LWT - Food Science and Technology 75 (2017) 640e647

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LWT - Food Science and Technology

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Semisweet chocolate as a vehicle for the probiotics Lactobacillus

acidophilus LA3 and Bifidobacterium animalis subsp. lactis BLC1:
Evaluation of chocolate stability and probiotic survival under in vitro
simulated gastrointestinal conditions
Marluci P. Silva a, Fabricio L. Tulini a, Júlia F.U. Marinho a, Marcella C. Mazzocato a,
Elaine C.P. De Martinis b, Valdecir Luccas c, Carmen S. Favaro-Trindade a, *
a
Departamento de Engenharia de Alimentos (ZEA), Faculdade de Zootecnia e Engenharia de Alimentos (FZEA), Universidade de Sa ~o Paulo (USP),
Pirassununga, Sa~o Paulo, Brazil
b
Departamento de Ana lises Clínicas, Toxicolo
gicas e Bromatolo
gicas (DACTB), Faculdade de Ci^
encias Farmac^ ~o Preto (FCFRP), Universidade
euticas de Ribeira
~o Paulo (USP), Ribeira
de Sa ~o Preto, Sa~o Paulo, Brazil
c
Centro de Tecnologia de Cereais e Chocolates (CEREAL CHOCOTEC), Instituto de Tecnologia de Alimentos (ITAL), Campinas, Sa ~o Paulo, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: Semisweet chocolate pleases a broad range of consumers, but it is an underexplored food matrix among
Received 12 May 2016 probiotic products. Thus, this study aimed to evaluate semisweet chocolate as a vehicle for probiotics
Received in revised form (Lactobacillus acidophilus LA3 and Bifidobacterium animalis subsp. lactis BLC1). The chocolates were
2 August 2016
evaluated by aw, pH, surface color and morphology, hardness, microbiological quality, sensorial accep-
Accepted 8 October 2016
tance and probiotic viability. Besides, probiotic survival in simulated gastric and intestinal fluids (SGF/SIF)
Available online 11 October 2016
was evaluated before and after application in chocolate. Samples presented pH values around 6 and aw
below 0.6. Free probiotics populations were reduced after exposure to SGF/SIF, while no significant
Keywords:
Viability
reduction was detected in probiotic populations incorporated into chocolate. After 120 days of storage at
Cocoa 25
C, probiotic populations in chocolate were reduced by only 1.4 and 0.7 logarithmic cycles, respec-
Phenolics tively. Considering these results and that all samples were very well accepted by panelists, semisweet
Sensorial acceptance chocolate can be considered a good vehicle for probiotics.
Hardness © 2016 Elsevier Ltd. All rights reserved.

1. Introduction Probiotic functionality may be enhanced when these microor-

ganisms are incorporated in food, since interaction with food in-
The World Health Organization (WHO) and the Food and Agri- gredients can protect microbial cells during the passage through
culture Organization (FAO) define probiotics as “live microorgan- the gastrointestinal tract (Ranadheera, Baines, & Adams, 2010;
isms that when administered in adequate amounts confer health Vinderola, Binetti, Burns, & Reinheimer, 2011). In this context,
benefits to the host” (Food and Agriculture Organization & World dairy products are considered a good vehicle for probiotics, and
Health Organization, 2002). However, probiotics have to survive they have been widely used by the food industry. However, part of
to food processing and throughout the gastrointestinal tract, chal- the world population do not consume those products due to lactose
lenging the food industry to find new alternatives for incorporating intolerance, milk allergy or even due to diets that restrict the use of
these microorganisms in food. Several parameters such as high animal protein. To overcome this problem, semisweet chocolate
temperatures, pH variation and oxygen may affect probiotic sur- may be an alternative to dairy products. Furthermore, chocolate has
varo-Trindade, Heinemann, & Pedroso, 2011; Tripathi &
vival (Fa other beneficial properties such as recognized antioxidant activity
Giri, 2014). (Gadhiya, Patel, & Prajapati, 2015). Moreover, phenolic compounds
in chocolate may also play an important role in delaying oxidative
stress in probiotics, which is one of the main causes of probiotic
death in food, leading to improved viability and extended shelf life
* Corresponding author. Faculdade de Zootecnia e Engenharia de Alimentos,
(Bialonska et al., 2010; Curiel, Munoz, & Lopez de Felipe, 2010;
Universidade de S~ ao Paulo, Avenida Duque de Caxias Norte, 225, 13635-000
Pirassununga, S~
ao Paulo, Brazil. Pereira, Almeida, de Jesus, da Costa, & Rodrigues, 2013). Accord-
E-mail address: [email protected] (C.S. Favaro-Trindade). ing to Gadkari and Balaraman (2015), chocolate is a potential source

http://dx.doi.org/10.1016/j.lwt.2016.10.025
0023-6438/© 2016 Elsevier Ltd. All rights reserved.
 M.P. Silva et al. / LWT - Food Science and Technology 75 (2017) 640e647 641

of catechins, epicatechins and type B procyanidins, which may 2.2.2. Semisweet chocolate preparation
contribute to the antiplatelet effects of this product. The chocolate was prepared at the Cereal Chocotec pilot plant
Chocolate is a suspension of particles, mainly sugar and cocoa, in (Instituto de Tecnologia de Alimentos, Campinas, Brazil) using a
the lipid matrix of cocoa butter. One of the main properties that solution of 47% sugar (w/w), 43% cocoa liquor, 10% cocoa butter,
makes chocolate very pleasant to consumers is its ability to melt in 0.3% soy lecithin and 0.2% PGPR. The cocoa liquor and sugar were
the mouth, despite being a solid product during storage (Luccas, mixed, and the mass was refined using a roller mill (Draiswerk,
Bonomi, & Kieckbusch, 2014). GMBH, Germany). After this step, samples were collected to eval-
The preparation of chocolate involves several steps that may uate maximum particle size (mm), as described by Luccas and
have deleterious effects on probiotic viability. Thus, the incorpo- Kieckbusch (2006). This control is essential because chocolates
ration of probiotics into chocolate is performed after the tempering with large particles may cause a gritty sensation in the mouth. Next,
step to avoid any deleterious effect of temperature on bacterial the mixture was submitted to a conching process in a jacketed
cells. Chocolate storage is another critical point, since semisweet mixer (INCO, Germany) for 16 h at 65
C. The cocoa butter and PGPR
chocolates are stored at room temperature for up to 6 months, were added, respectively, 2 and 1 h before ending the conching
depending on the type of packaging and processing, which may step. The tempering of chocolate was performed manually using
interfere with probiotic viability in the product. spatulas in a room with controlled temperature. During this step,
According to Lahtinen, Ouwehand, Salminen, Forssell, and the tempering index was constantly checked using the tempering
Myllarinen (2007) and Pedroso, Dogenski, Thomazini, index device, and the tempering process was repeated if index
Heinemann, and Fa varo-Trindade (2013), cocoa butter can protect values were not within the range of 4e6.
probiotic microorganisms. Furthermore, the lipid matrix probably Probiotic cells were prepared as described in Section 2.2.1 and
protects bacterial cells from water and Hþ ions. Other studies have added to chocolate after the tempering step at a ratio of 1010 cfu for
also verified the potential of milk and dark chocolate, under each 100 g, resulting in a product with 108 cfu/g. At this step, three
refrigeration temperature, for incorporating probiotics (Foong, Lee, formulations of chocolate were prepared: (i) LA3, containing only
Ramli, Tan, & Ayob, 2013; Lalicic-Petronijevic et al., 2015; Mandal, L. acidophilus LA3; (ii) BLC1, containing only B. animalis subsp. lactis
Hati, Puniya, Singh, & Singh, 2013; Nebesny, Zyzelewicz, Motyl, & BLC1; (iii) control, without probiotics. The pre-crystalized chocolate
Libudzisz, 2007; Possemiers, Marzorati, Verstraete, & Vand de was then poured into polyethylene molds and placed in a cooling
Wiele, 2010). However, until 2015, no data was published about tunnel adjusted to 10
C for 30 min. The chocolate bars were
probiotic viability in semisweet chocolates stored at room manually demolded, wrapped in aluminum foil and kept at 20
C
temperature. for 15 days to stabilize the crystal lattice. After this step, the
Chocolates containing probiotics may be potential candidates chocolate bars were kept at 25
C for up to 120 days.
for new functional foods due to the combined health benefits of
probiotics and chocolate phenolic compounds. Thus, the aim of the 2.3. Characterization of probiotic chocolates and evaluation of their
present study was to incorporate the probiotics Lactobacillus aci- stability
dophilus LA3 and Bifidobacterium animalis subsp. lactis BLC1, into
semisweet chocolate. Moreover, the influence of probiotics on 2.3.1. Physicochemical characterization
chocolate quality, the influence of chocolate on probiotic viability 2.3.1.1. Water activity (aw) and pH. Chocolate bars were finely
during storage and survival under in vitro simulated gastrointes- divided and analyzed using an Aqualab device (Decagon Devices,
tinal conditions were also evaluated. USA) to measure aw, and expressed as values from zero to one
(maximum aw). To evaluate final chocolate pH, 10 g of chocolate
bars were finely divided, stirred for 20 min in 100 ml of deionized
2. Material and methods water and analyzed using a potentiometer. These analyses were
performed monthly for 120 days.
2.1. Materials
2.3.1.2. Determination of total phenolic and fat contents. The prep-
L. acidophilus LA3 and B. animalis subsp. lactis BLC1, in freeze- aration of defatted chocolate samples was performed as described
dried form, were kindly provided by Sacco Brasil (Campinas, Brazil). by Adamson et al. (1999) and Alan ~on, Castle, Siswanto, Cifuentes-
Aside from probiotics, for semisweet chocolate preparation, the Go mez, and Spencer (2016). Initially, 1 g of chocolate was defat-
following materials were used: sugar (Unia ~o, Brazil), cocoa liquor ted using 10 ml of n-hexane, and the mixture was homogenized by
(Barry Callebaut, Brazil), cocoa butter (Barry Callebaut, Brazil), soy agitation and sonication for 5 min, followed by a centrifugation step
lecithin (Bunge, Brazil) and polyglycerol polyricinoleate (PGPR, at 2465g for 5 min. This procedure was performed twice and the
Danisco, Brazil). samples were air-dried to remove the residual of n-hexane.
Following, to extract the phenolic compounds, 1 g of defatted
chocolate was added into 10 ml of ethanol aqueous solution (80% v/
2.2. Production of probiotic chocolate v) and homogenized by sonication for 10 min, followed by centri-
fugation at 4108g for 5 min. The extraction of phenolic com-
2.2.1. Probiotic inoculum pounds was performed twice. Next, total phenolic content was
The probiotic inoculums were prepared as described by Okuro, determined according to Singleton, Orthofer, and Lamuela-
Thomazini, Balieiro, Liberal, and F avaro-Trindade (2013), with Raventos (1999) and Souza et al. (2014). For this, 0.25 ml of the
modifications. LA3 and BLC1 were cultivated three times in MRS chocolate extract was mixed with 2 ml of distillated water and
broth for 18 h at 37
C. Cells were harvested by centrifugation 0.25 ml of FolineCiocalteu reagent. After 3 min at room tempera-
(5752g for 10 min at 10
C), washed with 2% sodium citrate and ture, the mixture was added of 0.25 ml of saturated sodium car-
adjusted to ca. 1010 cfu/ml with the same solution by measuring the bonate solution (Na2CO3) and homogenized. The tubes were placed
absorbance at 600 nm (correlated with agar plate counts). Next, the in a water bath at 37
C for 30 min to complete the reaction and the
bacterial suspension was centrifuged (5752g for 10 min at 10
C) absorbance was measured at 750 nm (Hach DR 2800, USA). The
to remove the sodium citrate solution, and the cell pellet (con- total content of phenolic compounds was determined using gallic
taining 1010 cfu/g) was used to prepare the semisweet chocolate. acid as standard, and the results were expressed as mg of gallic acid
642 M.P. Silva et al. / LWT - Food Science and Technology 75 (2017) 640e647

equivalents (GAE) per g of chocolate. plates were incubated for 48 h at 37
C, and single colonies were
The fat content in chocolates was determined using the Bligh enumerated. This analysis was performed monthly for 120 days.
and Dyer (1959) method, which consists in the extraction of
lipids with chloroform, methanol and water in a ratio of 1:2:0.8, 2.3.2.3. Evaluation of probiotic survival under in vitro simulated
respectively. The amount of lipids in the samples was determined gastrointestinal conditions. The incorporation of probiotics in
by removing an aliquot of 5 ml from the chloroform fraction, fol- chocolate may change their ability to survive in the gastrointestinal
lowed by evaporation of the solvent in a stove at 100
C for 40 min. environment. Thus, L. acidophilus LA3 and B. animalis subsp. lactis
The recipient was cooled in a desiccator under vacuum, and then BLC1 were evaluated with regard to survival under in vitro simu-
the remaining material was weighed to determine the total fat lated gastrointestinal conditions, as described by Gbassi,
content. Vandamme, Ennahar, and Marchioni (2009), with modifications.
One milliliter of probiotic culture or 1 g of probiotic chocolate was
2.3.1.3. Surface color. The surface color of chocolate was analyzed added to 9 ml of simulated gastric fluid (SGF: NaCl 9 g/l, pepsin 3 g/
using a colorimeter (HunterLab Model MiniScan XE, Reston, USA), l, pH 1.8). After 120 min of incubation at 37
C under constant
which can be useful to evaluate the occurrence of the fat bloom agitation (100 rpm), 10 ml of simulated intestinal fluid (SIF: NaCl
phenomenon. The parameters “L”, “a” and “b” were measured and 9 g/l, pancreatin 10 g/L, trypsin 10 g/L, bile salts 3 g/L, pH 6.5) were
used to calculate the whiteness index (WI) with the formula (1) added to the samples and incubated for 180 min at 37
C under
described by Lohman and Hartel (1994), in which higher values constant agitation (100 rpm). Aliquots of 1 ml were withdrawn
of WI indicate that the chocolate surface is more white than from the beginning (t ¼ 0) until the end (t ¼ 300 min) of the
chocolates with lower WI values. This analysis was performed experiment, at intervals of 60 min, for bacterial enumeration, as
monthly for 120 days. previously described in Section 2.3.2.2. All bacterial enumerations
were performed in triplicate, and the assay was performed on the
h i0:5
first day of storage.
WI ¼ 100  ð100  LÞ2 þ a2 þ b2 (1)

2.3.3. Sensorial acceptance

Probiotic chocolates were submitted to consumer acceptance
testing after 20 days of storage, according to Meilgaard, Civille, and
2.3.1.4. Hardness. Chocolate hardness was evaluated using a tex-
Carr (1999), with 100 untrained panelists who evaluated the at-
turometer (TA XT Plus Texture Analyzer, Extralab, Brazil), as
tributes of taste, aroma, texture and overall acceptance. This sen-
described by Afoakwa, Paterson, Fowler, and Veira (2008), using
sory evaluation was accomplished in the laboratory using
samples prepared as pieces of 40 mm  25 mm x 7 mm (L x W x H).
individual booths and under fluorescent light. The samples were
This analysis was performed monthly for 120 days.
placed in plastic plates coded with three-digit random numbers,
and served one at time. Panelists were instructed to rinse the palate
2.3.1.5. Surface morphology assessment by scanning electron micro-
after each sample. A 9-point structured hedonic scale was used,
scopy (SEM). Scanning electron microscopy (SEM) was used to
with “1” as “dislike extremely” and “9” as “like extremely.”
evaluate the chocolate surface, as described by Afoakwa, Paterson,
Fowler, and Vieira (2009) and Luccas et al. (2014). This analysis was
2.4. Statistical analyses
performed on the first and last days of storage (120 days).
All experiments were performed as independent triplicates, and
2.3.2. Microbiological analyses the results were evaluated by analysis of variance (ANOVA) fol-
2.3.2.1. Microbiological quality of chocolate. Enumeration of total lowed by Tukey's post-test (95% confidence interval), using the SAS
and thermotolerant coliforms (Escherichia coli) and the detection of v9.1.3 program (Statistic Analysis Software, SAS Institute Inc., USA).
Salmonella spp. were carried out according to methods previously
reported in the literature (Andrews, Flowers, Silliker, & Bailey, 3. Results and discussion
2001; Kornacki & Johnson, 2001, pp. 69e82). This analysis was
performed on the first day of storage. In the present study, it was evaluated the potential of semisweet
chocolate as a new matrix to incorporate probiotics. Lactobacillus
2.3.2.2. Probiotic viability during chocolate storage. Probiotic bac- acidophilus LA3 and Bifidobacterium animalis subsp. lactis BLC1
teria may be affected during chocolate processing and storage, so were chosen to be applied in semisweet chocolate because they are
the enumeration of these microorganisms during storage is commercial strains, which means that they were previously studied
essential. Five grams of each chocolate sample were diluted in with regard the beneficial properties and safety. Moreover, both
45 ml of 2% sodium citrate previously heated at 48
C. After ho- species have been extensively studied by other authors that re-
mogenization for 2 min in stomacher, aliquots of 1 ml were with- ported the health benefits and safety of several strains (Parvez,
drawn, serially diluted and inoculated in MRS agar. Next, inoculated Malik, Kang, & Kim, 2006; Salminen et al., 1998). In addition,

Table 1
Water activity (aw) of chocolates stored at 25
C for up to 120 days (mean ± standard deviation).

Formulation Days

0 30 60 90 120
bB aAB bB bB bA
Control 0.36 ± 0.05 0.40 ± 0.02 0.38 ± 0.01 0.37 ± 0.01 0.44 ± 0.03
aA aB aB bB aA
LA3 0.51 ± 0.00 0.44 ± 0.02 0.43 ± 0.01 0.41 ± 0.00 0.52 ± 0.01
bC aBC aBC aB aA
BLC1 0.40 ± 0.03 0.44 ± 0.03 0.43 ± 0.02 0.45 ± 0.01 0.56 ± 0.00

LA3 chocolate was produced with Lactobacillus acidophilus LA3 and BLC1 chocolate was produced with Bifidobacterium animalis subsp. lactis BLC1. Control chocolate was
produced without probiotics.
Values with the same upper case letter in a row and values with the same lower case letter in a column are not statistically different (p > 0,05).
 M.P. Silva et al. / LWT - Food Science and Technology 75 (2017) 640e647 643

Table 2 although all formulations presented aw values below 0.6, which

Values of pH for chocolates stored at 25
C for up to 120 days (mean ± standard may contribute to the microbiological safety of chocolate because
deviation).
very few microorganisms can multiply under such conditions. The
Formulation Days control formulation presented lower values of aw when compared
0 30 60 90 120 to values detected in probiotic chocolate, which could be attributed
to the use of bacterial cells obtained from a liquid suspension.
Control 5.70 ± 0.04 5.74 ± 0.04 5.76 ± 0.02 5.71 ± 0.05 5.75 ± 0.05
LA3 5.66 ± 0.06 5.74 ± 0.03 5.73 ± 0.02 5.74 ± 0.05 5,73 ± 0.04 Vesterlund et al. (2012) evaluated the impact of aw in crushed
BLC1 5.72 ± 0.03 5.82 ± 0.02 5.75 ± 0.02 5.72 ± 0.02 5.78 ± 0.03 flaxseed matrix by mixing probiotics in crushed flaxseed followed
LA3 chocolate was produced with Lactobacillus acidophilus LA3 and BLC1 chocolate by drying in a laboratory incubator at 57
C. Those authors reported
was produced with Bifidobacterium animalis subsp. lactis BLC1. Control chocolate that low values of aw (ca. 0.11) extended the viability of Lactobacillus
was produced without probiotics. rhamnosus GG, while the same probiotic in a product with 0.43 of
Values in rows and columns are not statistically different (p > 0,05). aw was very unstable and reduced approximately 3.7 log cfu/g after
4 months of storage. Although the results were different from the
ones in the present study, the main reason for these differences is
these commercial strains have different energetic metabolisms,
the complexity of chocolate matrix, which is composed of various
since L. acidophilus LA3 is a microaerophilic microorganism, and
ingredients that may protect the probiotics.
B. animalis subsp. lactis is an anaerobic microorganism, which is
Another parameter that may affect chocolate characteristics and
interesting to investigate and compare their application in choco-
the probiotic viability is the pH, which may affect protein function,
late. The probiotic chocolates were produced and characterized as
transport patterns and cellular bioenergetics (Krulwich, Sachs, &
described below.
Padan, 2011). Thus, in the present study, the pH of chocolates was
evaluated during 120 days of storage at 25
C, and the results are
3.1. Characterization of chocolates presented in Table 2. All chocolate formulations presented pH
values in a narrow range (5.66e5.82), which were not statistically
3.1.1. Physicochemical characterization of chocolates different (p > 0.05). In addition, pH values remained unchanged
The aw is one of the main factors that may affect probiotic throughout the storage period (p > 0.05). These results revealed
viability in food because it indicates the amount of water available that probiotic bacteria did not change the pH, indicating that they
to the microorganisms (Vesterlund, Salminen, & Salminen, 2012). were metabolically inactive throughout storage at room tempera-
Thus, the aw was measured monthly in all chocolate formulations ture, probably due to the low aw, as previously discussed. According
stored at 25
C for 120 days, as previously described, and the results to Lahtinen, Gueimonde, Ouwehand, Reinikainen, and Salminen
are presented in Table 1. All chocolate formulations presented a (2005), probiotics may stay metabolically inactive during the
slight increase in aw throughout 120 days of storage (p < 0.05), storage of probiotic products, which may even influence bacterial
enumeration for these products.
Chocolates produced in this study were also characterized with
Table 3 regard the total phenolic and total fat contents, and the results are
Total content of phenolic and fat of chocolate stored at 25
C (mean ± standard presented in Table 3. No differences (p > 0.05) were observed
deviation).
among samples with regard the total phenolic and total fat con-
Formulation Total phenolic (mg gallic acid Total content tents, since all chocolate formulations were prepared with same
equivalent/g chocolate) of fat (%) proportions of ingredients, differing only in the incorporation of
Control 16.98 ± 0.32 31.34 ± 0.66 probiotics cultures. Total fat content was ca. 31.5%, while the total
LA3 16.69 ± 0.49 31.60 ± 0.46 phenolic content was ca. 17 mg GAE/g of chocolate. These values
BLC1 16.52 ± 0.67 31.58 ± 0.58
were similar to the results reported by Miller et al. (2006), which
LA3 chocolate was produced with Lactobacillus acidophilus LA3 and BLC1 chocolate analyzed the total phenolic and fat contents of commercial choc-
was produced with Bifidobacterium animalis subsp. lactis BLC1. Control chocolate
olates sold in the Unites States of America. According to those au-
was produced without probiotics.
Values in columns are not statistically different (p > 0,05).
thors, total fat content of semisweet chocolate ranged from 29.0% to

Fig. 1. Hardness (left) and whiteness index (right) of probiotic semisweet chocolates stored at 25
C for up to 120 days. LA3 chocolate was produced with Lactobacillus acidophilus
LA3, and BLC1 chocolate was produced with Bifidobacterium animalis subsp. lactis BLC1. Control chocolate was produced without probiotics. Significant increases (p < 0.05)
compared to baseline are highlighted with an asterisk.
644 M.P. Silva et al. / LWT - Food Science and Technology 75 (2017) 640e647

Fig. 2. Micrographs obtained by scanning electron microscopy of chocolates stored at 25
C for up to 120 days, at 1000 magnification. A, C and E correspond to the 1st day of
storage of control (chocolate produced without probiotics), LA3 (chocolate produced with Lactobacillus acidophilus LA3) and BLC1 (chocolate produced with Bifidobacterium animalis
subsp. lactis BLC1), respectively. B, D and F correspond to the 120th day of storage of control, LA3 and BLC1 chocolates, respectively.
 M.P. Silva et al. / LWT - Food Science and Technology 75 (2017) 640e647 645

avoid this defect is not possible, since the previous processes

employ high temperatures for long periods, which may affect cell
viability.

3.1.2. Microbiological analyses

Before the sensory evaluation of new foods, it is necessary to
analyze the microbial quality of samples to guarantee the safety of
panelists. In addition, these analyses are important to guarantee
that other bacteria will not interfere with the viability of probiotics
in chocolate. In the present study, the enumeration of total and
thermo-tolerant coliforms (Escherichia coli) and the detection of
Salmonella spp. were performed for chocolates. According to the
results, coliform populations were below the detection limit of the
method (<3 MPN/g), and Salmonella spp. were not detected in 25 g
of sample. These results indicate that the probiotic chocolates
Fig. 3. Lactobacillus acidophilus LA3 and Bifidobacterium animalis subsp. lactis BLC1
produced in this study were safe for consumption.
populations in chocolates stored at 25
C for up to 120 days. No significant reduction The viability of probiotics in chocolates was evaluated
(p < 0.05) was detected in any sample or period. throughout 120 days of storage at 25
C and, according to Fig. 3,
there was no significant reduction in L. acidophilus LA3 or
B. animalis subsp. lactis BLC1 populations. However, B. animalis
29.8%, and the total phenolic content was ranged from 11.8 to subsp. lactis BLC1 presented the highest viability, approximately 7.7
€
12.9 mg GAE/g of product. Similarly, Gültekin-Ozgüven, Berktaş, log cfu/g, while L. acidophilus LA3 was 7.3 log cfu/g. The presence of
€
and Ozçelik (2016) evaluated the total phenolic content of cocoa high fat content (31%) in chocolate was efficient to protect the
powder during the manufacturing steps. Those authors reported B. animalis subsp. lactis BLC1 of water phase, and possibly of at-
that the roasting of cocoa beans was responsible for the loss of mospheric oxygen, considering the anaerobic metabolism of this
about 65% of phenolic compounds. However, despite the loss of probiotic. Nebesny et al. (2007) produced probiotic dark chocolate
phenolic compounds during the process, cocoa powder presented with Lactobacillus casei and Lactobacillus paracasei and stored the
high values of phenolic compounds. samples for one year at 30, 18 and 4
C. Those authors reported
Hardness is a physical parameter in chocolate that may affect similar results to those presented here: the bacterial populations
the acceptance by the consumers, and it is closely related to the remained stable (106e107 cfu/g) throughout the storage period. In
ingredients and tempering process. In the present study, hardness contrast, Erdem et al. (2014) reported survival of approximately
was evaluated throughout the storage period of probiotic choco- 105 cfu/g for Bacillus indicus HU36 used to produce synbiotic dark
lates. According to the results presented in Fig. 1, the addition of chocolate.
probiotics did not affect the texture of chocolates when compared The results from the present study indicate that the pH of
to controls in the beginning of the storage period. However, BLC1 approximately 5.7 and the low aw (ca. 0.44) kept probiotics in a low
and control formulations presented a significant increase in hard- metabolic state. In addition, high fat content and phenolic com-
ness (p < 0.05) after 120 days of storage. Recently, Foong et al. pounds (antioxidant compounds responsible for reduced oxidative
(2013) produced dark chocolates with and without probiotics, stress) may also help to maintain the viability of probiotics in
and reported that chocolate without probiotic was harder than chocolates (Pedroso et al., 2013; Tzounis et al., 2011).
probiotic chocolate. However, tempering processes were different Food matrices may provide additional protection to probiotics
in both preparations, which could have influenced hardness in during passage through the gastrointestinal tract (Ranadheera
control samples. et al., 2010). In the present study, probiotic survival under in vitro
Fat bloom is a phenomenon that may occur in chocolate and it simulated gastrointestinal conditions was evaluated before and
has been studied extensively, although the complete mechanism is after incorporation into semisweet chocolate, and the results are
not well understood (Sonwai & Rousseau, 2010). Chocolates with presented in Fig. 4. According to the results, L. acidophilus LA3 and
fat bloom present a whitish layer on the outer surface, which can be B. animalis subsp. lactis BLC1 populations were reduced by 2.9 and
correlated with the whiteness index (WI). In the present study, 4.1 log cfu/g at the end of the assay, respectively, when evaluated as
probiotics chocolates and controls were evaluated with regard to free cultures. However, no significant reduction was detected for
WI throughout 120 days of storage, and the results are presented in both probiotic population under the same conditions when cells
Fig. 1. WI increased throughout the storage period in probiotic were analyzed after incorporation into the chocolate. The main
chocolates and controls, indicating the occurrence of fat bloom reason for this increased resistance is the interaction between the
probably due to tempering procedures, which is crucial for the probiotics and chocolate ingredients such as fat phenolic com-
development of this phenomenon. In addition to WI, micrographs pounds, which may protect the cells during the digestion and
obtained by SEM also revealed the occurrence of fat bloom in storage. Possemiers et al. (2010) also reported high survival rates
chocolates after 120 days of storage at 25
C, in all samples, as for Lactobacillus helveticus (91%) and Bifidobacterium longum (80%)
presented in Fig. 2. incorporated into milk chocolate, when samples were evaluated for
At the beginning of storage, the granulometry of samples pre- survival under in vitro simulated gastrointestinal conditions. These
sented similar structures. However, at the end of storage, the sur- data indicate that semisweet chocolate is a potential food matrix to
face of samples presented fat crystals, which migrated from protect probiotic cells during passage through the gastrointestinal
chocolate matrix to the surface of the product. In chocolate samples tract.
containing probiotics, larger crystals were detected when Some mechanisms of interaction between the chocolate matrix
compared to the control sample. The occurrence of this phenom- and probiotics have been proposed by different authors to explain
enon may be related to the addition of probiotics during the the protective effects of this product. One of them is the antioxidant
tempering process, which may influence the recrystallization of activity of phenolic compounds presents in cocoa liquor, which may
lipids. Besides that, the addition of probiotics before tempering to avoid the oxidative stress, thereby reducing cell death (Maukonen
646 M.P. Silva et al. / LWT - Food Science and Technology 75 (2017) 640e647

Fig. 4. Survival of Bifdobacterium animalis subsp. lactis BLC1 (left) and Lactobacillus acidophilus LA3 (right) under in vitro simulated gastrointestinal conditions. Cells were evaluated
as free cultures or incorporated into semisweet chocolate, and exposed to simulated gastric fluid (0e120 min) followed by simulated intestinal fluid (120e300 min). Significant
reductions (p < 0.05) on probiotic populations compared to initial populations were highlighted with an asterisk. No significant reduction was detected on probiotic populations
when they were incorporated into the chocolates.

& Saarela, 2015; Tzounis et al., 2011). Another possible interaction among all formulations, indicating that the addition of probiotics
occurs with probiotics and cocoa butter, depending on the hydro- did not influence the parameters evaluated in the present study.
phobicity profile of the cell wall of the probiotics, which may The hedonic scale used by panelists to evaluate the samples ranged
contribute to the release of the microorganisms into the intestine from 1 to 9, where “1” meant “dislike extremely” and “9” meant
during fat digestion. In this context, cocoa butter was even inves- “like extremely”, considering that the lowest score was 7.28 ± 1.38,
tigated as encapsulant material to protect probiotics, leading to it is possible to infer that all products were very well accepted. All
satisfactory results (Lahtinen et al., 2007; Pedroso et al., 2013). formulations presented overall acceptance above to 7.5. Further-
Furthermore, the high content of sugar in chocolate (47%) may more, no difference statically was observed about the attributes
buffer the gastrointestinal fluids during the digestion (Ranadheera evaluated in each sample.
et al., 2010).
4. Conclusions
3.2. Evaluation of sensory properties
L. acidophilus LA3 and B. animalis subsp. lactis BLC1 were suc-
Probiotic semisweet chocolates and controls were evaluated by cessfully incorporated into semisweet chocolate, which was
100 consumer panelists with regard to color, taste, aroma, texture revealed to be a potential vehicle for these probiotics by keeping
and overall acceptance, and the results are presented in Fig. 5. bacteria viable for up to 120 days at 25
C and by increasing bac-
According to the results, no significant difference was detected terial survival under in vitro simulated gastrointestinal conditions.
Both strains presented the same behavior (p > 0.05) during storage
and exposure to in vitro simulated gastrointestinal fluids, resulting
in high viability at the end of the assays. These results may be partly
attributable to the physical and chemical properties of semisweet
chocolate such as slightly acidic pH, low aw, high fat content and the
presence of phenolic compounds. In addition, probiotic chocolates
were very well accepted by a group of 100 panelists, indicating the
potential of these products in the marketing of functional foods.

Acknowledgements

The authors thank the Sa ~o Paulo Research Foundation for the

scholarships awarded (#2014/10754-5 and #2014/14540-0), the
Coordination for the Improvement of Higher Education Personnel
(CAPES, #23038.0011232/2014-67) and the National Council for
Scientific and Technological Development (CNPq, #462493/2014-8)
for financial support. Favaro-Trindade C.S. thanks CNPq for the
Fig. 5. Radar graphic with scores of probiotic chocolates evaluated by panelists with productivity grant (306708/2015-9). The authors also thank the
regard to the color, taste, aroma, texture and overall acceptance, using a hedonic scale Center of Cereal and Chocolate Technology of the Institute of Food
ranging from 0 to 9. LA3 chocolate was produced with Lactobacillus acidophilus LA3,
and BLC1 chocolate was produced with Bifidobacterium animalis subsp. lactis BLC1.
Technology (ITAL, Brazil) for technical assistance and support, Barry
Control chocolate was produced without probiotics. Values were not statistically Callebaut Brazil for the donation of raw materials and Sacco Brasil
different (p > 0.05). for the donation of probiotic cultures.
 M.P. Silva et al. / LWT - Food Science and Technology 75 (2017) 640e647 647

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