Food Hydrocolloids xxx (2016) 1e8

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Food Hydrocolloids
journal homepage: www.elsevier.com/locate/foodhyd

Physico-chemical characterization and evaluation of bio-efficacies

of black pepper essential oil encapsulated in hydroxypropyl-beta-
cyclodextrin
Jaruporn Rakmai a, Benjamas Cheirsilp a, Juan Carlos Mejuto b, Ana Torrado-Agrasar c,
ndara c, *
Jesús Simal-Ga
a
Department of Industrial Biotechnology, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai Campus, Hat Yai 90112, Thailand
b
Department of Physical Chemistry, Faculty of Science, University of Vigo, Ourense Campus, Ourense E32004, Spain
c
Department of Analytical Chemistry and Food Science, Faculty of Science, University of Vigo, Ourense Campus, Ourense E32004, Spain

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

Article history: Encapsulation of essential oils with cyclodextrins can protect their active compounds from environ-
Received 24 October 2016 mental conditions and improve their aqueous solubility, hence increasing their functional capabilities as
Received in revised form additives. The purpose of this study was to characterize the physico-chemical properties and bio-
11 November 2016
efficacies, antioxidant and antibacterial activities, of the encapsulated black pepper essential oil in
Accepted 11 November 2016
Available online xxx
hydroxypropyl-b-cyclodextrin (HPbCD), in comparison with the major ingredient in the oil, b-car-
yophyllene. The difference in encapsulation efficiency of the pure compound and the black pepper oil
results from the presence of other components in the black pepper oil such as limonene, d-3-carene and
Keywords:
Black pepper oil
pinene. Although the inclusion complexes increase their stability, they gave slightly lower antioxidant
Hydroxypropyl-beta-cyclodextrin activity as a result of the HPbCD was blocking the functional groups of active compounds during reaction
Encapsulation characterization with DPPH radicals. Instead, after encapsulated in HPbCD, the antibacterial activity of black pepper oil
Encapsulated oil bio-efficacy was improved by 4 times against both S. aureus and E. coli.
© 2016 Elsevier Ltd. All rights reserved.

1. Introduction
Black pepper (Piper nigrum L.) is considered the king of spices
because of its pungent of piperine (Srinivasan, 2007). It can be used
for different purposes such as medicine, human dietaries, pre-
servatives and biocontrol agents (Awen, Ganapati, & Chandu, 2010;
Hussain, Naz, Nazir, & Shinwari, 2011; Srinivasan, 2007). It has been
already reported that essential oil from black pepper possesses
antioxidant (Singh, Marimuthu, Catalan, & de Lampasona, 2004)
and antimicrobial activities (Dorman & Deans, 2000). Black pepper
oil is basically composed of terpenes which have been found to be
b-caryophyllene, limonene, d-3-carene and pinene (Menon,
Padmakumari, & Jayalekshmy, 2003; Singh et al., 2004). The ma-
jor component of black pepper oil was found to be b-caryophyllene
(Menon et al., 2003; Singh et al., 2004). Nevertheless, some active
compounds in essential oils are sensitive towards the chemical
* Corresponding author.
E-mail addresses: jarnarak@hotmail.co.th (J. Rakmai), benjamas.che@psu.ac.th
(B. Cheirsilp), xmejuto@uvigo.es (J.C. Mejuto), agrasar@uvigo.es (A. Torrado-
Agrasar), jsimal@uvigo.es (J. Simal-G
andara).
modification under effect of some external factors such as: tem-
perature, light, oxygen etc. (Dima et al., 2014). Besides, to apply
with food products, an extremely low flavor threshold of essential
oils can drastically change the sensory properties of foods, and
highly water insoluble may have limited contact with pathogens
(Kalemba & Kunicka, 2003).
The use of cyclodextrins for the essential oils encapsulation can
protect the active compounds of essential oils from environmental
conditions (Hedges, Shieh, & Sikorski, 1995, pp. 60e73; Qi &
Hedges, 1997, pp. 231e243) and improve the aqueous solubility of
essential oils for increasing their capacity to functionalize the
products in which it is used as additive (Helena & Cabral, 2010).
Cyclodextrin (CD) are cyclic oligosaccharides consisting of gluco-
pyranosyl units linked by a-(1,4) bonds (Schmann & Schollmeyer,
2002). The widely used natural cyclodextrins are a-, b- and g-
cyclodextrin consisting of 6, 7 and 8 glucopyranose units, respec-
tively. The cyclodextrin molecules have a unique structure with a
hydrophobic cavity and a hydrophilic surface which can form in-
clusion complex with a wide variety of guests. Among those cy-
clodextrins, b-cyclodextrin is the most widely applicable kind
because of its suitable cavity size for common guests with

http://dx.doi.org/10.1016/j.foodhyd.2016.11.014
0268-005X/© 2016 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Rakmai, J., et al., Physico-chemical characterization and evaluation of bio-efficacies of black pepper essential
oil encapsulated in hydroxypropyl-beta-cyclodextrin, Food Hydrocolloids (2016), http://dx.doi.org/10.1016/j.foodhyd.2016.11.014
2 J. Rakmai et al. / Food Hydrocolloids xxx (2016) 1e8
molecular weights between 200 and 800 g/mol and its availability
and reasonable price (Waleczek, Marques, Hempel, & Schmidt,
2003). In some cases, there is a need to enhance water solubility
of b-cyclodextrin by adding the hydroxyalkyl groups on the b-
cyclodextrin surface. A hydroxyalkylated or hydroxypropyl-b-
cyclodextrin derivative (HPbCD) is relatively high aqueous solubi-
lity with low toxicity and satisfactory inclusion ability (Garnero,
Zoppi, Genovese, & Longhi, 2010).
The purpose of this study was to characterize the physico-
chemical properties and bio-efficacies, antioxidant and antibacte-
rial activities, of the encapsulated black pepper essential oil in
hydroxypropyl-b-cyclodextrin (HPbCD).
2. Materials
2.1. Microorganisms
The indicator bacteria used for testing antimicrobial activity
were Staphylococcus aureus (representative for gram-positive bac-
teria) and Escherichia coli (representative for gram-negative bac-
teria). These strains were provided from the Microorganisms
Collection of Department of Industrial Biotechnology, Faculty of
Agro-Industry, Prince of Songkla University, Thailand. The indicator
bacteria were cultivated in Tryptic Soy Broth (TSB) and incubated at
37
C with shaking at 200 rpm for 24 h. All bacteria were main-
tained at 20
C in 25% (v/v) glycerol.
2.2. Chemicals
Hydroxypropyl b-cyclodextrin (HPbCD), 1, 1-diphenyl-2-
picrylhydrazyl (DPPH) and Butylated hydroxytoluene (BHT) were
purchased from Sigma-Aldrich (Steinheim, Germany). Black pepper
oil was purchased from Botanicessence (Bangkok, Thailand). b-
caryophyllene was purchased from Sigma-Aldrich (Steinheim,
Germany).
3. Methods
3.1. Encapsulation of black pepper essential oil
Black pepper oil was encapsulated in HPbCD through inclusion
complex formation. The inclusion complex was prepared via the
freeze-drying method (Karathanos, Mourtzinos, Yannakopoulou, &
Andrikopoulos, 2007). The 0.5 g of black pepper oil was slowly
added to an aqueous solution of hydroxypropyl-b-cyclodextrin
(HPbCD) (5 g of HPbCD in 25 mL of water). The mixture was left in a
sealed container under stirring at room temperature (~25
C) and
protected from the light for 24 h. The encapsulated particles dis-
solved in the solution were separated from the unencapsulated
particles by filtration through 0.45-mm PTFE filters (IC Millex-LH,
Millipore, Billerica, MA). The resulting solution containing encap-
sulated particles was frozen at 20
C and lyophilized at 50
C
and 1.09 Pa in a Labconco Freeze Dryer-5 (Kansas City, MO) for
approximately 48 h or until all moisture had been sublimated. The
lyophilized powder was washed with acetonitrile (to remove par-
ticles attaching on the HPBCD surface) and dried in a low temper-
ature incubator at 25
C. The product was stored in sealed container
inside a desiccator until use. The encapsulation efficiency was
calculated compared to the standard curve of black pepper oil,
taking into consideration the non-encapsulated oil, according to
the method described by Bae and Lee (2008).
3.2. Characterization of encapsulated black pepper oil
All the following methods were selected with the purpose of
Please cite this article in press as: Rakmai, J., et al., Physico-chemical characterization and evaluation of bio-efficacies of black pepper essential
oil encapsulated in hydroxypropyl-beta-cyclodextrin, Food Hydrocolloids (2016), http://dx.doi.org/10.1016/j.foodhyd.2016.11.014
understanding changes in particle morphology by SEM, in molec-
ular structure by FT-IR and UVeVis spectrophotometry, and in the
stability by phase solubility studies of the black pepper oil with
encapsulation in HPbCD.
3.2.1. Morphological examination
The particle morphology of the encapsulated black pepper oil
was examined using a Quanta 250 Scanning Electron Microscope
(SEM) (Quanta 250, Netherland). The samples (powder of encap-
sulated black pepper oil and free HPbCD) were fixed on aluminum
stubs with double adhesive tape and vacuum coated with a fine
layer of gold before viewing under 500 times magnification. Ob-
servations were carried out with voltage acceleration of 15 Kv
(Guimaraes et al., 2015).
3.2.2. FT-IR analysis
The FT-IR spectra of free HPbCD, free black pepper oil and
encapsulated black pepper oil were collected from 400 to 4000 cm1
using a Nicolet 550-II FT-IR spectrophotometer (Nicolet, USA) with 32
scans at a resolution of 4 cm1. The samples (powder of free HPbCD
and encapsulated black pepper oil) were diluted with potassium
bromide (KBr) powder at a mass ratio of 1:100. Then they were
ground and pressed to discs of diameters of 8 mm. A drop of black
pepper oil sample was spread on a piece of KBr window uniformly
then nipped with another piece of KBr window. FT-IR spectra were
analyzed by the spectrophotometer software (OMNIC 5.2) (Gomes,
Petito, Costa, Falcao, & Araujo, 2014; Wang, Li, Si, & Chen, 2011).
3.2.3. UVevis spectroscopy analysis
The formation of black pepper oil-HPbCD inclusion complex was
demonstrated by a UVevis spectrophotometer (Biochrom, LIBRA-
S22, England). Black pepper oil (0.5 mg/mL) was dissolved in
acetonitrile. The HPbCD, physical mixture (HPbCD:black pepper
oil ¼ 4:1 in a mass ratio) and encapsulated black pepper oil (in-
clusion complex) were prepared in acetonitrile (5 mg/mL), and
then the mixture was shaken for 10 min. The supernatant was
separated by centrifugation and then diluted by 100 times in
acetonitrile, and scanned in the range of 200e400 nm to obtain the
UVevis absorption spectrum (Liu et al., 2013; Wang et al., 2011).
3.2.4. Phase solubility study
Classification of inclusion complex formation and stability of the
inclusion compounds were evaluated by phase solubility study
(Higuchi & Connors, 1965). An excess of black pepper oil was added
to 10 mL aqueous solutions of HPbCD ranging in concentration from
0 to 10 mmol/L and incubated at 25 and 35
C for 24 h with shaking
at 200 rpm. The solution was filtered through 0.45-mm PTFE filters
(IC Millex-LH, Millipore, Billerica, MA) prior to measurement to
remove any insoluble material. The quantity of black pepper oil
remaining in solution was measured spectrophotometrically at
205 nm and compared to a standard curve of a major component of
black pepper oil, b-caryophyllene. The quantity of black pepper oil
in the solution was plotted against HPbCD concentration.
The inclusion complex formation was classified according to the
pattern of the plot between concentration of HPbCD and solubilized
guests with the presence of HPbCD in solution. The stability con-
stant, Ks (L/mol), was calculated from slope and intercept of the plot
following Eq. (1):
slope
Ks ¼ (1)
intercept  ð1  slopeÞ
Where: Ks (L/mol) is a stability constant, intercept (mmol/L) is the
dissolved guest in the aqueous complexation medium when no
cyclodextrin is present.
 J. Rakmai et al. / Food Hydrocolloids xxx (2016) 1e8
3.3. Evaluation of antioxidant activity of encapsulated black pepper
oil
The free black pepper oil and the encapsulated black pepper oil
were exposed to sunlight for 12 h. The antioxidant activity of the
tested samples was determined by 1, 1-diphenyl-2-picrylhydrazyl
(DPPH) radical scavenging assay (Brand-Williams, Cuvelier, &
Berset, 1995) with some modifications. An ethanolic solution of the
radical DPPH (0.12 mM) was prepared and protected from light. The
stock solutions of the encapsulated black pepper oil or free black
pepper oil were prepared in 50% DMSO and kept in the dark. A 500 mL
of black pepper oil or inclusion complex in several concentrations
(5e50 mg/mL) was mixed with 500 mL of a 0.12 mM ethanolic DPPH
solution. Concentration of encapsulated black pepper oil was based
on encapsulation efficiency. The mixture was kept in darkness for
60 min and the absorbance was measured at wavelength of 515 nm.
The DPPH scavenging (%) was established according to Eq. (2):
 
AðTested SampleÞ eA ðBlank SampleÞ
%DPPH scavenging ¼ 100 
A ðControlÞ
Where: A(Tested Sample) is the absorbance of the sample with DPPH;
A(Blank Sample) is the absorbance of the sample without DPPH;
A(Control) is the absorbance of DPPH without sample.
The antioxidant activity of free black pepper oil and the
encapsulated black pepper oil was compared to the chemical
antioxidant, butylated hydroxytoluene (BHT).
3.4. Evaluation of antimicrobial activity of encapsulated black
pepper oil
Antimicrobial activity of free and encapsulated black pepper oil
was performed against S. aureus and E. coli by broth microdilution
assay (Hill, Gomes, & Taylor, 2013). The 20 mL of microbial sus-
pension (cultured at 37
C/24 h and diluted to yield 105 CFU/mL)
was added to 180 mL of Muller-Hinton Broth (MHB) containing
various concentrations of free and encapsulated black pepper oil
ranging from 2 to 2000 mg/mL in each well of microtiter plate
(amount of encapsulated black pepper oil was based on encapsu-
lation efficiency). For preparation of the tested samples, the
encapsulated black pepper oil was diluted directly in the culture
broth, while the free black pepper oil was prepared as aqueous
microemulsions containing 10% (w/w) ethanol and 1% (w/w)
Tween 20 (Basheer, Noordin, & Ghareeb, 2013).
Negative control wells were prepared with sterile culture me-
dium (Muller-Hinton Broth) containing tested samples (free and
encapsulated black pepper oil). Positive control wells were prepared
with microbial suspension inoculating in culture medium (MHB)
and culture medium containing Tween 20 and ethanol in the same
concentration as black pepper oil microemulsion. The microplates
were incubated at 37
C for 24 h and the turbidity was determined
by a microplate reader (PowerwaveX Biotek, USA) at 600 nm. The
MICs of free and encapsulated black pepper oil were recorded as the
lowest concentration where no visible growth (0.05 changed in
OD600) was observed in the wells after 24 h incubation.
The viability of the test microorganisms in the culture broth,
which showed no turbidity at (OD600) was determined by trans-
ferring and spreading the culture broth on Muller-Hinton Agar and
incubated at 37
C for 24 h. The lowest concentrations of free and
encapsulated black pepper oil required to completely destroy the
Please cite this article in press as: Rakmai, J., et al., Physico-chemical characterization and evaluation of bio-efficacies of black pepper essential
oil encapsulated in hydroxypropyl-beta-cyclodextrin, Food Hydrocolloids (2016), http://dx.doi.org/10.1016/j.foodhyd.2016.11.014
3
test microorganisms (no growth on the agar plate) after incubation
were reported as minimum bactericidal concentrations (MBC). All
determinations were done in triplicate on two separate runs.
3.5. Statistical analysis
The data was calculated with mean values and standard de-
viations (mean ± SD) from triplicate trials. Statistical significance of
the results was evaluated by one way ANOVA (Analysis of Variance)
with Duncan’s multiple range tests (P < 0.05) using the Statistical
Package for Social Sciences (SPSS) version software 10.
4. Results and discussion
4.1. Encapsulation efficiency of black pepper oil
Encapsulation efficiencies of black pepper oil and its major
 100 (2)
component, b-caryophyllene, were evaluated and showed in
Table 1. This difference in encapsulation efficiency of the pure
compound and the black pepper oil would result from the presence
of other components in the black pepper oil such as limonene, d-3-
carene and pinene (Singh et al., 2004) which have also high affin-
ities for HPbCD. These components then competed with the major
compounds for inclusion complex formation with the HPbCD.
Similar observation has been reported by Hill et al. (2013). In
their study, the encapsulation efficiencies of cinnamon oil and clove
oil were 41.72% and 77.74%, respectively. The encapsulation effi-
ciencies of major components including trans-cinnamaldehyde in
cinnamon oil and eugenol in clove oil were also examined and
showed higher encapsulation efficiency of 84.70% and 90.15%,
respectively.
4.2. Characterization of encapsulated black pepper oil
4.2.1. Morphological examination
Since it has been reported that inclusion complex formation
changed the morphology of HPbCD, the morphology of the inclu-
sion complex was then observed to characterize the evidence of
inclusion formation (Guimaraes et al., 2015). The particle
morphology of the free HPbCD and the encapsulated black pepper
oil was observed under a Scanning Electron Microscope (SEM).
Fig. 1 shows SEM images of free HPbCD and encapsulated black
pepper oil at 500 times magnifications. The free HPbCD composed
of irregular-shaped particles (Fig. 1a). The particle shape and
morphology of the corresponding inclusion complex were similar
to that of free HPbCD (Fig. 1b). On the other hand, the particle size of
the free HPbCD was much larger than that of the encapsulated
Table 1
Encapsulation efficiency value determined of black pepper oil and b-caryophyllene
encapsulated in HPbCD.
Compound Encapsulation efficiency (%)*
Encapsulated black pepper oil 50.55 ± 1.57a
Encapsulated b-caryophyllene 85.30 ± 2.38b
*
Values given are averages of triplicate samples (n ¼ 3) ± standard deviations.
a,b
Indicate significantly different values (p < 0.05).
4 J. Rakmai et al. / Food Hydrocolloids xxx (2016) 1e8
(a)
Fig. 1. SEM micrographs of free HPbCD (a) and black pepper oil-HPbCD inclusion complex (b) at 500 times magnification. The samples used were in powder form derived from
inclusion complex formation via freeze-drying technique.
product. It indicates the hydrogen bonding of the free HPbCD
molecules interact with each other in solution (Le Bas & Rysanek,
1987; Batzdorf & Muller-Goymann, 1993) producing the cluster of
HPbCD. Therefore the freeze-dried product of free HPbCD shows
larger size than that of inclusion complex. Similar changes in
morphology and size of HPbCD due to inclusion complex formation
have been previously reported by Guimaraes et al. (2015). Smaller
particles of inclusion complex were also observed in complexation
of carvacrol with b-cyclodextrin compared to the free b-
cyclodextrin.
4.2.2. FT-IR analysis
FT-IR technique can be used to investigate the variation of shape,
intensity and position of peaks (Szente, 1996; Zeng, Fang, & Ji,
2012). The FT-IR spectra of black pepper oil, HPbCD and inclusion
complex show in Fig. 2. The FT-IR spectrum of black pepper oil
(Fig. 2a) consisted of the prominent absorption bands at 2954 cm1,
2923 cm1 and 2865 cm1 for CeH stretching vibration of meth-
ylene group, 1638 cm1 for HeOeH bending, 1447 cm1 for CeH
scissoring vibration, 1369 cm1 for symmetrical deformation vi-
bration of CH3, 886 cm1 for CeH deformation vibration and
789 cm1 for SeC absorption. However, the FT-IR spectrum of the
encapsulated black pepper oil (Fig. 2c) showed that no character
similar to the free black pepper oil. All bands of black pepper oil
spectrum were totally obscured by HPbCD bands (Fig. 2b). It was
possible that black pepper oil entered the cavity of HPbCD and
inclusion complex was formed.
The inclusion complex formation of b-cyclodextrin was also
investigated by Liu et al. (2013) using FT-IR analysis. The absorption
bands of b-caryophyllene were not detected in the spectrum of
inclusion complex. The changes were related to the inclusion
complex formation of b-cyclodextrin and the guests which whole
of guest could be contained in the cyclodextrin cavity. Similar re-
sults have been reported by Wang, Luo, and Xiao (2014). In their
study, the inclusion complex formation of soybean lecithin and b-
cyclodextrin was determined by FT-IR. All the absorption bands of
soybean lecithin encapsulated in b-cyclodextrin were obscured by
b-cyclodextrin spectrum showing that inclusion complex of b-
cyclodextrin and soybean lecithin was formed. On the other hand,
the prominent absorption band at the 1738 cm1 region of red bell
pepper pigments encapsulated in b-cyclodextrin was observed
from FT-IR spectrum showing that some region of the guest was not
contained in the cavity of b-cyclodextrin (Gomes et al., 2014).
4.2.3. UVevis spectroscopy analysis
As the inclusion complex formation with cyclodextrin changes
Please cite this article in press as: Rakmai, J., et al., Physico-chemical characterization and evaluation of bio-efficacies of black pepper essential
oil encapsulated in hydroxypropyl-beta-cyclodextrin, Food Hydrocolloids (2016), http://dx.doi.org/10.1016/j.foodhyd.2016.11.014
(b)
the original visible or UV absorption spectra of the guest (Szejtli,
1982, pp. 115e122) and sometimes the spectra of the guests
appear in line of cyclodextrin (Liu et al., 2013), the formation of
inclusion complex of HPbCD and black pepper oil was evaluated
using a UVevis spectrophotometer.
In Fig. 3, the absorption spectrum of black pepper oil was
recorded with absorption peaks at 200 nm, 205 nm and 214.5 nm
for d-3-carene, b-caryophyllene and limonene, respectively (Liu
et al., 2013). The maximum absorption peak at 205 nm was
ascribed to b-caryophyllene (Spectrum 3). The spectra of the
physical mixture of HPbCD with black pepper oil (Spectrum 4) and
with b-caryophyllene (Spectrum 5) accorded with UV spectra of
black pepper oil (Spectrum 2) and pure b-caryophyllene (Spectrum
3), respectively. When the active compounds in black pepper oil or
the pure compound (b-caryophyllene) were entrapped into the
cavity of HPbCD, the absorption peaks of the compounds also dis-
appeared in the spectrum of the inclusion complex (Spectrum 6
and 7). After extraction from the complex, the observable peaks of
entrapped compounds in black pepper oil could be seen (Spectrum
8). The spectrum of encapsulated compounds from black pepper oil
show absorption peaks at 205 nm and 214.5 nm indicating b-car-
yophyllene and limonene, respectively. The UV spectrum indicated
that the chemical components of black pepper oil were successfully
entrapped in the HPbCD. However, the encapsulation efficiency of
active compounds of black pepper oil was much lower than those of
its pure compound. This was likely because the competition of
major active compound among other components in black pepper
oil has occurred during inclusion complex formation.
4.2.4. Phase solubility study
Phase solubility study is generally performed to evaluate the
stability and to classify the inclusion complex when they are in the
solution. The phase solubility profiles can be obtained from the
interaction between the guests (encapsulated compounds) and the
hosts (cyclodextrins or derivatives) in the solution. In solution, a
fundamental parameter such as stability constant (Ks) of inclusion
complex formation can be used to evaluate the stability of the in-
clusion complex. The Ks can be acquired from the linear portion of
the phase solubility diagram (the plot of host concentration against
guest concentration dissolved in the solution). Types of inclusion
complexes can be considered from the plot curves.
Since the y-intercepts are concentrations of the guests solubi-
lized in the solution without the hosts (Yuan, Jin, & Li, 2008), the y-
intercepts increased with temperature indicated that the water
solubility of inclusion complexes increased with increasing tem-
perature (Table 2). A linear relationship between the amount of
 J. Rakmai et al. / Food Hydrocolloids xxx (2016) 1e8 5

(a)

(b)

(c)

Fig. 2. FT-IR spectra of black pepper oil (a), hydroxypropyl-b-cyclodextrin (HPbCD) (b) and black pepper oil-HPbCD inclusion complex (c).

dissolved black pepper oil or b-caryophyllene and the concentra-
tions of HPbCD in this study with slope 1 was classified as a typical
AL-type (type A reveals an inclusion complex formation where the
amount of encapsulated compounds increase as the HPbCD con-
centration increases, subscript L indicates a 1:1 molecular ratio
formation of soluble complexes) (Higuchi & Connors, 1965). As the
majority of encapsulated compounds are mono- and sesquiterpe-
noids and phenylpropane derivatives of an average molecular
weight of 120e160 g/mol, a 1:1 complex formation is observed
(Szente & Szejtli, 2004). The molar ratio of host to guest molecules
is usually 1:1 for inclusion complexes formed in solution, except for
complexes with long-chain or bifunctional guest molecules (e.g.
guest molecules having two aromatic rings on opposite sides of a
small central molecule segment).
In aqueous system, black pepper oil and b-caryophyllene show
difference in stability of complex form with the Ks of 104.5 and
132.8 L/mol at 25
C, respectively. This might be because of the
other components in black pepper oil might compete to HPbCD
form complex with b-caryophyllene. The decreases in Ks values
with increasing temperatures were expected for exothermic pro-
cesses (Hill et al., 2013).
Similar observations have been reported by Hill et al. (2013) and
Kamimura, Santos, Hill, and Gomes (2014). The water solubility of
trans-cinnamaldehyde, eugenol, cinnamon bark extract and clove
bud extract samples increased with increasing temperatures while
the Ks value of the samples decreased with increasing temperature
(Hill et al., 2013). Kamimura et al. (2014) reported that water sol-
ubility of the pure carvacrol increased and the Ks value decreased
with increasing temperatures.
4.3. Evaluation of antioxidant activity of encapsulated black pepper
oil
Fig. 4 shows antioxidant activity in term of DPPH scavenging

Please cite this article in press as: Rakmai, J., et al., Physico-chemical characterization and evaluation of bio-efficacies of black pepper essential
oil encapsulated in hydroxypropyl-beta-cyclodextrin, Food Hydrocolloids (2016), http://dx.doi.org/10.1016/j.foodhyd.2016.11.014
6 J. Rakmai et al. / Food Hydrocolloids xxx (2016) 1e8

100

% DPPH sca v en gin g

80
BO_before
60 BO_after

40 BC_before
BC_after
20
BHT
0
0 10 20 30 40 50
Concentration ( g/mL)
Fig. 3. UV absorption spectra of HPbCD (1), black pepper oil (2), b-caryophyllene (3),
physical mixture of black pepper oil and HPbCD (4), physical mixture of b-car- Fig. 4. DPPH radical scavenging (%) of free black pepper oil (BO) and black pepper oil-
yophyllene and HPbCD (5), black pepper oil-HPbCD inclusion complex (6), b-car- HPbCD inclusion complex (BC) at several concentrations (5e50 mg/mL) before and after
yophyllene-HPbCD inclusion complex (7), encapsulated compounds extracted from exposure to sunlight for 12 h. Reaction temperature: room temperature (28 ± 2
C).
black pepper oil-HPbCD inclusion complex (8). Reaction time: 60 min. Concentration of inclusion complex based on the encapsulation
efficiency. Values given are in triplicate (n ¼ 3).

capacity (%) of free and encapsulated black pepper oil compared to

a synthetic chemical antioxidant, butylated hydroxytoluene (BHT),
Before exposure to sunlight
at concentration ranged from 5 to 50 mg/mL. The DPPH scavenging
60 After exposure to sunlight
capacity of both free and encapsulated black pepper oil samples
increased with increasing sample concentrations. It should be
noted that the free HPbCD did not show antioxidant activity.
% DPPH s cavenging

Black pepper oil shows antioxidant activity with 54% DPPH

scavenging (50 mg/mL black pepper oil) (Fig. 5). It was established
that the components responsible for the antioxidant activity are b- 40
caryophyllene, limonene and a-pinene (Zengin & Baysal, 2014). b-
caryophyllene, a major component of black pepper oil, was found to
give a weak DPPH scavenging activity (Zengin & Baysal, 2014).
Limonene, a minor composition, has been reported to give a
moderate antioxidant activity and another component, a-pinene,
also possesses a moderate antioxidant property (Dai, Zhu, Yang, &
20
Qiu, 2013).
The inclusion complexes gave slightly lower antioxidant activity
than the activity of the free black pepper oil (Fig. 4). It was a result
of the HPbCD was blocking the functional groups of active com-
pounds during reacting with DPPH radicals (Kamimura et al., 2014). 0
However, the inclusion complexes have been reported to increase
the stability of the essential oils (Hedges et al.,1995, pp. 60e73; Qi & Black pepper oil Inclusion complex
Hedges, 1997, pp. 231e243).
The DPPH radical scavenging capacity of black pepper oil dras- Fig. 5. DPPH radical scavenging (%) of free black pepper oil and black pepper oil-
tically decreased after 12 h exposure to sunlight (Fig. 4). At the HPbCD inclusion complex (50 mg/mL) before and after exposure to sunlight for 12 h.
Reaction temperature: room temperature (28 ± 2
C). Reaction time: 60 min. Con-
sample concentration range of 5e25 mg/mL, the DPPH scavenging
centration of inclusion complex based on the encapsulation efficiency. Values given are
capacity decreased around 42e48%, while the decreasing of in triplicate (n ¼ 3).
30e39% was found at higher concentration range (30e50 mg/mL).
Figs. 4 and 5 show that the encapsulation of black pepper oil in
4.4. Evaluation of antibacterial activity of encapsulated black
HPbCD could protect the active components against the effect of
pepper oil
light. The stability of encapsulated black pepper oil was improved
from the free black pepper oil by 18e24%.
Table 3 shows minimum inhibitory concentration (MIC) and

Table 2
Phase solubility parameters and stability constants (Ks) of encapsulated black pepper oil and b-caryophyllene at specified temperatures.

Inclusion complex Temperature (
C) Intercept Slope Ks (L/mol) R2

Black pepper oil-HPbCD complex 25 1.442 0.131 104.5 0.978

Black pepper oil-HPbCD complex 35 1.751 0.149 100.0 0.984
b-caryophyllene-HPbCD complex 25 3.643 0.326 132.8 0.996
b-caryophyllene-HPbCD complex 35 4.541 0.341 114.0 0.949

Please cite this article in press as: Rakmai, J., et al., Physico-chemical characterization and evaluation of bio-efficacies of black pepper essential
oil encapsulated in hydroxypropyl-beta-cyclodextrin, Food Hydrocolloids (2016), http://dx.doi.org/10.1016/j.foodhyd.2016.11.014
 J. Rakmai et al. / Food Hydrocolloids xxx (2016) 1e8
Table 3
Minimum inhibitory and bactericidal concentration (MIC, MBC) against Staphylococcus aureus and Escherichia coli for both free black pepper oil and inclusion complex.
Antimicrobial compound S. aureus
MIC (mg/mL)
Black pepper oil 1000
Black pepper oil-HPbCD complexa 250
a
Values are based on the actual concentrations of black pepper oil encapsulated in the HPbCD (calculated from encapsulation efficiency).
minimum bactericidal concentration (MBC) values of free and
encapsulated black pepper oil. Black pepper oil exhibited antibac-
terial activity against S. aureus and E. coli with the MIC values of
1000 mg/mL and 2000 mg/mL, respectively. The results also indi-
cated that a representative for gram positive bacteria, S. aureus, was
more sensitive to the tested samples than E. coli, a representative
for gram negative bacteria. This was because the external surface of
outer membrane of the gram negative bacteria composing of li-
popolysaccharides and proteins better tolerates the tested samples.
Moreover, the O-side chains of the lipopolysaccharides of the gram
negative bacteria have a hydrophilic surface protecting the hydro-
phobic molecules to enter the bilayer (Zengin & Baysal, 2014).
After encapsulated in HPbCD, the antibacterial activity of black
pepper oil was improved by 4 times against both S. aureus and
E. coli. Inclusion complex formation with HPbCD could increase
aqueous solubility and improve antimicrobial efficacy at lower
concentrations of encapsulated compounds (Hill et al., 2013). As the
primitive sites for antimicrobial action were found at the cell
membrane and inside the cytoplasm, HPbCD may enhance the
accession of essential oils to these regions by improving water
solubility of essential oils (Wang et al., 2011).
5. Conclusions
The particle shape and morphology of the corresponding in-
clusion complex were similar to that of free HPbCD. Instead, the
particle size of the free HPbCD was much larger than that of the
encapsulated product by hydrogen bonding of the free HPbCD
producing the cluster of HPbCD. The characteristic FT-IR and UV
bands of the black pepper oil disappeared in the spectrum of the
inclusion complex, since the oil entered the cavity of HPbCD. The
difference in encapsulation efficiency of the pure compound and
the black pepper oil would result from the presence of other
components in the black pepper oil such as limonene, d-3-carene
and pinene. By this reason, in aqueous system, black pepper oil and
b-caryophyllene show difference in stability of complex form with
the Ks of 104.5 and 132.8 L/mol at 25
C, respectively. Although the
inclusion complexes increase their stability, they gave slightly
lower antioxidant activity as a result of the HPbCD was blocking the
functional groups of active compounds during reacting with DPPH
radicals. After encapsulated in HPbCD, instead, the antibacterial
activity of black pepper oil was improved by 4 times against both
S. aureus and E. coli.
Acknowledgments
This work was financially supported by the Graduate School of
Prince of Songkla University and Thai Government under Grant No.
AGR560387S.
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Please cite this article in press as: Rakmai, J., et al., Physico-chemical characterization and evaluation of bio-efficacies of black pepper essential
oil encapsulated in hydroxypropyl-beta-cyclodextrin, Food Hydrocolloids (2016), http://dx.doi.org/10.1016/j.foodhyd.2016.11.014