Carbohydrate Polymers 126 (2015) 168–174

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Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol

New product development: Cellulose/egg white protein blend fibers

Marta Tomczyńska-Mleko a , Konrad Terpiłowski b , Stanisław Mleko c,∗
a
Institute of Plant Genetics, Breeding and Biotechnology, University of Life Sciences in Lublin, Akademicka Street 15, 20-950 Lublin, Poland
b
Department of Physical Chemistry-Interfacial Phenomena, Maria Curie Skłodowska University, M. Curie Skłodowska Sq. 3, 20-031 Lublin, Poland
c
Department of Milk Technology and Hydrocolloids, University of Life Sciences, Skromna 8, 20-704 Lublin, 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 research was to form mixed cellulose/egg white isolate (EWI) fibers. Cellulose was dissolved
Received 12 January 2015 in the Schweitzer’s reagent. The blend fibers were obtained by simultaneous cellulose fiber formation
Received in revised form 4 March 2015 and acid-induced gelation of EWI in 33% sulphuric acid solution. Increased storage modulus was noted
Accepted 6 March 2015
for the blend fibers in comparison to the cellulose fibers. EWI alone formed fibers which were composed
Available online 13 March 2015
of microfibers with the average diameter of about 80 nm. Cellulose fibers had a loose microstructure
with about 10 ␮m gaps and rough surface. The addition of EWI caused that the surface of the fiber was
Keywords:
even more rough with a tendency to form microfibers, which were not observed for cellulose alone.
Egg white
Fiber
EWI protein molecules had the tendency to bridge the voids between cellulose microfibers. Protein in
Cellulose the blend fibrils formed more branched aggregates than in the EWI fibrils, which was probably caused
Schweizer’s reagent by interactions with copper ions. Both in cellulose and cellulose/EWI fibrils, the cellulose crystallized in
Microstructure cellulose II monoclinic system. Reduction in C OH groups was noted, which was probably caused by
interactions between the cellulose and proteins molecules. EWI/cellulose interactions caused formation
of ␤-sheet type structures.
© 2015 Elsevier Ltd. All rights reserved.

1. Introduction (Bryant & McClements, 1998). There is no research carried out on

Egg white powder is one of the most popular and convenient
sources of protein. Together with whey protein it is considered as
the most valuable protein with a high content of essential amino
acids. The main difference in functional properties of these protein
powders is mineral salt content. So far there has been many whey
protein isolates with low minerals content. Preheating of whey
protein isolate dispersions at the temperatures above denatur-
ation (ca. 70 ◦ C) allows to obtain whey protein polymers, i.e. linear
aggregates, which are stable at ambient temperatures (Barbut &
Foegeding, 1993; Alting, Hamer, de Kruif, & Visschers, 2000). The
addition of ions causes a “shielding effect” which changes the elec-
tric interactions between molecules. A special variation of the effect
is the addition of hydronium ions in an acidification process. Lin-
ear protein aggregates have more neutral charge and additional
hydrophobic and ionic interactions lead to additional aggregation,
this time in a more spherical form. When the protein concentration
is high enough—a three-dimensional matrix of the gel is formed
∗ Corresponding author. Tel.: +48 81 4623347; fax: +48 81 4623400.
E-mail addresses: martamleko@tlen.pl (M. Tomczyńska-Mleko),
terpil@poczta.umcs.lublin.pl (K. Terpiłowski), dairywhey@tlen.pl (S. Mleko).
ions induced egg white protein gelation as so far available egg white
protein isolates had too high minerals concentration and they gel at
the preheating process (Holt et al., 1984; Croguennec, Nau, & Brule,
2002). Already at low protein concentrations (30 g/L) a turbid gel is
formed. A desalting step is necessary to reduce the ionic strength
to about 3 mM and thus formation of possibly fibrillar structures is
possible (Weijers, Velde, Stijnman, Pijpekamp, & Visschers, 2006).
Lately some emphasis has been put on obtaining composite
polysaccharide-protein fibers due to their potential application as
packaging materials. Edible nanofibres were fabricated for the first
time from blend solutions of cellulose acetate in 85% acetic acid and
egg albumen in 50% formic acid by electrospinning (Wongsasulak,
Patapeejumruswong, Weiss, Supaphol, & Yoovidhya, 2010). Zhang,
Li, and Yu (2011) prepared novel cellulose/soy protein isolate fibers
using a direct dissolving approach.
The obtained fibers have potential application as a carrier for the
sustained release of drugs and in tissue engineering (Xu, Huang,
& Wang, 2013). The controlled release of aloe extract from the
fibers was applied to achieve the efficacies of antibacteria, dimin-
ishing inflammation, moistening skin and anti-ultraviolet light
which could be used for atopic dermatitis. Incorporation of metal
ions: silver, copper, silver, titanium or zinc in cellulose fibers
result in functional products offering wide range of applications.

http://dx.doi.org/10.1016/j.carbpol.2015.03.008
0144-8617/© 2015 Elsevier Ltd. All rights reserved.
 M. Tomczyńska-Mleko et al. / Carbohydrate Polymers 126 (2015) 168–174 169

Specialized properties such as antimicrobial, antifungal, self clean-

ing or UV protective can be achieved (Emam, Manian, Siroka, &
Bechtold, 2012).
Traditional chemical for direct cellulose dissolving is the
Schwiezer’s reagent, an aqueous ammoniacal solution of copper(II)
hydroxide. Some modifications of this method are still used in
industry and analytical chemistry (Burchard, Habermann, Klüfers,
Seger, & Wilhelm, 1994). Cellulose fiber is formed from the solu-
tion in 33% sulphuric acid. This method has never been used to
obtain cellulose/protein mixed fibers. In the following research a
new commercial egg white isolate with a low mineral content will
be used. In the egg white isolate all original egg white proteins were
preserved. The low mineral content in the isolate was achieved
using a method patented by the producer. Preliminary research
showed, that it does not gel at preheating protein dispersions up
to 6%. Gelation can be induced by changing electrical charge of the
obtained aggregates.
The aim of the research was to form mixed cellulose/egg
white protein fibers using direct dissolving of the cellulose in
the Schweitzer’s reagent and obtaining the fibers by simultaneous
forming of the cellulose fiber and acid-induced gelation of egg white
proteins in the sulphuric acid solution.

2. Experimental Fig. 1. Influence of frequency on storage and loss moduli of discs precipitated in
33% sulphuric acid.

2.1. Materials
Egg white isolate—EWI (88.1% protein) was obtained from
Kewpie Corporation (Tokyo, Japan). Protein concentration was
determined by the Kjeldahl method (AOAC, 1995). Mineral analysis
of the isolate was performed by an atomic absorption spectrome-
try using a Varian Spectra 280 FS (Varian, Inc., Palo Alto, USA). The
results were compared with the content of calcium, magnesium,
sodium and potassium in the dried egg white albumin presented
by U.S. Department of Agriculture (USDA, 2015) (Table 1).
Cellulose (MN-Cellulose powder 300, 10 ␮m particle size) was
purchased from Macherey, Nagel & Co, Düren (Germany).
2.2. Preparation of samples
2.2.1. Pre-heating of egg white isolate dispersion
Egg white isolate (6% protein w/w) was hydrated in distilled
water by mixing using a magnetic stirrer. Dispersions were heated
in water bath for 30 min at 80 ◦ C. After heating the dispersions were
cooled down in tap water.
2.2.2. Preparing of Schweizer’s reagent and obtaining fibers
100 cm3 of 25% aqueous ammonia solution (kept in a refrigera-
tor at 4 ◦ C) was added to 5 g of Cu(OH)2 (Aldrich, technical grade).
The solution was mixed for 60 min and used immediately for cel-
lulose dissolution. 3 g of cellulose was added to 30 g of Schweizer’s
reagent and mixed for 30 min. After this time 15 g of distilled
water or preheated egg white isolate dispersion was added. The
concentration of cellulose in the final solutions was 6.25% and
the concentration of EWI in the blend with cellulose was 1.875%.
The solutions were degassed for about 15–20 min using a vac-
uum pump. Deaerated solutions of preheated EWI, cellulose or
Table 1
Content of minerals in Kewpie egg white isolate and dried egg white
albumin—(DEWA).
Ca [mg/kg] Mg [mg/kg] Na [g/kg] K [g/kg]
Kewpie 198 ± 11 214 ± 19 1.57 ± 0.09 1.59 ± 0.06
DEWA 890 720 12.38 11.16
% DEWA 22.2 29.7 12.7 14.2
cellulose/EWI blend were put in a syringe with a needle and
injected slowly to a solution of 33% sulfuric acid. During the injec-
tion the dispersion precipitated into a fiber which was wound onto
a baguette. The fiber was rinsed in 1000 cm3 of distilled water,
1000 cm3 of 5% solution of ammonia and again in 1000 cm3 of
distilled water using a nylon strainer with 1 mm openings. For rhe-
ological measurements discs were obtained. The fibers were dried
for 15 h at 40 ◦ C. The fibers were dried for 48 h at 30 ◦ C and the
content of copper was evaluated by an atomic absorption spectrom-
etry using a Varian Spectra 280 FS (Varian, Inc., Palo Alto, USA). All
the experiments were replicated three times and the experiments
results were reproducible.
2.3. Dynamic oscillatory measurements
Samples were prepared as 2.5 mm thick discs precipitated in 33%
sulfuric acid and rinsed in 1000 cm3 of distilled water, 1000 cm3 of
5% solution of ammonia and again in 1000 cm3 of distilled water.
Rheological properties of wet discs were measured after an access
of water was removed using filter paper. The samples (35 mm
diameter and 2.5 mm thick) were prepared using a chirurgical
scalpel. Dynamic rheological measurements were performed using
the RS300 (ThermoHaake, Karlsruhe, Germany) rheometer with a
serrated parallel steel plate geometry (35 mm diameter, 2 mm gap
size) to limit the potentiality of sliding effects. The samples were
analyzed by frequency sweeps in the 0.1–10 Hz range in the lin-
ear viscoelastic region (at 0.01strain evaluated previously by strain
sweeps). All the measurements were performed at 21 ◦ C.
2.4. Scanning electron microscopy (SEM)
Samples of the fibers were fixed by immersion in 2.5% glu-
taraldehyde solution in 0.1 M sodium cacodylate buffer. The
samples were dehydrated in serial dilutions of ethanol and acetone
and dried at the critical point in liquid carbon dioxide. Preparations
were coated with gold using a vacuum evaporator EMITECH K550x
(Emitech, Ashford, United Kingdom). Then they were viewed and
photographed using a scanning electron microscope VEGA II LMU
(Tescan, Canberra, USA).
170 M. Tomczyńska-Mleko et al. / Carbohydrate Polymers 126 (2015) 168–174

Fig. 2. Scanning electron micrographs of fibrils: (a) cellulose, (b) cellulose fractured cross-section, (c) cellulose surface, (d) EWI, (e) EWI fractured cross-section, (f) EWI
surface, (g) cellulose/EWI blend, (h) cellulose/EWI blend fractured cross-section, (i) cellulose/EWI blend surface.

2.5. Transmission electron microscopy (TEM) 2.6. X-ray diffraction (XRD)

Samples of the fibers were cut with a scalpel into 5 mm long
pieces, fixed for 3 h at 4 ◦ C with 3% (v/v) glutaraldehyde and 2%
(w/v) paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7)
and then washed with phosphate buffer. Samples were post-fixed
in 0.5% (w/v) OsO4 for 1 h at 4 ◦ C. The samples were dehydrated
in serial dilutions of ethanol and acetone and dried at the criti-
cal point in liquid carbon dioxide. Dried fibers were embedded in
graded series of EPON (Embed 812, EMS, England) diluted in pro-
pylene oxide. Blocks were polymerized at 60 ◦ C for 12 h, then thinly
sliced (90 nm thick) using an ultramicrotome Reichert Ultracut S
(Leica Microsysteme, Wien, Austria). Ultrathin cuts were stained
using Reynolds lead citrate and uranyl acetate (Reynolds, 1963).
Microscopy observations were carried out in an FEI Tecnai Spirit
G2 microscope (FEI, The Netherlands) at an acceleration voltage of
120 kV.
The Empyrean powder diffractometer (PANalytical,
Netherlands) was used to record the X-ray diffraction (XRD)
patterns. The samples were investigated at room temperature for
10 s over the 2 theta range from 25◦ to 50◦ in steps of 0.02◦ . The
results were analyzed using High Score Plus Software (PANalytical,
Netherlands).
2.7. Infrared (IR-RAMAN) spectroscopy
The instrumentation used in this study was a Ranishaw InVia
Raman Microscope (Renishaw, Wotton under Edge, U.K.). Exci-
tation was provided using a 785 nm semiconductor laser with
300 mW output and about 30 mW at the sample. The microscope
objective used in the measurement was ×50, in a 180◦ backscat-
ter collection configuration. The obtained spectra were processed
 M. Tomczyńska-Mleko et al. / Carbohydrate Polymers 126 (2015) 168–174 171

(smoothed and baseline corrected) using the Wire 3.4 Renishaw

software.

3. Results and discussion

3.1. Rheological properties of precipitated discs

Fig. 1 shows frequency sweeps for cellulose, egg white isolate

and their mixture precipitated in 33% sulphuric acid. The lowest
values of storage and loss moduli were noted for EWI gels. The addi-
tion of this protein to the obtained cellulose using the Schweizer’s
method caused an increase in the moduli values, although the final
values of storage moduli at 10 Hz were very similar. All material
behaved as weak gels with the storage modulus value several times
higher than the loss modulus and the tendency of increase with the
increasing frequency. Higher values of storage modulus observed
for mixed cellulose/EWI precipitates are probably caused by inter-
actions between cellulose fibers and protein.

3.2. Microstructure of the fibrils

Fig. 3. Transmission electron micrograph of EWI microfibril.

EWI protein molecules formed gel with fibrous microstructure,
which had the tendency to bridge the voids between cellulose
microfibers (Fig. 2h). Intra- and intermolecular junctions were
observed previously by Lodha and Netravali (2005) for mixed
cellulose/soy protein isolate fibers. Cellulose fibers obtained in cur-
rent research had a loose microstructure with about 10 ␮m gaps
(Fig. 2b). Cellulose fibers surface had a rough structure (Fig. 2c).
The addition of EWI caused that the surface of the fiber was even
rougher with a tendency to form microfibers, which were not
observed for cellulose alone (Fig. 2i). The egg white isolate used
in the current research had a very low mineral content in compar-
ison to the standard dried egg white (Table 1). Preheating of 6%
protein EWI dispersion did not cause gelation and the pre-heated
dispersion was a material for “cold gelation” process, which is pos-
sible when the repulsive forces between negatively charged protein
molecules are shielded by cations. Obtaining EWI gels at ambient
temperatures using a low mineral content isolate was shown in the
previous research (Tomczyńska-Mleko, Nishinari, & Handa, 2014).
In current research the trigger agent to obtain gel were hydronium
ions of sulfuric acid. The obtained fibers were composed of fine-
stranded gels with a very subtle microstructure and a very smooth
surface (Fig. 2d–f). A similar microstructure was observed for ion-
induced egg white gels at pH 9.0 (Croguennec et al., 2002). Both at
low and high pH globular proteins form fine-strained gels because
of high repulsive forces between positively (low pH) or nega-
tively (high pH) charged protein molecules (Foegeding, Bowland,
& Hardin, 1990). Fig. 3 shows the TEM micrograph of the obtained
EWI fibers which are composed of microfibers with the average
diameter of about 80 nm. Similar linear aggregates were observed
for the acid-induced egg white protein gels using cryo-transmission
electron microscopy (Weijers et al., 2006). In another research TEM
micrographs showed that the fibrils formed for ovalbumin at pH 2
had a contour length of 200 nm, which was in the same order of
magnitude for all ionic strengths (Veerman, de Schiffart, Sagis, &
van der Linden, 2003). Forming a fibrillar microstructure is charac-
teristic of many globular proteins of both animal and plant origin.
Lately micrometer-long fibrillar aggregates were observed after pea
protein solutions had been heated at pH 2.0 (Munialo, Martin, van
der Linden, & de Jongh, 2014). For the EWI/cellulose fibers a very
interesting microstructure was revealed using TEM (Fig. 4). Pro-
tein in blend fibrils formed more branched aggregates than in EWI
fibrils. It was probably caused by interactions with copper ions
(Norkus, Vaiciuniene, Vuorinen, & Macalady, 2004). Regions with
different protein aggregation in the fibrils were found (Fig. 4a). Egg
white albumin is attached to the surface of the cellulose and creates
junction zones (Fig. 5). Zhang et al. (2011) hypothesized that in the
blend solution, the soy protein isolate partly destroyed the inter-
action between solvent and cellulose chains by a much stronger
interaction between amide groups of the protein, and the exposed
hydroxyl groups of cellulose prompted the formation of a new
physical cross-linking network with a bigger pore size than that

Fig. 4. Transmission electron micrograph of cellulose/EWI fibril: (a) an interface layer, (b) protein aggregation at the left side of the interface (a), (c) protein aggregation at
the right side of the interface (a).
172 M. Tomczyńska-Mleko et al. / Carbohydrate Polymers 126 (2015) 168–174

but part of the copper is complexed in EWI. Digestion of the pro-

Fig. 5. Scanning electron micrograph of the cellulose/EWI blend fibril surface.
of pure cellulose itself. The same mechanism is probably observed
for cellulose/egg white protein blends.
In current research the addition of EWI to cellulose resulted in
the microstructure with larger voids and rougher surface than for
cellulose fibrils (Fig. 2h–i vs. b–c). It seems that the fibrils could be
used as a carrier for the sustained release of copper in the human
organism. Cellulose is not metabolized in the human digestive tract,
tein would result in even more porous structure and release of
the copper. As copper is a heavy metal with very limited human
daily intake, the content of the element in the dried fibers was
evaluated using an atomic absorption spectrometry. Cellulose/EWI
fibers contained copper in the form of Cu2+ ions in the concen-
tration 0.229%, i.e. 79 mM. According to WHO the daily dietary
requirements are 1.3 mg/day and the safe upper copper intake
for adults is 10 mg daily (WHO, 1996; Trumbo, Yates, Schlicker,
& Poos, 2001). As copper is involved in etiology and therapy of
different illness as e.g. Menkes and Wilson disease, supplemen-
tation of foods with copper may be in some cases indispensable
(Navarra et al., 2009). The patients could take up to 4.37 g of cellu-
lose/EWI preparation daily. There are certainly some commercially
available supplements of copper, but the advantage of obtained
fibrils could be very slow release of copper ions in the stomach, as
copper ions are mostly inside of the fibrils and only egg white will
be hydrolyzed in the stomach (Tomczyńska-Mleko & Mleko, 2014).
Cellulose will create a backbone for ions release. In this form copper
ions have a potential to change many important cellular func-
tions and right now it is difficult to predict all applications (Santo,
Taudte, Nies, & Grass, 2008). Metallic-based materials are also
used to enhance antimicrobial, mechanical and barrier properties,

Fig. 6. X-ray diffractograms recorded on: (a) cellulose fibril, (b) cellulose/EWI blend fibril.

Fig. 7. X-ray diffractograms of the cellulose fibrils impurities.

 M. Tomczyńska-Mleko et al. / Carbohydrate Polymers 126 (2015) 168–174
Fig. 8. IR-RAMAN spectra of cellulose, egg white isolate (EWI) and cellulose + egg
white isolate fibrils.
and to prevent the photodegradation of plastics (Grace, Bajpai, &
Chand, 2009). Heavy metals are effective antimicrobials in the form
of different salts, oxides, colloids and complexes (Llorens, Lloret,
Picouet, & Fernandez, 2012b). Hassan, Amna, Yang, El-Newehy, and
Al-Deyab (2012) prepared CuO nanocrystals as a promising mate-
rial for antimicrobial applications. Cellulose/copper composites
173
synthesized by in-situ reduction of copper sulphate adsorbed on
cotton fibres showed excellent antifugal activity (Llorens, Lloret,
Picouet, & Fernandez, 2012a). Chattopadhyay and Patel (2010)
used nanosized colloidal copper to impregnate cotton fabrics. The
treatments of nano copper colloidal solution on cotton not only
improved its antimicrobial efficiency but also increased the tensile
strength of the fabric. Further research is needed to optimize the
mechanical properties of the fibrils to be applied in textile indus-
try, e.g. to create textiles with high resistance to microbiological
degradation.
3.3. Structural characterization of the fibers
Both in cellulose and cellulose/EWI fibrils, the crystal cel-
lulose was found. The XRD diffractogram showed a crystalline
peak at 2 = 21–23◦ characteristic for cellulose II monoclinic sys-
tem reported earlier by other researchers (Koyama, Helbert, Imai,
Sugiyama, & Henrissat, 1997; Hult, Iversen, & Sugiyama, 2003;
Nazir, Wahjoedi, Yussof, & Abdullah, 2013) (Fig. 6). The diffrac-
togram of the cellulose/EWI fibrils is far more complicated than
cellulose II alone, which shows that only a part of the cellulose
crystallized in the cellulose II monoclinic system. Additionally
interactions between cellulose and whey proteins caused changes
in the diffractogram. The peak at 2 = 11.9◦ of the blends dis-
appeared, suggesting that intermolecular hydrogen bonding of
cellulose was destroyed by strong interaction between EWI and
cellulose molecules (Fig. 6). Similar effect was observed by Wu,
Wang, Wang, Bian, and Li (2009) for cellulose/soy protein isolate
blend films. We additionally found, that extra peaks in the XRD
data presented in Fig. 6 come from impurities, i.e. microcrystals
of triammonium hydrogen disulfate and ammonium cupric sulfate
hexahydrate (Fig. 7).
Fig. 8 presents the IR-RAMAN spectra recorded on the fibers.
There was a higher peak intensity of the ␤-sheet structure at
976 cm−1 in the experimental spectra of the blend indicating
that EWI/cellulose interactions caused further formation of ␤-
sheet type structures. For cellulose/EWI blend fibrils the peak at
1078 cm−1 representing C OH groups is much smaller than for
cellulose alone, which is probably caused by interactions of cel-
lulose with the egg white proteins (Ngarize, Adams, & Howell,
2004). A strong band observed in cellulose at 1347 cm−1 which
corresponds to bending vibrations of C OH groups is divided and
decreased in the blend which suggests reduction in these groups
caused by interactions between cellulose and proteins molecules
(Lobo & Bonilla, 2005). In cellulose spectrum there is no band at
1113 cm−1 , which is characteristic of cellulose I but not of cellulose
II and amorphous cellulose (Nelson & O’Connor, 1964). For proteins
spectra the most important are the amide I vibration, representing
stretching of the C O bonds, and the amide II vibration, showing
deformation of the N H bonds and stretching of the C N bonds.
The characteristic amide I vibration band was found at 1670 cm−1
and the amide II one at 1462 cm−1 (Fig. 8). The absence of band
at 1692 cm−1 characteristic for the native state of protein proves
that EWI protein is denatured. Bands characteristic of C N and C C
stretching were observed at 1051 and 1078 cm−1 .
4. Conclusions
For the first time mixed cellulose/EWI fibers were obtained.
Future research will be concentrated on investigation of release
kinetics of the copper from the preparation in an artificial stomach
and optimization of the mechanical properties of the fibrils. Fibrils
with good tensile properties could be applied in textile industry, e.g.
to create textiles with high resistance to microbiological degrada-
tion.
174 M. Tomczyńska-Mleko et al. / Carbohydrate Polymers 126 (2015) 168–174
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