Eur Food Res Technol (2010) 231:181–188
DOI 10.1007/s00217-010-1263-1
ORIGINAL PAPER
Egg white lysozyme puriWcation with a chitin–silica-based aYnity
chromatographic matrix
Federico J. Wolman · Guillermo J. Copello · Andrea M. Mebert ·
Alexandra M. Targovnik · María V. Miranda · Agustín A. Navarro del Cañizo ·
Luis E. Díaz · Osvaldo Cascone
Received: 24 December 2009 / Revised: 8 March 2010 / Accepted: 15 March 2010 / Published online: 2 April 2010
© Springer-Verlag 2010
Abstract A composite biosorbent retaining non-
covalently bound chitin in between layers of a silicon oxide
matrix was assessed for lysozyme puriWcation from undi-
luted egg white. The matrix can be shaped with big size and
high density, thus allowing its eYcient separation from the
egg white after the adsorption step. The lysozyme-depleted
egg white can follow its usual commercialisation route.
A surface area of 142 m2/g and a total pore volume of
0.295 cm3/g were calculated from the nitrogen sorption iso-
therms. Its water content was 78.3%. The matrix structure
is the result of the polysaccharide addition to the polymeri-
sation mixture, which is known to inXuence the condensa-
tion process, leading to a material with characteristic
properties. A maximum capacity of 117.1 § 9 mg lyso-
zyme/g and a dissociation constant of 0.73 § 0.15 mg/mL
were calculated from the Langmuir isotherm. A lysozyme
puriWcation batch process from undiluted egg white was
developed, where 87% of the lysozyme was removed from
the egg white and the matrix was easily recovered by a sim-
ple Wltration through a strainer. The overall yield of the pro-
cess was 64% with a puriWcation factor of 20.
Keywords Egg white · Lysozyme · Chromatographic
matrix · Extraction · PuriWcation
F. J. Wolman · G. J. Copello · A. M. Mebert · A. M. Targovnik ·
M. V. Miranda · A. A. Navarro del Cañizo · L. E. Díaz ·
O. Cascone (&)
Facultad de Farmacia y Bioquímica,
University of Buenos Aires, Junín 956 (1113),
Buenos Aires, Argentina
e-mail: ocasco@ffyb.uba.ar
Introduction
An important challenge of protein downstream processing
is to have chromatographic matrices with high adsorption
capacity, high aYnity, eYcient elution of the adsorbed pro-
tein and good hydrodynamic behaviour. Besides, the per-
formance/cost ratio is critical when the usefulness of the
process is assessed [1]. With respect to hydrodynamic
parameters, not all commercial matrices are useful when
the raw starting material is complex. The presence of par-
ticulate material, the high viscosity and the presence of fat
bring about the need of clariWcation steps or conditioning of
the samples.
On the other hand, for puriWcation processes in batch, in
addition to contact time optimisation, matrix recovery after
the adsorption step becomes diYcult when the raw material
is not clear and its viscosity is high. To overcome this prob-
lem, diVerent approaches such as magnetic particles were
developed with success at a laboratory scale [2]. However,
they are expensive and their adsorptive capacity is rather
low; therefore, the beneWt/cost ratio is not too attractive. In
this work, a novel chromatographic matrix made of a
copolymer of chitin and silicon oxide was developed.
Chitin (Poly -(1 ! 4)-2-acetoamido-2-deoxy-D-glucose)
is the second more abundant polymer in nature. It is syn-
thesised by a great variety of species, being the exoskeleton
of arthropods the main source [3]. In spite of being a poly-
mer similar to cellulose, it has an acetamide group in posi-
tion C-2 instead an hydroxyl group, thus giving it unique
properties. Chitin is biodegradable, biocompatible, has high
chemical resistance, low immunogenicity and low cost [4];
these attributes make it a very interesting material for
chromatographic processes. Its deacetylated derivative—
chitosan—has also been used for chromatographic
purposes [5–7].
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182
From the chromatographic point of view, mechanic
resistance of the matrix must be adequate, especially for
batch processes where stirring is required. Sol–gel chemis-
try avoids the cross-link reagents that block reactive groups
of the polymer, which usually brings about a decrease in
the amount of immobilised ligand and, therefore, a concom-
itant decrease in protein adsorption capacity [8]. Sol–gel
method has been widely used in the obtention of polymeric
hybrid materials containing inorganic and organic moieties.
This is because of the mild conditions of the polymerisation
reaction, which include room temperature operation and
polymerisation at physiological compatible pH, among
other advantages [9–11]. Recently, the sol–gel method was
used for obtaining gels, xerogels and Wlms composed of
polysaccharides and silicon oxide [9, 12–15]. Examples of
polysaccharides immobilised in silicon oxide networks are
chitosan, carrageen, cyclodextrin and carboxymethylcellu-
lose [16, 17]. Spicules of R. Wbulata are an excellent example
of a naturally occurring silica–chitin composite responsible
for the observed high mechanical stability and resistance
[18]. In the present study, a composite biosorbent retaining
non-covalently bound chitin in between layers of a silicon
oxide matrix was assessed for lysozyme puriWcation from
egg white.
Lysozyme has a potent antimicrobial eVect due to the
hydrolysis of the  unions between muramic acid and
N-acetyl glucosamine present in the microbial walls. It
Wnds a lot of applications in food and medical areas. Egg
white is the main source of lysozyme, containing around
3.5% of this protein. Other main proteins present in egg
white are ovoalbumin, conalbumin, ovotransferrin, G2 and
G3 globulins and Xavoprotein.
Most lysozyme puriWcation processes take advantage of
the high isoelectric point of the protein by using ion
exchange chromatography [19–22]. Other processes make
use of the aYnity interaction between lysozyme and
N-acetyl-D-glucosamine monomers of chitin [23–25] or its
aYnity for dyes [26–28]. However, in almost all of these
processes dilution of the egg white is required in order to
obtain high adsorption capacity, thus preventing its further
utilisation as food additive.
We herein present a novel chromatographic matrix with
high lysozyme adsorption capacity and excellent mechani-
cal resistance. It can be shaped with big size and high den-
sity, thus allowing an eYcient separation of the matrix from
the egg white after the adsorption step. The lysozyme-
depleted egg white can follow its usual commercialisation
route.
In this work, we characterise chemically the copolymer
chitin–silicon oxide, we assessed its thermodynamic
parameters related to lysozyme adsorption and propose a
process for lysozyme separation and puriWcation from undi-
luted egg white.
123
Eur Food Res Technol (2010) 231:181–188
Materials and methods
Materials
Tetraethoxysilane (TEOS) and chitin (molecular weight
400,000) were from Fluka (Buchs, Switzerland and USA,
respectively). Lysozyme was from Sigma–Aldrich (Saint
Louis, MO, USA). All other reagents were AR grade.
Matrix preparation
A 5% chitin hydrogel was prepared by a modiWcation of the
procedure described previously for cellulose [29]. BrieXy, a
5% chitin solution was prepared by dissolving chitin powder
in methanol saturated with CaCl2. Then, 10 mL chitin solu-
tion was added to 200 mL distilled water under vigorous stir-
ring. Afterwards, the suspension was centrifuged to collect
the hydrogel in 10 mL. Finally, the hydrogel was washed
with 0.2 M potassium hydrogen phthalate, pH 5. A TEOS
solution was prepared by sonicating (Transonic 540 sonica-
tor, 35 kHz) a mixture of 10 mL TEOS, 0.6 mL 0.05 M HCl
and 2 mL water for 30 min at 20 °C. Equal volumes of TEOS
solution and hydrogel were then mixed. Polymerisation took
place at 25 °C and the matrix was sectioned in 2 £ 2 mm
beads. Prior to the adsorptive step, the matrix was washed 3
times (8 h each) with 50 mM sodium phosphate buVer, pH
8.0, 50 mM NaCl, to allow its ageing and to eliminate the
remaining alcohol released by the TEOS hydrolysis.
Matrix characterisation
Infrared spectrum: ATR-FTIR transmission spectra were
acquired in the range of 4,000–650 cm¡1 using a Fourier
transform infrared spectrometer (FT-IR) with a Xat-plate
attenuated total reXectance (ATR) (Perkin Elmer, Spectrum
One IR). The matrix was previously dried at 60 °C for 24 h
to preclude water-related bands interference.
Scanning electron microscopy-energy dispersive X-ray
analysis: Samples were analysed using a Zeiss Supra 40
microscope for scanning electron microscopy (SEM).
Elemental analyses were carried out by using an energy dis-
persive analyser (EDS) (Oxford Instruments).
Nitrogen adsorption isotherm: Before adsorption, the
sample was degassed at 50 °C for 24 h under high vacuum.
Nitrogen adsorption and desorption isotherms were per-
formed in duplicate at 77.7 K employing nitrogen Mathe-
son spectroscopic grade. The speciWc surface area (SBET)
and total pore volume (TPV) were estimated by the
Brunauer–Emmett–Teller (BET) and Barrett–Joyner–
Halenda (BJH) methods, respectively.
Matrix water content: Matrix water content was deter-
mined with a moisture analyser at constant temperature
(120 °C) (MX-50, A&D Company, Tokyo, Japan).
Eur Food Res Technol (2010) 231:181–188
Pure lysozyme adsorption isotherm
About 50 mg matrix was soaked in 1 mL lysozyme solu-
tions (0.125–10 mg/mL in adsorption buVer: 50 mM
sodium phosphate buVer, pH 8.0, 50 mM NaCl). The
suspensions were gently agitated overnight at 20 °C to
allow the system to reach its equilibrium. Lysozyme con-
centration in supernatants at equilibrium was then mea-
sured spectrophotometrically at 280 nm. The equilibrium
concentration of lysozyme bound to the matrix per unit of
total matrix volume was calculated spectrophotometrically
as the total amount of enzyme present at the beginning of
the experiment less the amount still in the soluble phase at
equilibrium.
Adsorption kinetics of pure lysozyme
1 g matrix was soaked in 10 mL lysozyme solution (3 mg/
mL in adsorption buVer). The suspension was gently agi-
tated and samples were taken at 0, 30, 60, 90, 120, 180,
240, 300, 360, 420, 480, 540, 600 and 1,440 min. Lyso-
zyme concentration in the supernatants was measured spec-
trophotometrically at 280 nm. The amount of protein bound
to the matrix was calculated as the amount present at the
beginning less the amount still present in the soluble phase.
Lysozyme adsorption kinetics from egg white
About 1 g matrix was incubated with 10 mL egg white for
24 h at room temperature with gentle agitation. Samples
were taken at 0, 30, 60, 90, 120, 180, 240, 300, 360, 420,
480, 540, 600 and 1,440 min. Adsorbed lysozyme concen-
tration was calculated by measurement of lysozyme activity
as the diVerence between the amount of lysozyme in the
egg white and its amount in the supernatant.
Elution studies
About 50 mg matrix was saturated with lysozyme by incu-
bating them overnight in adsorption buVer containing
10 mg/mL lysozyme. After three gentle washing steps with
adsorption buVer, they were soaked in the elution solution
during 8 h at room temperature.
Ten desorptive solutions (1 mL volume each) were
assayed, two of them previously published: (1) 0.1 M
sodium carbonate/bicarbonate buVer, pH 9.6, 0.3 M NaCl
[21]; (2) 0.1 M acetic acid [23]; (3) 2 M NaCl in adsorption
buVer (4) 0.5 M NaSCN in adsorption buVer (5) 25% ethyl-
ene glycol in adsorption buVer (6) 2 M NaCl + 25% ethyl-
ene glycol in adsorption buVer (7) 0.5 M NaSCN + 25%
ethylene glycol in adsorption buVer (8) 2 M NaCl in 0.1 M
sodium carbonate/bicarbonate buVer, pH 9.6 (9) 0.5 M
NaSCN in 0.1 M sodium carbonate/bicarbonate buVer, pH
183
9.6 and (10) 2 M NaCl + 25% ethylene glycol in 0.1 M
sodium carbonate/bicarbonate buVer, pH 9.6.
In all adsorption and elution experiments, determina-
tions were performed in triplicate, and the results expressed
as the average § standard deviation.
Lysozyme puriWcation from undiluted egg white
About 1 g matrix was incubated with 10 mL egg white for
10 h at room temperature with gentle agitation. Adsorbed
lysozyme concentration was calculated as the diVerence
between the amount of lysozyme in the egg white and its
amount in the supernatant after adsorption. After three
10 mL-washing steps with adsorption buVer, the enzyme
was eluted with 5 mL 0.1 M acetic acid or 0.1 M sodium
carbonate/bicarbonate buVer, pH 9.6, + 0.3 M NaCl or 2 M
NaCl + 25% ethylene glycol in 50 mM sodium phosphate
buVer, pH 8.0. After regeneration with 0.05 M NaOH, the
matrix was utilised for two new adsorptive cycles.
Total protein was calculated by the Bradford method [30].
Lysozyme activity measurement
Lysozyme concentration was calculated through the mea-
sure of its enzyme activity. Lysozyme activity was deter-
mined by its lytic action on Micrococcus lysodeikticus cells
according to Gorin et al. [31]. One lysozyme unit is deWned
as the amount of enzyme causing a decrease of 0.001 per
minute in the absorbance at 450 nm (pH 7.0, 30 °C), using
a suspension 1 mg/mL M. lysodeikticus as the substrate, in
1 mL reaction mixture.
SDS–PAGE
Runs were performed with a 15% gels and stained with
Coomassie Blue under standard conditions. Samples
(eluted fractions) were previously subjected to solvent
exchange to 50 mM sodium phosphate buVer, pH 8.0, using
an Amicon centrifugal ultraWlter (cut oV 5,000 kDa, Milli-
pore, Billerica, MA, USA). Purity was calculated by using
the Scion Image software (Scion Corporation, Frederick,
Maryland, USA).
Results and discussion
Matrix characterisation
Matrix ATR-IR spectrum is shown in Fig. 1. Intense bands
at 790, 950 and 1,070 cm¡1 correspond to symmetric Si–O–Si
bond stretching, Si–OH bond stretching and asymmetric
Si–O–Si bond stretching, respectively, due to SiO2 poly-
meric network generated by TEOS polymerisation [32, 33].
123
184
Fig. 1 ATR-IR spectrum of the matrix. Data were acquired in the
range 4,000–650 cm¡1 using a Fourier transform infrared spectrometer
with a Xat-plate attenuated total reXectance. The matrix was previously
dried a 60 °C for 24 h
Less intense bands accounting for chitin structure can be
seen at 1,660–1,620 cm¡1, corresponding to the amide I
band. This band—formed by a doublet at 1,655 and
1,625 cm¡1 (C=O and C–N stretching, respectively)—
appears unresolved in the ATR spectra. The bands of amide
II (1,540 cm¡1, N–H stretching) and amide III
(1,370 cm¡1) are hardly detected in this spectrum.
Peaks corresponding to nitrogen and carbon atoms were
observed in the EDS spectra (not shown), thus conWrming
the presence of chitin in the matrix. It could also be seen the
peaks corresponding to the presence of oxygen and silicon.
Matrix topography is shown in the SEM image of
Fig. 2, where is evident a rather homogeneous material
with no observable agglomerates of chitin. The matrix
structure is the result of the polysaccharide addition to the
Fig. 2 SEM images of the matrix. Scanning electron microscopy
images of the matrix
123
Eur Food Res Technol (2010) 231:181–188
polymerisation mixture, which is known to inXuence the
condensation process, leading to a material with character-
istic properties [34].
The inXuence of chitin in the polymerisation process is
evidenced in the pore structure of the matrix. Thus, the
nitrogen sorption isotherms correspond to those of the mes-
oporous materials (type IV), while typical TEOS matrices
show isotherms of microporous material (type II) [35].
Moreover, it could also be seen the hysteresis loop present
in some mesoporous materials [35, 36]. A surface area of
142 m2/g and a total pore volume of 0.295 cm3/g can be
calculated from the nitrogen sorption isotherms. Water con-
tent of the matrix was 78.3%.
Pure lysozyme adsorption isotherm
Figure 3 shows the lysozyme adsorption isotherm. Data
were analysed according to Langmuir and Freundlich mod-
els, and Table 1 shows the thermodynamic parameters cal-
culated from them.
The maximum capacity achieved—117 mg lysozyme/g
matrix—is very high when compared with the values
obtained by other authors. Bayramoglu et al. [37], working
with chitosan beads modiWed by grafting of a methacrylic
acid polymer, reported an adsorption capacity of 66 mg/g.
This diVerence could be due to the method used for increas-
ing the mechanical resistance of the chitosan beads.
120
100
80
q* (mg/g)
60
40
20
0
0 1 2 3
c* (mg/ml)
Fig. 3 Lysozyme adsorption isotherm. About 50 mg matrix was
soaked in 1 mL lysozyme solutions (0.125–10 mg/mL in adsorption
buVer: 50 mM sodium phosphate buVer, pH 8.0, 50 mM NaCl). The
suspensions were gently agitated overnight at 20 °C to allow the sys-
tem to reach its equilibrium. Lysozyme concentration in supernatants
at equilibrium was then measured spectrophotometrically at 280 nm.
The equilibrium concentration of lysozyme bound to the matrix per
unit of total matrix volume was calculated as the total amount of
enzyme present at the beginning of the experiment less the amount still
in the soluble phase at equilibrium. Standard deviation of each point is
lower than 3%
Eur Food Res Technol (2010) 231:181–188
Table 1 Calculated thermodynamic parameters for pure lysozyme
adsorption to TEOS-chitin matrix
Langmuir constants Freundlich constants
2 2
Qmax (mg/g) Kd (mg/mL) R N Kf
117.1 § 9 0.73 § 0.15 0.98 0.49 § 0.05 58 § 3
Adsorption capacity was expressed as the mass of adsorbed protein per
mass unit of matrix (mg/g)
There are two ways for increasing the mechanical resis-
tance of chromatographic beads: (1) cross-linking of the
bead material by means of bifunctional reagents (2) use of
sol–gel chemistry. The problem with the Wrst approach is
that the functional groups engaged in the cross-link become
not available for attachment of ligands or direct protein
adsorption, thus decreasing the adsorption capacity of the
matrix. Sol–gel chemistry, instead, increases the mechani-
cal resistance of the beads without any decrease in the
adsorption capacity. Taking into account that lysozyme
concentration in egg white is 0.3%, the Kd obtained from
the isotherm is more than four times lower than lysozyme
concentration in egg white, this assuring a good matrix
adsorptive capacity utilisation for batch mode processes.
It is expected that the interaction between them would be
speciWc as N-acetyl glucosamine being chitin monomer and
the substrate of lysozyme. Langmuir model is based on the
assumption that the adsorption takes place up to the forma-
tion of a homogeneous monolayer [38], this could explain
the better adjustment to the Langmuir model. On the other
hand, the equilibrium data were also consistent with the
Freundlich model which adjusts better to materials with
heterogeneous adsorption sites. This is probably because
other interactions than speciWc enzyme–substrate could be
present in this complex matrix where the protein could
interact in an unspeciWc way with many sites.
Lysozyme adsorption kinetics from a pure lysozyme
solution and from egg white
Figure 4 shows the lysozyme adsorption kinetics on the
chitin matrix from a lysozyme pure solution and from egg
white. For pure lysozyme, a plateau is reached only after
240 min. The mesoporous nature of the matrix probably
accounts for this slow adsorption. For this reason and due
to the characteristics of the starting material, assays for
lysozyme puriWcation from egg white were performed in
the batch mode. In the case of lysozyme adsorption from
egg white, a plateau is reached after 600 min. The larger
time to reach the plateau respect to adsorption from a pure
lysozyme solution can be explained taken into account the
high viscosity of the egg white, which limits the protein
185
100
Lysozyme Bound (%)
80
R
60
0.95
40
20
0
0 200 400 600 800 1000 1200 1400 1600
Time (min)
Fig. 4 Adsorption kinetics of pure lysozyme and lysozyme from
undiluted egg white. About 1 g matrix was incubated with 10 mL lyso-
zyme solution (3 mg/mL in adsorption buVer) and with 10 mL egg
white for 24 h at room temperature with gentle agitation. Samples
were taken at 0, 30, 60, 90, 120, 180, 240, 300, 360, 420, 480, 540, 600
and 1,440 min. Adsorbed lysozyme concentration was calculated as
the diVerence between the amount of lysozyme in the initial solution
(pure lysozyme) and in egg white and its amount in the supernatant.
(Wlled circles) Pure lysozyme solution (open circles) Lysozyme from
egg white. Standard deviation of each point is lower than 3%
molecular diVusion and, therefore, slows the adsorptive
process.
From Fig. 4 is evident that 10 h is an adequate time for
adsorption of approximately 80% of the lysozyme present
in the egg white, for the egg white/matrix ratio of 10/1
(volume/mass). Taking into account that the egg white is
going to be utilised for food purposes, it is reasonable that
part of the lysozyme remains in the egg white, as it is its
natural antimicrobial preservative.
Elution of the adsorbed lysozyme
Ten eluents were assayed: two of them were previously
reported for elution of lysozyme from aYnity matrices:
0.1 M acetic acid [25] and 0.3 M NaCl in 0.1 M sodium
carbonate/bicarbonate buVer, pH 9.6 [23]. The eYciency of
the elution solutions is shown in Table 2.
Results in Table 2 evidence that various eluents are able
to elute 100% of the lysozyme adsorbed. However, eluents
including NaSCN in their composition aVected the matrix
integrity thus impairing its further utilisation. For this
reason, they were discarded. It can also be concluded that
there are electrostatic and hydrophobic components in the
lysozyme/matrix interaction. Three eluents were selected
for developing a puriWcation process: 0.1 M acetic acid, 0.3
M NaCl in 0.1 M sodium carbonate/bicarbonate buVer, pH
9.6, and 2M NaCl + 25% ethylene glycol in 50 mM sodium
phosphate buVer, pH 8.0.
123
186 Eur Food Res Technol (2010) 231:181–188

Table 2 Performance of the

Eluent Elution
diVerent eluents for lysozyme
percentage
elution from the matrix
0.3 M NaCl in 0.1 M sodium carbonate/bicarbonate buVer, pH 9.6 100 § 3
0.1 M acetic acid 100 § 3
2 M NaCl in 50 mM sodium phosphate buVer, pH 8.0 44 § 2
0.5 M NaSCN in 50 mM sodium phosphate buVer, pH 8.0 23 § 1
25% ethyleneglycol in 50 mM sodium phosphate buVer, pH 8.0 81 § 3
2 M NaCl + 25% ethyleneglycol in 50 mM sodium phosphate buVer, pH 8.0 100 § 2
0.5 M NaSCN + 25% ethyleneglycol in 50 mM sodium phosphate buVer, pH 8.0 100 § 3
2 M NaCl in 0.1 M sodium carbonate/bicarbonate buVer, pH 9.6 100 § 4
0.5 M NaSCN in 0.1 M sodium carbonate/bicarbonate buVer, pH 9.6 32 § 3
2 M NaCl + 25% ethyleneglycol in 0.1 M sodium carbonate/bicarbonate buVer, pH 9.6 100 § 4

Table 3 Performance of the puriWcation process by using three diVerent eluents as regards yield
Eluent Eluted Yield SpeciWc activity of the PuriWcation
(IU) (%) eluted fraction (IU/mg protein) factor

0.1 M acetic acid 2,388,000 64 51,913 20

0.3 M NaCl in 0.1 M sodium carbonate/ 1,688,000 45 15,432 6
bicarbonate buVer, pH 9.6
2M NaCl + 25% ethylene glycol, pH 8 2,008,000 54 51,487 20
Egg white had 3,712,000 IU. Adsorbed lysozyme was 3,255,000, equivalent to 87% of the amount of lysozyme contained in the egg white utilised
as starting material
SpeciWc activity of lysozyme in egg white = 2,620 IU/mg protein
SpeciWc activity of lysozyme remaining in the supernatant after adsorption = 381 IU/mg protein
In all cases, standard deviation was lower than 3%

Matrix reuse
Lysozyme adsorption capacity remained unchanged after 3
cycles of adsorption/desorption using a 10 mg/mL pure
lysozyme solution as the sample.
When a regeneration step with 0.05 N NaOH was
included between each puriWcation cycle, lysozyme adsorp-
tion capacity did not undergo any decrease, thus evidencing
a good chemical stability of the matrix.
Lysozyme puriWcation from undiluted egg white
Table 3 shows the results obtained when lysozyme was
puriWed directly from egg white.
According to the adsorption kinetics results, puriWcation
experiments were performed in the batch mode. It is evi-
dent the eYcacy of the adsorptive performance of the
matrix: 87% of the lysozyme was removed from the egg
white and the matrix was easily recovered by a simple
Wltration through a strainer.
Most lysozyme puriWcation processes reported require a
previous egg white dilution or some kind of conditioning
before the chromatographic step, thus precluding its further
utilisation in the food industry. In our case, egg white can
be used for pastry after lysozyme depletion. Other signiWcant
advantage of our approach is the easy recovery of the chro-
matographic matrix by sedimentation after the adsorption,
washing and elution steps, this due to its size and density as
well as to its high mechanical resistance. Small beads usually
require centrifugation or Wltration steps to recover the matrix,
thus adding an extra cost to the process. Moreover, a fraction
of chromatographic matrix always is lost at that time.
From the comparison of results in Tables 2 and 3 is evi-
dent the better elution eYcacy of 0.1 M acetic acid as
regards to recovery of lysozyme adsorbed and purity of the
Wnal product. The high puriWcation factor obtained after
elution with 0.1 M acetic acid indicates a diVerential elu-
tion of the proteins adsorbed to the matrix, thus allowing a
high puriWcation degree of lysozyme in only one step and
starting from undiluted egg white. However, from Table 3
is also evident a decrease in the elution eYcacy when egg
white is the starting material instead of pure lysozyme, this
suggesting that some adsorbed biomolecules from egg
white could impair the total removal of the adsorbed lyso-
zyme. Despite this fact, a 64% yield for this puriWcation
process, and taking into account the nature of the starting
material, is comparable with the yields obtained by other
authors [25, 27, 37].
After three lysozyme puriWcation cycles from egg white
and the corresponding regeneration cycles with 0.05 N

123
Eur Food Res Technol (2010) 231:181–188
Fig. 5 SDS–PAGE. Lane 1, molecular weight markers (10, 19, 26, 34,
43, 55, 72, 95, 130 and 170 kDa); lane 2, fraction eluted with 2 M
NaCl + 25% ethylene glycol, phosphate buVer pH 8.0; lane 3, fraction
eluted with 0.3 M NaCl in 0.1 M sodium carbonate/bicarbonate buVer,
pH 9.6; lane 4, fraction eluted with 0.1 M acetic acid; lane 5, egg white
supernatant after lysozyme adsorption; lane 6, egg white; lane 7, lyso-
zyme standard
NaOH, there was not any decrease in the matrix adsorption
capacity.
Figure 5 shows the SDS–PAGE of the diVerent steps of
the puriWcation process. Purity of lysozyme eluted with
0.1 M acetic acid was 90% while that of the lysozyme
desorbed with 0.3 M NaCl in 0.1 M sodium carbonate/
bicarbonate buVer, pH 9.6, was only 51% and that of the
lysozyme desorbed with 2M NaCl and 25% ethylene gly-
col, pH 8, was 88%. This was conWrmed by the appearance
of several protein bands accompanying to that of lysozyme
in the last eluents.
Several authors reported aYnity processes for lysozyme
puriWcation from egg white. Yilmaz et al. [27] and Arica
et al. [28], reported dye membrane aYnity chromatography
methods, and Ruckenstein and Zeng [25] utilised chitin
membranes. Safarik and Safarikova [2] reported a puriWca-
tion process with a magnetic chitin matrix. Though this
matrix can be easily recovered by a magnetic Weld after
adsorption, the lysozyme capacity of the matrix is only
2.5 mg lysozyme/mL matrix. In all these cases, egg white
dilution was necessary for the adsorption step.
Bayramoglu et al. [37] reported an 87% yield with a
purity of 94% for lysozyme puriWcation from diluted egg
white by using ion exchange beads of cross-linked chitosan
grafted with an acrylic acid polymer. Unfortunately, egg
white dilution precludes the further commercialisation of
this product, thus increasing the costs.
Chang and Chang [39] utilised the ion exchange matrix
streamline SP for lysozyme puriWcation from undiluted egg
white and achieved a 97.5% yield with a puriWcation factor
of 11. In comparison with these authors, our process pro-
vides an economical matrix of high mechanical resistance
187
that allows obtaining lysozyme of high speciWc activity
(51,000 IU/mg).
Grasselli et al. [26] developed a dye aYnity method by
membrane chromatography based on the interaction of
lysozyme with Red HE 3-B. The maximum binding capac-
ity of the support was 26 mg/mL and the purity of lyso-
zyme was 88% starting from undiluted egg white. On the
other hand, Ghosh and Cui [40] proposed a puriWcation
method from egg white powder based on two ultraWltration
steps. A 99% pure lysozyme was obtained with high pro-
ductivity (21,874 U m¡2 s¡1). However, the pretreatment of
the starting material and the cost of the membrane technol-
ogy put some doubts on its industrial utilisation.
The matrix herein presented has a low cost and easy
manufacture.
Conclusion
The process developed allows recovery and puriWcation of
lysozyme from undiluted egg white with a high adsorption
capacity matrix of low cost and high mechanic resistance.
For these reasons, this process is a potential alternative for
industrial purposes.
Acknowledgments This work was supported by grants from the
Agencia Nacional de Promoción CientíWca y Tecnológica de la Repúb-
lica Argentina, the Universidad de Buenos Aires and the Consejo Nac-
ional de Investigaciones CientíWcas y Técnicas (CONICET). FJW,
OC, GJC, MVM and LED are researchers of the CONICET.
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