Journal of Membrane Science and Research 9 (2023) 562862

Journal of Membrane Science & Research

journal homepage: www.msrjournal.com

Research Paper

Isolation of Lysozyme from Chicken Egg White by Surface Charged Membranes

Nandala Shiva Prasad 1, 2, 3, Aarti Tallam 1, Namita Roy Choudhury 3, Sundergopal Sridhar 1, 2, *, Suresh K. Bhargava 3

1
Membrane Separations Laboratory, Process Engineering and Technology Transfer Division, CSIR - Indian Institute of Chemical Technology, Hyderabad,
India-500007.
2
Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, Uttar Pradesh, India.
3
Royal Melbourne Institute of Technology (RMIT), Melbourne, VIC 3001, Australia.

Article info
Received 2022-10-02
Revised 2022-12-27
Accepted 2023-01-31
Graphical abstract
Available online 2023-01-31

Keywords
Sulfonated polyethersulfone
Polyethylene glycol
Antifouling
Lysozyme separation
Morphology

Highlights
• Negatively charged membranes were fabricated using
sulfonated PES.
• Permeation flux was enhanced under cross-flow over
dead-end filtration.
• sPES membranes exhibited a low fouling tendency for
protein adsorption.
• Semi-batch protein isolation demonstrated selective
separation of Lysozyme.

Abstract
The fabrication of innovative and resourceful ultrafiltration membranes for the separation of protein mixtures is significantly required, principally in the food and pharmaceutical
manufacturing sectors. The current research focuses on the preparation of surface-charged membranes by polyethersulfone (PES)/ sulfonated polyethersulfone (sPES) membranes
blended with different molecular weight cut-off (MWCO) polyethylene glycol (PEG) for the separation of Lysozyme from natural chicken egg white (CEW). The synthesized
membranes were characterized by scanning electron microscopy (SEM), atomic force microscopy (AFM), contact angle, and porosity. By examining the morphological changes
and the effect of physicochemical parameters, the optimized process condition to attain maximum lysozyme separation was determined. In the semi-batch mode of operation,
the sPES-PEG400 membrane exhibited maximum Lysozyme transmission and high fouling resistance. The outcomes of the present study led to an in-depth understanding of the
interrelationship between the PES/sPES with PEG, which sheds light on the use of membrane technology in complex protein mixture separation.
© 2023 FIMTEC & MPRL. All rights reserved.
1. Introduction

Downstream processing and isolation of proteins have gained proteins. Indeed, packed bed column chromatography is widely practiced
tremendous attention in the last two decades due to their vast industrial commercially for large-scale protein separation, where the operational
applications, especially in the pharmaceutical [1-3] and food industries and regeneration costs are the primary concern due to slow mass transfer,
[4-6]. Purification of proteins involves intensive separation techniques high process pressure drops, and lengthy operational procedures [5,13].
such as electrophoresis [7,8], packed bed column chromatography [9], and Therefore, the rapid expansion in the biotechnology, pharmaceutical, and food
ion exchange chromatography [10-12]. On the other hand, the techniques industry demands a cost-effective large-scale protein purification technique.
used in the laboratory procedures for protein purification are proficient in Lysozyme is one of the most widely consumed proteins in food and
processing small quantities. However, laboratory processes are complicated pharmaceutical applications as a preservative agent [5,14], anti-microbial
to scale up as they involve complex instrumentation to yield high-purity [1,15], anti-inflammatory [4,16], anticancer drug [2,3,17-19], respectively.

* Corresponding author: sridhar11in@yahoo.com (S. Sridhar)

DOI: 10.22079/JMSR.2023.562862.1568

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Additionally, lysozyme acts as an antibacterial protein, which can cleave the 2. Materials and methods
peptidoglycan layer of the bacteria cell wall by disrupting ß-linkages between
the N-acetyl-D-glucosamine and N-acetylmuramic acid [5,20]. Furthermore, 2.1. Materials
Lysozyme has potential applications in human cancer chemotherapy in
addition to the anticancer drug [21,22]. Polyethersulfone (PES) (35 kDa) and Polyethylene glycol (PEG) with
Traditionally, Lysozyme is extracted from chicken egg white (CEW) by MWCO 400 and 6000 Da polymers were procured from Solvay Specialties
hybrid processes combining crystallization, precipitation, chromatography, India Private Limited, Mumbai, India, for membrane synthesis and as pore-
and adsorption [23]. Nevertheless, these methods are generally tedious, forming agents, respectively. Sulfuric acid, Triethyl phosphate (C6H15O4P)
complicated, and costly for large-scale production of Lysozyme. Besides the (TEP), 1,1,2,2-tetrachloroethane (C2H2Cl4), Methanol, N-Methyl-2-
low concentration of Lysozyme in crude CEW became a crucial challenge in pyrrolidinone (NMP) chemicals were purchased from SD fine chemicals,
the development of separation techniques since large volumes of process Hyderabad, India. Dipotassium hydrogen phosphate (K2HPO4), Potassium
streams need to be handled to produce a reasonable amount of pure Lysozyme dihydrogen orthophosphate (KH2PO4), Bradford reagent, Tris, Glycine,
[21]. In recent times, various innovative techniques have been proposed TEMED, Ammonium persulfate, Acrylamide, and Bis-acryl amide obtained
[5,24-29], in which the membrane separation process is one of the emerging from Sigma Aldrich, USA. The sodium hydroxide (NaOH), hydrochloric acid
technologies. Ultrafiltration being a powerful tool for bio-separation, the (HCl), and sodium chloride (NaCl) chemicals were procured from Avra
significant advantage is that proteins can be easily separated based on the size Chemicals, Hyderabad, India. Coomassie blue staining solution was procured
exclusion principle and are free from foreign particles. Ghosh and Cui have from Bio-Rad Laboratories (India) Private Limited, India, and used in
used the commercial polysulfone membranes of 25 and 50 kDa MWCO and Bradford protein assay to estimate protein concentration. The deionized water
studied the effect of different functional parameters (pH, pressure, system with TDS < 2 ppm was used for sample preparation and experimental studies
hydrodynamics) for the separation of Lysozyme from CEW. High purity of in the laboratory using the RO membrane cascade system. The laboratory
Lysozyme of 98.7% was achieved through a 25 kDa membrane at a glassware, including burettes, conical flask, measuring jar, beakers, etc., for
transmembrane pressure of 80 kPa and pH of 11 [30]. In addition, the the preparation of solutions, was supplied by Borosil, Hyderabad, India.
variation of purity levels of Lysozyme with the pre-treatment of 50 kDa
polysulfone membrane using myoglobin was also discussed. The separation 2.2. Sulfonation of PES using Triethyl phosphate and Sulfuric acid
of Lysozyme was 26% superior by using a pre-treated membrane with a
purity of 96% at 120 kPa pressure [21]. Among the protein purifications, The sulfonated PES ultrafiltration (UF) membrane was prepared by phase
membranes prepared from PES have gained special importance as it provides inversion method using triethyl phosphate (TEP) and sulfuric acid, where
high rigidity, a high degree of molecular immovability, and superior strength. TEP acts as a catalyst. The sulfonating agent was prepared using 5 ml triethyl
Conversely, membrane fouling is the major drawback faced by PES- phosphate in 10 ml sulfuric acid in a 1:2 ratio at 4˚C and kept aside for 24
based ultrafiltration membranes due to their hydrophobicity and surface hours. On the other hand, 20 g of PES was dissolved in 100 ml of 1,1,2,2-
roughness [31,32]. The adsorption and deposition of protein molecules on the tetrachloroethane at room temperature (27±2ºC) [21] and continuously stirred
membrane surface lead to the formation of a foulant layer. It is widely at a rate of 450 RPM to get a homogenous solution. Then, 15 ml of the
acknowledged that increasing hydrophilicity and decreasing surface sulfonating agent was slowly added to the polymer solution at a 0.125 ml/min
roughness improve the membrane's antifouling characteristic properties [33]. rate under continuous stirring at 75˚C for 2 h (Scheme S1). The mixture was
Several roots have been adopted to enhance the separation characteristic permitted to proceed for 120 minutes and terminated by precipitating the
properties of the PES membranes, which include surface coating, sulfonation, solution into an ice-cold bath. The precipitate was sieved and washed with
and blending with hydrophilic additives such as (polyvinyl pyrrolidone methanol for 1 h, followed by drying at 65 ˚C under a hot air oven for 4 h.
(PVP), polyethylene glycols (PEG), polyethylene oxide (PEO)) [31,34,35]. Further, to neutralize the SPES, the dried polymer was dissolved in 104 ml of
On the other hand, the separation properties of a membrane are 1-methyl-2-pyrrolidinone (NMP), as per the polymer weight. The dissolved
significantly influenced by surface pore size and cross-sectional morphology. polymer was precipitated into DI water followed by sieving and washing until
The wet phase inversion process is the most extensively used technique for reached neutral pH and dried at 70 ˚C for 20 h. This SPES polymer was used
fabricating asymmetric membranes. The kinetic parameters such as in the fabrication of ultra-porous cation exchange membranes.
solvent/non-solvent demixing rates and thermodynamic parameters, including
polymer/additive-solvent interactions, were well controlled to obtain the 2.3. Preparation of PES and SPES-UF membrane
desired membrane morphology and separation selectivity. Additionally, the
polymer dope solution composition, coagulation bath temperature, and PES and sPES membranes with different porosities were fabricated by
additive size alter the membrane morphology [36]. PEG has been used widely the non-solvent-induced phase separation (NIPS) technique called the wet
as a pore former due to its dispersion with membrane materials and is quite phase inversion process. NIPS technique is widely practiced to prepare
well miscible with solvents as well as non-solvents. Mohammad et al. various morphologies by controlling the thermodynamic characteristics of the
investigated the effects of PEG concentrations, molecular weight, and demixing process. In the present study, the key preparation parameters are the
coagulation bath temperature on the morphology of the PES membranes [37]. type of additives and the temperatures of the coagulation bath. The additives
They found that the increasing PEG concentration, and PEG molecular weight such as PEG_400 and PEG_6000 Da were used as pore-forming agents, and a
(MW), increased the demixing rate of solvent/non-solvent and enabled the combination of varying coagulation bath temperatures was employed to
formation of macrovoids in the membrane structure. Lin et al. have studied enhance the pore density and control the pore size of the resulting
the effect of different PEG molecular weights on ultrafiltration membrane membranes. Initially, 25 wt% of PES and SPES polymer solution were
properties and performance and found that the morphology of surface pore prepared by dissolving 25 g of polymer in 75 g of NMP and stirring for 4 h at
size increased with increasing PEG MW [38]. From the literature, the role of room temperature (28±2ºC). The obtained polymer solution was cast on a
PEG as a pore former and the demixing rate has well understood in polyester (PE) non-woven fabric support using a doctor's blade with an air
controlling the membrane morphology. However, limited articles have been gap of 120 µm to achieve the desired membrane thickness. After that, the
reported on the rationalization of membrane morphology in the selective coated film was immediately immersed in DI water bath at a specified
separation of CEW proteins. coagulation bath temperature. The same procedure was followed by adding
The present study aims to develop hydrophilized and enhanced 6% of PEG_400 or PEG_6000 additives to the polymeric solution and
antifouling membranes to isolate Lysozyme from CEW. PES/sPES varying the non-solvent (DI water) bath temperature from 10 to 45ºC to
membranes with different porosities were fabricated using PEG (400 and obtain various configurations. The combination of membrane denotations is
6000 kDa) additive and varying the coagulation bath temperature. The provided in Table 1. The procedure for preparing PES and SPES membranes
morphological changes of the prepared membranes were investigated using using the NIPS method is schematically represented in Fig. 1(a). The prepared
scanning electron microscopy (SEM), Atomic force microscopy (AFM), membranes were washed thoroughly with DI water to remove the trace of
whereas the hydrophilicity of the membranes was evaluated using contact solvent on the membrane surface and then dried at ambient conditions.
angle measurements. Subsequently, the performance of these membranes was
investigated in obtaining pure Lysozyme under the optimized condition and
antifouling properties.

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Table 1
Combinations of membrane fabrication

Coagulation bath
Membrane ID Polymer (g) Additive, weight (g) Solvent, weight (g)
temperature (°C)
PES_25 M1 PES, 25 - NMP, 75 25
sPES_25 M2 sPES, 25 - NMP, 75 25
PES_PEG400_25 M3 PES, 25 PEG400, 1.5 NMP, 79.5 25
sPES_PEG400_25 M4 sPES, 25 PEG400, 1.5 NMP, 79.5 25
PES_PEG6000_25 M5 PES, 25 PEG6000, 1.5 NMP, 79.5 25
sPES_PEG6000_25 M6 sPES, 25 PEG6000, 1.5 NMP, 79.5 25
sPES_PEG400_10 M7 sPES, 25 PEG400, 1.5 NMP, 79.5 10
sPES_PEG400_45 M8 sPES, 25 PEG400, 1.5 NMP, 79.5 45
sPES_PEG6000_10 M9 sPES, 25 PEG6000, 1.5 NMP, 79.5 10
sPES_PEG6000_45 M10 sPES, 25 PEG6000, 1.5 NMP, 79.5 45

2.4. Phosphate Buffer preparation

In the present study, the phosphate buffer solution was prepared by
(a)
mixing the dipotassium phosphate (A) and potassium dihydrogen phosphate
solutions (B). Individual solutions of A and B are prepared by dissolving
87.09 g and 68.045 g in 500 ml DI water to obtain 0.5 L of 1M dipotassium
phosphate and potassium dihydrogen phosphate, respectively. Further, to
achieve a 7.4 pH of the buffer, 19.8 ml of solution A was added to 80.2 ml of
solution B, followed by diluting 10 times to obtain 0.1 M phosphate buffer.

2.5. Egg white solution preparation

The supernatant egg white solution was prepared for the ultrafiltration
process, where a 100 ml homogenized egg white was diluted with 900 ml of
0.1M phosphate buffer solution, filtered, and centrifuged at 12000 rpm for 15
min. The collected supernatant contains a lipid-free protein solution, a source
for Lysozyme in the present study.

2.6. Experimental studies

2.6.1. Batch studies

(b)
Lysozyme extraction experiments were conducted to separate Lysozyme
from egg white solution using PES and SPES-UF in dead-end and cross-flow
filtration under batch mode operation. The supernatant solution of egg white
was used as feed. The schematic representation of the batch mode
experimental setup was shown in Fig. 1(b), where the feed tank of 5 L
capacity was connected to the membrane module with a membrane-active
surface area of 95 cm2, through a peristaltic pump. The jacketed glass vessel
was used as a feed tank to maintain the feed temperature. The permeate was
collected at one end of the module and the reject was recycled back to the
feed tank. The pressure gauge was fixed at the reject streamline to measure
the applied pressure across the membrane. The pressure on the membrane
surface was built by restricting the reject stream flow rate using a needle
valve.
Before starting each experiment, the experimental setup was circulated
with phosphate buffer to remove the previous experimental traces from the
system. The jacketed feed tank was filled with the prepared egg white
solution as feed, which was maintained at 10 ± 2 ºC temperature with the help (c)
of coolant circulation. The feed was fed to the membrane module in two
different configurations of dead-end and cross-flow manner at 120 rpm with
15 psi pressure. In dead-end filtration, the feed flow is perpendicular to the
membrane surface, whereas, in cross-flow filtration, the feed flows parallel to
the membrane surface. The permeation flux was estimated by measuring the
volumetric flow rate of permeate and samples were analyzed qualitatively and
quantitatively. The initial screening of the synthesized membrane was done in
batch mode experiments, where the separation characteristics such as
permeation flux, hydraulic resistance, and protein concentration in permeate
were estimated. Further, experiments were carried out to test the robustness of
membrane performance in the continuous extraction of Lysozyme in semi-
batch mode.

Fig. 1. Schematic representation of (a) fabrication of PES/sPES membranes, (b) dead-

end and cross-flow mode of ultrafiltration, (c) semi-batch operation for continuous
extraction of Lysozyme from CEW.

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2.6.2. Semi-batch studies where, Ww and Wd are weights of wet and dry membranes in g, ρw is the
density of water in g/cm3, A is the membrane-active surface area in cm2, and δ
The semi-batch experiments were conducted to continuously extract
is the membrane thickness in cm.
Lysozyme from the feed egg white solution. The semi-batch studies were
performed with the selected membrane from the preliminary screening based 2.7.6. Pure water flux (PWF) and fouling studies
on selective separation of Lysozyme under bench-scale. The schematic of the
semi-batch mode ultrafiltration system was provided in Fig. 1(c), where an All the membranes synthesized were analyzed for their performance and
additional buffer solution tank was introduced to the feed tank of the batch fouling characteristics based on the pure water flux. At first, the permeation
mode experimental setup. The buffer solution was continuously added to the test was performed by passing DI water through the membrane sample at 20
feed tank in order to maintain the constant feed volume. The feed tank level psi, and the time taken for collecting every 10 mL of permeate was recorded.
was continuously monitored using a level sensor, which activates the buffer In general, fouling studies are carried out to govern the life period of the
feeding pump (pump 2). membranes. The sample membranes were primarily immersed in egg white
supernatant for 7 days to investigate the antifouling behavior. After that, the
2.6.3. Protein Adsorption studies membranes were cleaned with DI water, and the pure water permeation test
was carried out similarly. The pure water flux through the membranes is
The protein adsorption experiments were performed to ascertain the
calculated using Equation 2.
adsorbed amount of protein on the membranes. These experiments were
conducted by submerging the membranes in a supernatant solution.
Q
Rectangular pieces of membranes (24cm2 area) were engulfed in vials Jw = (2)
containing 40g of egg white supernatant solution (10-fold) in 0.1M phosphate A
buffer of pH = 7.4. The vials were then kept on the shaker at 120 RPM at
room temperature (RT) with constant stirring. Then for every 24 h, the where, Jw is the pure water flux (L/m2.h), Q is the permeate volumetric flow
supernatant solution was collected until the membranes got saturated and the rate (L/h) and A is the effective area of the membrane (m2).
concentration of protein in the supernatant solutions was determined using a
2.7.7. Hydraulic resistance
UV-Vis spectrophotometer at 280 nm. The amounts of protein adsorbed on
the membranes were estimated from the change in UV absorption of protein Hydraulic resistance is a key element that defines the productivity of any
in the supernatant solution before and after the adsorption. The final results pressure-driven membrane process. The membrane performance at different
are the average of three measurements for each membrane [39]. operating pressures and its optimization are important factors for
ultrafiltration operations. The hydraulic resistance exerted by the membrane
2.7. Membrane characterization (Rm) is an intrinsic property determined by pure water flux. Hydraulic
resistance is assessed by first plotting the pure water flux as a function of
2.7.1. FTIR transmembrane pressure. The slope of this curve gives the ratio of water flux
The surface chemistry of chemical bonds and functional groups of the to the transmembrane pressure that states the hydraulic permeability, and the
PES/sPES membrane before and after protein adsorption were examined by inverse of it describes the hydraulic resistance of the membrane. The relation
Fourier transform infrared spectroscopy (FT-IR) using a PerkinElmer of hydraulic resistance in terms of flux and pressure is shown in Equation 3.
spectrum 100 FTIR spectrophotometer, Boston, MA, USA. The spectrum was
measured in the wavelength region of 650 – 4000 cm−1 at ambient PT
Rm = (3)
temperature. J w

2.7.2. SEM where Rm is membrane hydraulic resistance (m-1), PT is the transmembrane

The cross-sectional morphology of neat PES/sPES and PEG-loaded pressure (N/m2), and µ is the DI water viscosity (N.s/m2).
membranes were analyzed by scanning electron microscopy (SEM) with
2.7.8. Ion Exchange Capacity (IEC)
Quanta 200 model SEM instrument. Before the SEM analysis, the membranes
were freeze fractured in liquid nitrogen to shield the cross-sectional To evaluate the ion exchange capacity (IEC), the sample membrane of 10
morphologies and then coated with iridium of 5 nm thickness to minimize x 10 cm2 was immersed in 1 mol/dm3 HCl solution. Then, the excess HCl was
surface charging. thoroughly removed by washing the membrane with DI water. Subsequently,
the sample was dipped in 1 mol/dm3 NaCl solution, where the protons were
2.7.3. AFM topography replaced with the Na+ ion on the membrane surface. The released protons
The three-dimensional surface topographical changes of prepared from the membrane were estimated by titrating with NaOH solution (0.01
membranes were analyzed using atomic force microscopy (AFM, Asylum mol/dm3), where phenolphthalein was used as an indicator. Afterward, the
Research MFP-3D Infinity). The relative surface roughness of the membranes membrane was washed with DI water and dried by placing it in the oven
was collected by examining the membrane with a resolution of 256 x 256 and maintained at 50 ℃ for about 10 h. The weight of the dry membrane was
a scanning area of 5 X 5 µm under tapping mode. measured, and then the IEC was estimated using Equation 4.

CNaOH  VNaOH
2.7.4. Contact angle (CA) measurement IEC = (4)
Wdry
The wetting ability of the PES/sPES membranes' surface was
characterized using Dino-Lite Basic AM211 digital microscope model CA
analyzer, Taiwan. The CA measurements were performed at 25 °C using the where C and V are the concentration (mol/dm3) and volume consumed (dm3)
sessile drop method by placing a 3-μL droplet of water on the dry membrane of NaOH, respectively, and Wdry is the membrane dry weight (g).
sample; noted within 5 s after dropping to achieve accurate values.
2.8. Analytical Studies
2.7.5. Porosity 2.8.1. SDS-PAGE for characterization
To estimate the bulk porosity of the membranes, firstly, the sample The SDS–PAGE was performed using 12% polyacrylamide gel. The
membranes (10 x 10 mm) were soaked in deionized water for about 24 h. polyacrylamide gel was made by adding resolving gel (pH–8.8; 5ml) and
Later, the leftover water droplets on the surface were wiped off with soft stacking gels (pH -6.8; 2ml). First, the resolving gel (pH–8.8) was prepared
tissue and measured using a balance. Successively, the samples were dried in by adding 1575µl water, 2000µl ABA(Acrylamide/Bis-acrylamide) (30%
a hot air oven maintained at 60 ℃ for 8 h to diminish the upshot of lasting acrylamide), 1300µl Tris buffer (pH–8.8), 50µl Sodium Dodecyl Sulphate
additives and solvents on porosity measurement. The corresponding weights (SDS; 10%), 50µl Ammonium Persulfate (10%), 25µl stain free and 2µl
of dry membranes were recorded. The bulk porosity (ε) of the primed TEMED (N, N,N′,N′-Tetramethyl ethylenediamine) and it was poured in
membranes was estimated by a most regularly used gravimetric method between plates. Afterward, 2ml stacking gel was prepared by adding 1400µl
(Equation 1) [40]. water, 330µl ABA (30% acrylamide), 250µl Tris buffer (pH – 6.8), 20µl SDS
(10%), 20 µl APS (10%) and 2µl TEMED and it was also poured above the
Ww − Wd
=  100 (1) resolving gel. Then 30µl sample was added to the 10µl loading dye and
w  A   heated at 100°C for 10 min for protein denaturation. Afterward, the samples
were loaded on a prepared polyacrylamide gel and an experiment was

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performed. After that, the gel was kept on UV light to detect protein. Then the (water) and it was assumed that the entire void volume is filled with a wetting
gel was stained, and after some time, it was de-stained to identify the protein. agent. However, the estimated void volume using this technique deviates
from the true void volumes. This analysis at times provides porosity estimates
2.8.2. Bradford protein assay to large magnitude for membranes showing ample hydrophilic nature, while it
In the Bradford protein assay [41], the protein concentrations in each underestimates for the partial hydrophobic membranes. On the other hand, the
sample were prepared by adding 1µl of each protein sample in 99µl water. digital image processing tool gives realistic porosity values by skimming the
After that, 100µl Bradford reagent was added to each sample. A blank was high-resolution SEM images. As such it considers the actual geometry of the
prepared by adding 100µl water and 100µl Bradford reagent as reference. pores irrespective of the membrane characteristics such as hydrophilicity,
After 2 minutes, the absorbance of each sample was recorded using hydrophobicity, and membrane swelling. Overall, the ImageJ tool is a
spectrophotometrically at 562 nm. The protein concentration was calculated relatively accurate model for estimating porosity. From overall observations
from the standard curve prepared using bovine serum albumin as standard. of cross-sectional morphology and porosity of neat membranes, the sPES
displays approximately identical features to that of PES.
The morphologies of the additive doped membranes prepared from 6
3. Results and discussion wt% of PEG400 (Fig. 2(c) and (d)) and PEG6000 (Fig. 2(e) and (f)) revealed
significant development in the pore structure. In the presence of PEG pore
former, the asymmetric morphology of PES and sPES membranes led to a
3.1. FT-IR characterization
symmetric structure. In general, the addition of various types of pore-forming
Fig. S1 Shows the FTIR spectra of PES and sPES. From the FT-IR agents results in considerable changes in bulk morphology and porosity
spectra, the signals at 1,072, 1578, and 2923 cm−1 were attributed to aromatic compared to neat polymeric membranes [47]. The hydrophilic PEG additive
C-C stretching and bending along with C-H stretching vibrations, plays an important role in enhancing porosity, where increased pore diameters
respectively, commonly found in polyethersulfone [42,43]. The asymmetric and reduced length of the microporous layer have been observed. Especially
and symmetric stretching vibrations of sulfone (O=S=O) group signals were in the case of PEG6000 microporous layer almost disappeared, which led to
observed at 1,298 and 1148, whereas the asymmetric aryl ether (C–O–C) was symmetric pore stretching throughout the cross-section. The maximum %
identified at 1,237, respectively. Additional shoulder appeared at 1,027 cm -1, porosity was achieved for PES and sPES membranes prepared in the presence
representing the characteristic peak of the symmetric aromatic -SO3H of PEG400 and found to be 70.10 and 72.11, respectively. Additionally,
stretching vibrations, consistent with the results reported by Noel Jacob et al. increased cross-sectional pore diameters were observed due to the high
[42]. With the introduction of the -SO3H group on the aromatic ring during demixing rate of PEG with water.
the sulfonation, a change in C=C stretching vibration appeared at 1,662, Contact angles of PES and sPES membrane were measured to evaluate
which confirms the substitution of the sulfone group on the benzene ring. the surface wettability and hydrophilic interactions, which directly correlated
to the effect on the pure water flux (PWF) and antifouling tendency [48]. A
3.2. Preparation conditions and membrane structures reduction in the contact angle indicates an increase in the hydrophilicity of the
membrane surface. Certainly, neat PES is known to be a hydrophobic
In the present work, various porosity membranes were fabricated using polymer and tends to foul quickly. The measured contact angles are illustrated
the non-solvent-induced phase separation (NIPS) method for the selective in Fig. 2(g). The contact angles of neat PES (M1) and sPES (M2) were found
separation of Lysozyme from CEW. Generally, the membrane's physical to be 81.2⁰ and 71.7⁰, respectively. The reduction in the contact angle of sPES
morphology and porosity strongly depend on the cast conditions, including compared to the PES can be attributed to the presence of sulfone functional (-
solvent/non-solvent demixing rate, additives, cast film thickness, and SO3H) groups on the membrane surface. The sulfone group exhibits strong
viscosity of the cast solution. Several studies have been reported to prepare hydrogen bonding and Van der Waals interactions with polar groups [42,49].
different morphologies of the membrane by controlling the thermodynamics From Fig. 2(g), it is clearly evident that there is a significant decrease in the
of the demixing rate [44,45]. The coagulation bath temperature gives contact angle of PES and sPES membranes in the presence of a PEG additive.
excellent flexibility in altering the demixing rates; the higher the temperature, The surface porosity ascribes to the increase in the hydrophilicity of PEG-
the higher the demixing rate, which leads to wide pore diameters and vice- doped membranes. Noel Jacob et al., studied the effect of PEG200 additive on
versa. On the other hand, the size of the additive also influences the the PSf/SPES blend membranes. Their study revealed that the surface
morphology of the resulting membrane. To understand the influence of the porosity of the membranes has a strong effect on the contact angle due to
additive size, two different molecular weight PEG (400, 6000) solutions were capillary force acting underneath the water drop, which leads to penetration
employed as a pore-forming agent during the membrane preparation. To [42]. Furthermore, the lowest contact angle of 65.8⁰ was observed for the
control the demixing rate, the non-solvent coagulation bath temperature was sPES membrane cast in the presence of PEG400. Therefore, sPES membranes
varied from 10 - 45 ℃. demonstrate increased hydrophilicity compared to the PES membranes, and
low fouling tendency [42,48].
3.2.1. Effect of pore-former on PES and sPES membranes The three-dimensional surface topographic analysis and roughness
The microscopic and topographic morphologies were obtained using parameters of the PES and sPES membranes were characterized using AFM
SEM and AFM to investigate morphological changes of PES and sPES mapping. The surface roughness parameters such as mean roughness (Sa),
membranes in the presence and absence of PEG additives, which were cast at RMS roughness (Sq), and Maximum grain size height (Sz) are essential to
ambient temperature. The cross-sectional morphology and corresponding assess the fouling tendency of the prepared membranes for protein
surface topographic images of the prepared membranes are presented in Fig. purification [48]. The roughness factors of the AFM images were analyzed
2(a-f). Fig. 2(a),(b) represents the morphology of neat PES followed by sPES using Gwyddion (version 2.60), a modular program used for data
membranes, whereas Fig. 2(c),(d), and (e),(f) show the morphological visualization and analysis of SPM (scanning probe microscopy) images. The
changes between PES and sPES membranes in the presence of PEG400 and relative surface parameters of the membranes were investigated in a 5 x 5 µm2
PEG6000 additive, respectively. area of membranes and are summarized in Table 2. From Fig. 2(a) and (b)
The cross-sectional morphology of the PES and sPES membranes cast in and Table 2, it can be observed that the relative roughness of the sPES has
ambient conditions resulted in a similar asymmetric structure comprising two decreased when compared to the neat PES membrane. This may be because
distinguished micro and macro void layers. The asymmetric structure of the the sulfonic acid groups present on the sPES exhibited strong hydrophilic
neat PES and sPES membrane exhibited a typical length of microvoids interaction during the phase inversion [50]. Wen et al. fabricated antifouling
ranging from 10 – 12 µm above the finger-like macro void layer. Apart from PES ultrafiltration membranes by blending sulfonated polysulfone (sPSf) as a
similar morphological features of the membranes, the top microporous layer copolymer. Their study found that the roughness of the blend membranes
displayed variation in the average pore diameter, which is summarized in exhibited a decreased trend as the degree of sulfonation increased. On the
Table 2. On the other hand, the porosity of the membranes was estimated other hand, it also recognized that incorporating additives in the dope
using both gravimetric and cross-sectional SEM image analysis. The detailed solutions would increase the surface roughness of the prepared membranes
procedure for the porosity measurements was done by image processing using compared to the neat polymer [33,36,47,48]. However, the sPES_PEG400_25
ImageJ software as reported by Sun et al. [46]. The estimated % porosity of membrane exhibited much lower surface roughness than that of other PEG-
the membranes is summarized in Table 2. It can be observed that there is a doped membranes. Whereas the PEG6000 loaded PES and sPES membranes
noticeable difference in the percentage porosities assessed by the gravimetric demonstrated higher surface roughness, which would have high tendency of
method and ImageJ tool, besides heeding commensurate trends of divergence. surface fouling.
Conventionally, in gravimetric analysis, the total void volume of a membrane
is indirectly measured with the occupied volume of the pore-wetting agent

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(g)

Fig. 2. The cross-sectional morphology and corresponding surface topographic images of PES/sPES membranes (a) M1, (b) M2, (c) M3, (d) M4, (e) M5, and (f) M6, (g) Static water
contact angles of the PES and sPES membranes.

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Table 2
Porosity and roughness parameters of the PES and sPES membranes

Avg top layer micro Porosity (ε, %) Roughness (nm)

Membrane void diameter
Gravimetric Image analysis Sa Sq Sz
(µm)
PES (M1) 1.44 ± 0.41 43.56 63.91 14.99 21.14 193.0
PES_PEG400_25 (M3) 2.61 ± 0.80 63.32 70.10 16.27 22.10 171.3
PES_PEG6000_25 (M5) 2.73 ± 0.99 58.56 68.22 21.54 29.73 266.9
sPES (M2) 1.75 ± 0.66 45.23 66.82 5.76 7.93 81.7
sPES_PEG400_25 (M4) 3.01 ± 0.74 68.23 72.11 8.27 11.05 102.0
sPES_PEG6000_25 (M6) 2.76 ± 0.73 58.33 69.42 50.74 59.81 442.5

3.2.2. Effect of coagulation bath temperature sulfonated PES possessing a negative charge, IEC values of the sPES
Another influencing parameter on the membrane morphology is the membrane indicate surface charge negativity. Subsequently, the negatively
coagulation bath conditions. The demixing rate of solvent and non-solvent charged surface membranes show affinity towards the positively charged
rates could be precisely controlled by changing the temperature condition of protein during the separation. In addition, the high molecular weight proteins
the non-solvent bath. Cross-sectional SEM images of sPES membranes cast in with a negative charge will show a low tendency of fouling on the negatively
the presence of PEG additives and coagulation bath temperatures of 10 and 45 charged membranes [50].
℃ are presented in Fig. 3. As per Fig. 3(a)-(d), the rise in coagulation bath
temperature from 10 ℃ to 45 ℃ ensued in giant macro voids formation.
Furthermore, it was noticed that the membrane cast in the coagulation bath
temperature of 10 ℃ resulted in an asymmetric structure with micro and
macro void layers due to the slow demixing rates. On the other hand, the
membranes fabricated at 45 ℃ created larger macro void volumes within the
bulk matrix of the membranes under the influence of rapid demixing
phenomena [45]. In contrast, at lower temperatures, due to the slow demixing
rates initially leading to a formation of a tighter microporous layer afterward,
the nucleation occurs slowly in the bulk matrix. Consequently, the
membranes cast at lower temperatures despite the presence of PEG additives
tend to form an asymmetric layer throughout the cross-section. These results
are in good agreement with the previous reports [36,44,45,51].

3.3. Performance evaluation using PWF and hydraulic resistance

The PWF is one of the vital characterizations to demonstrate the
morphological changes in the performance of prepared membranes. The
calculated PWF and corresponding hydraulic resistance of the M1-M10
membranes are summarized in Table 3. PWF of the neat PES membrane
exhibits a very low value of 5.67 Lm-2h-1bar-1, whereas the neat sPES
demonstrated 20.50 Lm-2h-1bar-1. Although both neat membranes have similar
asymmetric morphology, sPES showed higher PWF than the PES membrane.
The increase in the PWF was attributed to the higher hydrophilic character of
the sulfone groups of sPES, confirmed by the contact angle analysis. Table 3
shows significant enhancement in the PWF of PES and sPES membranes
when cast in the presence of a PEG additive at ambient conditions (i.e., M3-
M6). Furthermore, the membranes fabricated in the presence of PEG400
showed higher PWF compared to the PEG6000 due to the higher porosity and
hydrophilic characteristic property.
On the other hand, the sPES membrane, when cast at temperatures of 10
to 45℃ in the presence of PEG400 and PEG6000, shows PWF values of
73.57, 107.46, 167.98 L m-2h-1bar-1 and 57.23, 63.10, 153.81 L m-2h-1bar-1,
respectively, indicating a rise in PWF values with increase in coagulation bath
temperature. The observed trend in PWF values is consistent with the
morphological analysis of SEM images presented in section 3.2. As the
temperature of the coagulation bath increases, the formation of macro voids
increases due to high demixing rates [44]. Consequently, the hydraulic
resistance of pure water permeation decreases with the increase in the
porosity of the membranes (Table 1).

3.4. Ion exchange capacity (IEC) of PES and sPES membranes

IEC is an essential factor for analyzing the charge density of a membrane.
In the present study, the IEC values of the prepared membranes were
estimated using the titration method described elsewhere [52]. The calculated
IEC values are presented in Table 3. IEC of the membrane cast from neat PES
and PES/PEG blend membranes were considerably constant and exhibited a
value of 0.04 ± 0.01, consistent with the value presented by Klaysom et al.
[52]. In contrast, the IEC values of the sPES and sPES/PEG blend membranes Fig. 3. Cross-sectional SEM images of sPES membranes cast in the presence of PEG
additives and coagulation bath temperatures of 10 and 45 ℃ (a) M7, (b) M8, (c) M9, and
showed a significant difference and increased with the increase in the porosity
(d) M10
of the membrane. The enhanced IEC of the sPES membranes might be
attributed to the exposure of an expanded number of sulfonated groups when
the porosity of the membrane increases. The maximum IEC was found to be
1.137 mmol eq/g for the membrane cast from sPES with PEG400 at a
coagulation bath temperature of 45 ℃ (M8). Due to -SO3- groups of the
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Table 3 sPES membranes is significantly improved compared with PES membranes.

PWF, Hydraulic resistance, and IEC values of PES and sPES membranes As we discussed in section 3.2.1., the hydrophilicity and electrostatic
interaction of proteins with the sulfonic functional groups of the sPES
Hydraulic
PWF IEC improved the permeation flux compared to the PES.
Membrane resistance
(L m-2h-1bar-1) (mmol eq/g) On the other hand, the total protein content in the permeate significantly
Rm (x 1012 m-1)
varied from M1-M10 along with the difference in the feed flow patterns (Fig.
PES_25 (M1) 5.67 98.37 0.030
4(b)). From the visual observation of Fig. 4(b), the total protein content of
sPES_25 (M2) 20.50 27.20 0.625 permeate samples collected from M4, M6, M8, and M10 membranes is
relatively high compared to the rest of the membranes. However, the protein
PES_PEG400_25 (M3) 10.93 51.00 0.040 concentration estimated from the Bradford assay does not reveal permeation
sPES_PEG400_25 (M4) 107.46 5.19 0.854 quality. Thus, the samples were analyzed using the electrophoresis technique.
The results in SDS-PAGE, which are presented in Fig. 4(c) and (d), reveal
PES_PEG6000_25 (M5) 9.46 58.96 0.045 that the high total protein in the permeate sample of M6, M8, and M10 is due
sPES_PEG6000_25 (M6) 63.10 8.83 0.656 to the presence of Ovalbumin and Overtransferrin (clear broad bands has been
observed). As discussed earlier, M8 and M10 were cast under 45 ℃ due to
sPES_PEG400_10 (M7) 73.57 7.58 0.540 the high demixing rates of solvent/PEG additive with the non-solvent
sPES_PEG400_45 (M8) 167.98 3.32 1.137 resulting in wider surface pores. Whereas the morphological changes of M4
(Fig. 2(d)) and M6 (Fig. 2(f)) significantly influence the size of the PEG
sPES_PEG6000_10 (M9) 57.23 9.74 0.477 additive. From the above CEW permeation flux and protein analysis, it can be
concluded that the membrane cast in the presence of PEG6000 fails to retain
sPES_PEG6000_45 (M10) 153.81 3.62 0.757
higher molecular weight proteins. In contrast, the protein band of Ovalbumin
and Overtransferrin in the permeate of the M4 membrane is found to be
3.5. Lysozyme separation from CEW relatively small and almost disappeared when operated under cross-flow
CEW solution was chosen as a source for the isolation of Lysozyme, filtration. It is noteworthy that the permeate samples of the dead-end and
which is the most acceptable and naturally available. However, the isolation cross-flow modes were collected after one hour after the start of the operation.
of Lysozyme from CEW is challenging as the concentration of Lysozyme is During the course, feed volume is decreased as the permeate is collected,
minimal among the CEW proteins. The major protein components of CEW leading to an increase in the viscosity and concentration of total protein. To
and properties such as molecular weight and iso-electric points are presented minimize the feed concentration fluctuation on the M4 membrane
in Table S1. Ovalbumin, Ovotransferrin, and Ovomucoid are high molecular performance, a semi-batch model was configured where the feed volume is
weight proteins compared to Lysozyme and are the major contributors of maintained constant.
CEW proteins, comprising about 54%, 12%, and 11% of the total proteins,
3.5.2. Semi-batch mode isolation of Lysozyme
respectively [5]. Lysozyme and Ovomucoin are present at approximately 3.5
% of each in the total protein [23]. As described above, the ultrafiltration experiment of the M4 membrane
In ion-exchange column chromatography and pH-graded gel, a protein was performed by maintaining the constant feed volume. The SDS-PAGE of
can be separated from a crude mixture based on the difference in the iso- permeate samples collected in the 30 min of time intervals are shown in Fig. 4
electric points. The iso-electric point of a protein indicates the pH value (e). Remarkably, at constant feed volume, the dynamic permeation of the M4
where the protein has a neutral net charge (zero). At the same time, protein membrane demonstrated selective separation of Lysozyme from the complex
carries a net negative charge above the iso-electric point and vice-versa [5]. CEW solution. Notably, the membrane morphology and electrostatic
Moreover, it is also reported that the iso-electric points play an essential role interaction between surface and protein are rationally correlated. At pH 7.4,
in the adsorption process where the pH conditions strongly influence the the negatively charged high molecular weight proteins have weaker
adsorption capacity of the substrate [5,39]. From Table S1, the iso-electric interaction with the negatively charged M4 surface and prevent permeation
points of higher molecular weight proteins in the CEW are less than 6.1, through the membrane. The clear and thick 14 kDa band of continuously
besides Lysozyme being 10.7 [53]. Chang et al. studied the effect of pH on extracted permeate sample indicates successful isolation of Lysozyme with
the batch adsorption of Lysozyme from CEW at pH ranging from 4 to 12. The the membrane fabricated using sPES/PEG400 under ambient coagulation
results revealed that the adsorption selectivities of hydrolyzed conditions. Hence, the morphology of the membrane with the conjugated
polyacrylonitrile (PAN-COOH) and bromoacetic acid functionalized effect of electrostatic interactions and optimization of the extraction process
polyacrylonitrile (PAN-BrA) electrospun fibers at pH 9 presented 2.37 and led to the high purity of Lysozyme in the permeate.
2.43 folds, relatively higher than at pH 5, respectively. Similarly, Fang et al.
studied the batch adsorption capacity of sulfonated polysulfone membranes at 3.6. Protein adsorption studies and membrane fouling evaluation
pH 7.4 for selective adsorption of synthetic Lysozyme solutions [39]. Table
S1 shows that Lysozyme possesses a net positive charge in phosphate buffer The antifouling performance of PES/sPES membranes was assessed
with a pH of 7.4, while higher molecular weight proteins (Ovalbumin, using PWF analysis and protein adsorption. Fig. 5(a) shows that the PWF of
Ovotransferrin, and Ovomucoid) have a net negative charge. Thus, pH 7.4 M1-M10 membranes after soaking for 7 days in the supernatant CEW
was chosen as the buffer media for the CEW crude mixture during the solution significantly decreased due to the deposition of proteins on the
isolation of Lysozyme through affinity base ultrafiltration. The strategic membrane surface. Furthermore, the relative change in protein concentration
approach effectively separates and minimizes the protein accumulation on the in the supernatant during the static adsorption of membranes is illustrated on
membrane surface due to the electrostatic repulsion. the secondary axis of Fig. 5(a). The PES membranes adsorbed a considerable
amount of protein on the membrane surface, clearly evident from the
3.5.1. Effect of flow patterns on the Lysozyme extraction from CEW reduction in the supernatant protein concentration (Fig. 5(a)), whereas the
protein uptake on the sPES membranes was minimal. Briefly, the fouling
The ultrafiltration experiments were carried out in the dead-end and
tendency of sPES is lower than that of PES membranes. It has to be noted that
cross-flow configurations to assess the membrane performance in the
the surface roughness and hydrophilicity of membrane and electrostatic
isolation of Lysozyme from CEW. Fig. 4(a) shows the flux values of M1-
interactions with proteins are the primary factors responsible for the
M10 membranes, and Fig. 4(b) represents the corresponding total protein
antifouling characteristics [36,43]. Topographic (Fig. 5(b) and (c)) and
concentration in the permeate. Additionally, sodium dodecyl sulfate-
surface morphologies (Fig. 5(d) and (e)) of M1 and M4 membranes, revealed
polyacrylamide gel electrophoresis (SDS-PAGE) analysis was performed to
the formation of protein layer. The AFM topography showed that the neat
qualitatively analyze dead-end (Fig. 4(c)) and cross-flow (Fig. 4(d)) permeate
PES heavily adsorbed protein, whereas the sPES (M4) exhibited a low
samples. Fig. 4(a) shows that in membrane M1-M10, the permeation flux is
adsorption tendency. As discussed earlier in section 3.2.1., the surface
enhanced when operated under a cross-flow configuration compared to the
roughness of the sPES membranes is substantially lower than the PES.
dead-end filtration. This is because the cross-flow pattern of feed significantly
Additionally, electrostatic repulsion force is offered between the negative
reduces the concentration polarization of high molecular weight protein on
charge of sulfonic groups with the negatively charged proteins, which further
the surface of the membrane [54]. The flow direction in cross-flow filtration
reduces the fouling tendency of sPES membranes.
is parallel to the membrane surface, whereas it is perpendicular in dead-end
filtration. The perpendicular flow of feed accumulates the retained proteins on
the membrane surface, leading to a fall in the permeation flux. Furthermore, it
is also recognized that dead-end flow filtration has a high fouling tendency
compared to the cross-flow configuration [55]. Additionally, the flux of the
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 N. S. Prasad et al. / Journal of Membrane Science and Research 9 (2023) 562862

(a) (b)

(d)
(c)

(e)

Fig. 4. Purification of Lysozyme from CEW crude solution using PES/sPES membranes (a) Permeation flux, (b) total protein, SDS-PAGE analysis of (c) dead-end, (d) cross-flow
permeate, and (e) Evaluation of Lysozyme purity under semi-batch mode

A more comparative and deeper understanding of the foulant layer on the M4 showed a lower intensity of fouled layer. In order to understand the
membranes were analyzed using EDS mapping (Fig. 5(f) and (g)) and ATR- fouling tendency of sPES membranes, ATR-FTIR spectra were collected to
FTIR analysis (Fig. 5(h)). Gorzalski et al. studied elemental analysis of the identify the functional groups of the adsorbed protein [57]. Fig. 5(h)
foulant layer using energy-dispersive X-ray spectroscopy (EDS), Rutherford’s illustrated functional group stretching at 1660-1670 cm-1, 1547 cm-1 of Amide
backscattering spectrometry (RBS), and X-ray photoelectron spectroscopy I and Amide II of the adsorbed CEW proteins [58]. Furthermore, ATR-FTIR
(XPS) evaluated whether the three techniques yielded consistent results for is a semi-quantitative technique that provides information on the amount of
fouled membrane composition [56] and recommended that EDS as an protein adsorbed on the membrane surface. It is evident that the M4
appropriate method to evaluate the overall elemental composition of the membrane indicates a low binding of protein compared to that of M2, M6,
foulant layer. The results obtained from EDS were consistent with the SEM M8, and M10 membranes.
and AFM analysis, where the protein adsorbed spots were enriched with
calcium (Ca) and oxygen elements [56]. However, the elemental mapping of

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 N. S. Prasad et al. / Journal of Membrane Science and Research 9 (2023) 562862

(d) (e)

(f) (g)

(h)

Fig. 5. (a) PWF of M1-M10 membranes before and after protein purification and relative protein concentration, AFM surface topography (b) neat PES (c) M4 after adsorption, the
surface morphology of (d) neat PES (e) M4, EDS elemental analysis of Ca (f) neat PES (g) M4 (h) FTIR spectra of adsorbed membranes

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 N. S. Prasad et al. / Journal of Membrane Science and Research 9 (2023) 562862

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Supplementary Information

Isolation of Lysozyme from Chicken Egg White by Surface Charged Membranes

N. Shiva Prasad1,2,3, Aarti Tallam1, Namita Roy Choudhury3, S. Sridhar1,2*, Suresh K Bhargava3

1 Membrane Separations Laboratory, Process Engineering and Technology Transfer Division,

CSIR - Indian Institute of Chemical Technology, Hyderabad, India-500007.
2Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, Uttar Pradesh, India.
3 Royal Melbourne Institute of Technology (RMIT), Melbourne, VIC 3001, Australia.

* Corresponding author; E-mail ID: sridhar11in@yahoo.com (Dr S. Sridhar)

Scheme S1 Stoichiometric reaction of sulfonated polyethersulfone

Figure S1 FTIR spectra of PES and SPES

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Table S1 CEW protein composition and properties

S.NO. CEW Protein Composition (Abeyrathne et al., Molecular weight Iso-electric point (Awadé and Efstathiou, The net charge of protein at 7.4 pH
2013) (kDa) 1999) Buffer

1 Ovalbumin 54% 45 4.6 Negative

2 Ovotransferrin 12% 76 6.1 Negative

3 Ovomucoid 11% 28 4.1 Negative

4 Lysozyme 3.50% 14.4 10.7 Positive

5 Ovomucin 3.50% 254 4.5 Negative

6 Avidin 0.05% 15.6 10-10.5 Positive

7 Cystatin 0.05% 13.3 9.3 Positive

8 Ovomacroglobulin 0.50% 184.7 4.5-4.7 Negative

9 Ovoflavoprotein 0.80% 32-26 4.0-4.1 Negative

10 Ovoglycoprotein 1.00% 24.4 3.9 Negative

11 Ovoinhibitor 1.50% 49 5.3 Negative

Figure S2 AFM surface topography of M4 and M8 membranes

Abeyrathne, E. D. N. S., Lee, H. Y., and Ahn, D. U. (2013). Egg white proteins and their potential use in food processing or as nutraceutical and pharmaceutical
agents—A review. Poultry Science, 92(12), 3292-3299. https://doi.org/https://doi.org/10.3382/ps.2013-03391
Awadé, A. C., and Efstathiou, T. (1999). Comparison of three liquid chromatographic methods for egg-white protein analysis. Journal of Chromatography B:
Biomedical Sciences and Applications, 723(1), 69-74. https://doi.org/https://doi.org/10.1016/S0378-4347(98)00538-6

15