Journal of Food Engineering xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Journal of Food Engineering

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

Encapsulation of elderberry extract into phospholipid nanoparticles

Anna Bryła a,⇑, Grazyna
_ Lewandowicz b, Wojciech Juzwa b
a
Institute of Chemical Technology and Engineering, Poznan University of Technology, 4 Berdychowo Street, 60-695 Poznan, Poland
b
Department of Biotechnology and Food Microbiology, Poznan University of Life Sciences, 48 Wojska Polskiego Street, 60-627 Poznan, Poland

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

Article history: The aim of the work was to compare the suitability of different lecithins for nanoencapsulation of elder-
Received 14 November 2014 berry extract in liposomes for the purpose of food enrichment. Lecithins extracted from egg yolk, soybean
Received in revised form 16 July 2015 and sunflower were tested. Vesicles were prepared by thin lipid film hydration technique followed by
Accepted 18 July 2015
freeze and thaw procedure. Liposomal particles were separated from unencapsulated material by gel per-
Available online xxxx
meation chromatography. Encapsulation efficiency, zeta potential, as well as size distribution were deter-
mined for the liposome characterization. Moreover, flow cytometry was used to examine the diversity of
Keywords:
structure in liposome populations. It was found that all of the three tested lecithins were proper for
Liposome
Elderberry extract
encapsulation of elderberry extract into liposomes. Soybean lecithin proved to be the material with
Encapsulation the best stability for the formation of liposomes. The zeta potential absolute value of those nanocapsules
was the highest ( 36.4 mV), and their Z-average size (205 nm) was the smallest. Encapsulation efficiency
in this case was of 25%. Moreover, population of soybean lecithin liposomes was the most uniform both in
terms of size and structure. By using both sunflower and egg yolk lecithins significantly higher encapsu-
lation efficiency could be achieved, however those liposomes were less stable. Moreover their popula-
tions were more diverse both in terms of size and structure.
Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction 3-glucoside, and cyanidine 3-rutinoside (Veberic et al., 2009;

Sambucus nigra, commonly called Elder, is a widespread plant,
occurring from temperate to subtropical parts of America, North
Africa, Europe and Asia. It is a deciduous shrub with 5–9 leaflets,
pinnate leaves, which is maximum 6 m high. It bears small, either
white or cream clusters of flowers in late spring, and early summer.
Its dark purple berries, of about 6 mm diameter each, ripen in late
summer (Charlebois et al., 1995; Kołodziejczak and Drozd _ zal,
_ as (S)-sambunigrin, (R)-prunasin, (R)-holacin, and (S)-zierin
2011). The relatively high content of bioactive compounds has
been the reason behind increasing interest in elderberries. The
abundance of antioxidants (anthocyanins, phenolics), proteins,
sugars, organic acids, vitamin A, vitamin C, bioactive compounds
in Sambuci fructus (elderberry fruits) makes them a valuable raw
material for biotechnology, food technology, pharmacy and medi-
cine (Ochmian et al., 2009). The fruits contain relatively low
amount of sugars combined with high levels of organic acids.
Moreover, they are a rich source of cyanidine based anthocyanins,
mostly cyanidine 3-sambubioside, but also cyanidine-3-sambubio
side-5-glucoside, cyanidine 3, 5-diglucoside, cyanidine
⇑ Corresponding author.
E-mail addresses: anna.bryla@doctorate.put.poznan.pl (A. Bryła), gralew@up.
poznan.pl (G. Lewandowicz), w_juzwa@up.poznan.pl (W. Juzwa).
Jordehaim et al., 2007). As they have been traditionally used for
years they have gained consumers trust. However, there are certain
possible obstacles in full utilization of those plants in food technol-
ogy and conventional medicine. Firstly, in some plants hydrogen
cyanide may be released through hydrolysis of cyanoglycosides
or cyanolipids. Cyanogenic glucosides are found in numerous plant
species (Jones, 1997). Sambucus nigra fruits contain cyanogens such
(Buhrmester et al., 2000; Jensen and Nielsen, 1973). Proper harvest
timing or heat treatment of fruits may minimalize toxic com-
pounds level (Kaack et al., 2006; Wierzbicki, 2002). Short-term
heating seems to be the best way to eliminate the problem
(Jimenez et al., 2014). Secondly, most of bioactive compounds are
susceptible to degradation due to thermal, mechanical, domestic
processes and storage conditions. Diverse interactions may occur
in food matrix. These, may positively or negatively affect antioxi-
dant activity, but the mechanisms have not been elucidated yet
(Ioannou et al., 2012). Thirdly, a significant part of bioactive com-
pounds content is not absorbed at a satisfactory rate. The uptake of
antioxidants, especially flavonoids of high molecular mass, from
the gastrointestinal tract is inefficient (Odeberg et al., 2003). The
bioavailability of these substances can be improved by encapsula-
tion. This process also protects the core material by enhancing its

http://dx.doi.org/10.1016/j.jfoodeng.2015.07.025
0260-8774/Ó 2015 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Bryła, A., et al. Encapsulation of elderberry extract into phospholipid nanoparticles. Journal of Food Engineering (2015),
http://dx.doi.org/10.1016/j.jfoodeng.2015.07.025
2 A. Bryła et al. / Journal of Food Engineering xxx (2015) xxx–xxx
stability (Mozafari et al., 2008a, 2008b). Although, many microen-
capsulation methods have been previously reported (Bo et al.,
2006; Guorong et al., 2007), nanoencapsulation provides more sur-
face area, better stability, less impact on products matrix proper-
ties (Odeberg et al., 2003). Therefore, successful attempts to
enclose bioactive compounds into liposomes are known
(Baomiao et al., 2011; Zhou et al., 2014).
Liposomes are nano-sized spheres, composed of a phospholipid
bilayer membrane. They are suitable carriers not only for lipophilic
compounds, but also for hydrophilic ones, encapsulation of which
is usually problematic. Liposome preparation method was
described for the first time in 1965 by Bangham and his coworkers
(Bangham et al., 1965). The technique of hydration of thin lipid
film was documented in the mentioned study. It has been widely
used until now (Meure et al., 2008). Also, many methods of
post-formation processing are known. They are usually carried
out to reduce either the number of bilayers or the size of obtained
nanocapsules. Among them, sonication (Papahadjopoulos and
Miller, 1967), freeze–thaw (Hope et al., 1985), hydration–dehydra-
tion (Shew and Deamer, 1985) should be named. The choice of
liposome preparation technique depends strongly on the required
effect. Since liposomes vary in size (from 20 nm to >1 lm) and
structure (uni, double, multilamellar, oligovescular), final appli-
cation must be defined. For oral administration vesicle diameter
values in the range of 100–200 nm are adequate, but the number
of bilayers should be reduced as much as possible to obtain satis-
factory level of intestinal absorption (Mozafari et al., 2008a,
2008b).
As liposomes are liquid crystals, they are formed in very specific
conditions. The choice of lecithin–water ratio and temperature is
dependent on the phospholipid used. Slight variations in those
parameters results in other forms such as micelles and reverse
micelles (Papahadjopoulos and Miller, 1967). Additionally, both
shell and core material compatibility is necessary for formation
of a stable system. Yet, the interaction between the two is not fully
understood. Thus, trial and error approach is common when lipo-
some shell composition is concerned.
Moreover, for food application, liposomes should be formed
using popular, relatively cheap raw materials, preferably ones that
are commonly used in nutrition. For example, various kinds of sur-
face active components of bulk oils, such as free fatty acids,
monoacylglycerols, diacylglycerols, phospholipids and polar
amphiphilic products of lipid oxidation. These substances
self-aggregate into associated colloids (Kittipongpittaya et al.,
2014). Phospholipids extracted from egg, soy, sunflower, or milk
are popular ingredients of food products (Mozafari et al., 2008a,
2008b; Taylor et al., 2005). These are found in commercial lecithin
preparations that typically contain also other components, such as
triglycerides, sterols, and free fatty acids. The composition of
lecithin ingredient influences the formation, stability, properties,
and functional properties of liposomes (McClements, 2015).
Among the factors that determine the properties of liposome
bilayer, particularly important is the composition of the material
used for its formation. The change in composition alters the rigidity
or fluidity and the charge of the bilayer. Of particular importance is
the structure of phospholipids. Unsaturated phosphatidylcholine
forms much more permeable and less stable bilayers than the sat-
urated phospholipids with long acyl chains (Akbarzadeh et al.,
2013). Phospholipids of natural origin differ significantly in com-
position especially in phosphatidylcholine content. Egg yolk
lecithin contains 73–74% of this component, whereas sunflower
and soybean only 41% and 21–22%, respectively (Stuchlik and
Zak, 2001; Hollo et al., 1993). Egg yolk lecithin contains signifi-
cantly less phosphatidylinositol (0.4–1%) than sunflower (23%)
and soybean (12–20%) lecithins. Moreover, egg yolk lecithin does
not contain phosphatidic acid, whereas sunflower and soybean
Please cite this article in press as: Bryła, A., et al. Encapsulation of elderberry extract into phospholipid nanoparticles. Journal of Food Engineering (2015),
http://dx.doi.org/10.1016/j.jfoodeng.2015.07.025
lecithin contain 3% and 5–6%, respectively. Much smaller differ-
ences in the phosphatidylethanolamine content are reported. Egg
and sunflower lecithins contain 17–19% of this phospholipid, in
soybean lecithin it amounts to 14–23%. Based on the diversity of
composition of lecithins from different sources, different proper-
ties of liposomes containing encapsulated elderberry extract were
to be expected, especially in terms of structure and stability. The
aim of this work was to compare the suitability of lecithins
obtained from egg yolk, soybean and sunflower for encapsulation
of elderberry extract into liposomes for the purposes of food
enrichment. The obtained liposomes were primarily characterized
by size, stability, and structure. Encapsulation efficiency was also
determined.
2. Materials and methods
2.1. Materials
Fresh elderberry fruits purchased on local market were directly
lyophilized and stored in dark, sealed containers at 18 °C.
Lyophilized fruits were used for extract preparation. For liposomes
preparation, three types of lecithin were used: soybean (Brenntag,
Poland), sunflower (Lasenor, Spain), and egg yolk one (Acros
Organics, Poland). Chloroform (POCH SA, Poland), Sephadex G-75
(Pharmacia Fine Chemicals AB Uppsale, Sweden), 1% phosphate
buffered saline (PBS) of pH = 7.4 (Sigma–Aldrich, Poland) were
used in experiments. To perform flow cytometry, liposomes were
labeled with Thiazole Orange (TO, Sigma Aldrich, Poland) and
DiD (Lipophilic Carbocyanine DiIC18, VybrantÒ Multicolor
Cell-Labeling Kit DiD, Life Technologies, Poland).
2.2. Preparation of elderberry extract
Fresh elderberry fruits were homogenized and lyophilized using
LMC-1 apparatus (Christ). The following parameters of the process
were applied: freezing ( 80 °C); pre-drying ( 18 °C), 0.12 mbar,
20 h; final drying (22 °C), 0.025 mbar, 4 h. The lyophilized fruits
were ground and subjected to extract preparation. The procedure
applied for preparation of the extract took into account the litera-
ture recommendations for preserving the biological activity of
elder fruits (Dembczyński et al., inpress-a, inpress-b). Thus, the
ground product was suspended in deionized water (1 g of dried
fruits per 10 ml of water), mixed using vortex shaker for 1 min,
incubated in an ultrasound water bath at 30 °C for 5 min, then
incubated at room temperature for 10 min. Further, the suspension
was centrifuged (10 min, 3500g). The obtained supernatant was
collected and the extraction was repeated for the sediment. 5
extraction cycles were performed. Extracts from all the extraction
cycles were combined. Dry matter was determined for the final
extract with the use of moisture analyser (Moisture Analyser
MAC 50/NH, Radwag), at a temperature of 120 °C. The dry matter
and pH value were 26.4% and 4.45%, respectively.
2.3. Nanoencapsulation
Liposomal particles were prepared using soybean, sunflower, or
egg yolk lecithin. Because of simplicity as well as documented
application in dairy industry thin lipid film hydration (Bangham)
technique was chosen (da Silva Malheiros et al., 2010; Malheiros
et al., 2010; Bizani et al., 2008). To that end, 1.13% solution in chlo-
roform was prepared for every lecithin separately. The organic sol-
vent was removed to complete dryness to form a lipid film using a
rotary evaporator under vacuum (Laborata 4000 Efficient,
Heidolph). The obtained phospholipid film was hydrated for 1 h
at 42 °C with elderberry fruit extract diluted tenfold with PBS.
 A. Bryła et al. / Journal of Food Engineering xxx (2015) xxx–xxx 3

Hydration was performed using rotary evaporator, spinning the threshold was set at the FSC signals. Data were acquired in a
flask in the water bath, under vacuum. Next, the liposome suspen- four-decade logarithmic scale as height signals and analyzed with
sion was frozen (N2 liquid bath) and thawed (42 °C water bath) 10 the FACS DIVA software (Becton Dickinson).
times.
Liposomes obtained by Bangham method followed by freeze
2.7. Statistical analysis
and thaw (FAT) procedure were separated from unencapsulated
material by gel permeation chromatography (GPC). Sephadex
One-way analysis of variance (ANOVA) was conducted indepen-
G-75 was used as a stationary phase, since it provides proper sep-
dently for each dependent variable. Post-hoc Tukey HSD multiple
aration and minimal level of lipid retention in the column
comparison test was used to identify statistically homogeneous
(Ruysschaert et al., 2005). The sample was eluted with 135 ml of
subsets a = 0.05. Statistical analysis with Statistica 10 (StatSoft,
1% PBS (3  column’s volume). All the fractions obtained by GPC
Inc., 2011) software.
were tested for turbidity at k = 340 nm (Specord 205, Analytic
Jena) to confirm presence of liposomes. Three batches of liposomes
were prepared for each lecithin. The measurements of zeta poten- 3. Results and discussion
tial, size distribution and encapsulation efficiency were performed
separately for each of them. 3.1. Encapsulation efficiency

2.4. Encapsulation efficiency determination
To estimate the encapsulation efficiency (EE), the absorbance of
each fraction collected during GPC was measured at k = 550 nm.
This wavelength was chosen because it corresponds to the maxi-
mum of absorption determined for the investigated extract.
Encapsulation efficiency is the ratio of the sum of the absorbance
values of the fractions rich in liposomes to the sum of the absor-
bance values of all the tested fractions. It is expressed as a
percentage.
2.5. Zeta potential and size distribution measurement
Average particle diameter, size distribution and zeta potential
were performed using Zetasizer (Nano series, Malvern
Instruments) equipped with a 5 mW helium/neon laser.
Measurement were performed at 21 °C for 200 s each, with a
detection angle of 90°. The obtained data were calculated using
cumulant technique, where Gaussian normal distribution is
applied. Results were given as Z-average size which is the mean
value of the hydrodynamic diameter of particles and polydispersity
index PDI which is a measure of the width of the particle size dis-
tribution. Liposomes were analyzed directly after preparation and
after storage at 4 °C in darkness in a sealed container for 2 or
4 weeks.
2.6. Flow cytometry
The structure of the particles was studied using a flow cytome-
ter BD FACS Aria™III (Becton Dickinson). The following fluorescent
dyes were used for liposomes staining: Thiazole Orange (TO) and
DiD. 5 ll DiD was added to 1 ml sample and incubated for
30 min at 37 °C (Thermomixer Compact, Eppendorf). Next, the
samples were thrice centrifuged (t = 4 min, 4000 rpm). Each time
the liposomes suspension was rinsed with PBS. Finally, 2 ll of
1 lM TO in DMSO were added to 1 ml sample and incubated for
10 min at 37 °C (Thermomixer Compact, Eppendorf). The configu-
ration of flow cytometer was as follows: 70 lm nozzle and 70 psi
sheath fluid pressure. 1% PBS filtered through 0.22 lm was used
as sheath fluid. The particles were characterized by two
non-fluorescent parameters: forward scatter (FSC) and side scatter
(SSC), and two fluorescent parameters: green fluorescence (FITC
detector) from the Thiazole Orange reagent collected using a
530/30 band pass filter and red fluorescence (APC detector) from
the DiD reagent collected using a 660/20 band pass filter. A
488 nm blue laser and 633 nm red laser were employed in excita-
tion of TO and DiD fluorochromes, respectively. Flow cytometry
analyses were performed using logarithmic gains and specific
detector settings (10,000 events were recorded per analysis). The
Encapsulation efficiency (EE) is one of the most important
parameters characterizing the process of nanoencapsulation.
When conventional methods for preparation of liposomes are used,
vesicles are both suspended in and filled with aqueous solution of
the encapsulated substance. The aqueous medium surrounding the
liposomes is replaced with an appropriate buffer in the later stage
of the process. Thus, the EE of water soluble compounds into lipo-
somes is reported to be less than 10%, regardless of the liposome
type (Fresta et al., 1993; Elorza et al., 1993; Sasaki et al., 1987)
or membrane properties (El Maghraby et al., 2001). However, it
was proved, that the EE can be significantly increased by employ-
ing proper production technique (Tomoko and Fumiyoshi, 2005). In
our experiment, vesicles obtained by Bangham method followed
by FAT procedure were separated from unencapsulated material
by gel permeation chromatography (GPC). Sephadex G-75 was
used in accordance to Ruysschaert and co-authors, who claim, that
it provides proper separation and minimal level of lipid retention
in the column (Ruysschaert et al., 2005). All the fractions obtained
by GPC were tested for turbidity at k = 340 nm. The results are
illustrated in Fig. 1. Particles were eluted from the column from
the largest to the smallest ones. The peak covering fractions num-
bered 5–15 corresponded to the presence of wanted nanoparticles.
Slight increase of the absorbance values for fraction numbered 20
and above indicated the unencapsulated elderberry extract. The
highest EE of 69% was achieved for sunflower lecithin, lower for
egg yolk lecithin (48%) and the lowest of 25% for soybean lecithin
liposomes. Encapsulation efficiency of 100% is achievable if the
encapsulated material is soluble in the liposome membrane.
Passive encapsulation of water-soluble substance occurs during
formation of a liposome when a droplet is trapped by the enclosing
Fig. 1. Turbidity determination for fractions obtained by GPC for k = 340 nm.

Please cite this article in press as: Bryła, A., et al. Encapsulation of elderberry extract into phospholipid nanoparticles. Journal of Food Engineering (2015),
http://dx.doi.org/10.1016/j.jfoodeng.2015.07.025
4 A. Bryła et al. / Journal of Food Engineering xxx (2015) xxx–xxx

Table 1
Dynamic light scattering and zeta potential data of liposomes obtained from different lecithins directly after preparation and after storage at 4 °C in darkness, in a sealed
containers, for 2 or 4 weeks.

Time (weeks) Z-average size (nm) Polydispersity index PDI Zeta potential (mv)
0 2 4 0 2 4 0
Sunflower lecithin 378 ± 79A,a,b 341 ± 85A,a,b 323 ± 94A,a,b 0.63 ± 0.13B,c 0.48 ± 0.15B,c 0.42 ± 0.12B,c 20.9 ± 1.9d
Egg yolk lecithin 503 ± 12A,a 387 ± 85A,a 433 ± 129A,a 0.54 ± 0.13B,c 0.47 ± 0.11B,c 0.50 ± 0.15B,c 31.6 ± 2.3e
Soybean lecithin 205 ± 57A,b 195 ± 39A,b 166 ± 35A,b 0.49 ± 0.14B,c 0.36 ± 0.07B,c 0.36 ± 0.08B,c 36.4 ± 2.9e

The means with different superscripts are significantly different (P < 0.05).

membrane. The encapsulation effectiveness generally does not
exceed 30% when the latter mechanism is observed (Akbarzadeh
et al., 2013). However, the EE of water soluble substances can be
improved by employing pH gradients (Arcadio and Cullis, 1995).
The distinctively high encapsulation efficiency obtained with sun-
flower lecithin is not related to the high content of specific phos-
pholipid components, rather to appropriate proportions between
them.
and Miller, 1967). Polydispersity index (PDI) is a dimensionless
parameter derived from cumulant analysis and it stands for the
width of Gaussian size distribution (ISO 22412:2008). Its values
lower than 0.1 indicate monodispersity of a sample. Statistical
analysis proved that these differences were not of statistical signif-
icance. Moreover, polydispersity index of all liposome suspensions
did not change significantly during storage.

3.2. Size and stability

As liposome dispersions are not thermodynamically stable their

application as nanoscale containers requires control of their stabil-
ity. It can be reliably characterized by dynamic light scattering
(DLS) and zeta potential measurement (Xu, 2008; Sabin et al.,
2006; Heurtault et al., 2003). It is believed that, the zeta potential
is a fundamental parameter estimating repulsion forces between
liposomes and, consequently, stability of colloidal systems. Zeta
potential of liposomes in the range from 30 mV to 30 mV indi-
cates their adequate stability even during long term storage
(Laouini et al., 2011; Wagner et al., 2002). Our results showed that
(Table 1), soybean and egg yolk lecithin liposomes were the most
stable. Zeta potential values above |30| pointed at a satisfactory
level of stability of these liposomes. Sunflower lecithin liposomes
significantly differed in zeta potential values, which indicated
lower stability.
The size of liposomes is related to their structure, but changes in
their dimensions indicate instability of dispersed colloidal systems.
Particle sizes were observed to increase before macroscopic
changes appeared (Heurtault et al., 2003). Primary and the most
stable parameter determined by DLS is Z-average size, defined as
a ‘‘harmonic intensity averaged particle diameter’’ (ISO
22412:2008) and can be also expressed as the intensity based har-
monic mean. All the resulting populations of liposomes were of
Z-average size higher than 100 nm (Table 1), which suggests at
least double-bilayer structure of vesicles. The biggest Z-average
size of particles was observed for egg yolk lecithin liposomes, the
smallest for soybean lecithin liposomes. The sizes of sunflower
lecithin liposomes did not differ significantly from those prepared
with soybean or egg yolk lecithins. The smallest dimension of lipo-
somes formed using soybean lecithin corresponded to the smallest
phosphatidylcholine and highest phosphatidic acid contents
(Stuchlik and Zak, 2001). In the structure of phosphatidylcholine
a quaternary ammonium cation is present, permanently charged,
independent of the pH of the solution. Moreover, in contrast to
other phospholipids, phosphatidic acid does not contain amino
groups in its structure. Thus, soybean lecithin contains overall less
amino motifs which could explain structural differences in the
formed liposomes. However, the means of the Z-average size of
all types of liposomes did not change significantly during storage.
Size distribution, beside zeta potential and vesicle size, is
another important parameter characterizing stability of colloidal
dispersion. A population of liposomes should be as homogeneous Fig. 2. Size distribution for (A) soybean lecithin, (B) sunflower lecithin, (C) egg yolk
as possible to avoid undesirable coalescence (Papahadjopoulos lecithin liposomes population during storage.

Please cite this article in press as: Bryła, A., et al. Encapsulation of elderberry extract into phospholipid nanoparticles. Journal of Food Engineering (2015),
http://dx.doi.org/10.1016/j.jfoodeng.2015.07.025
 A. Bryła et al. / Journal of Food Engineering xxx (2015) xxx–xxx 5

Fig. 3. Dot plots demonstrating the populations of liposomes obtained using soybean lecithin (A), sunflower lecithin (B), egg yolk lecithin (C). SSC-A – Side Scatter Channel;
FSC-A – Forward Scatter Channel; FITC-A – Green Fluorescence Signal; APC-A – Red Fluorescence Signal.

Please cite this article in press as: Bryła, A., et al. Encapsulation of elderberry extract into phospholipid nanoparticles. Journal of Food Engineering (2015),
http://dx.doi.org/10.1016/j.jfoodeng.2015.07.025
6 A. Bryła et al. / Journal of Food Engineering xxx (2015) xxx–xxx
While the mean values presented in Table 1 allow to analyze
changes in liposomes population only roughly, more detailed infor-
mation concerning changes in particles size and distribution are
provided in Fig. 2. The graphs show intensity distribution of parti-
cle size, which is the first order result from DLS experiment. The
intensity distribution is weighted according to the scattering inten-
sity of each particle fraction or family (ISO 22412:2008). The values
are proportional to the sixth power of its diameter (Raleygh’s
approximation) (Pecora, 1985). Freshly prepared samples from
soybean lecithin demonstrated two main peaks of intensity
(Fig. 2A). One, narrow peak corresponded to relatively homoge-
nous population of smaller particles. The other, broader corre-
sponded to bigger aggregates which were relatively scarce.
During storage, the share of smaller particles increased signifi-
cantly while the aggregates became less numerous yet bigger in
terms of size. Freshly prepared vesicles from sunflower lecithin
were more heterogeneous (Fig. 2B). The initial curve revealed
two main peaks: a wide one covering the main population, and a
narrow one covering smaller particles. During storage, aggregation
occurred, and the main peak became wider. Particles obtained with
egg yolk lecithin turned out to be the most diverse in terms of size
among all tested samples (Fig. 2C). At first, the curve revealed the
presence of particles of broad size range. During storage, the large
aggregates subpopulation eventually disappeared giving place to
small and medium size particles.
When size distribution is concerned, it should be noted that the
applied Bangham method for preparation of liposomes, although
followed by freeze and thaw procedure gave polydisperse popula-
tions of liposomes. Moreover, rather low repeatability was
observed. It is possible that, application of other procedures for
manufacture of liposomes, for example methods employing mem-
brane technology, could provide results that would allow a more
precise differentiation of the raw materials (Pham et al., 2012).
3.3. Flow cytometry
Liposomes vary in size and structure. The homogeneous distri-
bution of the characteristics of the population is highly desirable
due to satisfactory stability of the system. Moreover, the vesicle
structure determines its application. The multilamellar liposomes,
rich in phospholipid bilayers are suitable for lipophilic compounds
encapsulation, while those with extensive aqueous compartments
are adequate as hydrophilic compounds carriers (Tomoko and
Fumiyoshi, 2005). Papers describing attempts to apply flow cytom-
etry to the analysis of selected features distribution in a liposome
population are scarce, however in contrast to DLS study, it is not
a standard procedure (Sato et al., 2006). In our study flow cytom-
etry was used to examine the diversity of structure and properties
of liposomes (Fig. 3). The unique fluorochromes combination: one
penetrating the liposome (thiazole orange) and the other binding
to the liposome shell (DiD) were used in the experiment.
Nanoparticles were initially defined based on scattered laser light
signals: SSC-A, Side Scatter, corresponding to the complexity and
FSC-A, Forward Scatter, corresponding to the size of the vesicles.
For soy lecithin liposomes (Fig. 3A, chart SSC-A vs FSC-A) one
wide subpopulation of liposomes can be distinguished in term of
size and two in terms of structure. Furthermore chart SSC-A vs
FITC-A revealed three subpopulations differing in water phase con-
tent. Regions P1 and P2 were quite similar while P3 apparently
stood out, but it is sparse. Furthermore, chart SSC-A vs APC-A
showed, that the soybean liposomes population was heteroge-
neous in terms of phospholipid content.
Similarly, sunflower liposomes population (Fig. 3B) was also
divided into two main subpopulations in terms of structure.
Based on chart SSC-A vs FITC-A, three subpopulations with differ-
ent water phase content can be distinguished, as in the case of
Please cite this article in press as: Bryła, A., et al. Encapsulation of elderberry extract into phospholipid nanoparticles. Journal of Food Engineering (2015),
http://dx.doi.org/10.1016/j.jfoodeng.2015.07.025
soybean lecithin liposomes. However, as far as phospholipid phase
is concerned, they reveal less heterogeneous structure (SSC-A vs
APC-A).
Egg yolk lecithin liposomes (Fig. 3C) were the most complex
population in terms of both structure and size (SSC-A vs FSC-A).
According to water phase content, regions P1 and P2 are still the
main subpopulations, but previously defined P3 is absent.
Instead, a widely disseminated subpopulation appeared (SSC-A vs
FITC-A). In terms of phospholipid content, egg yolk lecithin lipo-
somes are the most heterogeneous among all the tested samples
(SSC-A vs APC-A).
Flow cytometry results indicated the highest homogeneity of
soybean lecithin liposomes. Only a minor percent of object stands
out from the main characteristic. Liposomes formed by sunflower
lecithin showed higher heterogeneity. However, they seem to be
more homogenous in terms of phospholipid content than soybean
lecithin liposomes. Flow cytometry also indicated high structural
heterogeneity of egg yolk lecithin liposomes. It should be pointed
out that evaluation of structural diversity of liposomes population
is impossible using DLS measurements, which are recognized as a
standard in liposomes studies.
4. Conclusions
Lecithins originated from egg yolk, soybean as well as sunflower
can be used for encapsulation of elderberry extract into liposomes
intended to be used in food products enrichment. However, none
of the investigated lecithins could be recommended as the best
raw material based on all the investigated parameters of obtained
liposomes. The highest EE of 69% was achieved for sunflower
lecithin. However, it was accompanied by the lowest stability.
Using the two other lecithins, it was possible to form liposomes
with similar, and satisfactory, stability. However, these capsules
were characterized by different dimensions and structure.
Liposomes formed using egg yolk lecithin are about twice larger
than those formed with soybean lecithin. The structure of lipo-
somes formed with egg yolk lecithin is also the most complex.
Liposomes obtained with soybean lecithin are most uniform in
terms of structure.
The applied Bangham method for preparation of liposomes,
although followed by freezing and thawing procedure gives poly-
disperse populations of liposomes with rather low repeatability.
Therefore it should not be recommended to researches that want
to precisely differentiate the applicability of different raw
materials.
The applied flow cytometry method has proven to be very use-
ful for the analysis of the structure of liposomes. Using this tech-
nique it was possible indicate heterogeneity of liposomes
populations more comprehensively than using DLS measurements,
which are recognized as a standard in liposomes studies.
Acknowledgement
The work was financially supported by the grant POIG
01.01.02-00-061/09 ‘‘Bioactive food’’ implemented within the
Programme Innovative Economy 2007–2013.
References
Akbarzadeh, A., Rezaei-Sadabady, R., Davaran, S., Woo Joo, S., Zarghami, N.,
Hanihefpour, Y., Samiei, M., Kouhi, M., Nejati-Koshki, K., 2013. Liposome:
classification, preparation, and applications. Nanoscale Res. Lett. 8, 102.
Arcadio, C., Cullis, P.R., 1995. Recent advances in liposomal drug-delivery systems.
Curr. Opin. Biotechnol. 6, 698–708.
Bangham, A.D., Standish, M.M., Watkins, J.C., 1965. Diffusion of univalent ions
across the lamellae of swollen phospholipids. J. Mol. Biol. 13 (1), 238–252.
 A. Bryła et al. / Journal of Food Engineering xxx (2015) xxx–xxx
Baomiao, D., Xiaoming, Z., Khizar, H., Shiqin, X., Chengsheng, J., Mingyong, X.,
Chengmei, L., 2011. Preparation, characterization and the stability of ferrous
glycinate nanoliposomes. J. Food Eng. 102, 202–208.
Bizani, D., Morrissy, J.A.C., Dominguez, A.P.M., Brandelli, A., 2008. Inhibition of
Listeria monocytogenes in dairy products using the bacteriocin-like peptide
cerein 8A. Int. J. Food Microbiol. 121, 229–233.
Bo, S., Wenli, Y., Yaping, Z., Xiaoyong, L., 2006. Study on microencapsulation of
lycopene by spray-drying. J. Food Eng. 76, 664–669.
Buhrmester, R.A., Ebinger, J.E., Seigler, D.S., 2000. Sambunigrin and cyanogenic
variability in population of Sambucus canadensis L. (Caprifoliaceae). Biochem.
Syst. Ecol. 28, 689–695.
Charlebois, D., Byers, P.L., Finn C.E., Thomas L.A., 1995. Elderberry: Botany,
Horticulture, Potential. Horticultural Reviews. John Wiley & Sons Inc, USA,
vol. 17, 4, pp. 213–280.
Da Silva Malheiros, P., Daroit, D.J., Brandelli, A., 2010. Food applications of liposome-
encapsulated antimicrobial peptides. Trends Food Sci. Technol. 21, 284–292.
_
Dembczyński, R., Białas, W., Olejnik, A., Kowalczewski, P., Drozyńska, A., Jankowski,
T., 2015a. Extraction of anthocyanins from black carrot, chokeberry,
blackcurrant and elderberry. In Polish: Ekstrakcja antocyjanów z korzenia
czarnej marchwi, owoców aronii, czarnego bzu i czarnej porzeczki. Zywność, _ Ochmian, I., Oszmiański, J., Skupień, K., 2009. Chemical composition, phenolics, and
Nauka Technologia, Jakość, in press.
_
Dembczyński, R., Białas, W., Olejnik, A., Kowalczewski, P., Drozyńska, A., Jankowski,
T., 2015b. Purification of anthocyanins from black carrot, chokeberry,
blackcurrant and elderberry with the of preparative chromatography. In
Polish: Separacja antocyjanów z korzenia czarnej marchwi, owoców aronii,
czarnego bzu i czarnej porzeczki za pomoca˛ chromatografii preparatywnej.
_
Zywność, Nauka Technologia, Jakość, in press.
El Maghraby, G.M.M., Williams, A.C., Barry, B.W., 2001. Skin delivery of 5-
fluorouracil from ultradeformable and standard liposomes in vitro. J. Pharm.
Pharmacol. 53, 1069–1077.
Elorza, B., Elorza, M.A., Frutos, G., ChantreIn Polis, s.J.R., 1993. Characterization of 5-
fluorouracil loaded liposomes prepared by reverse-phase evaporation of
freezing-thawing extrusion methods: study of drug release. Biochim. Biophys.
Acta 1153M, 135–142.
Fresta, M., Villari, A., Puglisi, G., Cavallaro, G., 1993. 5-Fluorouracil: various kinds of
loaded liposomes: encapsulation efficacy, storage stability and fusogenic
properties. Int. J. Pharm. 99, 145–156.
Guorong, S., Liqun, R., Huazhong, Y., Hua, X., Guoping, P., Sang, L., Chen, Y., 2007.
Yeast-cell-based microencapsulation of chlorogenic acid as a water-soluble
antioxidant. J. Food Eng. 80, 1060–1067.
Heurtault, B., Saulnier, P., Pech, B., Jacques-Emile Proust, J.-E., Benoit, J.-P., 2003.
Physico-chemical stability of colloidal lipid particles. Biomaterials 24, 4283–
4300.
Hollo, J., Peredi, J., Ruzics, A., Jeranek, M., Erdelyi, A., 1993. Sunflower lecithin and
possibilities for utilization. J. Am. Oil Chem. Soc. 70 (10), 997–1001.
Hope, M.J., Bally, M.B., Webb, G., Cullis, P.R., 1985. Production of large unilamellar
vesicles by a rapid extrusion procedure. Characterization of size, distribution,
trapped volume and ability to maintain a membrane potential. Biochim.
Biophys. Acta 812, 55–65.
Ioannou, I., Hafsa, I., Hamdi, S., Charbonnel, C., Ghoul, M., 2012. Review of the effect
of food processing and formulation on flavonol and anthocyanin behavior. J.
Food Eng. 111, 208–217.
International Standard ISO22412 Particle Size Analysis-Dynamic Light Scattering,
International Organisation for Standarisation (ISO), 2008.
Jensen, S.R., Nielsen, B.J., 1973. Cyanogenic glucosides in Sambucus nigra L. Acta
Chem. Scand. 27, 2661–2662.
Jimenez, P., Cabrero, P., Basterrechae, J.E., Tejero, J., Cordoba-Diaz, C., Cordoba Diaz,
M., Girbes, T., 2014. Effects of short-term heating on total polyphenols,
anthocyanins, antioxidant activity and lectins of different parts of dwarf elder
(Sambucus ebulus L.). Plant Foods Human Nutr. 69(2), 168–174.
Jones, D.A., 1997. Why are so many plants cyanogenic? Phytochemistry 47 (2), 155–
162.
Jordehaim, M., Giske, H.N., Andersen, O.M., 2007. Anthocyanins in Caprifoliaceae.
Biochem. Syst. Ecol. 35, 153–159.
Kaack, K., Christensen, L., Hughes, M., Eder, R., 2006. Relationship between sensory
quality and volatile compounds of elderflower (Sambucus nigra L.) extracts. J.
Eur. Res. Technol. 223, 57–70.
Kittipongpittaya, K., Panya, A., McClements, D.J., Decker, E.A., 2014. Impact of free
fatty acids and phospholipids on reverse micelles formation and lipid oxidation
in bulk oil. J. Am. Oil Chem. Soc. 91, 453–462.
Please cite this article in press as: Bryła, A., et al. Encapsulation of elderberry extract into phospholipid nanoparticles. Journal of Food Engineering (2015),
http://dx.doi.org/10.1016/j.jfoodeng.2015.07.025
7
Kołodziejczak, B., Drozd _ zal,
_ K., 2011. Właściwości przeciwutleniaja˛ce kwiatów i
owoców bzu czarnego pozyskiwanego ze stanu naturalnego. Zywność. _ Nauka.
Technologia. Jakość 4 (77), 36–44.
Laouini, A., Jaafar-Maalej, C., Sfar, S., Charcosset, C., Fessi, H., 2011. Liposome
preparation using a hollow fiber membrane contactor – application to
spironolactone encapsulation. Int. J. Pharm. 415, 53–61.
Malheiros, P.S., Daroit, D.J., Silveira, N.P., Brandelli, A., 2010. Effect of nanovesicle-
encapsulated nisin on growth of Listeria monocytogenes in milk. Food Microbiol.
27, 175–178.
McClements, D.J., 2015. Encapsulation, protection, and release of hydrophilic active
components: potential and limitations of colloidal delivery systems. Adv.
Colloid Interface Sci. 219, 27–53.
Meure, L.A., Foster, N.R., Dehghani, F., 2008. Conventional and dense gas techniques
for the production of liposomes: a review. AAPS PharmSciTech 9 (3), 798–809.
Mozafari, M.R., Khosravi-Darani, K., Borazan, G.G., Cui, J., Pardakhty, A., Yurdugul, S.,
2008a. Encapsulation of food ingredients using nanoliposome technology. Int. J.
Food Prop. 11, 833–844.
Mozafari, M.R., Johnson, C., Hatziantoniou, S., Demetzos, C., 2008b. Nanoliposomes
and their applications in food nanotechnology. J. Liposome Res. 18, 309–327.
firmness of small blach fruits. J. Appl. Botany Food Qual. 83, 64–69.
Odeberg, J.M., Lignell, A., Pettersson, A., Hoglund, P., 2003. Oral bioavailability of the
antioxidant astaxanthin on humans is enhanced by incorporation of lipid based
formulations. Eur. J. Pharm. Sci. 19, 299–304.
Papahadjopoulos, D., Miller, N., 1967. Structural characteristics of hydrates liquid
crystals. Biochim. Biophys. Acta 75067, 624–638.
Pecora, R., 1985. Dynamic Light Scattering: Application of Photon Correlation
Spectroscopy. Plenum Press, 1985.
Pham, T.T., Jaafar-Maalej, C., Charcosser, C., Fessi, H., 2012. Liposome and noisome
preparation using a membrane contactor for scale-up. Colloids Surf. B 94, 15–
21.
Ruysschaert, T., Marque, A., Duteyrat, J.L., Lesieur, S., Winterhalter, M., Fournier, D.,
2005. Liposomes retention in size exclusion chromatography. BMC Biotechnol.
5 (11).
Sabin, J., Prieto, G., Ruso, J.M., Hidalgo-Alvarez, R., Sarmiento, F., 2006. Size and
stability of liposomes: a possible role of hydration and osmotic forces. Eur. Phys.
J. E 20, 401–408.
Sasaki, H., Mutsukawa, Y., Hashida, M., Sezaki, H., 1987. Characterization of
alkylcarbomaoyl derivatives of 5-fluorouracil and their application to
liposomes. Int. J. Pharm. 36, 147–156.
Sato, K., Obinata, K., Sugawara, T., Urabe, I., Yomo, T., 2006. Quantification of
structural properties of cell-sized individual liposomes by flow cytometry. J.
Biosci. Bioeng. 102 (3), 171–178.
Shew, R.L., Deamer, D.W., 1985. A novel method for encapsulation of
macromolecules in liposomes. Biochimica et Biophisica Acta 816, 1–8.
Stuchlik, M., Zak, S., 2001. Lipid-based vesicle for oral drug delivery. Biomed. Pap.
142 (2), 17–26.
Taylor, T.M., Davidson, P.M., Bruce, B.D., Weiss, J., 2005. Liposomal nanocapsules in
food science and agriculture. Crit. Rev. Food Sci. Nutr. 45, 587–605.
Tomoko, N., Fumiyoshi, I., 2005. Encapsulation Efficiency of water-soluble and
insoluble drugs in liposomes prepared by the microencapsulation vesicle
method. Int. J. Pharm. 298, 198–205.
Veberic, R., Jakopic, J., Stampar, F., Schmitzer, V., 2009. European elderberry
(Sambucus nigra L.) rich in sugars, organic acids, anthocyanins and selected
polyphenols. Food Chem. 114, 511–515.
Wagner, A., Vorauer-Uhl, K., Katinger, H., 2002. Liposomes produced in a pilot scale:
production, purification and efficiency aspects. Eur. J. Pharm. Biopharm. 54,
213–219.
Wierzbicki, A., 2002. Wild elderberry: sourcing and application. In: Polish: Dziki bez
czarny-pozyskiwanie surowca i jego zastosowanie. Wiadomości Zielarskie, 4,
pp. 8–9.
Xu, R., 2008. Progress in nanoparticles characterization: sizing and zeta potential
measurement. Particuology 6, 112–115.
Zhou, W., Liu, W., Zou, L., Liu, W., Liu, Ch., Liang, R., Chen, J., 2014. Storage stability
and skin permeation of vitamin C liposomes improved by pectin coating. Colloid
Surfaces: Biointerfaces 117, 330–337.