2018 - Physicochemical Properties and

European Food Research and Technology
https://doi.org/10.1007/s00217-018-3035-2
ORIGINAL PAPER
Physicochemical properties and antioxidant activities of chocolates

enriched with engineered cinnamon nanoparticles
Dimas Rahadian Aji Muhammad1,2 · Arifin Dwi Saputro1,3 · Hayley Rottiers1,4 · Davy Van de Walle1 ·
Koen Dewettinck1
Received: 11 November 2017 / Revised: 6 January 2018 / Accepted: 7 January 2018

© Springer-Verlag GmbH Germany, part of Springer Nature 2018
Abstract
The use of nanocapsules to overcome incompatibility between bioactive compounds and food matrices targeting fortifica-
tion has been widely acknowledged. This study provides a novel method to enhance the nutritional properties of chocolate
by employing lyophilised colloidal nanoparticles made of a combination of shellac, xanthan gum and cinnamon extract.
Lyophilised colloidal nanoparticles containing cinnamon extract (LCNP-CE) were prepared by an anti-solvent precipita-
tion method followed by freeze drying. Cinnamon extract was loaded into nanoparticle to entrap the aroma of the cinnamon
extract; thereby, the cinnamon extract can be incorporated in the chocolate to expand its bioactive profile without altering
its sensorial characteristic. LCNP-CE was formulated into white and milk chocolate in multilevel ratios (0–2% w/w). The
results show that the fortification of milk and white chocolates by LCNP-CE significantly improved the total phenolic content
and antioxidant activity of the chocolates without remarkable changes in the fineness and melting profile properties. Even
though slight changes in the hardness, flow behaviour and colour have been observed, the enriched chocolates are likely in
the range of acceptable values. Encapsulation has a positive impact on preventing flavour alteration on the cinnamon enriched
chocolates; however, a drawback in the release behaviour of the cinnamon extract from the chocolate was observed.
Keywords Nanoparticle · Chocolate · Cinnamon · Antioxidant
Introduction syndromes [2, 3]. Cocoa beans (Theobroma cacao L.), the
main ingredients of chocolate, are naturally rich in polyphe-
The attention of food industry toward beneficial bioactive nol compounds. However, they significantly decrease or pos-
components of food has rapidly been increasing since the sibly undergo conversion to their epimers during chocolate
last decades [1]. Polyphenols have been obtaining great manufacturing in which mainly in fermentation and roasting
interest due to their potential use as antioxidant and prom- [4–7]. It was also noted by Di Mattia et al. [8] that conching
ising role in preventing the human body from metabolic can cause the loss of bioactive compounds of chocolate. An
even more vivid demonstration regarding this problem was
reported by Schinella et al. [9] who showed low levels of
Dimas Rahadian Aji Muhammad catechin, epicatechin, procyanidin B1 and procyanidin B2
dimasrahadianaji.muhammad@ugent.be; in cocoa powder between 0.01 and 0.12% (w/w).
rahadiandimas@uns.ac.id
In tackling those issues, food scientists have been focus-
1
Laboratory of Food Technology and Engineering, Faculty sing on two principal approaches: (a) preserving the bioac-
of Bioscience Engineering, Ghent University, Coupure links tive cocoa components during the chocolate manufacturing
653, Ghent 9000, Belgium by performing some process modifications and (b) enriching
2
Department of Food Science and Technology, Universitas chocolate with polyphenol compounds derived from other
Sebelas Maret, Jl. Ir Sutami 36A, Surakarta 57126, Indonesia plant substrates. Extracts of mangosteen pericarp, nettle
3
Department of Agricultural Engineering, Universitas Gadjah and raspberry leaves as well as goji berry, black mulberry,
Mada, Jl. Flora 1, Yogyakarta 55281, Indonesia peanut skins and various herbs and spices have been suc-
4
Laboratory of AgriFing, Department of Applied Biosciences, cessfully incorporated in chocolate to improve its phenolic
Faculty of Bioscience Engineering, Ghent University, content and antioxidant activity [10–16]. Herbs and spices
Valentin Vaerwyckweg 1, Ghent 9000, Belgium
13
Vol.:(0123456789)
are attractive ingredients to be incorporated in chocolate, incorporated in the chocolate without altering its sensorial
due to their unique flavour, high polyphenol content and characteristic.
potential biological activity [17, 18]. Nanoparticles containing cinnamon extract have been
Among spices, cinnamon (family Lauraceae, genus successfully obtained by means of a nano-encapsulation
Cinnamomum) is one of the most promising sources of technique using anti-solvent precipitation method adopted
polyphenols and antioxidant properties [18, 19]. Phenolic from Patel et al. [50]. The incorporation of the nanoparti-
constituents identified in cinnamon are vanillic acid, caf- cles in chocolate is hypothesised to additionally expand the
feic acid, p-hydroxybenzoic acid, p-hydroxybenzaldehyde, bioactive profile without significantly changing the qual-
p-coumaric acid, gallic acid, proanthocyanidin, ferulic ity attributes of the chocolate. This study therefore aims at
acid, quercetin, syringic acid, sinapic acid, tannic acid, and investigating the impact of lyophilised colloidal nanoparti-
trans-cinnamic acid [20–25]. Cinnamon also contains a wide cles containing cinnamon extract (LCNP-CE) on the quality
spectrum of volatile compounds such as cinnamaldehyde, attributes and physicochemical properties of chocolate.
eugenol, linalool and limonene [26–31]. Nowadays, phy-
tochemicals derived from cinnamon are of medical inter-
est based on the fact that cinnamon has been widely used Materials and methods
in traditional remedy around the world since ancient times.
Recently, the role of cinnamon and its constituents as physi- Preparation of LCNP-CE
ologically active components in the diet is being increasingly
acknowledged [32]. A considerable number of recent stud- Cinnamon extract was prepared from cinnamon bark (Cin-
ies showed that cinnamon offer anti-cancer and anti-tumour, namomum burmannii Blume) by the following step: 10 g of
anti-mutagenic, anti-inflammatory, anti-diabetes, and anti- cinnamon powder was extracted in 100 ml ethanol and then
viral activity [33–37]. Due to its unique composition and its stirred for 48 h at 20 °C. Afterwards, the mixture was filtered
potential health effects, cinnamon has been used to improve by vacuum filter (Laboport, KNF Neuberger, Inc, USA). The
the health-promoting properties in foods, such as coffee and solvent was removed using rotary evaporator under reduced
yoghurt [38, 39]. pressure (Laborota 4000 Heidolph, Germany) to obtain con-
Enrichment of cinnamon in chocolate has been reported centrated ethanolic cinnamon extract (cinnamon oleoresin).
to improve its phenolic content and antioxidant activity. The shellac colloidal particles containing cinnamon extract
However, the enrichment caused ramification on the melt- were prepared by anti-solvent precipitation method adopted
ing profile, rheological behaviour, textural characteristic and from Patel et al. [50]. Shellac fine powder (SSB 55 Astra FP)
sensorial properties of the chocolate [10, 40]. The altera- was obtained from SSB Stroever GmbH & Co. KG (Bremen,
tion to some quality attributes of the chocolate should be Germany). In brief, 1 g of cinnamon oleoresin and 1 g of
taken into account since it may substantially affect the prod- shellac powder were solubilised in 49 g of ethanol using a
uct functionality and acceptability. According to Fang and magnetic stirrer. Separately, 0.6 g of xanthan gum (Satiaxane
Bhandari [41], food fortification is a tremendously challeng- CX 931, Cargill France SAS) was prepared in 299.40 g of
ing process since it can pose some problems including stor- distilled water. The solution containing shellac and cinna-
age stability of the fortified food, incompatibility between mon extract was injected using a syringe into the xanthan
fortifier and food matrix and/or alteration of organoleptic gum aqueous solution to obtain a homogenous mixture. The
properties of the enriched food. In vivo related problems colloidal particle was formed by removing the solvent in a
such as bio-accessibility, release, and bioavailability of the rotary evaporator under vacuum conditions (Laborota 4000
additional functional ingredient are other serious issues in Heidolph, Germany). The evaporation was terminated when
the fortification of food [42, 43]. the weight of dispersion became equal to the weight of the
Currently, a number of publications have reported that initial stock of the solvent. To verify the size of the nano-
application of encapsulation in sub-micron scale has a sub- particle, a few drop of the colloidal dispersion was appro-
stantial impact in improving biological activity, controlling priately diluted in distilled water and then placed in Photon
the release of bioactive compound as well as masking unde- Correlation Spectroscope (PCS100M, Malvern Instrument
sirable odours and flavours [44, 45]. Nano-encapsulation Ltd, UK) using a temperature setting of 25 °C. It was con-
has also been reported to improve bioavailability and bio- firmed that the average size of the colloidal nanoparticles
accessibility of bioactive compounds [46–48]. Peters and was 191 nm. The colloidal dispersion was then lyophilised
his co-workers [49] reported that nanomaterials have been (Vaco 5-D Zirbus technology GmbH) and pulverised to
successfully applied as delivery system for lycopene, flavour obtain fine particles. The lyophilised colloidal nanoparti-
and dietary antioxidant supplement. In this study, cinnamon cles containing cinnamon extract (LCNP-CE) morphology
extract was loaded into nanoparticle to entrap the aroma of was visualised under a JSM-7100F TTLS LV TFEG-SEM
the cinnamon extract; thereby, the cinnamon extract can be (Jeol Europe, Belgium) equipped with a PP3000T device
13
Table 1 Physicochemical characteristic of the lyophilised colloidal the chocolate bars were transferred to a thermal cabinet at
nanoparticles containing cinnamon extract (LCNP-CE) 20 °C for 14 days to ensure proper maturation before further
Parameters Characteristics analyses. Chocolate containing non-encapsulated cinnamon
extract was also made by adding cinnamon extract directly to
Physical properties
the molten chocolate in equal amount with cinnamon extract
Water content (g/100 g) 4.67 ± 0.4
in the chocolate with 2% LCNP-CE.
Water activity, Aw < 0.027
Particle size distribution
Determination of antioxidant capacity
D(4,3) (µm) 49.6 ± 3.3
of the chocolates
D(3,2) (µm) 7.8 ± 2.8
D(v, 0.1) (µm) 10.9 ± 4.6
Extraction of antioxidant properties of the chocolate
D(v, 0.5) (µm) 34.7 ± 1.4
D(v, 0.9) (µm) 103.5 ± 6.5
White and milk chocolates enriched with 1 and 2% LCNP-
Antioxidant properties
CE were used for antioxidant evaluation. White and milk
Total phenolic content (mg epicatechin 98.5 ± 0.3
equivalent/g)
chocolates without LCNP-CE were also analysed as the con-
Ferric reducing antioxidant power activity (mM 725.0 ± 0.3
trol. The preparation of chocolate extracts was adopted from
ascorbic acid equivalent/g) Belščak et al. [51]. The sample (20 g) was washed three times
Total antioxidant activity (mg tannic acid 195.7 ± 3.5 with 100 ml of n-hexane to remove the fat content then air-
equivalent/g) dried in a dark condition during 24 h to evaporate residues
of n-hexane. The defatted chocolate (10 g) was extracted in
25 ml of solvent containing acetone (70%), distilled water
(29.8%), and acetic acid (0.2%) for 30 min in an ultrasonic
bath (Elmasonic P30H Elma Schmidbauer GmBH, Ger-
many). Afterwards, the mixture was centrifuged (Sigma
4K15 Sartorius AG, Germany) for 10 min at 3000 rpm. This
procedure was carried out twice, the supernatant was col-
lected and filtrated to remove the residual particles.
Total phenolic content
The total phenolic content was estimated according to the

method described by Muhammad and his colleagues [32].
The chocolate extract (200 µl) was added to 1 ml distilled
Fig. 1 a Physical appearance of lyophilised colloidal nanoparticles
water and 200 µl Folin–Ciocalteu reagent and then incu-
containing cinnamon extract (LCNP-CE) and b a Cryo-SEM image
of the LCNP-CE in ×20,000 magnification showing nanoparticles bated for 6 min. Afterwards the mixture was mixed well
in clustered formation. Scale bar in the Cryo-SEM image represents with 2.5 ml 7% Na2CO3 solution and 2.1 ml distilled water.
1 µm The solution was maintained for 90 min at ambient tem-
perature (20 °C). The absorbance was measured at 760 nm
(Quorum Technologies, East Sussex, UK). The character- in a UV–visible spectrophotometer (Varian Cary 50 Bio,
istics of the LCNP-CE are presented in Table 1 and Fig. 1. Agilent Technology). Total phenolic content was expressed
as micrograms of epicatechin equivalent per gram choco-
Formulation of the experimental chocolates late (µg ECE/g chocolate) by means of a standard plot of
epicatechin.
A microwave was used to melt white and milk choco-
late callets (Barry Callebaut Belgium NV). The molten Ferric reducing antioxidant power (FRAP) activity
chocolate was mixed with the LCNP-CE in various con-
centrations (0.5, 1, 1.5, and 2% w/w) at 35 °C using a Ste- The method described by Muhammad et al. [32] was used
phan Universal Machine UMC 5 (Stephan Food Service for the FRAP (Ferric Reducing Antioxidant Power) assay.
Equipment GmbH, Hameln, Germany). The chocolates Shortly, 2.5 ml phosphate buffer (0.2 M, pH 7) was added
were then manually tempered by a graduated chocolatier. into 1 ml of the sample, and then mixed with 2.5 ml of 1%
Afterwards, the tempered chocolates were each moulded potassium ferricyanide. The solution was incubated at 50 °C
into 100 mm × 24 mm × 10 mm bars and cooled at 12 °C for 30 min before adding 2.5 ml of 10% trichloroacetic acid.
for 2 h then stored at 18 °C for 2 h. After de-moulding, To separate larger aggregates, the solution was centrifuged
13
for 10 min at 6500 rpm. The supernatant was mixed well detectors (FIDs) were used for the odour components’ analy-
with distilled water and 0.1% FeCl3 in a ratio 5:5:1, and then sis of the chocolates. A discrimination test was performed
the absorbance was measured at 700 nm using a UV–vis- including chocolate control (without cinnamon extract),
ible spectrophotometer. A standard plot of ascorbic acid was chocolate containing LCNP-CE (encapsulated cinnamon
used to measure FRAP activity of the samples. The FRAP extract) and chocolate containing non-encapsulated cinna-
activity was recorded as mmol/l of ascorbic acid equivalent mon extract. The samples were cut into uniform shapes of
per gram chocolate (mmol/l AAE/g chocolate). 2.00 ± 0.02 g and placed in a 20 ml vial that was hermeti-
cally capped. The samples were allowed to melt by incuba-
Total antioxidant activity tion for 20 min at 60 °C and then continued by agitation
(500 rpm) for headspace generation. A 5000 µl headspace
Total antioxidant content was assayed using phosphomolyb- volume was injected into the e-nose system at an acquisition
denum method of Muhammad, et al. [32]. In brief, 0.5 ml time of 110 s. The measurements were done in duplicate
of the sample was added into 4.5 ml of the reagent solution for each sample and a discrimination test was performed.
(0.6 M sulphuric acid, 28 mM sodium phosphate and 4 mM Principal Component Analysis (PCA) map was built by the
ammonium molybdate). Afterwards, the mixture was incu- AlphaSoft software (v12.4, Alpha M.O.S., France) based on
bated at 95 °C for 90 min in a waterbath. The absorbance the presence of the volatile odorous molecules.
was measured at 695 nm using UV–visible spectrophotom-
eter after the sample reached room temperature. The total
antioxidant content was expressed as milligrams of tannic Determination of quality attributes
acid equivalent per gram chocolate (mg TAE/g chocolate). of the chocolates
DPPH (2,2-diphenyl-1-picrylhydrazyl) assay Particle size distribution (PSD)
As described by Muhammad et al. [32], chocolate extract The method described by Saputro et al. [52] was adopted
(100 µl) was added into 4 ml DPPH (0.01 mM), and then for determining the PSD of the chocolates. A Malvern Mas-
maintained in a dark condition for 30 min. Subsequently, the tersizer (Malvern Instruments Ltd, Worcestershire, UK)
absorbance was measured at 517 nm using UV–vis spectro- equipped with a 300 RF lens was utilised to measure parti-
photometer. Butylated hydroxyl anisole (BHA) and epicat- cles in the range of 0.05–900 µm. Isopropanol (10 ml) was
echin 100 µg/ml were used as the reference: used to dissolve the chocolate (0.5 g) and the solution was
(Absorbance of the control (Ac) − Absorbance of the sample (As)) × 100

%inhibiton = .
Absorbance of the control (Ac)
Release study subsequently heated in an oven (50 °C, 1 h). The sample
was shaken well before the measurement. D(v,0.9), D(v,0.5)
White chocolates enriched with 2% LCNP-CE was used for and D(v,0.1) denotes that 90, 50 and 10% of the particles are
the release study. The release of cinnamon extract from the smaller than the given value, respectively. D(4,3) indicates
chocolate was studied by adding 25 ml release medium to volume-weighed mean and D(3,2) represents Sauter diam-
5 g of sample. The release behaviour was investigated under eter. Specific surface area and span of the particles were
gastrointestinal condition using the pH change method (2 h also observed. The latter indicates distribution width of the
in pH 1.2 and continued further for 2 h in pH 7.4) adopted chocolates.
from Patel et al. [50]. Aliquot was periodically sampled and
the release of cinnamon was evaluated based on the total
phenolic content. Texture analysis of chocolate
Electronic nose (e-nose) analysis A texture analyser Instron 5942 (Norwood, MA, Canada)
equipped with a needle-shaped probe (ф = 1 mm) was used
Heracles II Electronic Nose (Alpha M.O.S., Toulouse, to analyse the hardness of the chocolate. The measurement
France) equipped with autosampler HS 100 (Toulouse, was triggered by a pre-load of 0.2N and the penetration was
France) was used to test the odour profiles of the choco- set at a rate of 2 mm/s to a depth of 5 mm into the chocolate
lates. A polar column (MXT-5-FID1) and non-polar col- bar. The hardness (N) was defined as the maximum force
umn (MXT-1701-FID2), followed by two flame ionization recorded during sample penetration [53].
13
Rheological measurements
√
C∗ = a∗2 + b∗2 ,
Chocolate flow behaviour was determined according to the √
protocol of ICA46 using an AR2000 rheometer (TA Instru- WI = 100 − (100 − L∗ )2 + a∗2 + b∗2 ,
ments) equipped with concentric DIN cylinder (cylinder:
42.00 mm; rotor outer radius: 14.00 mm; stator inner radius: √( ) ( ) ( )
15.00 mm; geometry gap: 5920 mm). Approximately 20 g of ΔE = L1∗ − L2∗ 2 + a∗1 − a∗2 2 + b∗1 − b∗2 2 .
chocolate was prepared in the cylinder after it was heated at
52 °C in the oven for 1 h. The sample was first sheared at 5/s Moisture content analysis
at 40 °C for 15 min before the test. The measurement of the
shear stress was conducted by performing ramp up (increas- The moisture content of the chocolate was determined by
ing the shear rate from 2 to 50/s) for 16 s/step, subsequently Karl Fischer titration method. The analysis was performed
by holding at 50/s for 60 s and continued by performing using the 719 Titrino apparatus (Metrohm, Switzerland)
ramp down (decreasing the shear rate from 50 to 2/s) for with Hydranal solvent (Riedel-de-Haen, 34812) and Hydra-
16 s/step. The flow data were fitted to the Casson model to nal titrant 5 (Riedel-de-Haen, 34801).
derive the Casson yield stress (σCA) and Casson viscosity
(ηCA). The difference between the ramp up and ramp down Statistical analysis
shear stress at 5/s was calculated as thixotropy [52].
SPSS 23.0 Software (SPSS Inc., Chicago, IL, USA) was used
Melting profile analysis for data analysis. Duncan’s Multiple Range Test (DMRT) to
test the differences of means of a one-way ANOVA. The
A differential scanning calorimeter (DSC Q1000 TA Instru- differences were considered significant at 0.95 confidence
ments, New Castle, USA) equipped with a refrigerated cool- level. Pearson test was performed to determine the correla-
ing system was used to analyse the melting profile of the tion coefficient between the nanoparticle percentages and the
chocolate. The DSC was calibrated with indium, azobenzene physicochemical characteristics of the chocolates as well as
and undecane, and aluminium cups were used. Approxi- the correlation coefficient among quality parameters of the
mately 5 mg of the chocolate slices were put in hermeti- chocolate.
cally sealed cups. The samples were equilibrated at 20 °C
followed by a heating step at a rate of 5 °C/min to 200 °C.
A linear baseline in the Universal Analysis 2000 software Results and discussion
version 4.7A (TA Instruments) was used to do the melting
peak integration. Peaks were characterised by onset (°C), Total phenolic content, antioxidant activity
maximum temperature (°C) and enthalpy (J/g) [53]. The and release behaviour
melting profile of chocolates was also compared with the
thermal behaviour of the LCNP-CE. The samples (± 1 mg) Due to its polyphenol constituents, potential antioxidant
were accurately weighed on to hermetic pan and then sealed. activities and prospective health benefits, cinnamon has
The analysis was performed at heating rate of 10 °C/min been used to improve functionality of food. In the making
from 20 to 200 °C. of polyphenol- and antioxidant-rich food, supplementation
using extracts of whole cinnamon is more recommendable
Colour measurement than using a large amount of single antioxidant, because high
dose of single antioxidant may have adverse effect on health
The colour of the chocolate was measured with a colorim- [2]. In this current work, the effects of LCNP-CE enrichment
eter (Minolta Model CM-2500D Spectrophotometer, Konica on physicochemical properties of white chocolate and milk
Minolta Sensing, Tokyo, Japan). The SCE mode (Specu- chocolate were evaluated. According to Meng et al. [57],
lar Component Excluded) values were recorded, as they polyphenol content and antioxidant activity of those two
have been claimed to be highly associated with the human types of chocolates were significantly lower than those of
eye reflection. The colour was expressed in terms of the dark chocolate. The addition of the LCNP-CE in the choco-
CIELAB system L* (lightness component), a* (green to late formulation was calculated based on the following data:
red component) and b* (blue to yellow component) [52]. (a) the minimum cinnamon extract-containing polyphenol
Chroma (C*), whiteness index (WI) and the degree of dif- (8.8%) administered in the body to have anti-inflammatory
ference between the chocolate containing cinnamon particle activity was 20 mg/kg body weight per day (based on the
and chocolate control (ΔE*) were calculated using the fol- study of Hong and his co-worker [34]); (b) average body
lowing equations [54–56]: weight used in this calculation is 70 kg; (c) Total phenolic
13
content of the engineered nanoparticle is 98.5 mg per gram Boeing et al. [58] who emphasised the strong correlation
LCNP-CE (Table 3); (d) chocolate intake per meal are among antioxidant parameters. The antioxidant activity of
20 g with three times meal per day. Based on a simulation, LCNP-CE in different test systems indicates that the LCNP-
LCNP-CE at the concentration of 2% in chocolate is likely CE has a wide spectrum of pathways for inhibiting oxida-
sufficient to fulfil minimum polyphenol level in a piece of tion. In many literatures, antioxidant has become closely
chocolate (20 g); so that a person with 70 kg body weight tied to health benefit because antioxidant plays a role in pre-
can obtain the beneficial effect of consuming the chocolate venting destructive chain reactions in the living system by
three times per day. To investigate the effect of the LCNP- scavenging the free radicals [32]. However, it is important to
CE on antioxidant properties and physicochemical proper- note that the knowledge on antioxidant activity can be used
ties of the chocolate, a gradual increase of the LCNP-CE for a preliminary prediction, but is insufficient to define the
level from 0% (control) to 2% was applied. overall health effects of the chocolate enriched with LCNP-
Figure 2 clearly shows that the incorporation of LCNP- CE. Structural variations in the phenolic compounds present
CE can improve total phenolic content and antioxidant in LCNP-CE should be considered since the molecular struc-
activity of chocolate regardless of the chocolate type. The ture is a key factor in their efficacy as free radical scavengers
percentage of LCNP-CE incorporated in the chocolates and as potential antioxidants in the human body.
was directly proportional to the antioxidant capacity of the The existence of the phenolic compound and antioxi-
chocolate when evaluated by FRAP assay, DPPH radical dant activity in the enriched chocolate further indicates the
scavenging analysis and phosphomolybdenum method. It stability of the polyphenol of cinnamon in the nanoparti-
was confirmed by Pearson’s correlation analysis (Table 2). cles to oxidation and degradation under mixing, tempering
Highly significant correlations (p = 0.01) between the and moulding applied in this study. According to Budryn
results of all employed antioxidant assays were also veri- et al. [59], a high temperature process causes great losses
fied (TPC-FRAP = 0.982, TPC-DPPH = 0.948, TPC- of the polyphenols contained in nanoparticle when evalu-
TAA = 0.817, FRAP-DPPH = 0.938, FRAP-TAA = 0.827, ated in bread, cookies, meat stuffing, mushroom stuffing,
DPPH-TAA = 0.875). There is an excellent agreement with nutty filling and caramel cottage; and thus suggests that
the findings reported by Belščak-Cvitanović et al. [11] and the nanoparticle containing polyphenols may be more
6 14
A c B c
( mM AAE/g chocolate)
12
Total phenolic content
( mg CE/g chocolate)
5
10
FRAP Activity
4 b b
8
3
a 6 a
2 c 4 c
b b
1 2 a
a
0 0
White Chocolate Milk Chocolate White Chocolate Milk Chocolate
Choc Control Choc LCNP-CE 1%" Choc LCNP-CE 2% Choc Control Choc LCNP-CE 1% Choc LCNP-CE 2%
100 4
C c D
Total antioxidant activity
c
( mg TAE/g chocolate)
80 c
b 3
Inhibition ( %)
b
60
2 a
c
40 a b
b
1 a
20
a
0 0
White Chocolate Milk Chocolate White Chocolate Milk Chocolate
Choc Control Choc LCNP-CE 1% Choc LCNP-CE 2% Choc Control Choc LCNP-CE 1% Choc LCNP-CE 2%
Fig. 2 a Total phenolic content (TPC), b FRAP activity, c DPPH formed separately for each type of chocolate. Mean values within
radical scavenging activity and d total antioxidant activity (TAA) of each type of chocolate with the same lowercase letter do not differ
chocolate enriched with lyophilised colloidal nanoparticles contain- significantly (p < 0.05)
ing cinnamon extract (LCNP-CE). The statistical analysis was per-
13
Table 2 Correlation coefficient between the presence of lyophilised colloidal nanoparticles containing cinnamon extract (LCNP-CE) and the
quality parameters of the chocolates
Parameters White chocolate Milk chocolate
Percentage of Particle size Moisture content Percentage of Particle size Moisture content
LCNP-CE D(v,0.9) LCNP-CE D(v,0.9)
Antioxidant properties
Total phenolic content 0.976** 0.935**
FRAP activity 0.987** 0.944**
DPPH radical scavenging activity 0.984** 0.965**
Total antioxidant activity 0.873** 0.971**
Particle size distribution
D(4,3) 0.126 0.405
D(3,2) 0.183 − 0.006
D(v,0.1) 0.251 − 0.153
D(v,0.5) 0.235 0.153
D(v,0.9) − 0.029 0.259
Span − 0.150 0.486
Specific surface area − 0.211 − 0.034
Moisture content 0.627* 0.410 0.765** 0.084
Hardness 0.930** 0.180 0.713** 0.774** − 0.155 0.677**
Flow properties
Casson yield value 0.849** − 0.084 0.461 0.058 − 0.081 − 0.346
Casson viscosity 0.995** − 0.001 0.641* 0.789** − 0.082 0.527*
Thixotropy 0.942** − 0.020 0.508 0.395 0.089 0.073
Melting properties
Tonset of cocoa butter − 0.369 0.386 0.056 − 0.028 0.214 − 0.293
Tmax of cocoa butter 0.278 − 0.124 0.052 − 0.128 0.432 − 0.193
Tstop of cocoa butter 0.408 − 0.323 − 0.019 0.000 − 0.122 0.205
Enthalpy of cocoa butter melting − 0.232 0.383 0.192 0.151 − 0.213 0.124
*Significant at p < 0.05; **significant at p < 0.01
suitable to be incorporated in food that do not need high

temperature during the manufacture. This study agrees
with this perspective; and therefore, nanoparticle supple-
mentation in chocolate in this study was designed without
involvement of heat treatment. 60
The total phenolic compounds assay was also used to

Cinnamon extract released (%)
50
study release profile of cinnamon extract from white choc-
olate. In this research, milk chocolate was not used as the 40
model to avoid the intervention of phenolic compounds White choc with non-encapsulated
30
from the milk chocolate leading to ambiguity. It is impor- cinnamon extract
tant to study the release behaviour of bioactive compounds 20

from food matrices, since nanomaterials studies focusing
on gastrointestinal tract transmission were still limited 10
White choc with LCNP-CE
[60]. The food matrix has a significant effect on the bio- 0
activity of a compound and the behaviour of a compound 0 40 80 120 160 200 240
Time (min)
in whole food might be substantially different from that
of the single pure compound [19]. According to Campos
et al. [61], in vitro release study is necessary and useful Fig. 3 Release profile of cinnamon extract from white chocolates as
a function of time studied in physiologically relevant pH values: in
for the prediction of in vivo kinetics. gastric pH (1.2) for initial 2 h followed by incubation in intestinal pH
(7.4) for additional 2 h
13
Figure 3 illustrates the release profile of cinnamon the nanoparticle to prevent alteration of chocolate quality in
extract from white chocolate. There was a burst release the case of cinnamon extract incorporation.
of around 7 and 17% in the first 15 min from the choco-
late enriched with LCNP-CE and non-encapsulated cin- Electronic nose profile
namon extract, respectively. It was followed by a gradual
increase in pH 1.2 until 120 min. When changing the pH An analysis related to aroma is essential to verify the effect
to 7.4, there was a sharp rise in the release of the poly- of encapsulation on masking the odour of cinnamon extract.
phenols for both samples. The release of the cinnamon An instrumental approach such as an e-nose is an appro-
extract from LCNP-CE and non-encapsulated form in the priate tool for verifying this hypothesis. To evaluate food
chocolate matrix after 240 min were approximately 24% aroma, the e-nose has some advantages including the objec-
and 54%, respectively. In this study, the release of phe- tivity, the possibility to standardize and the reproducibility
nolic compounds from encapsulated cinnamon extract was of the results [64]. The discrimination of the chocolates is
much lower than uncoated cinnamon extract in the choco- illustrated in Fig. 4.
late matrix. It indicates that encapsulation can reduce the The experimental data show differences between the
release of bioactive compounds from the chocolate matrix. chocolate samples. Based on the PCA plot, the samples were
This result is in accordance with Gibis et al. [62] who well-differentiated regarding the overall volatile composition
showed that the release rate of non-encapsulated bioactive with a high discrimination index of 88. As their clusters
compound was higher than the encapsulated one. This phe- are clearly separated, chocolate enriched with LCNP-CE
nomenon can be understood since the nano-capsule builds can be distinguished from chocolate enriched with non-
a second layer that may improve physicochemical stabil- encapsulated cinnamon extract. Milk chocolate enriched
ity (preventing aggregation and discharge of content), and with LCNP-CE are in the same quadrant as the milk choco-
subsequently can inhibit the polyphenols release from the late control. However, it is remarkable that white chocolate
matrix. In the context of release control, the ability of enriched with LCNP-CE can also be distinguished from
nano-capsule to inhibit the release of bioactive compounds white chocolate control. On the one hand, the result indi-
is an advantage, since an uncontrolled burst release can cates that encapsulation using shellac and xanthan gum can
be prevented. However, on the other hand, it may result a entrap specific aroma of the cinnamon extract by preventing
lower bioavailability, since the bioactive compounds are the release of the volatile compounds. However, on the other
still entrapped in either the capsule or the food matrix until hand, deviations on aroma profile between chocolate with
in the end of the digestion process. This drawback might LCNP-CE and chocolate with non-encapsulated cinnamon
occur in the case of the LCNP-CE incorporated in choco- extract are also observed. It illustrates that encapsulation
late. Moreover, in this study, the simulation duration was could not perfectly mask the aromatic compounds of the cin-
only 4 h resulting in around 60% of phenolic compounds namon extract. The list of key aromatic compound per PCA
of the uncoated cinnamon extract released from the choco- plot including the peak areas of the groups were provided by
late matrix. In real condition, the digestion might be more the e-nose system (data are not shown). From the polar and
than 4 h, and as a result, more phenolic compounds of non-polar column, it was observed that the white chocolate
uncoated cinnamon extract can be released. In this case, control contains 60 odour compounds. Meanwhile, the white
the release difference between LCNP-CE and uncoated chocolate with non-encapsulated cinnamon extract contains
cinnamon extract may become more significant. 91 odour compounds indicating that 31 compounds were
As aforementioned, the result from the release study can originally from cinnamon. It is notable that only 65 odour
be useful for predicting in vivo kinetics. Nevertheless, this compounds were perceived in the white chocolate with
result is actually not sufficient for claiming that the choco- LCNP-CE designating that 25 odour compounds from cin-
lates enriched with cinnamon are really beneficial for health. namon disappeared when encapsulating including coumarin,
In fact, bioaccessibility and bioavailability of a bioactive 1-tetradecanol, 2-methylthiophene, α-terpineol, benzalde-
compound in the digestion system are influenced by the ini- hyde, β-ionone, citronellal, ethyl phenylacetate, γ -nonal-
tial concentration of the compound, the food matrix and the actone, γ-terpinene, heptadecane, myrcene, and trans-car-
gastrointestinal conditions [63]. Therefore, studies on the veol. A similar phenomenon was also observed in the case
bioaccessibility, bioavailability and real effect of LCNP-CE of milk chocolate in which some odour compounds were
incorporated in chocolate in the body are interesting topics not detected in the milk chocolate enriched with LCNP-CE,
in the future. Our research group are working on the experi- but the compounds are identified in the milk chocolate with
mental setup of this study, and the result of the study will non-encapsulated cinnamon extract. There are two possible
be reported in another manuscript. Thus, this study contin- reasons: (1) the volatiles compounds are entrapped inside
ued focusing on the impact of the LCNP-CE on the quality the nanocapsule; (2) the volatile compounds lose during the
attributes of the chocolates to investigate the capability of encapsulation process.
13
Fig. 4 Discrimination of white and milk chocolates enriched with late enriched with non-encapsulated cinnamon extract, respectively.
LCNP-CE and non-encapsulated cinnamon extract. Code A, B, and C Each type of the chocolates has two replications directed by the num-
are chocolate control, chocolate enriched with LCNP-CE, and choco- ber 1 and 2
The results suggest that encapsulation can really have a in cinnamon might be an issue due to its potential toxicity.
positive impact on preventing aroma alteration on choco- Coumarin has non-genotoxic carcinogenic characteristic in
late enriched with bioactive compounds. Nevertheless, some some species of rodents, but it can cause liver damage in
compounds originating from cinnamon were still identified some other species. The European Food Safety Authority
in the chocolate enriched with LCNP-CE, such as cinna- (EFSA) recommends tolerable daily intake (TDI) of cou-
maldehyde, linalool and β-pinene. The chocolate flavour marin which is less than 0.1 mg/kg body weight [67]. To
may be altered due to the presence of those compounds. calculate the possible exposure of coumarin from the choco-
Cinnamaldehyde, linalool, and β-pinene can give cinnamon late containing LCNP-CE to the body, a simulation can be
spicy, strong sweet floral and terpene-like aroma [65, 66]. performed. For an example, a 70-kg person who consumes
However, those aroma compounds can only be perceived 60 g of chocolate/day (a dose giving an anti-inflammatory
by human sensory if the concentration of the compounds is effect according to the above calculation) with 7.7 mg cin-
higher than its threshold. Moreover, the fact that cinnamon namon extract/g chocolate and a coumarin concentration of
has been used in foods and beverages in many cultures in 0.92 mg/g cinnamon extract (based on analysis by Solaiman
the world due to its pleasant odour and flavour [32] can be a and Al-Zehouri [68]) would lead to a daily coumarin intake
reason why chocolates with LCNP-CE are expected to have of 6.1 µg/kg body weight. This means that the coumarin
an acceptable pleasant aroma. To confirm the effectiveness exposure is still below the TDI. Apart from that, the fact that
of encapsulation on masking the aromatic compound of cin- cinnamon in the form of extracts and oleoresins is generally
namon extract and the product acceptability, sensory evalu- recognised as safe (GRAS) [32] indicates that LCNP-CE can
ation may be required. Sensory evaluation can also help to be considered as a safe ingredient for food.
designate flavour difference among samples since the e-nose
technique only describes the presence of volatile compounds Particle size distribution (PSD)
in a headspace meaning that the e-nose analysis does not at
all consider different flavour impacts of the volatile mol- Particle size distribution (Table 3) is a pivotal parameter
ecules and has not much evidence in describing a perceived for determining the quality attribute of chocolate in term
flavour profile. of fineness which refers to mouthfeel. In addition to the
From the e-nose analysis, it was also observed that the direct influence on mouthfeel, particle size distribution
cinnamon extract contains coumarin. However, in the choc- has an indirect influence on the rheological behaviour,
olates enriched with LCNP-CE, coumarin was no longer hardness and colour [52, 69]. Although the acceptance
detected because of either entrapped inside the nanocapsule range for fineness is different for each region, particle sizes
or lost during the encapsulation process. Coumarin content in solid-eating chocolate should be below 30–35 µm to
13
Table 3 Fineness of chocolate enriched with lyophilised colloidal nanoparticles containing cinnamon extract (LCNP-CE)
Chocolate Derived diameter (µm) Distribution percentiles Specific surface Span
area (m2/m3)
D(3,2) D(4,3) D(v,0.9) D(v,0.5) D(v,0.1)
White chocolate
Choc control 3.1 ± 0.4a 11.0 ± 0.2a 24.3 ± 1.1a 8.4 ± 0.2a 1.7 ± 0.2a 1.95 ± 0.26a 2.7 ± 0.2a
Choc LCNP-CE 0.5% 3.3 ± 0.7a 12.3 ± 0.7a 27.4 ± 0.1b 8.4 ± 0.4a 1.6 ± 0.2a 1.88 ± 0.39a 3.1 ± 0.1a
Choc LCNP-CE 1% 3.3 ± 0.3a 11.4 ± 0.4a 25.1 ± 1.1ab 8.6 ± 0.2a 1.8 ± 0.1a 1.80 ± 0.15a 2.7 ± 0.2a
Choc LCNP-CE 1.5% 3.5 ± 0.7a 11.7 ± 0.4a 26.3 ± 0.9ab 8.5 ± 0.3a 1.7 ± 0.2a 1.80 ± 0.37a 2.9 ± 0.2a
Choc LCNP-CE 2% 3.4 ± 0.1a 11.6 ± 1.2a 24.7 ± 1.9ab 8.5 ± 0.2a 1.8 ± 0.1a 1.78 ± 0.44a 2.7 ± 0.2a
Milk chocolate
Choc control 1.3 ± 0.2a 11.9 ± 1.2a 29.3 ± 2.5a 7.7 ± 0.2bc 0.4 ± 0.1a 4.74 ± 0.59b 3.3 ± 0.3a
Choc LCNP-CE 0.5% 1.6 ± 0.1b 11.8 ± 0.7a 28.0 ± 1.7a 8.0 ± 0.5bc 0.6 ± 0.1b 3.79 ± 0.15a 3.4 ± 0.1ab
Choc LCNP-CE 1% 1.2 ± 0.1a 11.9 ± 1.6a 27.5 ± 2.5a 7.1 ± 0.2a 0.4 ± 0.1a 4.94 ± 0.35b 3.8 ± 0.2bc
Choc LCNP-CE 1.5% 1.3 ± 0.1a 13.3 ± 0.2a 31.2 ± 0.5a 7.4 ± 0.1ab 0.4 ± 0.1a 4.65 ± 0.13ab 4.2 ± 0.1c
Choc LCNP-CE 2% 1.4 ± 0.1ab 12.9 ± 1.2a 29.9 ± 1.9a 8.3 ± 0.3c 0.5 ± 0.1a 4.25 ± 0.44ab 3.6 ± 0.4a
The statistical analysis was performed separately for each type of chocolate. Different superscripts in the same column indicate significant differ-
ences (p < 0.05) among samples
avoid a gritty mouthfeel which is perceived as unpleas- Moisture content

ant by consumers [70–73]. In this research, the D(v,0.9)
range for the white and milk chocolate were 24.3–27.3 Incorporation of LCNP-CE significantly influenced moisture
µm and 27.5–31.2 µm, respectively. D(v,0.9) was directly content of chocolates. The increased moisture content of the
proportional to D(v,0.5), D(v,0.1), D(4,3) and D(3,2) in chocolates correlated positively and significantly with the
both white and milk chocolate. D(v,0.9), D(v,0.5) and addition of the LCNP-CE for both type of chocolate (Fig. 5;
D(v,0.1) denotes that 90, 50 and 10% of the particles are Table 2). The initial moisture content of the LCNP-CE was
smaller than the given value, respectively. D(4,3) indi- relatively high [4.6% (w/w)] compared to that of the choco-
cates volume-weighed mean and D(3,2) represents Sauter late [0.7 (w/w) and 0.9% (w/w) for white chocolate and milk
diameter. Specific surface area and span of the particles chocolate, respectively]. Therefore, it could be predicted
were also compared. The latter indicates relative distribu- that the addition of the LCNP-CE would induce a rise in
tion width of the chocolates. Even though the initial sizes the moisture content of the chocolates. However, the actual
of the LCNP-CE were relatively big (103.5 ± 6.5 µm), moisture contents of the chocolates were slightly higher than
no remarkable change in particle size distribution [in the the calculated ones. Discrepancies in the expected change of
value of D(3,2), D(4,3), D(v,0.9), D(v,0.5), D(v,0.1), spe- the moisture content of the enriched chocolates may be the
cific surface area, and span] was found. It may be due to consequence of hygroscopicity of the LCNP-CE as well as
a small quantity of the LCNP-CE added to the chocolate. the amorphous lactose presented in these chocolates. During
This result suggests that the presence of the particles up to the mixing, it might absorb moisture from the environment.
2% does not alter the quality characteristic of the choco-
late in term of fineness since the D(v,0.9) values of the
1.5
enriched chocolates were still below 35 µm. According to ab
b
Beckett [74], D(v,0.9) is one of the most prominent param- a

a
Moisture content
a
eters for particle size distribution as it could illustrate sen- 1.0
a ab
ab b b Choc Control
(% w/w)
sory character. Albeit statistical differences were found in Choc LCNP-CE 0.5%
Choc LCNP-CE 1%
some PSD parameters, it is noteworthy that the correlation 0.5
Choc LCNP-CE 1.5%
between the presence of LCNP-CE up to 2% and all of par- Choc LCNP-CE 2%
ticle size distribution parameters are insignificant both in 0.0
white and milk chocolate according to Pearson’s analysis White Chocolate Milk Chocolate
(Table 2). This outcome is in accordance with the results

obtained by Belščak-Cvitanović et al. [11] revealing that Fig. 5 Moisture content of chocolate enriched with lyophilised colloi-
the change in particle size did not depend on the con- dal nanoparticles containing cinnamon extract (LCNP-CE). The sta-
tistical analysis was performed separately for each type of chocolate.
centration of the lyophilised polyphenol-rich plant extract Mean values within each type of chocolate with the same lowercase
added into the chocolate. letter do not differ significantly (p < 0.05)
13
Hygroscopicity of the amorphous lactose has been described the Pearson’s correlation analysis that shows high correla-
by Torres et al. [75]; while that of freeze-dried particle has tions at p < 0.01 between: (a) LCNP-CE addition and hard-
been reported by Yamashita et al. [76]. Changes in the mois- ness with R2 = 0.930 and R2 = 0.774, (b) LCNP-CE addi-
ture content can substantively influence the quality attribute tion and moisture content with R2 = 0.627 and R2 = 0.765
of chocolate, such as textural and rheological properties [70, and (c) moisture content and hardness with R2 = 0.713 and
77, 78]. High level moisture content may also create particle R2 = 0.677 for white chocolate and milk chocolate, respec-
agglomeration in chocolate [79]. To test whether those find- tively. Moisture can really have an implication in the forma-
ings are true for our case, further analysis on hardness, flow tion of sugar network and may strengthen the aggregated
properties and microstructural properties were conducted. particle-to-particle network system resulting in a higher
breaking resistance [77, 82]. Another possible mechanism
Hardness is that the presence of LCNP-CE increased the solid volume
fraction of the chocolate control. Accordingly, more particle
Hardness, an essential quality attribute of chocolate that interactions could be formed resulting in a harder chocolate
is strongly related to sensory perception, is defined as the [83]. Even though the chocolates became harder along with
force required to attain a given deformation to the food the particle enrichment, the enriched chocolates are likely
sample. Hardness describes the stiffness and the degree of to be acceptable as the obtained values of hardness are still
‘snap’ of chocolate [80, 81]. Figure 6 shows in both white comparable with the chocolates in the study of Konar [84]
and milk chocolates the influence of LCNP-CE on their and Aidoo et al. [85].
hardness which was likely to a large extent caused by the Earlier studies have been undertaken to verify the influ-
rise in moisture content. This conclusions was based on ence of particle size distribution on textural properties of
chocolate [70, 86]. In this study, the relationship between
12
hardness and particle size distribution [D(v,0.9)] was
c also investigated; however, no significant correlation was
c d d b b b
b a
observed (Table 2). This study is in agreement with Belščak-
a
Cvitanović et al. [87] who supplemented freeze–dried net-
Hardness (N)
Choc Control
6
Choc LCNP-CE 0.5% tle extract up to 2% into chocolate formula. This outcome,
Choc LCNP-CE 1% therefore, supports the justification that the hardening of the
Choc LCNP-CE 1.5%
chocolate in this current research is strongly affected by the
Choc LCNP-CE 2%
rise in moisture content and may be also influenced by par-
0 ticle volume fraction.
White Chocolate Milk Chocolate
Rheological behaviour
Fig. 6 Hardness of chocolate enriched with lyophilised colloidal nan-
oparticles containing cinnamon extract (LCNP-CE). The statistical Rheological behaviour of chocolate is critical for pumping
analysis was performed separately for each type of chocolate. Mean
values within each type of chocolate with the same lowercase letter and moulding properties and for organoleptic perception
do not differ significantly (p < 0.05) [52, 78, 85]. In a sensorial perspective, for example, highly
A 10 Choc Control
B 10 a a a
a
a
b b Choc Control
Choc LCNP-CE 0.5% Choc LCNP-CE 0.5%
Flow behaviour parameter
a
Flow behaviour parameter
a a Choc LCNP-CE 1% Choc LCNP-CE 1%

Choc LCNP-CE 1.5% Choc LCNP-CE 1.5%
Choc LCNP-CE 2% Choc LCNP-CE 2%
5 5
c c
e
b c d b b
a ab a b bc bc c ab ab
a a ab
0 0
Casson Yield Casson Thixotropy (Pa) Casson Yield Casson Thixotropy (Pa)
Value (Pa) Viscosity (Pa s) Value (Pa) Viscosity (Pa s)
Fig. 7 Rheological behaviour of a white chocolate and b milk choco- separately for each parameter. Mean values within each type of
late enriched with lyophilised colloidal nanoparticles containing cin- chocolate with the same lowercase letter do not differ significantly
namon extract (LCNP-CE). The statistical analysis was performed (p < 0.05)
13
viscous chocolate may stick to the teeth and palate during stress required to initiate fluid flow), but only of the white
consumption. Figure 7 illustrates that the presence of LCNP- chocolate and their correlation was significant (Table 2).
CE influence the flow properties of the chocolates to some While the Casson viscosity and thixotropy of both white and
extents. Several factors contributing to flow behaviour of milk chocolate were substantially increased by the addition
chocolate are fat content, emulsifier content, particle size of LCNP-CE. However, it is worth to note that the increase
distribution, particle volume fraction, particle density and in Casson viscosity and thixotropy of milk chocolate is
moisture content [73, 74, 86, 88, 89]. As explained in the less substantial than that of white chocolate. The different
previous discussion, the presence of LCNP-CE increased composition of white and milk chocolate may be the main
the moisture content of the chocolate. Therefore, the change reason for this dissimilarity. Difference in rheology can be
in flow behaviour of the chocolates after incorporation of due to the difference in the amorphous and crystalline lac-
the LCNP-CE can reasonably be attributed to the moisture tose composition [91]. Particle size, amount and distribution
content; the higher the level of LCNP-CE incorporated in of fat, presence of solid particle, particle shape as well as
the chocolates, the higher the Casson viscosity (internal fric- particle–particle interaction of the ingredients are also key
tion of the chocolate determining coating thickness among determinant factors affecting flow behaviour [89, 92]. As
others). Table 2 shows that the correlation between moisture well stressed by De Clercq et al. [93], in the condition of
content and Casson viscosity are significant in both white large surface area, more surface is available to effectuate
and milk chocolate. interconnection of particles exhibiting a higher Casson yield
Different possible mechanisms of the thickening effect value as relatively higher stresses are needed to disrupt the
have been explained in earlier studies. Afoakwa et al. [70] network formed and to initiate flow. In the chocolate matrix,
explained that moisture on sugar particle surface increases particle size has less effect on viscosity than on Casson yield
friction among particles and promotes aggregating them to value. Milk chocolate has a larger surface area than white
form gritty lumps. According to Saputro et al. [79], the pres- chocolate (Table 3) indicating that the milk chocolate has a
ence of moisture reduces the availability of “free” fat which stronger particle–particle interaction accordingly resulting
is beneficial for a swift flow, because more fat is used to in a higher Casson yield value. It seems that the presence
coat the increased size of the sugar particles. As previously of the LCNP-CE was insufficient to change the extent of
discussed, LCNP-CE may influence the solid volume frac- particle–particle interaction of the milk chocolate, and so
tion of the chocolate. A higher particle volume fraction has the Casson yield value of the milk chocolate remains stable
more particle interactions, resulting in a higher viscosity. In in contrast to the white chocolate. Thus, the value of some
the literature, adjustment of flow behaviour can be carried rheological parameters was influenced by the combination of
out by the addition of fat and emulsifier, by the reduction the composition of the chocolate, the presence of moisture in
of inter-particle contacts and particle aggregation strength, the LCNP-CE and the changing of particle volume fraction.
and by increasing the maximum volume fraction, such as
the modification of particle arrangement, particle shape and Melting properties
particle size distribution [70, 72, 74, 78, 86]. Beckett [74]
stated that approximately 1% extra fat should be added into Determination of melting properties is of great importance
chocolate formula for every 0.3% extra moisture content in in chocolate since it has a strong association with sensorial
the chocolate to attain similar flow properties. perception. It illustrates the melting behaviour of chocolate
In addition to the moisture, the LCNP-CE materials may in the mouth. In this study, differential scanning calorim-
also contribute to the increase of the viscosity. As pointed etry (DSC) analysis records some melting properties of the
out by Habibi and Khosravi-Darani [90], xanthan gum has chocolate including Tonset (the temperature at which a spe-
pseudoplasticity properties, accordingly it is habitually cific crystal form starts to melt), Tmax (the temperature at
used as a thickening agent in many foods. Xanthan gum which melting rate is highest), width (peak width at half
may absorb moisture from the environment and chocolate height) and enthalpy (the amount of latent heat absorbed
matrix due to its hygroscopicity; therefore, the pseudoplastic during melting) [94].
effect is possible. The resinous constituent in the cinnamon Table 4 shows fat melting profile of white and milk
extract and shellac may also take part in the thickening of chocolate enriched with LCNP-CE, while Fig. 8 illustrates
the chocolates. An analogous hypothesis also spelled out by typical melting profiles of the chocolates which consists of
Belščak-Cvitanović et al. [11] in their study on the topic of three endothermic peaks. The first, second and third peak are
raspberry leaves extract-enriched chocolate. attributed to the melting of milk fat, cocoa butter and sugar,
The addition of LCNP-CE exhibits different phenomena respectively. The LCNP-CE depicts endothermic peaks at
in the flow properties parameters of white and milk choco- 55 and 150 °C which can be interpreted as the melting of
late. It can be observed that the presence of particles con- shellac and xanthan gum, respectively. In the LCNP-CE-
siderably increased the Casson yield value (the amount of enriched chocolate, the endothermic peaks of the LCNP-CE
13
Table 4 Fat melting profile Type Tonset (°C) Tmax (°C) Width (°C) Enthalpy (J/g)
of chocolate enriched
with lyophilised colloidal White chocolate
nanoparticles containing
Choc control 28.0 ± 1.6a 32.2 ± 0.5a 3.7 ± 0.8ab 29.8 ± 1.0a
cinnamon extract (LCNP-CE)
Choc LCNP-CE 0.5% 30.5 ± 0.5b 32.7 ± 0.2ab 2.7 ± 0.5a 31.3 ± 0.6b
Choc LCNP-CE 1% 29.7 ± 1.5ab 33.2 ± 0.5b 3.6 ± 0.8ab 29.2 ± 0.5a
Choc LCNP-CE 1.5% 27.6 ± 0.1a 32.7 ± 0.1a 4.1 ± 0.2b 28.2 ± 1.3a
Choc LCNP-CE 2% 27.4 ± 0.1a 32.7 ± 0.2a 4.1 ± 0.1b 29.5 ± 0.7a
Milk chocolate
Choc control 28.5 ± 1.0a 33.5 ± 0.4b 3.7 ± 0.3ab 29.9 ± 0.6a
Choc LCNP-CE 0.5% 29.1 ± 1.3a 33.9 ± 0.3b 4.1 ± 0.3b 31.2 ± 1.3a
Choc LCNP-CE 1% 28.1 ± 1.0a 32.9 ± 0.1a 3.6 ± 0.2ab 30.6 ± 0.4a
Choc LCNP-CE 1.5% 30.5 ± 1.1a 33.7 ± 0.2b 3.4 ± 0.3a 30.5 ± 0.6a
Choc LCNP-CE 2% 27.3 ± 0.1a 33.5 ± 0.1ab 4.0 ± 0.3ab 30.7 ± 1.0a
The statistical analysis was performed separately for each type of chocolate. Different superscripts n the
same column indicate significant differences (p < 0.05) among samples
Fig. 8 Typical DSC thermo- A B

grams of white chocolate (a)
LCNP-CE LCNP-CE
and milk chocolate (b) enriched
Relative heat flow (W/g)

with different levels of lyoph-
Relative heat flow (W/g)
Choc LCNP-CE 2% Choc LCNP-CE 2%

ilised colloidal nanoparticles
Choc LCNP-CE 1,5% Choc LCNP-CE 1.5%
containing cinnamon extract
(LCNP-CE) Choc LCNP-CE 1% Choc LCNP-CE 1%
Choc LCNP-CE 0,5% Choc LCNP-CE 0.5%
Choc Control Choc Control
20 40 60 80 100 120 140 160 180 20 40 60 80 100 120 140 160 180
Exo Temperature (°C) Exo Temperature (°C)
cannot be observed any longer. It can be understood that it is measurement resulting in different onset temperatures [52].
due to a little amount of the LCNP-CE suspended in cocoa This might be true as well in melting profile of the choco-
butter crystals in the chocolates. Even though the endother- lates in this study.
mic peaks of the LCNP-CE disappear, no clear trend on the
effect of LNCP-CE on the melting profile of the chocolates Appearance
was identified. Moreover, there were no significant correla-
tions between the presence of the LCNP-CE and all of the Appearance, including colour, is considered to be one of
melting profile parameters in both white and milk chocolate the essential parameters when defining general chocolate
(Table 2). It implies that the presence of the LCNP-CE has quality since it intensely influences the product acceptabil-
limited influence on the melting profile of the chocolates. ity [97]. Evaluation of chocolate surface appearance in this
This study shows that all the samples have a Tmax in the study was performed by colorimetric instrumental meas-
range of 32–34 °C indicating that the chocolates were well- urement methods. Figure 9 illustrates that the enrichment
tempered regardless of the LCNP-CE incorporation. It was of LCNP-CE substantially changed all colour parameters
detected that in some cases the Tonset subsequently altered of the white chocolate but not those of the milk chocolate.
the width and the enthalpy without showing a certain trend. One reason why this dissimilarity turned up is that the basic
The most likely cause of this occurrence is that the LCNP- colour of the LCNP-CE (L* = 59.1 ± 0.4; a* = 16.2 ± 0.0;
CE behave as impurities in the crystals and cause a slight b* = 23.47 ± 0.1) is relatively closer to the colour of the milk
change in the Tonset. Many theoretical and experimental chocolate (L* = 36.7 ± 0.3; a* = 11.5 ± 0.1; b* = 15.3 ± 0.1)
efforts have been made to study the effect of impurities on than to the colour of the white chocolate (L* = 83.6 ± 0.2;
phase-transition behaviour of fat including in crystallisation a* = − 0.4.±0.3; b* = 27.3 ± 0.4). Therefore, the white-
[95, 96]. Another possible reason is a thermal contact dis- ness index of the milk chocolate remained stable along with
parity between sample and DSC pan at the beginning of the the presence of the LCNP-CE. Moreover, the difference
13
100 A 100
L* B L*
a* a*
80 b* 80 b*
C* C*
60 60
40 40
20 20
0 0
Control 0.5% 1% 1.5% 2% Control 0.5% 1% 1.5% 2%
-20
Percentage of LCNP-CE in white chocolate Percentage of LCNP-CE in milk chocolate
100 40 Milk Chocolate

C Milk Chocolate D
White Chocolate
White Chocolate
E between sample
80 c c
Whiteness index
and control
60 b
20 a
40
20
ab ab a b
0 0
Control 0.5% 1% 1.5% 2% 0.5% 1.0% 1.5% 2.0%
Percentage of LCNP-CE in chocolate
Percentage of LCNP-CE in chocolate
E F
Fig. 9 Impact of LCNP-CE enrichment on the a appearance of white statistical analysis was performed separately for each type of choco-
chocolate; b appearance of milk chocolate; c whiteness index of late. Mean values within each type of chocolate with the same lower-
white and milk chocolate; d the difference in colour between enriched case letter do not differ significantly (p < 0.05). L* lightness; a* red-
chocolate and control; e picture of white chocolate enriched with ness; b* yellowness; C* chrome
LCNP-CE; f picture of milk chocolate enriched with LCNP-CE. The
in colour (∆E) between the milk chocolate with LCNP- LCNP-CE in milk chocolate is more practical than that in
CE enrichment up to 2% and the milk chocolate control is white chocolate.
trivial, in contrast to the white chocolate. It was found that
the ∆E of milk chocolate are less than 3 for all the samples,
while that of white chocolate are more than 3. According Conclusions
to Żyżelewicz et al. [98], colour differences are not obvi-
ous for the human or could be appreciated by the human Our study provides first experimental data on the applica-
eye depending of the hue in the condition of ∆E < 3. If ∆E tion of engineered nanoparticles in chocolate that was still
is higher than 3, the colour differences are obvious for the scarcely investigated. This research highlights the use of
human eye. This finding suggests that the enrichment of the lyophilised colloidal nanoparticles containing cinnamon
13
extract (LCNP-CE) as an effective method to increase poly- degradation through fermentation-like incubations. LWT Food
phenolic content and antioxidant activity in chocolates. Sci Technol 68:514–522
7. Żyżelewicz D, Krysiak W, Oracz J, Sosnowska D, Budryn G,
It was found that the polyphenols in LCNP-CE form has Nebesny E (2016) The influence of the roasting process condi-
slower release rate than in its free form. The most important tions on the polyphenol content in cocoa beans, nibs and choco-
finding is that nano-encapsulation has a positive effect on lates. Food Res Int 89(2):918–929
the aroma profile of cinnamon-enriched chocolate by reduc- 8. Di Mattia C, Martuscelli M, Sacchetti G, Beheydt B, Mastrocola
D, Pittia P (2014) Effect of different conching processes on
ing and preventing flavour alteration on chocolate enriched procyanidin content and antioxidant properties of chocolate.
with cinnamon extract. LCNP-CE incorporation did not Food Res Int 63:367–372
significantly change particle size distribution and melting 9. Schinella G, Mosca S, Cienfuegos-Jovellanos E, Pasamar M,
properties of the chocolates. Even though slight variations Muguerza B, Ramón D, Ríos JL (2010) Antioxidant properties
of polyphenol-rich cocoa products industrially processed. Food
in moisture content, hardness, and flow behaviour were Res Int 43(6):1614–1623
recorded, the enriched chocolates are likely to be in the 10. Albak F, Tekin AR (2014) The effect of addition of ingredients
range of acceptable. Those alterations may be prevented by on physical propertıes of dark chocolate during conching. Basic
adding LCNP-CE with an equal moisture content with the Res J Food Sci Technol 1(7):51–59
11. Belščak-Cvitanović A, Komes D, Benković M, Karlović S,
chocolate. Thus, this research serves as a model for the use Hečimović I, Ježek D, Bauman I (2012) Innovative formula-
of dried colloidal nanoparticles to enhance the nutritional tions of chocolates enriched with plant polyphenols from Rubus
properties of chocolates. Further study needs to be under- idaeus L. leaves and characterization of their physical, bioactive
taken to evaluate the acceptability of chocolate incorporated and sensory properties. Food Res Int 48(2):820–830
12. Belščak-Cvitanović A, Komes D, Durgo K, Vojvodić A, Bušić
with engineered nanoparticles. A (2015) Nettle (Urtica dioica L.) extracts as functional
ingredients for production of chocolates with improved bioac-
Acknowledgements This work was supported by the Directorate tive composition and sensory properties. ˪J Food Sci Technol
General of Higher Education, Ministry of Research, Technology, 52(12):7723–7734
and Higher Education, Republic of Indonesia (Grant number: 15.1/ 13. Dean LL, Klevorn CM, Hess BJ (2016) Minimizing the negative
E4.4/2015). Hercules Foundation is acknowledged for its financial flavor attributes and evaluating consumer acceptance of chocolate
support in the acquisition of the Scanning Electron Microscope JEOL fortified with peanut skin extracts. J Food Sci 81(11):S2824-S2830
JSM-7100F equipped with cryo-transfer system Quorum PP3000T and 14. Gültekin-Özgüven M, Karadağ A, Duman Ş, Özkal B, Özçelik B
Oxford Instruments Aztec EDS (Grant number: AUGE-09-029). (2016) Fortification of dark chocolate with spray dried black mul-
berry (Morus nigra) waste extract encapsulated in chitosan-coated
Compliance with ethical standards liposomes and bioaccessability studies. Food Chem 201:205–212
15. Morais Ferreira JM, Azevedo BM, Luccas V, Bolini HMA (2016)
Isosweetness concentrations of sucrose and high-intensity sweet-
Conflict of interest None. eners and antioxidant activity in white chocolate with functional
properties. Int J Food Sci Technol 51(9):2114–2122
Compliance with ethics requirements This article does not contain 16. Sim SYJ, Ng JW, Ng WK, Forde CG, Henry CJ (2016) Plant
any studies with human or animal subjects. polyphenols to enhance the nutritional and sensory properties of
chocolates. Food Chem 200:46–54
17. Viuda-Martos M, Ruiz-Navajas Y, Fernández-López J, Pérez-
Alvarez JA (2010) Spices as functional foods. Crit Rev Food Sci
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European Food Research and Technology

Physicochemical properties and antioxidant activities of chocolates

Received: 11 November 2017 / Revised: 6 January 2018 / Accepted: 7 January 2018

Keywords Nanoparticle · Chocolate · Cinnamon · Antioxidant

Total phenolic content

The total phenolic content was estimated according to the

DPPH (2,2-diphenyl-1-picrylhydrazyl) assay Particle size distribution (PSD)

(Absorbance of the control (Ac) − Absorbance of the sample (As)) × 100

*Significant at p < 0.05; **significant at p < 0.01

suitable to be incorporated in food that do not need high

The total phenolic compounds assay was also used to

tant to study the release behaviour of bioactive compounds 20

avoid a gritty mouthfeel which is perceived as unpleas- Moisture content

Beckett [74], D(v,0.9) is one of the most prominent param- a

(Table 2). This outcome is in accordance with the results

a a Choc LCNP-CE 1% Choc LCNP-CE 1%

Fig. 8 Typical DSC thermo- A B

Relative heat flow (W/g)

Choc LCNP-CE 2% Choc LCNP-CE 2%

Choc LCNP-CE 0,5% Choc LCNP-CE 0.5%

Choc Control Choc Control

100 40 Milk Chocolate

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