Food Chemistry 272 (2019) 619–627

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Rapid purification of lysozyme by mixed-mode adsorption chromatography T

in stirred fluidized bed
⁎
Kuei-Hsiang Chen, Shin-Ying Chou, Yu-Kaung Chang
Department of Chemical Engineering, Graduate School of Biochemical Engineering, Ming Chi University of Technology, New Taipei City 24301, Taiwan

A R T I C LE I N FO A B S T R A C T

Keywords: The commercial STREAMLINE Direct HST adsorbent was employed to investigate the adsorption characteristics
Lysozyme of lysozyme in complex chicken egg white (CEW). The effects of operating parameters, including adsorption pH,
Chicken egg white ionic strength, and hydrophobicity of liquids on binding capacity, were examined using a well-mixed contactor.
Purification To determine the elution strategy, the experiments were carried out in a small packed bed. The lysozyme was
Direct recovery
completely eluted by 0.5 M NaCl in 40 mM carbonate buffer (pH 12) at a high liquid velocity of 200 cm/h. The
Stirred fluidized bed adsorption
STREAMLINE HST adsorbent
effect of rotating speed on fluidization characteristics was further investigated by using a stirred fluidized bed
process. Smoother fluidization was observed when the rotating speed reached 200 rpm and lysozyme was di-
rectly recovered from highly viscous CEW in a single step with a high yield of 94.3% and a purification factor of
15.7.

1. Introduction
Chicken egg white (CEW) has been commonly used as a source of
lysozyme (Awadé & Efstathiou, 1999). The highly viscous property of
CEW is due to the presence of ovomucin, with a very high molecular
weight (Mw). Ovomucin (Mw ∼ 5.5–8.3 × 106 g/mol) is a glycoprotein
accounting for 2–4% of the total egg albumin protein and it is a key
component in maintaining the viscous nature of egg white (Awadé &
Efstathiou, 1999). Among the proteins in egg whites, lysozyme is a
minor component (∼3.4%) which consists of 129 amino acid residues
and has four disulfide bonds in one molecule. The pI value of lysozyme
(MW ∼ 14,300 g/mol) is ∼11.0. The percentages of the hydrophobic,
aromatic, and basic residues in a lysozyme molecule are ∼35, ∼10,
and ∼25%, respectively (Tsunoda et al., 2001). Hence, lysozyme has a
considerable hydrophobic property among these proteins in egg whites.
The complex CEW may cause difficulty in predicting the adsorption
behaviour for lysozyme due to the presence of large amounts of con-
taminating proteins in CEW (Awadé & Efstathiou, 1999; Wang, Lu, &
Cui, 2006).
It is well known that commercially available lysozyme has sig-
nificant potential applications for pharmaceutical and food industries,
and it can be produced from complex CEW using a combination of
various purification processes, such as precipitation (Roy, Rao, &
Gupta, 2003), membrane chromatography (Yilmaz, Bayramğlu, &
Arıca, 2005), ultrafiltration (Ghosh & Cui, 2001; Wang et al., 2006),
ion-exchange chromatography (Awadé, Moreau, Mollé, Brule, &
Maubois, 1994; Levison et al., 1992; Ming, Howell, Acosta, & Hubble,
1993), affinity chromatography (Chiang, Su, Tsai, & Tsao, 1993), re-
verse micellar and aqueous two-phase systems (Noh & Imm, 2005; Su &
Chiang, 2006), as well as expanded and/or fluidized bed chromato-
graphy (Noppe et al., 1996; Tong, Dong, & Sun, 2002). Among these
purification methods, packed bed adsorption chromatography has been
widely used for the purification of lysozyme (Awadé et al., 1994;
Levison et al., 1992; Ming et al., 1993). However, the crude CEW needs
to be clarified, using centrifugation and filtration, which are time-
consuming and may result in significant loss of the desired products,
before running the packed bed adsorption process.
During the past two decades, expanded bed adsorption (EBA) and/
or fluidized bed adsorption (FBA) have been demonstrated as efficient
techniques for direct capture of target proteins from unclarified feed-
stocks, as well as improved productivity and process economy
(Anspach, Curbelo, Hartmann, Garke, & Deckwer, 1999; Chase, 1994;
Hjorth, 1997; Thömmes, 1997). Clarification, capture, and initial pur-
ification steps can be achieved in a single step. Hence, EBA and/or FBA
techniques can shorten purification time and reduce production costs.
As noted in previous studies (Anspach et al., 1999; Chase, 1994), two
important key issues in the development of a direct recovery process of
enzyme from highly viscous feedstock are the specialized design

⁎
Corresponding author at: Center for Biochemical Engineering, Graduate School of Biochemical Engineering, Ming Chi University of Technology, 84, Gung-Juan
Road, Taishan Dist., New Taipei City 24301, Taiwan.
E-mail address: ykchang@mail.mit.edu.tw (Y.-K. Chang).

https://doi.org/10.1016/j.foodchem.2018.06.050
Received 15 January 2018; Received in revised form 17 May 2018; Accepted 11 June 2018
Available online 15 June 2018
0308-8146/ © 2018 Published by Elsevier Ltd.
K.-H. Chen et al.
Fig. 1. (a) A schematic representation of stirred fluidized bed adsorption
column: 1. Motor and control assembly, 2. Distributor plate, 3. Support net
(70 μm), 4. Agitator (diameter 2 cm, width 1.5 cm, height 3.5 cm), 5. Column
tube (i.d. 2.5 cm), 6. Adjustable plunger, 7. STREAMLINE Direct HST adsorbent.
(b) Basic structure of STREAMLINE Direct HST adsorbent with multi-functional
group.
column and high-density adsorbent.
Chang and Chang (2006) reported that the conventional EBA
column used to recover the highly viscous unclarified feedstocks
showed disadvantages, and hence, a new stirred fluidized bed adsorp-
tion (SFBA) column (YKC-SFBA, Fig. 1(a)) equipped with an agitator
has been developed (Chang & Chang, 2006). The SFBA column differs
in design and operational procedure from the conventional EBA
column. It can effectively directly purify the desired protein from highly
viscous unclarified feedstock. Moreover, high-density adsorbent can be
made, using a highly dense material, and hence, the expanded bed re-
mains stable over a wide range of liquid densities and viscosities, de-
spite being operated at high flow rates (Zhao, Yao, & Lin, 2009). For
example, the high-density core can be covered with a gel layer, such as
agarose-stainless steel, agarose-glass beads, and agarose-Nd-Fe-B
620
Food Chemistry 272 (2019) 619–627
(Jahanshahi, Partida-Martinezb, & Hajizadeha, 2008; Lihme, Zafirakos,
Hansen, & Olander, 1999; Miao, Lin, & Yao, 2005; Pålsson, Gustavsson,
& Larsson, 2000), dispersing high-density powder (for example, cellu-
lose-titanium oxide and cellulose-stainless steel powder) into the solid
phase (Tong & Sun, 2002; Xia, Lin, & Yao, 2007a, 2007b), and using
high-density porous material (for example, zirconium oxide and tita-
nium oxide) (Voute & Boschetti, 1999; Yun, Lin, Lu, Zhong, & Yao,
2004).
In this study, STREAMLINE Direct HST adsorbent was used to pro-
vide the required high density with a highly cross-linked agarose matrix
(4%) and an inert stainless-steel core (Charoenrat, Ketudat-Cairns,
Jahic, Enfors, & Veide, 2006; Jahic, Knoblechner, Charoenrat, & Enfors,
2006; Li, Xiu, Mata, Grande, & Rodrigues, 2006). HST adsorbent is a
mixed-mode adsorbent with multi-functional groups and has been
specialized in design to handle highly viscous feedstocks at moderate
conductivities (Charoenrat et al., 2006; Jahic et al., 2006; Li et al.,
2006). The influences of operating parameters, including adsorption
pH, conductivity, and hydrophobicity, on the binding capacity of ly-
sozyme on the mixed-mode HST adsorbent were first examined in a
well-mixed contactor before the SFBA was operated. Various elution
schemes for lysozyme were also investigated with clarified CEW in
packed beds. To date, direct recovery of lysozyme from highly viscous
CEW remains a challenge. Therefore, the aim of this study was to de-
velop a SFBA technique to directly purify lysozyme from highly con-
centrated CEW in a single step. A schematic diagram of the overall work
is shown in Fig. S1.
2. Materials and methods
2.1. Materials and apparatus
STREAMLINE Direct HST adsorbent (particle size 80–165 μm, av.
135 μm, mean particle density 1.80 g/cm3, apparent ionic
capacity ∼0.164 mmol Na+/ml adsorbent (Fig. 1(b)) and ÄKTAprime
chromatographic system were purchased from GE Healthcare (Uppsala,
Sweden). Micrococcus lysodekticus cells were obtained from Sigma (St.
Louis, MO, USA). All other chemicals used were of analytical grade. The
fresh chicken eggs were obtained from a local supermarket (New Taipei
City, Taiwan). A high-speed dispersion homogenizer (Model HG-300D,
dispersion element model K-20S, dimension 20 × 170 mm, rotating
speed 0–2600 rpm, power 250 W, 50/60 Hz) was obtained from Hsiang-
Tai Machinery Industry Co., Ltd., (New Taipei City, Taiwan). The re-
ciprocal shaker (Model B603D-L) was purchased from Firstek Scientific
Co. (Taiwan) and the rotary mixer was obtained from Labinco Ltd.
(model LD-79, the Netherlands). The small packed bed column (Bio-Rad
Econo column, 0.7 cm i.d., 10 cm length) was purchased from Bio-Rad
Labs. (Hercules, CA, USA) and the peristaltic pumps were purchased
from Tokyo Rikakikai Co., Ltd. (Model MP-3N, Japan). A UV–vis
spectrophotometer (Model Ultrospec 3100 pro, GE Healthcare Bios-
ciences, Uppsala, Sweden) measured the absorbance of protein samples.
The stirred fluidized bed adsorption column (2.5 cm i.d., 100 cm
length) (YKC-SFBA column, Fig. 1(a)) with a two-blade flat paddle was
of specialized design and constructed in our laboratory (Chemist Sci-
entific Corp., New Taipei City, Taiwan).
2.2. Egg white pre-treatment
The sticky CEW was separated from the fresh eggs. To obtain a
homogeneous and less viscous CEW for ease of use in column chro-
matography, pre-treatment of CEW was necessary (Li-Chan, Nakai, Sim,
Bragg, and Lo (1986)). In this experiment, a 1000 ml CEW solution was
transferred into the dispersion reactor (2500 ml) and controlled tem-
perature at 4 °C. The CEW suspension was homogenized at a rotating
speed of 3000 rpm for 30 min, using a high-speed dispersion homo-
genizer (∼30 W, 50/60 Hz) (Koumoulidis, Tiberius, & Sdoukos, 2001).
After dispersion homogenizer treatment, the apparent viscosity of
K.-H. Chen et al.
homogeneous CEW sample was measured, using a HAAKE VT 181
viscometer (Thermo Scientific, Thermo Fisher Scientific Inc, UK) at a
shear rate of 106 s−1 and controlled temperature of 25 °C.
2.3. Total protein determination and lysozyme activity assay
Total protein concentration was measured by the Bradford method,
using lysozyme as the standard (Bradford, 1976). The activity of lyso-
zyme was measured by lysis of 0.25 mg/ml M. lysodeikticus cells in
100 mM sodium phosphate (pH 6.24) at 25 °C (Shugar, 1952). One unit
(U) of lysozyme activity was defined as the decrease of OD450 by 0.001
per minute. All data represented are the mean values of triplicate
measurements, where the standard deviation was within 5% of the
mean value.
2.4. Choice of adsorption conditions for lysozyme in batch studies
The homogenized CEW was diluted to 20% (v/v) with 10 mM
concentration of relevant buffer (i.e., sodium acetate buffer, pH 4–5;
sodium phosphate buffer, pH 6–8; glycine-NaOH, pH 9–10; sodium
carbonate buffer, pH 11–12). A 1:5 dilution of crude CEW (con-
taminating proteins 6.15 mg/ml, lysozyme activity 5.60–7.69·104 U/ml,
specific activity 0.91–1.25·104 U/mg) at the relevant pH (4–12) was
used as a lysozyme source.
A 2 ml (1:1) adsorbent buffer slurry and 10 ml CEW at the relevant
buffer pH (4–12) were added to each flask (15 ml). The flasks were
sealed and mixed completely by a rotary mixer (20 rpm) for 3 h at 25 °C.
After this period had elapsed, the supernatants were removed. Both
concentration of contaminating proteins (mg/ml) and activity of lyso-
zyme (U/ml) in the supernatants collected in each flask were de-
termined. The binding capacity of HST adsorbent was calculated, using
a simple mass balance. Each experiment was performed, in triplicate,
under identical operating conditions, and the error of the measurements
was in the range of ± 5%. Subsequent experiments were conducted to
investigate the influences of hydrophobicity of process liquids (0–50%
ethylene glycol or glycerol) and ionic strength (0–1 M NaCl) at optimal
adsorption pH on the binding capacity for lysozyme to the HST ad-
sorbent.
2.5. Elution of lysozyme in packed bed
In the experiments, HST adsorbent (3 ml) was poured into Bio-Rad
Econo column (i.d. 0.7 cm) to give a settled bed height of 7.8 cm. The
column was then irrigated with 10 mM glycine-NaOH buffer (pH 10) at
a liquid velocity of 200 cm/h. After 30 ml buffer had passed through the
column, 10 ml of clarified CEW (6.18 mg/ml, 7.75·104 U/ml,
1.25·104 U/mg) were loaded onto the bed at the same superficial ve-
locity. Non-adsorbed components were then washed from the bed with
10 mM sodium carbonate pH 10 (15 ml) and the bound components
eluted by various elution schemes. Each elution scheme was carried out
at different liquid velocities (100–200 cm/h) by injection pulses (3–10
bed volumes, 30 ml) of the relevant eluent at the desired molarity.
These eluents were used to evaluate elution efficiency of lysozyme,
including sodium carbonate buffer (10–100 mM, pH 12), 0.5 M NaCl in
carbonate buffer (10–40 mM, pH 12), 50% EG (ethylene glycol) in
carbonate buffer (10–40 mM, pH 12), and 0.5 M NaCl/50% EG in car-
bonate buffer (10–40 mM, pH 12). Both, concentration of con-
taminating proteins and activity of lysozyme in the supernatants col-
lected at each stage were assayed. The elution efficiency (E, %) was
calculated by Eq. (1).
A
E (%) = E × 100
AB (1)
where AB is the binding activity of lysozyme (U/ml) and AE is the eluted
activity of lysozyme (U/ml).
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Food Chemistry 272 (2019) 619–627
2.6. Effect of rotating speed of agitator
Chicken egg white (CEW) was first separated from the fresh eggs.
After the high-speed dispersion homogenizer treatment and pH ad-
justment, the viscous CEW was then used in this study. The effect of
rotating speed of agitator (i.e., 0–200 rpm) on adsorption performance
of lysozyme and contaminating proteins from viscous CEW feedstock
was investigated in the SFB column (i.d. 25 mm). Two pumps, A and B,
were employed to control the process liquid velocity of adsorption
buffer (pH 10) and crude CEW solution, respectively, and to deliver the
two liquids at equal velocities during the adsorption process.
In this experiment, the HST adsorbent (∼100 ml) was poured into
the SFBA column to give a settled bed height of 20.0 ± 0.2 cm. The top
adaptor of the column was positioned at a height of ∼50 cm. Initially,
the adsorbent bed was equilibrated with adsorption buffer at the same
velocity of 245 cm/h by pumps A and B, corresponding to an overall
liquid velocity of 490 cm/h. When a stable fluidized bed was achieved
(e.g., the fluidized bed height was maintained at a constant level, two
times settled bed height, ∼40 cm), the agitator was then switched on
and the rotating speed was controlled at the desired value (i.e.,
0–200 rpm). Simultaneously, Pump B was switched to crude CEW so-
lution delivery and pump A continuously pumped the adsorption buffer
onto the column. During the adsorption stage, the two process liquids
were controlled at equal velocities and the fluidized bed was main-
tained at two times settled bed height. Fractions were collected
throughout the experiments and subsequently assayed for total protein
concentration and lysozyme activity.
2.7. Direct recovery of lysozyme, using stirred fluidized bed adsorption
chromatography
More detailed description of the standard operating procedures as
described in Chang and Chang (2006). The related operating conditions
for direct recovery of lysozyme from highly viscous CEW are shown in
Table 1. Experiments were carried out to assess the feasibility of using
STREAMLINE HST and YKC-SFBA column for the direct recovery of
lysozyme from highly crude CEW (26.2 mg, protein/ml, 3.80·105 U,
lysozyme/ml, in 20 mM glycine-NaOH buffer at pH 10). In the SFBA
column, the rotating speed was maintained at 200 rpm. Two pump
systems were used to control the liquid velocity of buffer and crude
CEW, respectively and operated to deliver the two liquids at equal
Table 1
Operating conditions for SFBA experiments carried out with HST adsorbent in a
specially designed YKC-SFBA column.
Adsorbent & Column STREAMLINE Direct HST
100 ml HST in YKC-SFBA column (i.d. 25 mm)
Settled bed height 20.0 ± 0.2 cm (Ho)
Fluidized bed operated at two-fold settled bed height
40.0 ± 2.0 cm (Hexp)
Feedstock Homogenized chicken egg white without dilution and pH
adjusted to 10 only (10 mM sodium carbonate buffer, pH 10)
Total protein concentration 26.2 ± 0.32 mg/ml
Lysozyme activity (3.80 ± 0.23)·105 U/ml
Operating procedures Operating conditions (25 C) (equal Overall liquid
(Fluidized bed velocity controlled by pumps A and B) velocity (cm/
only, 2xHo) h)
Equilibration 10 mM glycine-NaOH (pH 10, pumps 490
A and B)
Adsorption Chicken egg white (600 ml, pump B) 490–120 (av.
and buffer (600 ml, pump A) at pH 10 200)
Wash 10 mM glycine-NaOH (1200 ml, pH 120–490 (av.
10, pump A) 200)
Elution (1) (1) 0.5 M NaCl, (2) 50% EG, (3) 200
0.5 M NaCl, and 50% EG in sodium
carbonate buffer (10–40 mM, pH
12) (900 ml, pump A)
K.-H. Chen et al. Food Chemistry 272 (2019) 619–627

Fig. 2. (a) Effect of adsorption pH on binding capacity of lysozyme in a crude CEW solution: pH 4–5 (20 mM sodium acetate buffer); pH 6–8 (20 mM sodium
phosphate buffer); pH 9–10 (20 mM glycine-NaOH buffer); pH 11–12 (20 mM sodium carbonate buffer). Adsorbent-buffer slurry: 2.0 ml (1:1), unclarified CEW: 10 ml
(pH 4–12), rotary mixer: 20 rpm, 3 h at 25 °C. (b) Effect of adsorption pH at 30% ethylene glycol on binding capacity of lysozyme in a crude CEW solution: (1) pH 4–5
(20 mM sodium acetate buffer); (2) pH 6–8 (20 mM sodium phosphate buffer); (3) pH 9–10 (20 mM glycine-NaOH buffer); (4) pH 11–12 (20 mM sodium carbonate
buffer). Adsorbent-buffer slurry: 2.0 ml (1:1), unclarified CEW: 10 ml (30% ethylene glycol, pH 4–12), rotary mixer: 20 rpm, 3 h at 25 °C. (c) Effect of ethylene glycol
(0–50% in 20 mM glycine-NaOH buffer, pH 10) on binding capacity of lysozyme in a crude CEW solution. Adsorbent-buffer slurry: 2.0 ml (1:1), unclarified CEW:
10 ml (0–50% ethylene glycol, pH 10), rotary mixer: 20 rpm, 3 h at 25 °C. (d) Effect of NaCl concentration on binding capacity of lysozyme in a crude CEW solution.
Adsorbent-buffer slurry: 2.0 ml (1:1), unclarified CEW: 10 ml (0–0.5 M NaCl, pH 10), rotary mixer: 20 rpm, 3 h at 25 °C.

liquid velocities. During the experiments, all operating steps (namely,
adsorption, washing, and elution stages) were carried out in fluidized
bed mode only (∼two times settled bed height 40 cm). At the end of
adsorption stage, solid particulates were completely washed out with
adsorption buffer. After washing, a one-step elution scheme was used to
elute the adsorbed lysozyme. Fractions were collected throughout the
experiment and subsequently assayed for total protein concentration
and lysozyme activity. Eq. (2) was used to determine the recovery yield
(RY, %) of lysozyme from the adsorbent bed.
AE
RY (%) = × 100
AL
where AE and AL are the eluted and loaded activities of lysozyme (U/
ml), respectively.
3. Results and discussion
3.1. A new approach to SFBA process
The YKC-SFBA column has been employed in a lysozyme purifica-
tion process from low-viscosity CEW solution, using STREAMLINE SP-
type adsorbent (Chang & Chang, 2006). In a previous study, the CEW
powder was obtained from Sigma (St. Louis, MO, USA) and 15 mg/ml of
CEW with large-sized particulates was prepared as a feedstock. Before
running the YKC-SFBA column, the large-sized solid particulates
(> 3 mm) in the CEW solution need to be removed by a stainless filter
(pore size ∼ 3 mm) to avoid particles’ blockage in the column. It has
been confirmed that the UpFront EBA and STREAMLINE Direct columns
(Lihme et al., 1999; Hubbuch, Heboll-Nielsen, & Hobley, 2002;
Zafirakos & Lihme, 1999) would be blocked with large-sized solid
particulates (> 1 mm) in crude CEW, even though the concentration
used was low at 5 mg CEW/ml (Chang & Chang, 2006). In comparison,
commercial columns (e.g., STREAMLINE EBA, UpFront EBA, and STR-
EAMLINE Direct columns) have great difficulty in controlling for solid
particulates > 1 mm. Clarification, used in this work, was simplified
only to remove the large-sized particulates (> 3 mm). Hence, the
functionality of the newly designed YKC-SFBA column is superior to
that of conventional columns. A more detailed description of the SFBA
process system setup, handling, and performance can be found else-
(2)
where, as by Chang and Chang (2006).
Conventional STREAMLINE SP adsorbent (GE Healthcare) has a
particle size of 100–300 μm and mean density of 1.2 mg/ml. However,
because of its low density, the stability of an expanded bed may be
significantly affected when operated with viscous process liquids
(Hubbuch et al., 2002; Lihme et al., 1999; Zafirakos & Lihme, 1999).
STREAMLINE Direct HST adsorbent (GE Healthcare) has a smaller size
and a higher density than have conventional STREAMLINE EBA ad-
sorbents and can operate with higher viscosity liquids at higher liquid
velocity. Additionally, HST adsorbent has high salt-tolerance; hence, it
may avoid diluting high ionic strength and high viscosity feedstock. Use
of high-density adsorbents with appropriate size distribution can ensure
that the EBA process can be stably carried out at high flow rates without
losing adsorbent particles in the flow-through factions. This approach
provides significant benefits, such as reducing the mixing in liquid-solid
phases, decreasing process cycle time, improving product yield, and

622
K.-H. Chen et al.
avoiding the dilution of large volumes of feedstock (Anspach et al.,
1999; Chase, 1994; Hjorth, 1997; Thömmes, 1997).
3.2. Adsorption pH
It has been confirmed that the carboxylic groups on an HST ad-
sorbent display strongly acidic characteristics (i.e., pKa 1.95) (Chang,
Chou, Liu, & Tsai, 2007) and the adsorption performance of an HST
adsorbent is significantly influenced by working pH. Hence, the optimal
working pH had to be selected to achieve effective binding for lyso-
zyme. Over the range of adsorption, pH 4–12, investigated in this work,
the surface charge on lysozyme varied with adsorption pH and the
molecule had more positively charged amino groups at lower pH. Under
these circumstances, the HST adsorbent has fewer negatively charged
carboxylic groups.
Fig. 2(a) shows the effect of adsorption pH on binding capacity of
lysozyme to HST adsorbent in a crude CEW solution. The maximum
binding capacity (7.60·105 U/ml) was observed at pH 10. It may be
noted that the HST adsorbent contains more than one binding site for
ionic and hydrophobic interactions at a pH below pI 11.0 (Wang et al.,
2006). The minimum binding capacity for lysozyme was 3.40·105 U/ml
at pH 12. This may be caused by the repulsive electrostatic forces be-
tween HST and lysozyme.
As compared with the binding capacity of HST adsorbent for con-
taminating proteins, the maximum binding was at pH 5 and the binding
capacity was ∼44.7 mg/ml. The minimum binding capacity at pH 10
was ∼31.5 mg/ml. Generally, the contaminating proteins carry nega-
tive charges at pH above 7. At a higher pH of 12, the binding capacity
for contaminating proteins was 33.3 mg/ml. Hence, some strongly ionic
or hydrophobic interactions may be present. The differences in binding
capacity for contaminating proteins and lysozyme over an entire range
of pH vales may be caused by several factors, such as charge density of
HST, charge distribution and conformational structure of lysozyme, and
complex interactions between HST and lysozyme/contaminating pro-
teins. Based on the results, the optimal adsorption pH was found to be
10 and this was selected for further studies.
To optimize the design of an adsorption process, it is important to
establish the appropriate correlations for the equilibrium isotherms at
the optimal pH of 10. Two isotherm equations were used to analyze the
experimental data, namely Lamgmuir and Freundkich isotherm models
(Finette, Mao, & Hearn, 1997). The results are shown in Fig. S2 (sup-
plementary material). The experimental data of the equilibrium ad-
sorption studies for lysozyme and contaminating proteins followed the
Langmuir and Freundlich models, respectively. Based on the results, the
adsorption behaviour for lysozyme of the HST adsorbent was more
selective than that for contaminating proteins. The results indicated
that the mixed mode HST adsorbent was suitable for the purification of
lysozyme from CEW solutions.
3.3. Liquid hydrophobicity
It has been confirmed that the mixed-mode HST adsorbent has a
dual functionality with hydrophilic and hydrophobic properties
(Charoenrat et al., 2006; Jahic et al., 2006; Li et al., 2006). Hence, the
presence of a hydrophobic agent (e.g., ethylene glycol, EG) may de-
crease the polarity of adsorption buffer (Hofstee, 1973; Chang et al.,
2007), which was employed to reduce the hydrophobic interaction
between HST and lysozyme.
In this study, experiments were carried out with 30% EG at various
pH values. At adsorption pH below pI, the binding capacity in 30% EG
solution was lower than that in the buffer-only solution (Fig. 2(b)),
indicating the presence of competitive hydrophobic interactions during
the adsorption process. This might be due to the EG hindering the hy-
drophobic binding sites on HST adsorbent, resulting in less lysozyme
molecules adsorbed by HST adsorbent. The binding capacities for ly-
sozyme were reduced by 32.8% (pH 4) and 12.9% (pH 10), respectively
623
Food Chemistry 272 (2019) 619–627
as compared with the buffer-only systems.
At adsorption pH > pI value, the negative charge density of the
ionizable carboxylic groups on HST and lysozyme was significantly
increased. This may cause the electrostatic repulsion to overcome the
weakly hydrophobic interaction. Hence, the binding capacity decreased
dramatically from 4.08·105 U/ml (pH 11) to 6.29·104 U/ml (pH 12) in
the presence of 30% EG and was reduced by 18.7% (pH 11) and 81.6%
(pH 12) as compared with the adsorption in the absence of EG. The
hydrophobic interaction between HST and lysozyme was significantly
affected by the addition of 30% EG to the adsorption buffer.
Similar results were also observed for the adsorption of con-
taminating proteins and the binding capacity in 30%EG decreased from
39.1 mg/ml (pH 5) to 21.0 mg/ml (pH 12). When adsorption pH ap-
proached the pI value, the binding capacity was found to be 37.2 mg/ml
(pH 11). A multilayer adsorption process might occur, resulting in a
higher binding capacity at this pH value. The results revealed that the
binding capacity for contaminating proteins was less affected by the
addition of 30% EG to the adsorption buffer as compared with the
binding capacity for lysozyme. Hence, it was expected that more
strongly hydrophobic forces were present between HST and con-
taminating proteins.
Further experiments were carried out, investigating the effect of EG
concentrations in 10 mM glycine-NaOH (pH 10) on binding capacity of
HST for lysozyme and contaminating proteins. The binding capacity for
lysozyme and contaminating proteins decreased slightly from 7.60·105
to 4.75·105 U/ml and 31.5 to 28.8 mg/ml, respectively with increase in
EG concentration from 0 to 50% (Fig. 2(c)). The binding capacities were
decreased by 37.5% (lysozyme) and 17.0% (contaminating proteins).
However, the binding capacity for lysozyme and contaminating pro-
teins on the adsorbent remained high, probably due to the presence of
some strongly hydrophobic and/or ionic interactions. Hence, the pre-
sence of hydrophobic agent would cause the decrease of binding ca-
pacity of lysozyme on the HST adsorbent. This is due to the reduction of
hydrophobic interaction between HST and lysozyme. The hydrophobic
interaction plays an important role during the adsorption process.
3.4. Liquid ionic strength
The analysis of ionic strength effects on binding capacity, for lyso-
zyme in crude CEW, was carried out with various NaCl concentrations
(0–1 M, pH 10). Increase in NaCl concentration enhances both ionic
strength and conductivity of the liquid phase. This may lead to a de-
crease in hydrophilic and ionic interactions between HST and lysozyme,
resulting in a lower binding capacity. However, as NaCl concentration
rose above 0.5 M at pH 10, some deactivation of lysozyme occurred
during the adsorption process. Hence, the effect of NaCl concentration
(0–0.5 M) on lysozyme adsorption was investigated and the results are
shown in Fig. 2(d). As can be seen, the binding capacity decreased with
increasing NaCl concentration because of decrease in electrostatic in-
teraction between HST and lysozyme. The binding capacity for lyso-
zyme decreased from 7.60·105 U/ml (10 mM) to 1.52·105 U/ml (0.5 M)
and was reduced by ∼80%. Hence, changes in binding capacity may
contribute to the competitive adsorption for lysozyme between nega-
tively charged carboxylate groups of HST and negatively charged ions
of buffer.
3.5. Development of elution scheme for adsorbed lysozyme
3.5.1. General
As described Sections 3.3 and 3.4, the complex adsorption me-
chanisms between HST and lysozyme in CEW may result from the
combined effects of electrostatic and hydrophobic interactions. Hence,
a polarity-reducing agent, a solution of high ionic strength or changes
in buffer pH could possibly be incorporated into the elution buffer. In
these studies, the experiments were carried out with clarified CEW at a
liquid velocity of 200 cm/h in small packed beds (i.d. 0.7 cm, 3 ml
K.-H. Chen et al.
Fig. 3. Effects of various elution schemes on elution efficiencies of lysozyme and contaminating proteins. Column: Bio-Rad Econo 7/10 (i.d. 0.7 cm, STREAMLINE
HST, 3 ml), clarified CEW: 10 ml (6.18 mg/ml, 7.75·105 U/ml), liquid velocity: 200 cm/h. (a) Elution scheme: sodium carbonate buffer (10–100 mM, pH 12), 0.5 M
NaCl, (10–40 mM, pH 12), 50% EG (ethylene glycol, 10–40 mM, pH 12), and 0.5 M NaCl/50% EG (10–40 mM, pH 12), (b) 2–10 adsorbent bed volumes (VE/VB,
6–30 ml, 0.5 M NaCl, 40 mM, pH 12).
adsorbent).
In a column process, the losses of contaminating proteins and ly-
sozyme activity, during both adsorption and washing stages, were
83.2% and 11.0%, respectively. The binding capacities for lysozyme
and contaminating proteins were 7.32·105 U/ml and 10.4 mg/ml, re-
spectively. The relative elution efficiencies of contaminating proteins
and lysozyme for different elution schemes with elution volume (VE) of
30 ml were evaluated. These experimental results are discussed in the
following sections and shown in Fig. 3(a)–(b).
3.5.2. Effect of hydrophobicity on lysozyme elution
As a hydrophobic interaction is responsible for the binding me-
chanism, the presence of a hydrophobic agent, which can reduce the
hydrophobic interaction between HST and lysozyme in the elution
buffer (e.g., ethylene glycol, EG), may decrease liquid polarity as de-
scribed in Section 3.3.
For the elution scheme with 50% EG in various concentrations of
carbonate buffer (10–40 mM, pH 12), the adsorbed lysozyme was
completely eluted with 40 mM carbonate buffer. However, only 41.5%
of the adsorbed contaminating proteins were eluted from the adsorbent
bed, indicating that 50% EG at pH 12 as an eluent was not strong en-
ough to overcome the binding strength between HST and con-
taminating proteins. Based on the results, the purification factor of ly-
sozyme was 14.2, using 50% EG in 40 mM sodium carbonate buffer (pH
12) as an eluent.
3.5.3. Effect of ionic strength on lysozyme elution
It was expected that, when elution pH was increased to pH 12, the
negative charge density for lysozyme would rise. This may cause the
electrostatic repulsion to overcome some weakly hydrophilic and ionic
binding interactions. Moreover, the increase in both elution buffer and
salt concentrations may lead to a decrease in these interactions. This
would cause the adsorbed lysozyme to be more easily eluted from the
adsorbent.
For the elution scheme with various concentrations of carbonate
buffer (10–100 mM) at pH 12, the bound lysozyme was eluted to the
extent of 62–72%; however, only 14.2–31.0% of bound contaminating
proteins was removed from the adsorbent bed, indicating that the
electrostatic repulsion was not strong enough to overcome the binding
strength between HST and lysozyme/contaminating proteins. Based on
this study, the maximum purification factor (PF) of lysozyme was 14.7,
using 40 mM sodium carbonate buffer (pH 12) as an eluent.
624
Food Chemistry 272 (2019) 619–627
For the elution scheme with high ionic strength of salt (e.g., 0.5 M
NaCl) in various concentrations of carbonate buffer (10–100 mM, pH
12), the adsorbed lysozyme was completely eluted when the carbonate
buffer reached 40 mM, revealing that lysozyme was predominantly
bound by HST adsorbent via ionic interaction. Electrostatic repulsion
would overcome the weakly ionic and hydrophilic interactions between
HST and lysozyme. However, contaminating proteins removed from the
adsorbent bed were only 30.4%. It was apparent that some strongly
hydrophobic interaction might be involved for the binding mechanism
of contaminating proteins. From this study, 0.5 M NaCl in 40 mM so-
dium carbonate buffer (pH 12) as an eluent gave a recovery yield of
∼100% and purification factor of 19.5, Hence, it was selected for a
further optimization study.
3.5.4. Combination effect of ionic strength and hydrophobicity on lysozyme
elution
For the final elution scheme with a combination of 0.5 M NaCl and
50% EG in 10 mM carbonate buffer (pH 12), the adsorbed lysozyme was
also completely eluted from the bed; however, the removal of con-
taminating proteins remained low (approximately 48.2%). It was ap-
parent that there were some strong binding sites present on HST, which
bound contaminating proteins very tightly. Hence, the bound con-
taminating proteins cannot be eluted easily and completely. Lysozyme
was completed eluted with a recovery yield of ∼100% and purification
factor of 12.3.
3.5.5. Evaluation of elution efficiency for lysozyme
Based on our previous results, the adsorbed lysozyme was com-
pletely eluted from the adsorbent bed with an eluent volume of 30 ml at
a liquid velocity of 200 cm under the following conditions: (1) 0.5 M
NaCl in 40 mM carbonate buffer (pH 12), (2) 50% EG in 40 mM car-
bonate buffer (pH 12), and (3) 0.5 M NaCl-50% EG in 10 mM carbonate
buffer (pH 12). The elution efficiencies (E, ∼100%) in lysozyme purity
under the above elution schemes for purification factor (PF) are in the
order: 0.5 M NaCl (40 mM, pH 12, VE 30 ml, PF 19.5) > 50% EG
(40 mM, pH 12, VE 30 ml, PF 14.2) > 0.5 M NaCl-50% EG (10 mM, pH
12, VE 30 ml, PF 12.3) (Fig. 3(a)). The experimental results demon-
strated that both electrostatic and hydrophobic forces contributed to
the elution of lysozyme. The electrostatic repulsion was considered to
play a more important role during the elution process. Further experi-
ments conducted with 0.5 M NaCl (40 mM, pH 12) were used to in-
vestigate the effect of eluent volumes used (VE, 2–10 bed volumes,
K.-H. Chen et al. Food Chemistry 272 (2019) 619–627

6–30 ml) on the elution efficiency. The yield of eluted lysozyme was
found to increase as irrigation volume of eluent (VE) increased and then
approached a plateau value (∼100%) for 9-fold adsorbent bed volumes
(VB) of eluent (Fig. 3(b)). Hence, 9-fold column volumes were chosen as
the optimal volume of eluent (i.e., 0.5 M NaCl, 40 mM, pH 12) to be
useful in a further SFBA purification study.
3.6. Effect of rotating speed on adsorption performance of lysozyme
Previous experimental results confirmed that the degree of bed ex-
pansion in fluidized bed was not significantly affected by the fluidized
bed height, rotating speed, and aspect ratio of the column (< 5%)
(Chang & Chang, 2006). CEW viscosity could be effectively lowered by
high-speed dispersion homogenizer treatment, leading to the formation
of a homogeneous solution. In the present case, the viscosity of CEW
dropped to 3.09 mPa·s after the treatment.
The effect of rotating speed (0–200 rpm) on fluidization character-
istics was examined by careful measurements of liquid flow
visualizations, as shown in Fig. S3 (supplementary material).
In fluidized bed without agitation, liquid channelling and by-pas-
sing were obvious and the top surface of the bed appeared unstable.
Smoother fluidization was observed upon increasing rotating speed by
mechanical agitation; hence the degree of agglomeration of fine ad-
sorbent particles, channelling and by-passing of process liquid were
reduced, and a relatively more stable fluidized bed was achieved.
Obviously, agitation is an effective method to overcome the inter-
particle forces of fine particles in fluidization and to enhance their
fluidization stability with the viscous CEW as a feedstock. The effec-
tiveness of agitation for improving fluidization is strongly dependent on
the rotating speed employed. Therefore, an increase in rotating speed
enhances the stability of liquid flow, which contributes to the stability
of the fluidized bed and improves the fluidization quality of the entire
bed.
Fig. 4 shows the breakthrough profiles for lysozyme and con-
taminating proteins in a crude CEW at a settled bed height of ∼20 cm,
operated with various rotating speeds (i.e., 0–200 rpm). At rotating

Fig. 4. Effect of rotating speed on adsorption performance of (a) lysozyme activity and (b) contaminating proteins in SFBA process. Column: YKC-SFBA (i.d. 25 mm),
settled bed height: 20 cm (100 ml), fluidized bed height: 40 cm, adsorption buffer: 10 mM glycine-NaOH (pH 10), inlet crude CEW: 3.80·105 U/ml, 26.2 mg/ml (pH
10), flow rate ratio between CEW and adsorption buffer: 1:1, liquid velocity during adsorption: 490–120 cm/h, average liquid velocity: 200 cm/h. C is the con-
centration of total protein in out stream (mg/ml), Co is the inlet concentration of total protein (mg/ml), A is the activity of lysozyme in outlet stream (U/ml), and Ao is
the inlet activity of lysozyme (U/ml). (c) Purification of lysozyme from highly viscous CEW using HST adsorbent in SFBA process. Column: YKC-SFBA (i.d. 25 mm),
settled bed height: 20 cm (100 ml), fluidized bed height: 40 cm, adsorption buffer: 10 mM glycine-NaOH (pH 10), inlet crude CEW: 3.80·105 U/ml, 26.2 mg/ml (pH
10), flow rate ratio between CEW and adsorption buffer: 1:1, liquid velocity during adsorption: 490–120 cm/h, average liquid velocity: 200 cm/h. Elution: 0.5 M NaCl
in 40 mM sodium carbonate buffer (pH 12) at liquid velocity of 200 cm/h. A is the activity of lysozyme in outlet stream (U/ml), Ao is the inlet activity of lysozyme (U/
ml), C is the concentration of total protein in out stream (mg/ml), and Co is the inlet concentration of total protein (mg/ml). (d) SDS-PAGE analysis for purified
lysozyme samples from CEW: 1. Standard lysozyme (Sigma), 2. Fresh CEW sample, 3. CEW powder (Sigma), 4. Flow-through sample, 5. Wash sample, 6–7 Eluted
lysozyme sample, and 8. Standard lysozyme (Sigma).

625
K.-H. Chen et al.
speed < 200 rpm, the adsorption behaviour for lysozyme presented an
irregular breakthrough curve, due to the instability of the liquid flow
(Fig. 4(a)). On the other hand, the behaviour of fluid flow significantly
affected lysozyme adsorption performance. However, this adsorption
behaviour has less influence on the contaminating proteins, indicating
that the HST adsorbent was less selective for the adsorption of un-
desired proteins (Fig. 4(b)).
For a given SFB adsorption process, altering the flow rate would
contribute to maintaining a constant degree of bed expansion (i.e., two-
fold the settled bed height, ∼40 cm). When the adsorption buffer was
changed to highly viscous CEW, the liquid velocity was reduced from
490 to 120 cm/h during the adsorption stage. The average loading
velocity of CEW for each rotating speed was controlled at ∼200 cm/h.
No significant differences in breakthrough values of contaminating
proteins were found under these rotating speeds. The dynamic binding
efficiency obtained from frontal analysis was expected to be similar.
However, the breakthrough values of lysozyme were significantly af-
fected by the rotating speed of agitator. The results clearly indicated
that the dynamic binding efficiency was much higher at a rotating
speed of 200 rpm. Lysozyme molecules in CEW feedstock became much
more selective when using the mixed-mode adsorbent. The lower ad-
sorption efficiency at lower rotating speed in the SFB column was due
to the higher degree of liquid channelling and/or by-passing, which led
to insufficient contact time between the lysozyme molecule and the
adsorbent bed. Simultaneously, the lysozyme molecule does not have
enough time to diffuse into the binding sites of adsorbent beads. Hence,
a higher percentage of lysozyme was lost during adsorption at a lower
rotating speed.
In the SFBA process operated with different rotating speeds of agi-
tator, the differences in breakthrough values and/or dynamic binding
efficiency for lysozyme under similar operating conditions (e.g., inlet
concentration of CEW, column aspect ratio, degree of fluidized bed, and
process liquid velocity) were attributed to the stability of flow hydro-
dynamics and the residence time of process liquids. The flow stability of
the bed may be a significant impact factor of the adsorption process.
Hence, with increase in rotating speed, a higher percentage of lysozyme
was expected to be adsorbed. In the SFBA purification process, the
maximum loading volume of CEW was chosen to be 600 ml, corre-
sponding to six-fold the settled bed volume at a rotating speed of
200 rpm.
3.7. Direct recovery of lysozyme from highly viscous CEW using the SFBA
process
According to the results described above, the rotating speed was
operated at 200 rpm in a SFBA process. Two pumps were employed to
control the liquid velocity of adsorption buffer and crude CEW, re-
spectively and to deliver the two liquids at equal velocities during the
adsorption stage. In this experiment, the settled bed height was fixed at
20 cm and all operating stages were carried out in a fluidized bed only.
Owing to the high viscosity of CEW, the overall liquid velocity was
reduced from ∼490 cm/h to ∼120 cm/h during the adsorption stage
and the average velocity was approximately 200 cm/h. The residual
particulate material was completely washed out from the bed with the
Table 2
Purification of lysozyme from highly viscous CEW using SFBA technique. Column: YKC-SFBA (i.d. 25 mm), settled bed height: 20 cm, 100 ml, fluidized bed height:
40 cm, adsorption buffer: 10 mM glycine-NaOH (pH 10), inlet CEW: 3.80·105 U/ml, 26.2 mg/L, pH 10, 600 ml, flow rate ratio between CEW and adsorption buffer:
1:1, liquid velocity during adsorption: 490–120 cm/h, average liquid velocity: 200 cm/h. Elution: 0.5 M NaCl in 40 mM sodium carbonate buffer (pH 12) at a liquid
velocity of 200 cm/h.
Purification step Volume (ml) Total protein (mg) Total activity (U)
Crude CEW 600 (1.57 ± 0.02)·104 (2.28 ± 0.14)·108
Flow and wash 2400 (1.50 ± 0.02)·104 (1.36 ± 0.08)·107
Elution 900 (9.45 ± 0.12)·102 (2.15 ± 0.13)·108
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Food Chemistry 272 (2019) 619–627
adsorption buffer at 200 cm/h. The loss of lysozyme activity during
adsorption and washing stages was ∼5.98%, whereas the removal of
undesired proteins was ∼43.0%. In the elution stage, lysozyme was
recovered by 0.5 M NaCl in 40 mM sodium carbonate buffer (pH 12)
with a high yield of 94.3% and a purification factor of 15.7. The ex-
perimental results are shown in Fig. 4(c) and Table 2. Fig. 4(d) depicts
the SDS-PAGE analysis of lysozyme samples purified from CEW,
showing the molecular weight of lysozyme at ∼14.3 kDa. The eluted
lysozyme exhibited only one protein band, indicating high purity of
lysozyme and the specific activity of the purified lysozyme was de-
termined as 2.28·105 U/mg. Hence, the SFBA technique provided an
efficient single-step purification process for lysozyme from highly
turbid CEW.
3.8. Comparison with conventional techniques for the purification of
lysozyme
After the pre-treatment of CEW, centrifugation and filtration steps
are commonly used in conventional techniques to obtain a clarified
sample. Ion-exchange and dye-ligand affinity chromatography are po-
pularly used in purification of lysozyme from a diluted CEW sample,
either in packed bed or in aqueous two-phase systems. As shown in
Table S1 (supplementary material), some examples have been demon-
strated that the SFBA technique that is used in this work gives much
higher productivity in a single step than do conventional purification
techniques (Chang & Chang, 2006; Chiang et al., 1993; Su & Chiang,
2006; Tong et al., 2002; Yilmaz et al., 2005); hence, this technique is
eminently suitable for purification of lysozyme, especially from a highly
turbid CEW.
4. Conclusions
This study suggested that working pH was important for adsorption
of lysozyme to HST adsorbent in complex CEW. However, the binding
capacity is largely independent of ionic strength or hydrophobicity of
process liquids. The adsorbed lysozyme was completely eluted by three
elution schemes with salt of high ionic strength or a polarity-reducing
agent in a sodium carbonate buffer (pH 12). This finding was consistent
with the assumption that hydrophobic and ionic interactions between
HST and lysozyme were principal forces. Hence, the complex binding
mechanisms might involve multi-binding sites on HST adsorbent during
the adsorption process, which poses difficulty for the adsorbed lyso-
zyme and/or contaminating proteins to elute from the HST mixed-mode
adsorbent.
The effectiveness of agitation on improving fluidization stability is
strongly dependent on the rotating speed employed. Agitation was
found to significantly reduce both agglomeration of fine adsorbent
particles and channelling of the process liquids in the bed, thus im-
proving the fluidization stability of the bed. The work demonstrated
that hydrodynamic stability of the stirred fluidized bed can be achieved
by operation with highly turbid CEW at a rotating speed of 200 rpm.
Hence, the dense HST adsorbent is suitable for operating with highly
viscous CEW at a high fluidization velocity. The present results showed
successful single-step purification of lysozyme with a high yield of
Specific activity (U/mg) Purification factor (folds) Lysozyme yield (%)
(1.45 ± 0.07)·104 1 100
– – 5.96
(2.28 ± 0.11)·105 15.7 94.3
K.-H. Chen et al.
94.3% and a purification factor of 15.7.
Acknowledgement
The work has been supported by the Ministry of Science and
Technology of Taiwan (Grant numbers MOST 104-2622-E-131-006-CC3
and 105-2221-E-131-030).
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