
log units of both strains were present in chocolate after 6 months and the cell number of commercial
were present in chocolate after 6 months and the cell number of commercial Lb. plantarum 299v strain
Lb. plantarum 299v strain remained at the same level after 9 months of storage. The decrease of
remained at the same level after 9 months of storage. The decrease of bacterial cell number below the
bacterial cell number below the threshold of 6 log cfu/g in further storage period indicates that
Sensors 2018, 18, 2570 8 of 16

threshold of 6 log cfu/g in further storage period indicates that chocolate samples with Lb. planatrum
564 and commercial probiotic 299v cannot be called “probiotic chocolate” after 180 and 270 days of
storage, respectively.

3.3. Chemical Composition of Dark Chocolate

Table 2 shows the chemical composition of three dark chocolate variants. Presented data are the
mean values of three replicate trials. Results of statistical analysis showed that there was no statistically
significant difference (p > 0.05) in moisture, protein and fat content between control and probiotic
chocolate samples. Also, the slight difference in ash content between control and probiotic sample with
Lb. plantarum 299v was not statistically different (p > 0.05), nor was the slight difference in carbohydrate
content between control and probiotic chocolate with Lb. plantarum 564.
According to the results, it can be concluded that there was no statistically significant difference
(p > 0.05) in chemical composition between tested chocolate samples, which means that spray dried
probiotic bacteria did not make an impact on the main chemical composition of dark chocolate.
These important findings are in accordance with other literature data [24,51].

Table 2. Chemical composition of dark chocolates made with and without probiotic bacteria.

Chemical Composition (g/100g) *

Compositional Parameters
Control Variant Chocolate 564 Chocolate 299v
Moisture 0.82 ± 0.05 0.80 ± 0.05 0.81 ± 0.07
Protein 9.88 ± 0.08 9.86 ± 0.04 9.90 ± 0.06
Lipids 39.94 ± 0.07 39.94 ± 0.06 40.02 ± 0.09
Carbohydrate 24.85 ± 0.09 25.01 ± 0.1 24.84 ± 0.1
Ash 2.30 ± 0.04 2.33 ± 0.07 2.41 ± 0.07
* Values presented are the means of three replicate trials ± standard deviation.

3.4. Volatile Analysis

Volatile profile of control and probiotic dark chocolate is presented in Table 3.
In total, 36 volatile compounds were identified, consisting of mainly aldehydes (7), alcohols (6),
ketones (6), pyrazines (5), with lower amounts of lactones (3), acids (3), esters (2), terpenes (2), phenol
(1) and furan (1). Apart from two compounds (2-heptanol and isoamyl acetate), all the remaining
volatiles were present in all three chocolate variants, indicating that there were no major differences in
the volatile profiles between the samples, nor did the majority of volatile compounds differ significantly
(p > 0.05) between the experimental variants and the control. The major difference detected between
the samples was that the experimental samples (chocolate 564 and 229v) did not contain 2-heptanol
and the control sample did not contain isoamyl-acetate.
Figure 2 is a principal component analysis (PCA) bi-plot of the variation in volatile profile between
each sample in triplicate. The overall variance is 67%, of which 48% relates to PC1 and 19% to PC2.
It is apparent that the control sample replicates (A, B & C) are all on the negative side of PC1, while the
experimental probiotic samples (A, B & C replicates of chocolate 564 and 229v) are on the positive
side of PC1. Some variation between the three replicates is evident but that is to be expected with
HS-SPME using non-homogenous samples.
Overall, the experimental samples (chocolate 564 and 229v) were differentiated from the control
by containing higher abundances of isodihydrolavandulal, 2-penyl furan, hexanal, 2-ethyl-6-methyl
pyrazine, acetophenone, tetramethylpyrazine and 3-methyl-butanal. On the other hand, control
samples contained higher abundance of butanone, 2-nonanone, 2-ethyl-1-hexanol, d-octenolactone
and 2-methyl-3,5-diethylpyrazine.
Sensors 2018, 18, 2570 9 of 16

Table 3. Volatile compounds detected in dark chocolate with and without probiotic bacteria.

Volatile Compounds CAS No. Odour Descriptor Control Variant Chocolate 299v Chocolate 564
Abundance Levels *
Aldehydes
Isobutyraldehyde 78-84-2 Banana, malty, chocolate-like, cocoa 9.34 × 108 a 3.88 × 108 b 6.87 × 108 ab
3-Methyl-butanal 590-86-3 Malty, powerful, cheese, green, dark chocolate, cocoa 1.05 × 109 a 4.11 × 109 b 4.57 × 109 b
Hexanal 66-25-1 Green, slightly fruity, lemon, herbal, grassy, tallow 1.37 × 108 a 4.28 × 108 b 4.23 × 108 b
Benzaldehyde 100-52-7 Bitter almond, sweet cherry 2.26 × 108 a 3.84 × 108 a 6.25 × 108 a
Nonanal 124-19-6 Green, citrus, fatty, floral 4.02 × 108 a 1.76 × 108 a 1.72 × 108 a
Isodihydro-lavandulal 35158-25-9 Herbal, lavender, woody, green, blueberry, tomato 1.33 × 108 a 4.29 × 108 b 4.51 × 108 b
Ethyl Vanillin 121-32-4 Sweet, creamy, vanillia, caramellic 2.91 × 109 a 2.85 × 109 a 2.76 × 109 a
Alcohols
Ethanol 64-17-5 Dry, dust 1.88 × 109 a 1.91 × 109 a 1.82 × 109 a
2,3-Butanediol 513-85-9 Fruity, creamy, buttery 7.20 × 1010 a 5.18 × 1010 a 6.23 × 1010 a
1,6-Heptadien-4-ol 2883-45-6 Unknown 5.58 × 108 a 2.28 × 108 a 4.61 × 108 a
2-Heptanol 543-43-7 Fresh lemon grass herbal sweet floral fruity green 2.34 × 108 a 0b 0b
2-Ethyl-1-hexanol 104-76-7 Animal, Cardboard 2.20 × 108 a 6.35 × 107 b 2.32 × 107 b
Phenylethyl Alcohol 60-12-8 Unclean, rose, violet-like, honey, floral, spicy 4.35 × 108 a 3.84 × 108 a 4.25 × 108 a
Ketones
Acetone 67-64-1 Earthy, wood pulp, hay 1.63 × 109 ab 1.42 × 109 b 1.89 × 109 a
Butanone 78-93-3 Buttery, sour milk, etheric 1.10 × 109 a 4.09 × 108 b 5.35 × 108 b
Acetoin 513-86-0 Buttery, sour milk, caramel 3.13 × 109 a 1.33 × 109 a 2.74 × 109 a
2-Heptanone 110-43-0 Blue cheese, spicy, Roquefort 7.51 × 108 a 3.90 × 108 a 1.58 × 108 a
Acetophenone 98-86-2 Almond, musty, glue, orange blossom, sweet 1.18 × 108 a 1.68 × 108 b 1.88 × 108 b
2-Nonanone 821-55-6 Malty, fruity, hot milk, smoked cheese 8.12 × 108 a 2.72 × 108 b 3.93 × 108 b
Pyrazines
2-Ethyl-6-methylpyrazine 13925-03-6 Roasted potato 7.93 × 107 a 1.47 × 108 b 1.55 × 108 b
Trimethylpyrazine 14667-55-1 Chocolate, earthy 2.39 × 109 a 3.09 × 109 b 3.17 × 109 b
3,6-Cocoa pyrazine 13360-65-1 Patato, cocoa, roasted nutty 2.52 × 108 a 2.68 × 108 a 2.67 × 108 a
Tetramethyl-pyrazine 1124-11-4 Musty, nutty, chocolate, coffee, cocoa, lard, burnt 6.00 × 109 a 6.30 × 109 b 6.51 × 109 b
2-Methyl-3,5-diethylpyrazine 18138-05-1 Nutty, meaty, vegetable 2.10 × 108 a 1.52 × 108 b 1.55 × 108 b
Lactones
g-Butyrolactone 96-48-0 Creamy, oily, fatty nuances 1.64 × 109 a 6.26 × 108 a 1.35 × 109 a
Pantolactone compound 599-04-2 Unknown 1.63 × 108 a 1.06 × 108 a 7.88 × 107 a
d-Octenolactone 16400-69-4 Sweet, coconut-like 1.47 × 108 a 7.98 × 107 b 7.46 × 107 b
Sensors 2018, 18, 2570 10 of 16

Table 3. Cont.

Volatile Compounds CAS No. Odour Descriptor Control Variant Chocolate 299v Chocolate 564
Acids
Acetic acid 64-19-7 Vinegar, peppers, green, fruity floral, sour 8.07 × 109 a 1.12 × 1010 a 7.21 × 109 a
Isovaleric acid 503-74-2 Cheesy, sweaty, old socks, rancid, faecal, rotten fruit, goaty 1.16 × 1010 a 1.10 × 1010 a 1.08 × 1010 a
2-Methyl-butanoic acid 116-53-0 Fruity, waxy, sweaty-fatty acid 3.01 × 109 a 2.55 × 109 a 2.59 × 109 a
Esters
Isoamyl acetate 123-92-2 Banana, sweet, pear, apple peel 0a 4.12 × 108 b 4.32 × 108 b
Phenethyl acetate 103-45-7 Floral, rose-like 2.45 × 108 a 1.77 × 108 a 1.95 × 108 a
Terpenes
β-Pinene 127-91-3 Herbaceous 2.61 × 108 a 1.75 × 108 a 3.52 × 108 a
α-Limonene 138-86-3 Citrus 1.94 × 109 a 4.32 × 108 b 3.79 × 108 b
Phenols
Phenol 108-95-2 Medicinal 1.91 × 108 a 5.83 × 107 a 2.12 × 107 a
Furans
2-Pentylfuran 3777-69-3 Fruity, green, earthy, beany, vegetable, metallic 2.21 × 107 a 1.76 × 108 b 1.83 × 108 b
* Values presented are the means of three replicates; Values within a raw not sharing a common small superscript letter (a, b) differ significantly (p < 0.05).
Sensors 2018, 18, 2570 11 of 16
Sensors 2018, 18, x FOR PEER REVIEW 11 of 15

Figure 2. Principal component analysis (PCA) bi-plot of volatile compounds detected in

Figure
dark 2. Principal
chocolate with andcomponent
without analysis
probiotic(PCA) bi-plot
bacteria. of volatile are:
Compounds compounds detected
1. ethanol, in dark
2. acetone,
3. isobutanal, 4. butanone, 5. 3-methyl-butanal, 6. acetic acid, 7. acetoin, 8. hexanal, 4.
chocolate with and without probiotic bacteria. Compounds are: 1. ethanol, 2. acetone, 3. isobutanal,
9. butanone, 5. 3-methyl-butanal,
2,3-butanediol, 6. acetic
10. isoamyl acetate, 11. acid, 7. acetoin, 8. hexanal,
1,6-heptadien-4-ol, 9. 2,3-butanediol,
12. isovaleric 10. isoamyl
acid, 13. 2-heptanone,
14.acetate, 11. 1,6-heptadien-4-ol,
2-methyl-butanoic 12. isovaleric
acid, 15. 2-heptanol, acid, 13. 2-heptanone,
16. butyrolactone, 14. 2-methyl-butanoic
17. benzaldehyde, acid,
18. beta-pinene,
15. 2-heptanol, 16. butyrolactone, 17. benzaldehyde, 18. beta-pinene, 19. phenol, 20. 2-pentylfuran,
19. phenol, 20. 2-pentylfuran, 21. 2-ethyl-6-methylpyrazine, 22. trimethylpyrazine, 23. 2-ethyl-1-hexanol,
24.21.limonene,
2-ethyl-6-methylpyrazine, 22. trimethylpyrazine,
25. pantolactone compound, 26. acetophenone, 23. 27.
2-ethyl-1-hexanol, 24. limonene,
2-ethyl-3,6-dimethylpyrazine,
25. pantolactone compound, 26. acetophenone, 27. 2-ethyl-3,6-dimethylpyrazine,
28. tetramethylpyrazine, 29. 2-nonanone, 30. nonanal, 31. trans-5-methyl-2-isopropyl-2-hexen-1-al,
32.28. tetramethylpyrazine,
phenylethylalcohol, 29. 2-nonanone, 30. nonanal, 34.
33. 2-methyl-3,5-diethylpyrazine, 31. phenethyl
trans-5-methyl-2-isopropyl-2-hexen-1-al,
acetate, 35. d-octenolactone,
32. phenylethylalcohol,
36. ethylvanillin. 33. 2-methyl-3,5-diethylpyrazine, 34. phenethyl acetate, 35. d-octenolactone,
36. ethylvanillin.
3.5. Sensory Analysis
3.5. Sensory Analysis
Dark or plain chocolate contains no less than 35% cocoa but nowadays there is a trend for the
Dark or plain chocolate contains no less than 35% cocoa but nowadays there is a trend for the
chocolate production with higher percentage of cocoa (such as 55%, 70%, 85% and >85%). The more
chocolate production with higher percentage of cocoa (such as 55%, 70%, 85% and >85%). The more
cocoa the product contains, the less sugar it has and therefore the stronger intensity of cocoa flavour
cocoa the product contains, the less sugar it has and therefore the stronger intensity of cocoa flavour
and the more bitter the chocolate [12]. In this study, dark chocolate samples were produced with
and the more bitter the chocolate [12]. In this study, dark chocolate samples were produced with
75% cocoa.
75% cocoa.
The panellists have evaluated the appearance of all three chocolate variants (control and
The panellists have evaluated the appearance of all three chocolate variants (control and
experimental probiotic chocolates) by using a pair of small tongs for lifting the sample and the
experimental probiotic chocolates) by using a pair of small tongs for lifting the sample and the
appearance observation. Then by taking a square of each sample and after keeping it for 20 s in
appearance observation. Then by taking a square of each sample and after keeping it for 20 s in a
a mouth, the taste was analysed by identifying the gustatory, olfactory and physical sensations.
mouth, the taste was analysed by identifying the gustatory, olfactory and physical sensations. While
While gustatory sensations can register the bitterness, acidity and sugary content of a chocolate,
gustatory sensations can register the bitterness, acidity and sugary content of a chocolate, the
the olfactory sensations are more sensitive and can register the full aromatic palette of cacao, such as
olfactory sensations are more sensitive and can register the full aromatic palette of cacao, such as
grilled, roasted, spicy or smooth notes. Physical characteristics include chocolate texture, melting
grilled, roasted, spicy or smooth notes. Physical characteristics include chocolate texture, melting
quality, graininess or smoothness, as well as the whole mouth feel [12].
quality, graininess or smoothness, as well as the whole mouth feel [12].
After production and during the storage of probiotic dark chocolates, the metabolic activity of
After production and during the storage of probiotic dark chocolates, the metabolic activity of
probiotic bacteria could influence some textural properties, appearance, taste and aroma. Moreover,
probiotic bacteria could influence some textural properties, appearance, taste and aroma. Moreover,
the powder addition of spray dried bacteria could have an impact on texture with their granulation,
the powder addition of spray dried bacteria could have an impact on texture with their granulation,
appearance and texture. Also, powder of spray dried bacteria could affect the colour of final product.
appearance and texture. Also, powder of spray dried bacteria could affect the colour of final
Considering the fact that both chocolates had the highest probiotic cell number in first two months
product.
and that the cells of both probiotic bacteria stayed above 106 cfu/g in further 6 months of storage,
Considering the fact that both chocolates had the highest probiotic cell number in first two
months and that the cells of both probiotic bacteria stayed above 106cfu/g in further 6 months of
Sensors 2018, 18, 2570 12 of 16
Sensors
Sensors 2018,
2018, 18,
18, xx FOR
FOR PEER
PEER REVIEW
REVIEW 12
12 of
of 15
15

storage, the
the sensory
storage, sensory
sensory evaluation
the evaluation at
at these
at these two
evaluation two
two time
time points
these was points
time was
wasindescribed
described
points this paper.in
described this
inThe paper.
thissensory The
The sensory
paper. evaluation of
sensory
evaluation
evaluation of 2 months old samples was shown in Figure 3, while Figure 4 depicts the evaluation of 66
2 months of
old 2 months
samples old
was samples
shown inwas shown
Figure 3, in Figure
while 3,
Figure while
4 Figure
depicts the4 depicts
evaluationthe evaluation
of 6 months of
old
months
chocolate
months old
old chocolate
chocolate samples.
samples. samples.

Figure
Figure3. ◦ °C.
Figure 3.3.Sensory
Sensoryevaluation
Sensory evaluationof
evaluation ofofdark
darkchocolates
dark chocolateswith
chocolates with probiotic
with probiotic bacteria
probiotic after
bacteria after 60
after60 days
60days of
daysof storage
storageatat
ofstorage at2020
20 C.
°C.

Figure 4.
Figure
Figure Sensory
4. 4.
Sensory evaluation
Sensory
evaluation of
of dark
evaluation dark chocolates
of dark with
chocolates
chocolates probiotic
with
with bacteria
probiotic
probiotic after
bacteria
bacteria 180
afterafter days
180 180 of
of storage
daysdays at
at 20°C.
of storage
storage at
20°C.
◦
20 C.
After
After 60
60 days
days ofof storage,
storage, all
all three
three chocolate
chocolate samples
samples were
were evaluated
evaluated withwith very
very high
high marks
marks for
for
odour
odour and
and60
After taste.
taste. Maximum
daysMaximum quality
of storage,quality
all threeof control
ofchocolate chocolate
control chocolate was 92.75%,
was 92.75%,
samples were evaluatedwhile
while dark
withdark chocolates
verychocolates
high marks with
with
for
spray
spray dried
odour anddried Lb. plantarum
Lb.Maximum
taste. 564
564 and
plantarumquality and 299v gained
gained slightly
299v chocolate
of control slightly lower marks
lowerwhile
was 92.75%, dark(89.66%
marks (89.66%
chocolates and
and 89.06%,
89.06%,
with spray
respectively).
respectively). However,
However,
dried Lb. plantarum 564 probiotic
and 299vchocolate
probiotic chocolate variants
variantslower
gained slightly received
received
markshigher
higher scores
scores
(89.66% for sensory
for89.06%,
and quality
quality after
sensoryrespectively).after
180
180 days
However, of storage
days ofprobiotic (95.25%
storage (95.25%
chocolate for both
forvariants experimental
both experimental
received higher varieties),
varieties),
scoresreaching
reaching similar
similar
for sensory value as
valueafter
quality the
as the180control
control
days
(96.63%).
of storageBased
(96.63%). Based
(95.25% on
onforthe
the sensory
sensory
both analysis, it
it can
can be
analysis,varieties),
experimental concluded
bereaching
concluded that there
thereaswere
thatvalue
similar were not
not statistically
the control statistically
(96.63%).
significant
significant
Based on the differences
differences at
at the
the confident
sensory analysis, confident level
level of
it can be concludedof 0.05
0.05 between
between
that all
all three
there were three variants
variants during
during
not statistically 60
60 and
significant and 180
180 days
days
differences
of
of storage
at storage at 20 °C.
at 20 °C.
the confident Furthermore,
Furthermore,
level all analysed
all analysed
of 0.05 between all threesamples
samples
variantswere
were classified
classified
during 60 andin the
in 180 excellent
the excellent sensory quality
20 ◦ C.
sensoryatquality
days of storage
category.
category.
Furthermore, all analysed samples were classified in the excellent sensory quality category.
In
In this
this study,
study, sensory
sensory evaluation
evaluation showed
showed that that dark
dark chocolate
chocolate enriched
enriched with
with probiotic
probiotic bacteria
bacteria
had
had good marks for appearance and texture. Sandiness, a geometrical property that, if
good marks for appearance and texture. Sandiness, a geometrical property that, if pronounced,
pronounced,
could
could cause
cause texture
texture defects,
defects, has
has not
not been
been noticed
noticed in in both
both probiotic
probiotic chocolate
chocolate varieties
varieties during
during the
the
Sensors 2018, 18, 2570 13 of 16

In this study, sensory evaluation showed that dark chocolate enriched with probiotic bacteria
had good marks for appearance and texture. Sandiness, a geometrical property that, if pronounced,
could cause texture defects, has not been noticed in both probiotic chocolate varieties during the
storage. Also, probiotic dark chocolate had appropriate structure, hardness and good chewiness. Some
probiotic bacteria could produce components which may result in the appearance of some foreign
odour and taste, such as the so-called vinegar flavour, that may contribute negatively to the taste and
aroma of the product [52]. In this experiment, those negative characteristics have not been detected,
marking dark chocolates as samples with very good odour and taste during the whole 6 months
storage period.
Generally, it can be concluded that both probiotic dark chocolates had excellent sensory quality
and that encapsulated probiotic bacteria did not have an effect on aroma and texture of final products
in 6 months storage period.

4. Conclusions
This study demonstrated that encapsulated spray dried probiotic bacteria (both commercial
Lb. plantarum 299v and potential probiotic Lb. planatrum 564) had very good viability in dark chocolate
during storage period of 180 days at 20 ◦ C, while it significantly dropped after further storage of
360 days, reducing the shelf life of Chocolate 564 and 299v to six and nine months, respectively.
Nevertheless, encapsulated spray dried probiotic bacteria did not lead to the substantial disruption of
texture and aroma, showing an excellent sensory quality of probiotic dark chocolate after 180 days
of storage. Also, the chemical composition was not statistically different (p > 0.05) between control
and probiotic dark chocolate samples. Only minute differences were evident in the volatile profiles
between the probiotic and the control chocolate at 180 days but these related mainly to differences in
abundance of volatiles rather than actual differences in the type of volatile compounds present.
Hence, it can be concluded that the enrichment of dark chocolate with microencapsulated
probiotic bacteria gives a functional product with very good sensory and compositional characteristics.
Besides, this research has demonstrated that encapsulation by spray drying could provide adequate
protection of probiotic bacteria during production and storage of dark chocolate. The present study
provides grounds for further investigation on the possibility of using dark chocolate as a carrier
of probiotic bacteria into gastro-intestinal environment, where chocolate may be regarded as an
alternative way of supplying probiotics to consumers.

Author Contributions: Investigation, Z.R., M.M., N.M., S.S., K.K. and D.M.; Writing-Original Draft Preparation,
Z.R., M.M. and D.M.; Writing-Review & Editing, S.S. and K.K.
Funding: This research was funded by Ministry of Education, Science and Technological Development of Serbia
(Project No. 046009 and 046010).
Conflicts of Interest: The authors declare no conflict of interest.

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