Food Research International 51 (2013) 866–871

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Food Research International

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Short communication

Nanoencapsulation of date palm pit extract in whey protein particles generated

via desolvation method
Leila Bagheri, Ashkan Madadlou ⁎, Mohammadsaeed Yarmand, Mohammad E. Mousavi
Department of Food Science, Technology and Engineering, University College of Agriculture and Natural Resources, University of Tehran, Karadj, Iran

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

Article history: An alkaline solution of whey protein isolate was charged with absolute ethanol resulting in precipitation of whey
Received 18 November 2012 protein particles. The vacuum-dried particles were then dispersed either in water or aqueous ethanol.
Accepted 29 January 2013 Heat-treatment of whey proteins before desolvation process decreased the mean size of particles when
dispersed in aqueous ethanol from 280 nm to 183 nm. The range and mean size of particles prepared from
Keywords:
heat-treated protein solution when dispersed in water were 41–212 nm and 103 nm, respectively. Date palm
Desolvation
Date palm
pit aqueous extract was encapsulated inside the particulating heat-treated whey proteins during the desolvation
Encapsulation stage with encapsulation efficiency of ~78%. Extract-loaded particles had mean size of 163 nm in alcoholic
Whey protein nanoparticles dispersion and 92 nm in water dispersion. Scanning electron microscopy imaging showed spherical
nanoparticles aggregated in dry state. Fourier transform infrared spectroscopy suggested that extract and
whey proteins did not covalently bind. Heat-treatment of whey proteins before desolvation resulted in the
absence of denaturation endotherm in differential scanning calorimetry curve of extract-free particles. Extract
loading in particles interrupted the continuity of protein matrix causing the occurrence of mild glass transition
phenomenon in extract-loaded particles when heated.
© 2013 Elsevier Ltd. All rights reserved.

1. Introduction and potential benefits of these nutraceuticals (Bell, 2001). Nano-

Nanoparticles when used in biological and food systems may provide
superior characteristics to microparticles including unique quality,
improved sensory properties, extended flavor perception, better mouth-
feel, transparent appearance and enhanced processability (Moraru et al.,
2003). Therefore, nanobioparticles prepared from generally recognized
as safe (GRAS) biopolymers are increasingly nominated for various
applications in biomedical, pharmaceutical and nutraceutical industries
remembering their acknowledged biocompatibility.
Polyphenols act as metal scavengers, antimutagenes, and antimicro-
bial agents (Proestos et al., 2005). Their consumption may reduce the
risk of some chronic diseases such as cancer, cardiovascular disease,
chronic respiratory disease and diabetes (Arts, Van de Putte, &
Hollman, 2000; Scalbert & Williamson, 2000). However, incorporation
of polyphenols into foods may cause quality defects such as astringent
taste and increased haze in beverages. They can act as substrates for
browning reactions (Lesschaeve & Noble, 2005) resulting in undesirable
color changes in food products. Also, polyphenols and other bioactive
substances may undergo degradation and/or deterioration by food
processing operations and storage (e.g. heating, acidification, light and
oxygen) or in the gastrointestinal tract (acidic pH, enzymes, presence
of other nutrients). These drawbacks impose limits for application
⁎ Corresponding author.
E-mail address: Ashkan.madadlou@gmail.com (A. Madadlou).
encapsulation of bioactive molecules within appropriately structured
carriers may overcome these problems without adverse effect on senso-
ry characteristics and appearance of final product.
Desolvation technique is a straightforward, rapid and easily applica-
ble method to carry out the whole nanoparticle preparation procedure
in one pot. This technique requires only two miscible solvents without
involvement of destructing factors such as high shear rate, heating
and sonication that damage the tertiary structure of proteins. As well,
the procedure does not include toxic reagents and surfactants (Bilati,
Allémann, & Doelker, 2005). Gunasekaran, Ko, and Xiao (2007) used ac-
etone to desolvate β-lactoglobulin and then crosslinked the generated
nanoparticles by glutaraldehyde–ethanol mixture. Recently, fish oil
was encapsulated in zein nanoparticles desolvated by water from aque-
ous alcoholic solution (Zhong, Tian, & Zivanovic, 2009). Gülseren, Fang,
and Corredig (2012a) generated protein nanoparticles by adding etha-
nol as antisolvent to an alkaline solution of whey proteins, followed
by resolvation of particles through diluting the suspension by aqueous
buffers. The procedure was later used to encapsulate zinc chloride with-
in whey protein particles (Gülseren, Fang, & Corredig, 2012b). To the
best of authors' knowledge there is no report in the literature on encap-
sulation of polyphenols in protein nanoparticles prepared by anti-
solvent (desolvation) method. The objective of the present study was
therefore to encapsulate the aqueous extract of date palm pit as a rich
source of phenolic compounds within whey protein nanoparticles
obtained through desolvating by ethanol. The obtained extract-loaded

0963-9969/$ – see front matter © 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodres.2013.01.058
 L. Bagheri et al. / Food Research International 51 (2013) 866–871
particles are expected to be easily added to beverages with minimum
influence on sensory characteristics of food product.
2. Materials and methods
2.1. Materials
Sodium chloride (NaCl), sodium bicarbonate (NaHCO3), sodium
hydroxide (NaOH), hydrochloric acid (HCl), ethanol and Folin and
Ciocalteau's phenol reagent (mixture of phosphomolybdate and
phosphotungstate) were purchased from Merck (Darmstadt, Germany).
Lactose- and fat-free whey protein isolate (WPI) with 92% protein
content was a kind gift from Arla Food Ingredients (Viby J, Denmark).
Bi-distilled water was used throughout the study.
2.2. Preparation of date palm pit extract
Date palm fruit (Kabkab variety) pits were washed and air-dried at
50 °C for 4 h. The dried pits were milled using a heavy-duty grinder to
pass 1 mm screen, afterwards, 1 L boiling water was added to 50 g pit
powder and extraction was carried out at 30 °C for 7 h while shaken
at 100 rpm. The mixture was filtered through a series of Whatman filter
papers in order to remove all suspended materials. The extract was
freeze-dried and kept in dark glass bottles at −80 °C until use.
2.3. Measurement of phenolic content
Total phenolic content was measured using the Folin–Ciocalteau
method (Singleton & Rossi, 1965). Pit extract (30 μL) was mixed
with 2.37 mL deionized water and 150 μL Folin–Ciocalteau's
phenol reagent and allowed to stand at room temperature for
7 min, then 450 μL sodium bicarbonate (20% w/v) was added to
the mixture. After standing for 70 min at room temperature, absor-
bance was measured (Spectrophotometer BioQuest CE2502, Cecil
Instruments Ltd., UK) at 760 nm. Results were expressed as mg
gallic acid equivalents (GAE)/100 g sample (Shui & Leong, 2006).
Polyphenol content of extract was 1582 mg gallic acid equivalent
per 100 g dry weight at pH 6.25.
2.4. Preparation of extract-free and extract-loaded particles
WPI was dissolved (3% w/v) in 10 mM NaCl solution by stirring at
500 rpm at room temperature for 2 h; sodium azide (50 mg/L) was
added to prevent microbial growth. The solution was stored at 4 °C
for 12 h and filtered through 0.45 μm PVDF syringe filter (Whatman,
Germany) prior to use. Protein solution was heat-treated at 60 °C for
30 min (Qi & Onwulata, 2011) and its pH was adjusted to 9.0 with
2 M NaOH. The pH adjustment led to smaller particles based on pre-
liminary experiments. The solution was then charged with ethanol at
a rate of 1 mL min −1 while stirring at 500 rpm until became turbid.
The rate of ethanol addition was controlled carefully since it influ-
ences the size of generated particles (Langer et al., 2003). The amount
of ethanol added was approximately 3.3 mL per mL protein solution.
Nanoparticle suspension was centrifuged at 18,000 ×g (refrigerated
centrifuge model RS-20IV, Tomy Seiko Co., Ltd., Tokyo, Japan) for
10 min and obtained supernatant was used in measurement of
encapsulation efficiency. The resulting nanoparticles were then
vacuum dried at 60 °C and stored at − 80 °C until analyses.
For preparation of pit extract-loaded particles, WPI solution was
supplemented before pH adjustment with 0.045 g or 0.06 g extract
powder to obtain 1:20 or 1:15 mass ratio of extract to WPI, respectively.
The whole procedure for preparation and separation of nanocapsules
was performed the same as particles.
867
2.5. Particle size measurement
Size and polydispersity of particles and capsules were determined
by using a dynamic light scattering particle size analyzer (ZetaPALS,
Brookhaven Instruments Co., NY, USA). For this purpose, dried samples
were either dispersed in 10 mL ethanol at pH 9.0 or bi-distilled water
with pH 9.0 at ratio of 1:200 (w/v) and shaken continuously at room
temperature for 12 h using orbital incubator (Stuart®, S150, Guill
Bern Corporation Inc., Philippines) to allow complete hydration. To
investigate the influence of heat-treatment of WPI on particle size, a
sample was also prepared from non-heated WPI following the same
procedure as described. Particle size measurements were carried out
at 25 °C with laser beam operated at 657 nm and scattering angle of
90°. Each sample was read three times. Average sizes reported are the
volume-averaged diameters.
2.6. Scanning electron microscopy
The morphology of dry extract-free and extract-loaded nanoparticles
was observed with a scanning electron microscope (SEM, KYKY-
EM3200, KYKY Technology Development Ltd, China) operated at
24 kV. The surfaces of particles were sputtered with gold, observed
and photographed.
2.7. Particle yield and encapsulation efficiency
The weight of dried pellet of extract-free and extract-loaded
particles was used for calculating the particle yield as follows:
weight of dry pellet
Particle Yieldð%Þ ¼  100:
total weight of extract and WPI used for particles preparation
The efficiency of extract entrapment in particles was calculated as
the difference between phenolic content added to WPI solution before
desolvation stage and the content remained in the centrifugal superna-
tant. To determine the holding degree of encapsulated polyphenols
within particles, precipitated pellet was dispersed both in 70% ethanol
and bi-distilled water, mildly shaken for 30 min and re-precipitated
through centrifugation after which the supernatant was used for
polyphenol measurement.
2.8. Fourier transform infrared (FTIR) spectroscopy and thermal analysis
FTIR spectra of pit extract, WPI, extract-free and extract-loaded
particles were obtained with a Perkin Elmer 2000 FT-IR spectrometer
(Perkin Elmer Co., MA, USA) using the KBr disk method. Spectra were
obtained in transmission mode from 450 to 4500 cm −1 wavenumber
range.
The thermal analysis of samples was performed by using a cali-
brated differential scanning calorimeter (Star System DSC1, Mettler
Toledo, OH, USA). For this purpose, each specimen (5 mg) was heated
under nitrogen stream (20 mL/min) from 25 to 150 °C at rate of
10 °C min −1.
2.9. Statistical analysis
The data, reported as mean± standard deviation, are from experi-
ments conducted in triplicate. One-way analysis of variance (ANOVA)
was performed using SPSS (ver. 16) software. ANOVA was used to
check the assumptions of variance homogeneity and normality and
compare the treatment means. Differences among mean values were
examined by the least significant difference (LSD) and Duncan's test
at P b 0.05 significance level.
868 L. Bagheri et al. / Food Research International 51 (2013) 866–871

Table 1
Size and polydispersity of WPI nanoparticles and date pit extract-loaded nanocapsules.

Polydispersity Size distribution (nm) Diameter (nm) Sample

0.423 ± 0.016a 77–443 280.88 ± 29.61a Particles in ethanol (from non-heated WPI)
0.326 ± 0.022b 70–353 183.98 ± 20.66b Particles in ethanol (from heated WPI)
0.232 ± 0.011c 60–366 163.1 ± 10.43c Capsules in ethanol (from heated WPI)
0. 366 ± 0.006d 41–212 103.78 ± 15.09d Particles in water (from heated WPI)
0.296 ± 0.007e 20–241 92.89 ± 17.31e Capsules in water (from heated WPI)

Data are expressed as mean ± standard deviation for triplicate tests. Different superscripts in the same column indicate significant differences at P b 0.05.

3. Results and discussion assemblies by desolvation method. Zou, Li, Percival, Bonard and Gu
(2012) produced zein nanocapsules with particle yield of 76% by
3.1. Particle size desolvation method. Langer et al. (2003) fabricated human serum
albumin nanoparticles with yield of 65–95% by desolvation procedure.
The mean particle size, polydispersity index and size distribution It is clear from the results that heat-treating of whey protein isolate
range of extract-free and extract-loaded particles are reported in solution before desolvation increased the particle yield significantly
Table 1. All samples were of nanoscalar size indicating the successful (Table 2). As discussed earlier, partially denatured whey proteins by
generation of protein nano-associations via alcoholic desolvation. The heat are extensively interconnected, which boosted the particulation
repulsive forces among negatively charged protein particles at alkaline of molecules and thus a higher yield of precipitated pellet was
pH (Papiz et al., 1986) of dispersing medium that was aqueous ethanol recovered.
or water prevented from particle aggregation. As well, the alkalinity of Encapsulation of pit extract in WPI particles resulted in higher parti-
protein solution before desolvation stage might unfold the whey protein cle yields (Table 2) due probably to action of extract ingredients as base
native assemblies exposing their hidden thiol groups to undergo thiol– for protein arrangement during desolvation. However, a higher mass
disulfide interchanges. This led to enhancement of particulation and ratio of core to matrix-forming protein i.e. 1:15 caused a lower yield
inhibition of large aggregate formation (Gunasekaran et al., 2007). of particulation in comparison to a ratio of 1:20. A similar trend was
Heat treatment of WPI before desolvation stage resulted in significantly
smaller particles (Table 1). It is argued that heat-treating of WPI solution
at low temperature i.e. 60 °C might favor the unfolding of whey proteins
(Qi & Onwulata, 2011) into a state referred to as molten globule (Nicolai,
Britten, & Schmitt, 2011). This resulted in extensive exposure of hidden
groups and more interconnection of protein molecules during precipita-
tion, leading to generation of tinier assemblies. Eissa (2012) found that
the size of whey protein assemblies decreased with increasing temper-
ature in the range of 30–65 °C.
The type of dispersing medium influenced the size of extract-free
and extract-loaded particles significantly. Water-dispersed particles
and capsules were smaller than their aqueous ethanol-dispersed
counterparts (Table 1). Water probably well hydrated the desolvated
protein nanoassemblies causing the solubilization of a population of
whey proteins and thus fragmented the originally formed particles
to smaller offsprings. Gülseren et al. (2012a) reported sizes as small
as ~ 8.8–36 nm for WPI particles desolvated by ethanol and dispersed
in pH 3.0 buffer. The majority of amino groups (over 90%) are proton-
ated at low pH value (pH b 3) (Kumar, 2000) to form an extended mo-
lecular chain which probably accounts for the extremely small size of
particles reported by Gülseren et al. (2012a).
The polydispersity of all samples was less than 0.4 except for particles
prepared from non-heated WPI (Table 1). This manifests the importance
of preheating in obtaining more homogenous particles from desolvated
WPI. Encapsulation of date palm pit extract resulted in generation
of smaller and more monodisperse particles (Table 1) due either to
some chemical interactions between core and whey proteins resulting
in denser assemblies or action of extract ingredients namely phenolic
compounds as base for arrangement of protein molecules around those
during precipitation.

3.2. Morphology, particle yield and encapsulation efficiency

Fig. 1 shows the exemplar SEM images of nanoparticles and

extract-loaded particles. Samples had spherical morphology and in
dry state were clumped. A similar connection/aggregation phenome-
non was observed by Zhong and Jin (2009) in SEM images of freeze-
dried zein particles.
The yield of nanoparticles was higher than 50% and ~76% for
samples prepared from non-heated and heat-treated WPI, respectively Fig. 1. SEM images of WPI extract-free (S1) and pit extract-loaded (S2) particles from
(Table 2) implying in proper mass generation of associated protein heat-treated WPI.
 L. Bagheri et al. / Food Research International 51 (2013) 866–871 869

Table 2
Particle yield and encapsulation efficiency of WPI extract-free and date pit extract-loaded particles.

Encapsulation efficiency (%) Particle yield (%) Extract-to-WPI ratio Sample

– 52.23 ± 1.6a – Nanoparticles (from non-heated WPI)

– 77.45 ± 1.3b – Nanoparticles (from heated WPI)
78.33 ± 2.3a 95.43 ± 1.5c 1:20 Nanocapsules
70.26 ± 1.3b 90.23 ± 1.1d 1:15 Nanocapsules

Data are expressed as mean±standard deviation for triplicate tests. Different superscripts in the same column indicate significant differences at Pb 0.05.

observed for encapsulation efficiency at higher extract-to-WPI ratio. It is 2963 cm −1 in spectra of extract-free and extract-loaded particles
argued that protein–protein and polyphenol–protein interactions were were because of O\H and C\H stretching, respectively.
weakened with increasing extract-to-WPI ratio resulting in remaining In the FTIR spectrum of extract-loaded particle, the peak of
of more extract and protein in the supernatant instead of being WPI's hydroxyl group (3298 cm − 1) merges with that of phenolic
particulated and/or encapsulated. These results are consistent with hydroxyl groups (3400 cm − 1). The sharp peaks at 2963 cm − 1
those of Zou et al. (2012). It has been similarly reported for other poly- representing C\H stretching of WPI's CH3 and CH2 functional
meric matrices that over-loading of core may cause a decrease in encap- groups overlaid with the C\H stretching peak of extract's phenolic
sulation efficiency (Liu & Park, 2009; Shah, Pal, Kaushik, & Devi, 2009). rings methyl and isopropyl groups at 2962 cm − 1. The three peaks
Antisolvent precipitation of whey proteins to enclose polyphenols specific to the phenolic rings of extract at wavenumbers ranging
was significantly efficient as an encapsulation efficiency of >70% was from 1611 to 1443 cm − 1 disappeared with the overlaying effect
obtained (Table 2). It is remembered that pit powder was extracted of Amide I, Amide II and C\H bending of WPI at similar positions
by sole water and thus water-soluble phenolics present in the extract i.e. 1653 cm − 1, 1535 cm − 1 and 1450 cm − 1, respectively. The
were efficiently enclosed inside the associating protein assemblies peak due to polyflavonoids at 1285 cm − 1 for extract disappeared
during the desolvation stage by alcohol. Wu, Luo, and Wang (2012) by the overlaying effect of protein β-sheet structure at a similar po-
encapsulated volatile essential oils in desolvated zein and obtained an sition (1242 cm − 1). Peaks observed at 1062 cm − 1 for extract due
efficiency of 50%. The amount of zinc attached to WPI nanoparticles to asymmetrical C\O vibration merged with the peak at
was higher than 80% (Gülseren et al., 2012b). 1083 cm − 1 for C\OH in WPI. Peaks due to out-of-plane bending
When capsules were dispersed in bi-distilled water and shaken of C\H bonds in the benzene rings at 778 and 617 cm − 1
for relatively short time (30 min), a minor portion of phenolic disappeared by the overlaying effect of out-of-plane N\H wagging
compounds i.e. 3.5% ± 0.5 of total phenolics was recovered in centrif- vibration in WPI at similar positions (817 and 667 cm − 1).
ugal supernatant; while, no trace of polyphenols was detected in All information reflects that extract ingredients were physically
aqueous alcohol used for extract-loaded particle dispersion. This entrapped in the particle matrix without covalent interactions. Hy-
result confirms our hypothesis based on the particle size measure- drogen bonds, van der Waals and hydrophobic interactions might
ments that WPI desolvated particles unpack and fragment when dis- contribute in extract inclusion (He et al., 2011). Our results are in ac-
persed in water. cordance with previous observations that polyphenols interact with
proteins via hydrophobic interactions and hydrogen bonding
3.3. FTIR spectrum (Emmambux & Taylor, 2003; Taylor, Taylor, Belton, & Minnaar,
2009).
FTIR spectra of pit extract, WPI and WPI extract-free and
extract-loaded particles are shown in Fig. 2. The band at 3400 cm−1
for pit extract may assign to O\H stretching of the hydroxyl group of
acidic carboxyl group (COOH) on the benzene ring of phenolic acids.
Peaks observed at 1611, 1524, and 1450 cm−1 correlate to C_C
stretching of aromatic rings which are the typical functional groups of
phenolic compounds. Peak observed at 1285 cm−1 attributed to the
C\O in flavonoids of extract (Yazaki & Hillis, 1977). Peak observed at
1062 cm−1 attributed to the asymmetrical C\O vibration. The peaks
appeared at 778 and 617 cm−1attributed to out-of-plane bending of
the ring C\H bonds in the benzene rings (Silverstein, Webster, &
Kiemle, 2005) Peaks at 1535 and 1653 cm−1 in the FTIR spectra of
native WPI, extract-free and extract-loaded particles are attributed to
C_O stretching of Amide I band in α-helical component of
α-lactalbumin and β-lactoglobulin secondary structures (Bayler &
Purcell, 1986; Geara, 1999; Kretschmer, 1957) and Amide II band of
both C\N stretching and C\N\H in plane bending (Sessa, Mohamed,
& Byars, 2008), respectively. The appearance of a band at 1238 or
1242 cm−1 in spectra of extract-free and extract-loaded nanoparticles
in comparison to native WPI was assigned to β-sheet structure in pro-
tein secondary structures (Seo et al., 2010). Heat-treatment of whey
proteins before desolvation stage therefore modified the spatial confor-
mation of proteins resulting in the enhanced β-sheet structure.
Two peaks appeared at 1450 cm −1 and 1400 cm −1 in spectra of
native and particulated WPI due to C\H bending and C\N stretching,
respectively. Peaks due to N\H stretching, C\H stretching and O\H
bending vibrations of deionized carboxylic acid were observed at Fig. 2. FTIR spectra of date palm pit extract, native WPI, and WPI extract-free and
3300, 2963 and 1396 cm −1, respectively. Peaks at 3300 cm −1 and extract-loaded nanoparticles.
870 L. Bagheri et al. / Food Research International 51 (2013) 866–871
Fig. 3. DSC thermograms of native WPI (…), WPI extract-free (− − −) and
extract-loaded (22)nanoparticles.
3.4. Thermal behavior
The DSC scan (Fig. 3) of native WPI exhibited a large endother-
mic peak between 39 and 110 °C centered at 74 °C which is attri-
buted to heat-induced transitions occurring in α-lactalbumin and
β-lactoglobulin. Fitzsimmons, Mulvihilla, and Morris (2007) by using
a microcalorimeter observed an exotherm following the denaturation
endotherm for 3.0 wt.% WPI in 100 mM NaCl due to aggregation of pro-
tein molecules. In conventional fast scanning calorimeters, only endo-
thermic transitions are observed (Fitzsimmons et al., 2007) as they
occurred in the present study. It is also worthy to note that aggregation
of denatured protein molecules can occur solely in protein solutions;
while, the WPI powder analyzed in the present study could merely
undergo some heat-induced transitions in the conformational topology
of protein molecules.
Interestingly, no endotherm occurred during heat scanning of
particulated proteins most probably because of the heat treatment
already applied to proteins during particle preparation. A similar
result has been reported in preparation of WPI nanoparticles via
microemulsification of native WPI followed by heat gelation
(Zhang & Zhong, 2010).
Extract-free nanoparticles showed no apparent endothermic
peak when heated in DSC apparatus; however, several mild endo-
and exothermic peaks were recorded for extract-loaded particles.
The endotherms suggest that extract-loaded particles underwent
some glass transition phenomena during which the glassy particles
became rubbery by the thermal energy supplied. Particulation of
heat-treated whey proteins by antisolvent resulted in a homogenous
amorphous assembly. However, the matrix of assembled particles
was interrupted by the presence of core biomaterials including poly-
phenols. Therefore, the less organized assemblies of extract-loaded
particles tended to mobilize by lower thermal energy when thermal-
ly scanned during DSC analysis. The recorded small exotherms may
refer to a number of heat-induced complicated transitions in extract
ingredients.
4. Conclusions
WPI extract-free and pit extract-loaded particles were successful-
ly prepared at alkaline conditions. Heat-treatment of whey proteins
before desolvation process decreased the mean size and increased
the monodispersity and particle yield of extract-free and extract-
loaded particles. A noticeable efficiency for pit extract entrapment
inside the WPI particles was achieved. However, a higher mass
ratio of core to matrix-forming protein produced a lower yield of
particulation and encapsulation efficiency. It was argued that
because of over-loading protein–protein and polyphenol–protein
interactions were weakened at the higher extract-to-WPI ratio.
FTIR results evidenced no covalent attachment between pit extract
and WPI in extract-loaded particles.
Acknowledgements
The authors would like to thank Zam Zam Co. (Tehran, Iran) for
providing the financial support for this project.
References
Arts, I. C. W., Van de Putte, B., & Hollman, P. C. H. (2000). Catechin contents of
foods commonly consumed in The Netherlands. 1. Fruits, vegetables, staple
foods, and processed foods. Journal of Agricultural and Food Chemistry, 48,
1746–1751.
Bayler, D. M., & Purcell, J. M. (1986). FTIR examination of thermal denaturation and gel
formation in whey proteins. SPIE Fourier Transform Spectroscopy, 1145, 415–417.
Bell, L. N. (2001). Stability testing of nutraceuticals and functional foods. In R. E. C.
Wildman (Ed.), Handbook of nutraceuticals and functional foods (pp. 501–516).
New York: CRC Press.
Bilati, U., Allémann, E., & Doelker, E. (2005). Development of a nanoprecipitation
method intended for the entrapment of hydrophilic drugs into nanoparticles.
European Journal of Pharmaceutical Sciences, 24, 67–75.
Eissa, A. S. (2012). Newtonian viscosity behavior of dilute solutions of polymerized
whey proteins. Would viscosity measurements reveal more detailed molecular
properties? Food Hydrocolloids, 30, 200–205.
Emmambux, N. M., & Taylor, J. R. N. (2003). Sorghum kafirin interaction with various
phenolic compounds. Journal of the Science of Food and Agriculture, 83(5), 402–407.
Fitzsimmons, S. M., Mulvihilla, D. M., & Morris, E. R. (2007). Large enhancements in
thermogelation of whey protein isolate by incorporation of very low concentration
of guar gum. Food Hydrocolloids, 22, 575–586.
Geara, C. (1999). Study of the gelation of whey protein isolate by FTIR spectroscopy and
rheological measurements. M.Sc. thesis, Montreal, Canada: McGill University.
Gülseren, I., Fang, Y., & Corredig, M. (2012a). Whey protein nanoparticles prepared
with desolvation with ethanol: Characterization, thermal stability and interfacial
behavior. Food Hydrocolloids, 29, 258–264.
Gülseren, I., Fang, Y., & Corredig, M. (2012b). Zinc corporation capacity of whey protein
nanoparticles prepared with desolvation with ethanol. Food Chemistry, 135,
770–774.
Gunasekaran, S., Ko, S., & Xiao, L. (2007). Use of whey protein for encapsulation and
controlled delivery applications. Journal of Food Engineering, 83, 31–40.
He, L., Mu, C., Shi, J., Zhang, Q., Shi, B., & Lin, W. (2011). Modification of collagen with a
natural cross-linker, procyanidin. International Journal of Biological Macromolecules,
48(2), 354–359.
Kretschmer, C. B. (1957). Infrared spectroscopy and optical rotatory dispersion of zein,
wheat gluten and gliadin. The Journal of Physical Chemistry, 61(12), 1627–1631.
Kumar, M. N. V. R. (2000). Nano and microparticles as controlled drug delivery devices.
Journal of Pharmacy & Pharmaceutical Science, 3(2), 234–258.
Langer, K., Balthasar, S., Vogel, V., Dinauer, N., Briesen, H. V., & Schubert, D. (2003).
Optimization of the preparation process for human serum albumin (HSA)
nanoparticles. International Journal of Pharmaceutics, 257, 169–180.
Lesschaeve, I., & Noble, A. C. (2005). Polyphenols: Factors influencing their sensory
properties and their effects on food and beverage preferences. The American Journal
of Clinical Nutrition, 81(Suppl.), 330S–335S.
Liu, N., & Park, H. J. (2009). Chitosan-coated nanoliposome as vitamin E carrier. Journal
of Microencapsulation, 26, 235–242.
Moraru, C. I., Panchapakesan, C. P., Huang, Q., Takhistov, P., Liu, S., & Kokini, J. L. (2003).
Nanotechnology: A new frontier in food science. Food Technology, 57, 24–29.
Nicolai, T., Britten, M., & Schmitt, C. (2011). β-Lactoglobulin and WPI aggregates:
Formation, structure and applications. Food Hydrocolloids, 25, 1945–1962.
Papiz, M. Z., Sawyer, L., Eliopoulos, E. E., North, A. C. T., Findlay, J. B. C., Sivaprasadarao,
R., et al. (1986). The structure of beta-lactoglobulin and its similarity to plasma
retinol-binding protein. Nature, 324, 383–385.
Proestos, C., Bakogiannis, A., Psarianos, C., Koutinas, A. A., Kanellaki, M., & Komaitis,
M. (2005). High performance liquid chromatography analysis of phenolic
substances in Greek wines. Food Control, 16, 319–323.
Qi, P. X., & Onwulata, C. I. (2011). Physical properties, molecular structures, and protein
quality of texturized whey protein isolate: Effect of extrusion temperature. Journal
of Agricultural and Food Chemistry, 59, 4668–4675.
Scalbert, A., & Williamson, G. (2000). Dietary intake and bioavailability of polyphenols.
Journal of Nutrition, 130, 2073S–2085S.
Seo, J. A., Hédoux, A., Guinet, Y., Paccou, L., Affouard, F., Lerbret, A., et al. (2010).
Thermal denaturation of beta-lactoglobulin and stabilization mechanism by treha-
lose analyzed from Raman spectroscopy investigations. The Journal of Physical
Chemistry. B, 114(19), 6675–6684.
Sessa, D. J., Mohamed, A., & Byars, J. A. (2008). Chemistry and physical properties of
melt-processed and solution-cross-linked corn zein. Journal of Agricultural and
Food Chemistry, 56(16), 7067–7075.
Shah, S., Pal, A., Kaushik, V. K., & Devi, S. (2009). Preparation and characterization of
venlafaxine hydrochloride-loaded chitosan nanoparticles and in vitro release of
drug. Journal of Applied Polymer Science, 112(5), 2876–2887.
Shui, G., & Leong, L. P. (2006). Residue from star fruit as valuable source for functional
food ingredients and antioxidant nutraceuticals. Food Chemistry, 97, 277–284.
 L. Bagheri et al. / Food Research International 51 (2013) 866–871
Silverstein, R. M., Webster, F. X., & Kiemle, D. J. (2005). Spectrometric identification of
organic compounds (7th ed.). New York: John Wiley & Sons Inc.
Singleton, V. L., & Rossi, J. A., Jr. (1965). Colorimetry of total phenolics with
phosphomolybdic–phosphotungstic acid reagents. American Journal of Enology
and Viticulture, 16, 144–158.
Taylor, J., Taylor, J. R. N., Belton, P. S., & Minnaar, A. (2009). Kafirin microparticle
encapsulation of catechin and sorghum condensed tannins. Journal of Agricultural
and Food Chemistry, 57(16), 7523–7528.
Wu, Y., Luo, Y., & Wang, Q. (2012). Antioxidant and antimicrobial properties of essential
oils encapsulated in zein nanoparticles prepared by liquid–liquid dispersion method.
LWT — Food Science and Technology, 48, 283–290.
Yazaki, Y., & Hillis, W. E. (1977). Components of the extractives from Callitris columellaris
F. Muell. heartwood which affect termites. Holzforschung, 31(6), 188–191.
871
Zhang, W., & Zhong, Q. (2010). Microemulsions as nanoreactors to produce whey protein
nanoparticles with enhanced heat stability by thermal pretreatment. Food Chemistry,
119, 1318–1325.
Zhong, Q., & Jin, M. (2009). Zein nanoparticles produced by liquid–liquid dispersion.
Food Hydrocolloids, 23, 2380–2387.
Zhong, Q., Tian, H., & Zivanovic, S. (2009). Encapsulation fish oil in solid zein particles by
liquid–liquid dispersion. Journal of Food Processing and Preservation, 33, 255–270.
Zou, T., Li, Z., Percival, S. S., Bonard, S., & Gu, L. (2012). Fabrication, characterization, and
cytotoxicity evaluation of cranberry procyanidins-zein. Food Hydrocolloids, 27,
293–300.