Food Hydrocolloids 25 (2011) 1098e1104

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

Fabrication and characterization of TiO2/whey protein isolate nanocomposite film

Yanxia Li, Yanfeng Jiang, Fei Liu, Fazheng Ren, Guanghua Zhao, Xiaojing Leng*
CAU&ACC Joint-Laboratory of Space Food, Key Laboratory of Functional Dairy Science of Beijing and Ministry of Education, College of Food Science & Nutritional Engineering, China
Agricultural University, No.17 Qinghua East Road, Haidian, Beijing 100083, China

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

Article history: Composite films of whey protein isolate and TiO2 are formed through three simultaneous processes,
Received 23 August 2010 i.e., the self-assembly of proteineprotein, TiO2eTiO2, and the association of proteineTiO2. All the
Accepted 15 October 2010 processes could be controlled by adjusting TiO2 concentration in the blended system. The self-assembly
of proteineprotein molecules constituted the main network of the composite film. A low TiO2 concen-
Keywords: tration (<0.25%) dispersed the TiO2 nanoparticles in the protein matrix, reinforced the association of
Self-assembly
proteineTiO2, reduced the ability of UVC absorption, and promoted the fluorescence and tensile strength
Titanium oxide
of the composite films. In contrast, a high TiO2 concentration (>0.25%) enhanced the self-assembly of
Whey protein isolate
Edible film
TiO2eTiO2 nanoparticles, brought fluorescent quenching, and produced a decline of the tensile strength
and water vapor permeability. The transmittance of the visible, UVA, and UVB lights showed a first order
exponential decay relative to the TiO2 concentration.
Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction activities) of edible films (Chaurasia, Chand, & Bajpai, 2010;

As mentioned by many authors, edible biodegradable coatings
or packaging films for serving ware act as barriers of water, oxygen
and light-radiation to improve the food shelf time become more
and more attractive during the past decades (Meyers, Chen, Lin, &
Seki, 2008; Ruiz-Hitzky, Darder, Aranda, & Ariga, 2010; Seydim &
Sarikus, 2006). Among them, how to avoid food spoilage caused
by light-induced oxidation is one of the most concerns in food
industries. Edible films have been made with various biopolymers,
including soy, starch, myoglobin, and whey proteins (WP) (Avella
et al., 2005; Chen, Remondetto, Rouabhia, & Subirade, 2008;
Jiang, Li, Chai, & Leng, 2010; Qi, Honma, Ichihara, & Zhou, 2006).
The latter, a group which represents byproducts from cheese-
making, has received particular attention, because it has high
nutritional values and remarkable abilities to form emulsions,
foams, gels, and films (Foegeding, Davis, Doucet, & McGuffey,
2002). These abilities are mainly based on the protein’s self-
aggregation capacity, which, in turn, allows the formation of
different types of network microstructures. However, these films
only have limited abilities to block light.
Metal oxides are often used to improve or expand the func-
tional properties (such as anti-radiation and anti-microbial
* Corresponding author. Tel.: þ86 10 6273 7761; fax: þ86 10 6273 6344.
E-mail address: xiaojing.leng@gmail.com (X. Leng).
Pagella, Spigno, & De Faveri, 2002; Zhou, Wang, & Gunasekaran,
2009). Titanium dioxide (TiO2), an inert metal oxide, is a kind of
widely used food color additive. In compliance with the recom-
mended safe dosage (Ferin, Oberdörster, & Peney, 1992;
Oberdorster, Ferin, & Lehnert, 1994; Schulz et al., 2002), TiO2 has
been used extensively in food and cosmetic applications to block
light and give a white appearance (Feng et al., 2007; Song, Zhou,
Zhu, Ye, & Huang, 2007; Tao, Pan, Yan, Tang, & Zhu, 2007).
Moreover, due to its photocatalytic activities, TiO2 can be also used
to make biopolymer-based films to provide protection against
foodborne microorganisms and allergens in the presence of
ultraviolet radiation (Rajh et al., 1999; Zhou et al., 2009). However,
TiO2 particles are easy to aggregate and thus influence the prop-
erties of the film (Zhou et al., 2009). How to control particle
agglomeration and the physical properties of the films was
therefore the focus of the present work. The contributions of TiO2
particles in WPI composite films and in the film structural orga-
nization were analyzed using X-ray diffraction (XRD), light scat-
tering spectroscopy, scanning electron microscopy (SEM), Raman
spectroscopy and fluorescence spectroscopy. Concentration effects
of TiO2 nanoparticles and agglomerates on the physical properties
of the composite films were emphasized. For the terminology in
this work, “self-assembly” was used to describe a process in which
the particles formed an organized structure or pattern, but in
“self-aggregation” process the formed structure or pattern was
still disordered.

0268-005X/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodhyd.2010.10.006
 Y. Li et al. / Food Hydrocolloids 25 (2011) 1098e1104
2. Materials and methods
2.1. Materials
Whey protein isolate (WPI, 98 wt% protein) was obtained from
Davisco Foods Intl., Inc. (Eden Prairie, Minn., U.S.A.). TiO2 nano-
particles (anatase, purity > 98.5%) were purchased from Joint-
venture Shanghai Jiang Hu Industry Co., Ltd. (China). All other
reagents were of analytical grade. Distilled water was used for all
sample preparations.
2.2. Film preparation
WPI solutions (10%, w/w) were mixed for 2 h at pH 8 adjusted
with NaOH; then heated at 90  C for 30 min in a water bath.
Glycerol as plasticizer for film was added to the WPI solutions
before degassing under vacuum. The mass ratio of WPI/glycerol
was 2:1. Solutions were then cooled at room temperature, and
a vacuum was applied to remove dissolved air. TiO2 nanoparticles
were added at 0.10, 0.25, 0.50, 1.0 and 2.0% (TiO2/WPI mass ratio). In
order to have a good dispersion in protein solution, the addition of
TiO2 was very slow with intense stirring and ultrasonic dispersion
at the same time. Five grams of the film-forming solution was cast
onto a rimmed Plexiglas plate with 8 cm internal diameter. The WPI
film solution was dried at 22  3  C and 56  8% RH for 18 h. Dried
films were peeled and stored at 22  3  C and 56  8%.
2.3. Light transmittance measurements
Transmittance of the films was scanned in the range of
200e800 nm using a UVmini-1240 spectrophotometer (Shimadzu,
Kyoto, Japan). Film specimen was cut into a rectangle piece and
placed in a spectrophotometer test cell directly, and air was used as
reference (Pérez-Mateos, Montero, & Gómez-Guillén, 2009).
Measurements were performed at least in three replicates.
2.4. Morphology measurements
The morphology of the surface and the cross-section of the films
were examined by means of Scanning Electron Microscopy (High
resolution cold emission scanning electron microscope Hituchi
S-5500, Japan). Films were mounted on aluminum stubs using
high-purity silver paste and carbon paint. All samples were
examined using an accelerating beam at a voltage of 5 kV.
2.5. Size and crystal structure measurements
The measurements of particle size in film-forming solutions
were carried out with a Delsa-Nano Particle Analyzer (Beckman
Coulter Inc, USA). The measurements of the dried TiO2 nanoparticle
size were observed using Transmission Electron Microscopy (TEM,
Hitachi 7650B, Japan) operating at 80 kV. The crystal structures of
the pure TiO2 nanoparticles, WPI and TiO2/WPI composite films
were obtained using a D8 Advance Polycrystal X-Ray diffractometer
(Bruker Co., Germaby) with a nickel-filtered Cu Ka radiation beam
in the angular range of 6e70 (2q) at a voltage of 40 kV and current
of 40 mA.
2.6. FT-Raman spectroscopy
FT-Raman spectra were obtained using a LabRAM HR-800
spectrophotometer (HJY Inc., France). Spectral resolution was set at
2 cm1 and laser power at 0.3 mW. Raman spectra were produced
over the range of 0e3500 cm1. The spectra were analyzed using
Origin V8.0 software (Origin-Lab, Northampton, MA). The baseline
1099
of the spectra was approximated by a straight line using the points
enveloping the regions of amide I (1700e1600 cm1), amide II
(1580e1480 cm1) and amide III (1300e1230 cm1). Calculation of
Raman intensity ratio was based on the average of triplicate
measurements at least. The spectra herein reported were all-orig-
inal and were not smoothed but normalized using the phenylala-
nine peak near 1003 cm1 (Dàvila, Parés, & Howell, 2006; Ngarize,
Herman, Adams, & Howell, 2004).
2.7. Intrinsic fluorescence spectroscopy
Sample films were cut into strips in 45  9 mm and then
attached to one side of a colorimetric cup. The fluorescence of WPI
was measured at room temperature (25  1  C) using a Varian Carry
50 fluorospectrophotometer (Varian, USA). The measurements
were performed using an excitation wavelength of 290 nm and
emission spectra from 305 to 450 nm (Kulmyrzaev & Dufour, 2002;
Garimella Purna, Prow, & Metzger, 2005) with slits of excitation and
emission of 10.0 and 20.0 nm, respectively. Three scans were per-
formed on each sample.
2.8. Mechanical properties measurements
Film thickness was determined using a Digital micrometer
(Cheng-Du-Cheng-Liang Co., China). For each film, the values
obtained at ten different locations were averaged. The tensile and
elongation of the films were determined using a texture analyzer
TMS-Pro (FCT Co. Ltd., American) according to ASTM D638M
(ASTM, 1993). Sample films were cut into strips with 6 mm wide.
The ends of the strip were mounted between cardboard grips using
double-sided adhesive tape and the exposed film area was
40  6 mm. Initial grip separation was set at 50 mm and crosshead
speed was set at 1 mm/s. Tensile strength (TS, MPa) was calculated
by dividing the maximum load by the initial cross-sectional area of
the specimen. Elongation (E, %) was calculated as the percentage of
the change of the initial length of the specimen (50 mm) at the
point of a sample failure. Measurements were performed at least in
five replicates.
2.9. Water vapor permeability (WVP), moisture content (MC)
and film solubility (S)
The methods to determine WVP, MC and S of the films were
described in our previous work (Chai, Shang, Jiang, Ren, & Leng,
2010; Jiang et al., 2010). For WVP, the films were sealed onto
a polymethyl methacrylate cup filled with significant amount of
silica gel to make the air gap within the cell less than 5 mm. This
cup was placed in a Constant Temperature and Humidity Chamber
(Ning-Bo-Dong-Nan-Yi-Qi Co., China) at 22  1  C and 56  2% RH.
Measurements of each sample were performed at least in three
replicates. MC was determined by drying small filmstrips in oven at
105  C for 24 h. Eight MC measurements were conducted for each
sample, and the average was taken as result.
2.10. Statistical analysis
Statistical data were analyzed using Origin 8.0 and SPSS 16.0.
Statistics on a completely randomized design were performed
using the General Linear Models procedure with the One-way
analyses of variance (ANOVA). Duncan’s Multiple Range Test
(P < 0.05) was used to detect differences among film property
mean values.
1100 Y. Li et al. / Food Hydrocolloids 25 (2011) 1098e1104
3. Results and discussions
3.1. Film images and light transmittance
The photographs of one pure WPI film (a) and two TiO2/WPI
composite films (b) containing 0.1% TiO2 and (c) containing 2% TiO2
are shown in Fig. 1. The pure WPI film was transparent. The film
containing 0.1% TiO2 was translucent. The film containing 2% TiO2
was opaque with a white color. The variation of the transmittance
(T) of the WPI films containing different TiO2 concentrations in the
200e800 nm wavelength range is shown in Fig. 2. The inset image
shows the enlarged UV region (200e300 nm). In Fig. 2b, the
T variations of the films in the UVC region were plotted versus the
TiO2 concentrations in the composite systems. The inset images
represented the UVA, UVB, and visible regions, respectively, and the
vertical axis were in logarithmic form. Moreover, data were
extracted from Fig. 2a. The pure WPI film had 87e91% trans-
mittance in the visible region (400e700 nm) and 30e83% trans-
mittance in the UVA and UVB region (300e400 nm) but only less
0.06% transmittance in the UVC region (200e300 nm) (Fig. 2a). The
UVC block ability was mainly attributed to the UV-absorption
ability of the aromatic amino acid residues, tyrosine and trypto-
phan, in the protein structure. The addition of TiO2 led to the
decline of T in the visible, UVA, and UVB regions, and these declines
showed a first order exponential decay versus TiO2 concentration
(Fig. 2b, inset). However, T variation in the UVC region was
abnormal. Here, T reached the maximum values with an addition of
0.1% TiO2 (Fig. 2b). This phenomenon suggested that the UV-
absorption ability of tyrosine and tryptophan was modified due to
the changes of p electron energy levels caused by certain interac-
tions with TiO2 nanoparticles.
3.2. Scanning electron microscope (SEM) photographs
The SEM micrographs of the surface and cross sections of the
pure WPI and the TiO2/WPI composite films are shown in Fig. 3. The
surface morphology of the pure WPI film was smooth and
homogenous (Fig. 3a). With less than 0.25% TiO2, only small TiO2
particles were observed on the surface. When the TiO2 concentra-
tion was more than 1%, many large agglomerates (most between
0.5 and 5 mm) covered the whole surface of the film, which became
very coarse (Fig. 3b). The agglomerates were also found to be
connected together by the protein molecules, and the individual
particles became undistinguished (inset image in Fig. 3c).
The cross-section of the pure WPI film, where the microstruc-
ture network of the film was constituted with fine-stranded and
twisted wormlike chain, are shown in Fig. 3d. At pH 8, the nega-
tively charged protein molecules performed specific adsorptions
and self-assembled in a regular chain-shaped structure as denoted
in our previous work (Jiang et al., 2010). TiO2 nanoparticles are
Fig. 1. Photographs of the pure WPI film (a) and TiO2/WPI composite films (b: 0.1% TiO2; c: 2% TiO2).
prone to aggregation. However, with less than 0.25% TiO2, the
nanoparticles scattered in the matrix and associated with the
protein chains rather than self-aggregating (Fig. 3e).
The strong association between protein-particle happened not
only in the present case but also in other systems, such as in fetal
bovine serum (FBS)-TiO2 and human serum albumin (HAS)-TiO2
(Allouni, Cimpana, Hol, Skodvind, & Gjerdet, 2009). For the latter,
the electrostatic sites (such as carboxyl, ReCOOe) of the protein
served as bridges between the negatively charged protein and the
TiO2 surface (eTiþ4Oe) under physiologic conditions. When TiO2
concentration was more than 1.0%, the nanoparticles tended to
form large agglomerates. In this case, the network microstructure
of the protein films was seriously damaged and the whole
composite system became heterogenous with separated phases
(Fig. 3f). The enlarged figure (inset in Fig. 3f) showed that the
agglomerates that formed inside the film matrix were similar to
those on the surface (inset in Fig. 3c), i.e., in terms of the connection
of TiO2 agglomerates by protein molecules.
3.3. Light scattering analysis and X-Ray diffraction (XRD)
The light scattering measurements of WPI agglomerates with
TiO2 versus TiO2 concentrations in film-forming solutions at pH 8
and room temperature are shown in Fig. 4. In this figure, the
agglomerate size increased rapidly from 525 mm to 2850 nm when
TiO2 concentration increased from 0 to 0.25%. When TiO2 concen-
tration was more than 0.5%, the rate of size increase slowed down
and reached about 5140 nm with 2% TiO2. These data were in
accordance with the SEM observation, where the large TiO2
agglomerates were predominant in high TiO2 concentrations.
Hence, low TiO2 concentrations, where the small and individual
TiO2 particles were predominant, could better induce protein
agglomeration through proteineTiO2eprotein association. The XRD
patterns with the corresponding TEM or SEM micrographs of the
pure TiO2 nanoparticles, pure WPI film, and WPI/TiO2 composite
films were compared in Fig. 5. The inset image shows the TEM
(Fig. 5a) or SEM (Fig. 5beg) micrographs of the typical agglomer-
ates in the films. Using the pure TiO2 XRD pattern (Fig. 5a), an
anatase characteristic structure was found (2q ¼ 26, 38, 48, 55, 63,
70, and 75 ). The obtained lattice constants (Cullity & Stock, 2001),
a ¼ 3.7845 Å and c ¼ 9.5143 Å, coincided with the anatase lattice
TiO2 standard (JCPdS card nr 21-1272). Using Scherrer equation,
Dhk1 ¼ Kl/bhk1 Cosq (where K: shape factor of average crystallite; l:
wavelength of X-ray radiation; b: the full width at half-maximum of
the diffraction line; and q: Bragg’s angle), the crystallite size was
estimated to be 40e80 nm. This size distribution was in agreement
with the TEM measurements (40e100 nm). In Fig. 5, when the
concentration of TiO2 nanoparticles in the film was less than 0.25%,
XRD pattern was amorphous, but when the concentration
increased and reached 2%, the obtained pattern was almost the
 Y. Li et al. / Food Hydrocolloids 25 (2011) 1098e1104 1101

a 100 b 0.10
1000
UV-B and UV-A
0.2 WPI 100
80

T (%)
0.08

T [%]
UV-C 10
TiO2 0.1%
0.1 290 nm 1
60 270 nm 0.1
T (%) TiO2 0.25% 0.06 250 nm
0.0 0.5 1.0 1.5 2.0

T [%]
230 nm
40 0.0 100
200 250 300 TiO2 0.5% Visible
0.04

T [% ]
Wavelength (nm)

10 400 nm
20 500 nm
TiO2 1.0% 0.02 600 nm
700 nm
800 nm
1
0 TiO2 2.0% 0.0 0.5 1.0 1.5 2.0
0.00 TiO [%]

0 200 400 600 800 0.0 0.5 1.0 1.5 2.0

Wavelength (nm) TiO2 [%]

Fig. 2. Variations of the transmittance (T) of the TiO2/WPI composite films. a: T variation versus the wavelengths in the range of 200e800 nm. The inset image was for the enlarged
UV region (200e300 nm). b: T variation versus TiO2 concentrations in the UVC region. The inset images represented the UVA, UVB, and visible regions, respectively, and the vertical
axis were in logarithmic form. The data of Fig. 2b were extracted from Fig. 2a.

same as that of the pure TiO2. As depicted in the work of Jalava et al.
(1998), the primary particles of TiO2 was X-ray amorphous because
of the fractalic porous surface of the particles, but the large
agglomerates had evident characteristics of anatase crystallites. In
the present work, the results of the transmittance, SEM, light
scattering, and XRD tests suggested that it was the coverage of
protein on the surface of the TiO2 particles that reduced the crys-
tallization characteristics of TiO2 at low TiO2 concentrations. On the
other hand, self-assembled large TiO2 agglomerates formed at high
TiO2 concentration could recover their crystallization ability.
3.4. Raman spectroscopy analysis
FT-Raman spectroscopy was used to gain information about the
protein secondary structure and the molecular environment
around the protein side chains. The Raman spectra of the TiO2/WPI
composite films with different TiO2 concentrations are shown in
Fig. 6. The relative intensity values of the bands are shown in
Table 1. The strong bands in amide I at 1680 cm1 and in amide III at
1245 cm1, which are respectively assigned to the intermolecular
hydrogen-bonded anti-parallel b-sheet and b-sheet, indicated that
these structures were predominant in the secondary structure of
the proteins (Dàvila et al., 2006; Ngarize et al., 2004). The band at
1622 cm1 was assigned to the b-aggregation formed through
intermolecular hydrogen bonds for the heat-denatured proteins.
The band at 1680 cm1 showed a little split with 0.1% TiO2, while
the intensity of the band at 1622 cm1 increased slightly with 0.1%
TiO2, but decreased when TiO2 concentration was more than 0.25%.
These changes indicated the disruption of the original hydrogen
bonds and the formation of new stronger intermolecular hydrogen
bonds with 0.1% TiO2. When TiO2 concentration was more than
0.25%, the original structure was disturbed. For the band at
1245 cm1, the subtle differences were also believed to be related to
the presence of protein aggregation caused by TiO2.
The band located at 930 cm1 was assigned to the a-helix
content (Dàvila et al., 2006; Ngarize et al., 2004), the intensity of
which increased with the presence of 0.1% TiO2. The band at
1554 cm1 was an aromatic Raman mode, and the band at
760 cm1 was due to the indole-ring vibrations. Both bands were
assigned to the tryptophan residue, and their relatively high

Fig. 3. SEM micrographs of the surface and cross sections of the pure WPI and TiO2/WPI composite films. Surface: a, the pure WPI film; b, the WPI film containing 0.25% TiO2; c, the
WPI film containing 2% TiO2 (the inset image was for another location on the surface of the same film). Cross-section: d, the pure WPI film; e: the WPI film containing 0.25% TiO2; f:
the WPI film containing 2% TiO2 (the inset image was the enlarged agglomerates in the same image).
1102 Y. Li et al. / Food Hydrocolloids 25 (2011) 1098e1104

6000

4500
Size (nm)

3000

1500

0
0.0 0.5 1.0 1.5 2.0
TiO 2 (%)

Fig. 4. Light scattering measurements of WPI agglomerates with TiO2 versus TiO2
concentrations in film-forming solutions at pH 8 and room temperature.

intensities suggested that the hydrophobic residues of tryptophan
were in a buried environment with the presence of 0.1% TiO2. The
bands at 850 cm1 and 830 cm1 were assigned to the tyrosine
doublet bands, and the relative intensity ratio I855/I830 was used to
monitor the microenvironment around the tyrosine. The intensity
ratios are shown in Table 1, where this ratio attained a minimum
value with the presence of 0.1% TiO2. The intensity change of
644 cm1 was believed to be a reflection of the tyrosine side-chain
conformational changes (Liang, Chen, Chen, & Chen, 2006). More-
over, the decrease of tyrosine intensities suggested that these
residues were in a buried environment. The buried states of tryp-
tophan and tyrosine may explain why their UV-absorption ability
was reduced, as it was denoted in Section 2.1. The bands near
495 cm1 and 505 cm1 were due to the SeS stretching vibrations
of disulfide bonds in the all-gauche conformation, and the observed
increase in intensities with TiO2 demonstrated their contribution to
the formation of new networks through the interactions between
the sulfhydryl groups of WPI and the positively charged Ti4þewater
complex (Zhou et al., 2009).
3.5. Fluorescence spectroscopy analysis
Fluorescence can be used to identify the interactions among
protein and other molecules in conjugates or blends. The intrinsic
fluorescence of WPI depends on tyrosine and tryptophan residues
in the protein structures (Murillo Pulgari
cn, Molina, & Pardo, 2005).
Fig. 6. Raman spectra of the composite films with different TiO2 concentrations: a)
pure WPI; b) 0.1% TiO2; c) 0.25% TiO2; d) 0.5% TiO2; e) 1% TiO2; f) 2% TiO2.
The fluorescence spectra of the composite films with different TiO2
concentrations are shown in Fig. 7. The maximum emission wave-
length of the pure WPI film was at 341 nm, which could be slightly
affected by the film preparation procedures. With increasing TiO2
concentration from 0% to 0.1%, the fluorescence intensity increased
and attained a maximum value with a red shift of 18 nm. When TiO2
concentration continued to increase from 0.1% to 2%, a fluorescence
quenching and a red shift of 39 nm (from 341 to 380 nm) occurred.
The above phenomena may be related to the photocatalytic prop-
erties of TiO2. The photocatalytic activities and quenching effects of
TiO2 nanoparticles in the composite films are schematically illus-
trated in Fig. 8. TiO2 is a semiconductor. Its valence band is
composed primarily of the hybridized orbitals of Ti 3d with oxygen
2p, and the conduction band is the Ti 3d orbital. When TiO2 is
exposed to near-UV light, electrons (e) in the valence band are
excited to the conduction band leaving behind holes (hþ), and thus,
forming the eehþ pair (Banerjee, Gopal, Muraleedharan, Tyagi, &
Raj, 2006). In the present work, the obtained film should contain
water (Table 2) and oxygen dissolved in water and the protein
matrix. Irradiated TiO2 in the films could transfer an excited elec-
tron from the conduction band to the adjacent O2 molecules
(acceptor) and form superoxide radical anions ($O 2 ). Meanwhile,
an electron from the adjacent H2O molecules (donor) was obtained
for the empty holes in the valence band. Then, water was oxidized
and split into $OH and Hþ.
In the previous sections, the analysis of light transmittance
(Fig. 2b) and Raman spectroscopy (Fig. 6) indicated that TiO2
nanoparticles had a direct impact on tyrosine (Y) and tryptophan
(W) residues. In the case of high TiO2 concentration, the fluo-
rescence quenching could be caused by the electron transported

Table 1
Relative intensity values of the bands at the selected regions versus TiO2
concentrations.

Peak assignment TiO2 [%] 0 0.10 0.25 0.50 1.00 2.00

1
Wavenumber (cm )
SeS stretching 495 0.58 0.33 0.67 0.75 1.58 1.80
SeS stretching 505 0.42 0.25 0.33 0.92 0.75 1.11
Tyr side chain 644 0.12 0.12 0.44 0.92 1.50 1.80
Tyr doublet 850/830 1.20 0.90 1.30 1.20 1.10 1.01
Trp 760 0.45 0.44 0.41 0.25 0.17 0.20
a-helix 930 0.25 0.58 0.17 0.17 0.17 0.30
Fig. 5. Comparison of the X-Ray diffraction patterns with the corresponding TEM or
Amide III 1245 0.92 0.83 1.11 0.83 1.01 1.01
SEM micrographs of the pure TiO2 nanoparticles, pure WPI and TiO2/WPI composite
Try 1554 0.50 1.25 0.44 0.51 0.58 0.70
films: a) pure TiO2; b) pure WPI; c) 0.1% TiO2; d) 0.25% TiO2; e) 0.5% TiO2; f) 1% TiO2; g)
b-sheet 1622 0.25 0.42 0.11 0.08 0.08 0.12
2% TiO2. The inset image shows the TEM (a) and SEM (beg) micrographs of the typical
Amide I 1680 1.83 1.67 2.16 2.01 1.83 2.31
agglomerates in the films. The bars were 200 nm.
 Y. Li et al. / Food Hydrocolloids 25 (2011) 1098e1104 1103

1000 Table 2
WVP, Moisture and Solubility of the WPI and TiO2/WPI composite films.

750 0.05%
0.1% TiO2/WPI Thickness [mm] WVP  1010 MC [%] S [%]
0.25% [w/w, %] [g s1 m1 Pa1]
Intensity
0.025%
0.5% 0 102.01  2.02 a
3.19  0.08a 32.40  0.71a 17.14  0.47a
WPI
500 1% 0.1 100.02  2.88 a
3.28  0.08a 32.55  0.64a 16.97  0.81a
2% a
0.25 107.10  5.00 3.19  0.14a 36.44  0.64c 17.11  0.60a
a
0.50 101.02  3.80 3.19  0.11a 36.43  0.52c 16.04  0.40b
250 1.00 105.10  4.80 a
2.89  0.11b 34.56  1.13b 15.37  0.49c
a
2.00 109.01  3.00 2.92  0.06b 33.75  0.89b 17.15  0.81a
0 Means within a column followed by different superscript letters are significantly
300 350 400 450 different among samples (P < 0.05).
Wavelength(nm) MC ¼ moisture content; S ¼ Solubility; WVP ¼ Water Vapor Permeability.

Fig. 7. Fluorescence spectra of the composite films with different TiO2 concentrations.
The WPI concentrations were 8%.
from the excited tyrosine and tryptophan to TiO2 (Zhou et al.,
2009). The more the TiO2 nanoparticles were incorporated into
the film, the more quenching was found (Fig. 8b). The red shift
could be attributed to the result of protein unfolding and the
exposure of hydrophobic groups to water (Marangoni, Barbut,
McGauley, Marcone, & Narine, 2000). However, at a low concen-
tration of TiO2, i.e., from 0 to 0.1%, the limited holes of TiO2 were
occupied by the electrons of water molecules, which inhibited the
electron transfer from the excited WPI to TiO2. Moreover, the
protein quenching ability of oxygen (Lakowicz & Weber, 1973)
wasneutralized by the electron transfer from TiO2 to O2, and the
ability of the protein fluorescence emission was, thus, enhanced
(Fig. 8a).
3.6. Physical properties
The variation of the mechanical properties, e.g., tensile strength
(TS) and elongation (E) versus TiO2 concentration in the composite
film, is shown in Fig. 9. When TiO2 concentration varied from 0% to
0.5%, E was unchanged (P > 0.05), but when TiO2 was higher than
0.5%, E decreased from 96 to 27%. TS variation showed somewhat
different characteristics. As the TiO2 concentration was increased
into the range of 0.1e0.25%, TS increased and reached a maximum
value of 10.2 MPa. When TiO2 was higher than 0.25%, TS decreased
from 10.2 to 6.9 MPa. These data indicated that a high concentra-
tion of TiO2 reduced E and TS because the network microstructure
of the film was damaged by the large TiO2 agglomerates. In
a b
O2
·O2- Conduction band
e-
TiO2 Band gap TiO2
h+
·OH+H+ Valence band
+ Hole
H 2O
S2
contrast, a low TiO2 content (0.1e0.25%) could strengthen the film
tensile strength through proteineTiO2 association. Zhou et al.
(2009) also noticed that a high TiO2 concentration could decrease
the mechanical properties of the composite films but did not
discuss the effect of a low TiO2 concentration. The composite
system could be depicted by a three-dimensional bond matrix
constituted with metal oxide nanoparticles and protein chains. For
a given external strain, the bond sites were relaxed towards
mechanical equilibrium with their neighbours by a systematic
sequence of operations, which steadily reduced the net residual
force acting on each site (Termonia, 1990). The matrix bonds could
be broken according to their local stress. This bond matrix could be
strengthened through electrostatic attraction, hydrogen bonding,
or OeTieO bonding by adding an appropriate amount of TiO2
nanoparticles. However, this mechanically equilibrated system
could be wrecked by large TiO2 agglomerates, which could make
the bond matrix discontinuous and lead to catastrophic failure of
the film tensile strength and elongation.
The values of the water vapor permeability (WVP), moisture
content (MC), and solubility (S) of the films versus the quantity of
TiO2 in the composite system are shown in Table 2. The thickness of
the films was kept at about 105 mm in all cases (P > 0.05). WVP is
related to the micro-paths of water vapor in the network micro-
structure, S is related to the hydrophilicity of the materials, and MC
is a parameter related to the total void volume occupied by water
molecules in the network microstructure of the film. A significant
decrease of WVP (P < 0.05%) with the presence of a high concen-
tration of TiO2 (>0.5%) implied that the large water-insoluble TiO2
agglomerates were blocking the micro-paths in the network
microstructure. No regular variation of MC and S was seen with the
different TiO2 concentrations, which suggested that the present
TiO2 amounts (2%) were insufficient to alter MC and S of the
whole system.
Light
e-
16 120
E
h+
TS
+ Hole
12 90
Quenching
TS ( M Pa )

S1
W or Y
E (%)

S0 S0
Fluorescent light 8 60

TiO2 TiO2 4 30
Y
W W Y

0 0
Low TiO2 concentration High TiO2 concentration 0.0 0.5 1.0 1.5 2.0
Fig. 8. Schematic illustration of the photocatalytic activities and quenching effects of TiO2 (%)
TiO2 nanoparticles in TiO2/WPI composite films at a) low concentration of TiO2; b) high
concentration of TiO2. W: tryptophan; Y: tyrosine. The arrangement of W and Y in the Fig. 9. Variations of the mechanical properties (TS and E) of the TiO2/WPI composite
protein chains in the figure is arbitrary. films versus TiO2 concentrations.
1104 Y. Li et al. / Food Hydrocolloids 25 (2011) 1098e1104
4. Conclusions
By adjusting the concentration of TiO2 nanoparticles, the prop-
erties of the TiO2/WPI composite film, including light sensibility,
mechanical strength, and WVP, could be controlled through the
combination of the effects of proteineprotein and TiO2eTiO2 self-
assembly and the association of protein and TiO2. A low TiO2
concentration (0.1%) could: 1) reinforce the association of protein
and TiO2 nanoparticles, 2) reduce the ability of UVC absorption, 3)
raise the ability of fluorescence emission of tyrosine and tryptophan,
and 4) enhance the tensile strength of the film. In contrast, a high
TiO2 concentration led to: 1) the increase in size and crystallization
degree of TiO2 agglomerates, 2) the discontinuity of the film network
microstructure and further reduction of the tensile strength, elon-
gation, and WVP of the film, 3) fluorescence quenching, and 4)
reduced light transmittance of the film. Finally, with 1% TiO2 (FDA
maximum allowable amount for food), the obtained composite film
can block more than 70% visible light and more than 90% UV light.
Acknowledgements
This work was funded by National Science and Technology
Support Program (2006BAD27B04 & 2006BAD04A06). The authors
thank Mr. Jingzhi Wei (Analysis Center, Tsinghua University, Beijing,
China) and Mr. Yunjie Yan (Beijing national center for microscopy,
Tsinghua University, Beijing, China) for their technical assistances.
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