Effect of Polysaccharide Conformation on Ultrafiltration Separation Performance

Carbohydrate Polymers 260 (2021) 117830

Contents lists available at ScienceDirect

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

Effect of Polysaccharide Conformation on Ultrafiltration
Separation Performance
Severin Eder a, Patrick Zueblin a, Michael Diener b, Mohammad Peydayesh b, Samy Boulos a,
ăm a, *
Raffaele Mezzenga b, Laura Nystro
a

ETH Zurich, Department of Health Science and Technology, Institute of Food, Nutrition and Health, Laboratory of Food Biochemistry, Schmelzbergstrasse 9, 8092
Zurich, Switzerland
ETH Zurich, Department of Health Science and Technology, Institute of Food, Nutrition and Health, Laboratory of Food and Soft Materials, Schmelzbergstrasse 9, 8092
Zurich, Switzerland

b

A R T I C L E I N F O

A B S T R A C T

Keywords:
Ultrafiltration
Polysaccharide conformation
Polysaccharide separation
Molecular weight cut-off deviation
Glucose-based polysaccharide

The manifold array of saccharide linkages leads to a great variety of polysaccharide architectures, comprising
three conformations in aqueous solution: compact sphere, random coil, and rigid rod. This conformational
variation limits the suitability of the commonly applied molecular weight cut-off (MWCO) as selection criteria for
polysaccharide ultrafiltration membranes, as it is based on globular marker proteins with narrow Mw and hy­
drodynamic volume relation. Here we show the effect of conformation on ultrafiltration performance using
randomly coiled pullulan and rigid rod-like scleroglucan as model polysaccharides for membrane rejection and
molecular weight distribution. Ultrafiltration with a 10 kDa polyethersulfone membrane yielded significant
different recoveries for pullulan and scleroglucan showing 1% and 71%, respectively. We found deviations
greater than 77-fold between nominal MWCO and apparent Mw of pullulan and scleroglucan, while recovering
over 90% polysaccharide with unchanged Mw. We anticipate our work as starting point towards an optimized
membrane selection for polysaccharide applications.

1. Introduction
The global production of polysaccharides in nature considerably
exceeds the production volume of any other polymer. Polysaccharides
constitute the central carbon source for living organisms and provide a
basis for all life on our planet (Navard & Navard, 2012). In recent de­
cades, polysaccharides aroused great interest in research and across
various industries owing to their unique biological and physiological
properties, such as biocompatibility and –degradability paired with
atoxic characteristics (Muzzarelli, 2012). In particular, polysaccharide
purity becomes a crucial product criterion in applications involving
humans, such as biomedicine or food technology (Pinelo, Jonsson, &
Meyer, 2009). Furthermore, as the bioactive potential of

polysaccharides is distinctly related to their chemical structure and
molecular weight, selective purification processes become indispensable
(Wang et al., 2017).
Common purification techniques such as chromatography, evapo­

ration and ion exchange require resource-intensive operation and
maintenance, involve substantial investment costs, and lack scalability.
In comparison to these conventional separation and purification pro­
cesses, membrane filtration offers several merits, including high effi­
ciency, simple modification of operating variables and low energy
requirements (Cano & Palet, 2007; Chen et al., 2020). In addition,
membrane separation is especially suited for heat-sensitive bio­
molecules as it is operated at room temperature (RT) and without phase
transfer (Sun, Qi, Xu, Juan, & Zhe, 2011).

Abbreviations: α, Mark-Houwink parameter; AFM, atomic force microscopy; AN-scleroglucan, alkaline-treated and neutralized scleroglucan; ANS-scleroglucan,
alkaline-treated, neutralized and sonicated scleroglucan; Đ, dispersity index; DLS, dynamic light scattering; dn/dc, refractive index increment; HPAEC, high-per­
formance anion-exchange chromatography; HPSEC, high-performance size exclusion chromatography; HY, hydrosart; IEP, isoelectric point; LALS, low-angle light
scattering; Δ%Mw, percentage difference in Mw between the retentate and feed solution; Mn, number average molecular weight; Mw, weight average molecular weight;
MWCO, molecular weight cut-off; PAD, pulsed amperometric detection; PES, polyethersulfone; RALS, right-angle light scattering; RI, refractive index; RT, room
temperature; SSE, sum of squared error; ζ, zeta potential; [η], intrinsic viscosity.
* Corresponding author.
E-mail address: (L. Nystră
om).
/>Received 9 November 2020; Received in revised form 12 February 2021; Accepted 13 February 2021
Available online 17 February 2021
0144-8617/© 2021 The Authors.
Published by Elsevier Ltd.
This is an open
( />
access

article

under

the

CC

BY-NC-ND

license


S. Eder et al.

Carbohydrate Polymers 260 (2021) 117830

Over the past years, the advances in ultrafiltration technology have
led to the development of refined membranes, enabling selective sepa­
ration of saccharides with molecular weights as low as 3 kDa (Pinelo
et al., 2009; Sun et al., 2011). The rejection properties of ultrafiltration
membranes are reflected in the molecular weight cutoff (MWCO). Its
arbitrary definition comprises the lowest molecular weight at which
90% of the solute is retained by the membrane (Koros, Ma, & Shimidzu,
1996). Membrane manufacturers generally assign MWCOs using glob­
ular marker proteins for calibration, although an industry-wide standard
is still lacking (Scott, 1995). Since globular proteins fold to sphere-like
structures in solution, they present a narrow relation of molecular
weight to hydrodynamic volume, influencing the separation factor of
membrane filtration that is governed decisively by hydrodynamic vol­
ume (Pinelo et al., 2009). Unlike the linear sequences found in peptide
bonds, polysaccharides form diverse primary structures with a variety of
condensation linkages (Liu, Brameld, Brant, & Goddard, 2002). This

extra dimension of geometry conjunct with variable saccharide units
leads to a remarkable world of polymeric architecture (Atkins, 1985). It
is generally recognized that the conformation of polysaccharides in so­
lution comprises three distinct patterns with increasing rigidity:
compact sphere, random coil, and rigid rod (Harding, Abdelhameed, &
Morris, 2011). The respective glycosidic linkage geometry of a poly­
saccharide mainly defines its conformation in aqueous solution, which
in turn determines the hydrodynamic volume (M. Q. Guo, Hu, Wang, &
Ai, 2017). The polysaccharides pullulan and scleroglucan can be
considered as extreme representatives of their respective conforma­
tional cluster owing to their diverse glycosidic linkage patterns. Pul­
lulan, a linear water-soluble (1→4;1→6)-α-D-glucan produced by the
polymorphic fungus Aureobasidium pullulans, behaves as random coil in
aqueous solution (Nishinari et al., 1991). The rotational freedom pro­
vided by the α-(1→6)-linkages enables flexible folding along the poly­
mer chain (Gidley & Nishinari, 2009). Scleroglucan, a water-soluble
(1→3;1→6)-β-D-glucan produced by fungi of the genus Sclerotium
(Coviello et al., 2005), exhibits the (1→6)-linkage only in the sidechain
of D-glucopyranosyl residues attached to the (1→3)linked backbone
(Castillo, Valdez, & Farina, 2015). The alignment of scleroglucan strands
in aqueous solution results in a stiff rigid rod-like conformation (Slet­
moen & Stokke, 2008). Ultimately, distinct hydrodynamic volume and
spatial orientation of polysaccharides with comparable molecular
weight may restrict the applicability of MWCO as suitable selection
guide for polysaccharide ultrafiltration membranes. Numerous studies
reported discrepancies between the apparent and the nominal MWCO
provided by the manufacturer (Kim et al., 1994; Platt, Mauramo,
Butylina, & Nystrom, 2002). Platt et al. (2002) found apparent MWCOs
lower than the nominal when filtering polyethylene glycol solutions and
excluded fouling and concentration polarization as underlying cause.

Sun et al. (2011) observed a considerable loss of a polysaccharide
mixture subjected to different ultrafiltration membranes that should
have retained the investigated fraction according to the manufacturer’s
MWCO. However, governing factors for the discrepancies in membrane
separation obtained were not suggested.
So far, the conformation of polysaccharides in ultrafiltration appli­
cations was assessed mostly in terms of the resulting hydrodynamic
volume for membrane and MWCO selection. The ultimate effect of
polysaccharide conformation on membrane transport during ultrafil­
tration attracted little attention until now. Mathematical simulations
focusing on the adaptation of steric pore models to include capsularshaped molecules, or the assessment of the probability of elongated
shapes entering a membrane pore compared to spherical particles
showed the necessity to consider also conformation for a better under­
standing of ultrafiltration separation (Montesdeoca, Bakker, Boom,
Janssen, & Van der Padt, 2019; Vinther, Pinelo, Brons, Jonsson, &
Meyer, 2012). However, the systematic assessment of the effect of
polysaccharide conformation on the resulting ultrafiltration perfor­
mance remains yet unaddressed.
The present work seeks to describe the effect of polysaccharide

conformation on the separation performance of ultrafiltration mem­
branes with an empirical approach. For this purpose, pullulan and
scleroglucan as model polysaccharides were subjected to crossflow ul­
trafiltration with two membrane materials, namely Hydrosart (HY), a
low-binding regenerated cellulose material, and polyethersulfone (PES),
exhibiting various MWCO (2, 3, 5, and 10 kDa). Particular attention was
paid to the exclusion of any potential effect on membrane separation
beside the polysaccharide conformation. The requirements for the
polysaccharide model solution to guarantee an unambiguous assign­
ment of ultrafiltration variation owing to the effect of conformation

encompassed: (i) identical monomeric units; (ii) uniform distinct
conformation in aqueous solution; (iii) solubility and conformation
stability in aqueous solution; (iv) comparable weight average molecular
weight (Mw) with ΔMw ≈ 100 kDa. Moreover, state-of-the-art sizeexclusion chromatography coupled to light scattering and viscometer
detectors (HPSEC-triple detection), high-performance anion-exchange
chromatography with pulsed amperometric detection (HPAEC-PAD),
and high-resolution atomic force microscopy imaging (AFM) assured
comprehensive evaluation of the ultrafiltration retentate in terms of
polysaccharide yield, conformation, and molecular weight.
2. Materials & methods
2.1. Chemicals
Pullulan powder was purchased from Carbosynth (Berkshire, United
Kingdom). Scleroglucan powder was obtained from Elicityl (Crolles,
France). D-Glucose anhydrous (≥ 99.5%), sodium azide (NaN3; >99%),
sodium hydroxide (NaOH, ≥ 98%), sodium hypochlorite solution
(NaClO), sodium nitrate (NaNO3; ≥ 99.5%), D-sorbitol (99%) and tri­
fluoroacetic acid (TFA, >99.9%) were purchased from Sigma-Aldrich
(St. Louis, United States). Hydrochloric acid (HCl, >37%) was ob­
tained from VWR International (Radnor, United States). All solutions
were prepared with purified water using a Millipore MilliQ-system
(Billerica, United States).
2.2. Preparation of polysaccharide standard solutions
Pullulan was dissolved at RT under stirring for 1 h. Scleroglucan was
dissolved at 80 ◦ C under stirring for 24 h. We selected the mildest
possible conditions facilitating complete dissolution of both poly­
saccharides. Pullulan and scleroglucan exhibit different flexibilities in
their structure that affect the strength of intermolecular interactions and
thus require adapted dissolution procedures. Pullulan and scleroglucan
solutions were prepared at 0.1% (w/v) for polysaccharide character­
ization and at 0.025% (w/v) for ultrafiltration feed solutions. Poly­

saccharide solutions were prepared taking into consideration the purity
assessment of the crude polysaccharide powder (w/w) (see Section 2.5).
To unify the dispersity and to reduce the molecular weight to a com­
parable level with pullulan, scleroglucan feed solution was preliminary
treated with 0.2 M NaOH at RT for 10 min and subsequently neutralized
with HCl, followed by centrifugation at 9000 rpm for 15 min, resulting
in alkaline-treated and neutralized scleroglucan (AN-scleroglucan). The
solution was then subjected to ultrasonic treatment over a total duration
of 180 min with a probe sonicator (UP200H, Hielscher, Germany),
operated at 100% pulsation with 80% amplitude, resulting in alkalinetreated, neutralized and sonicated scleroglucan (ANS-scleroglucan).
During the sonication procedure, the solution was cooled in an ice bath
and kept under stirring to prevent heating. Prepared pullulan feed so­
lution was used without further treatment. All polysaccharide solutions
were filtered through a 0.45 μm Nylon filter prior to analysis or
ultrafiltration.
2.3. Crossflow ultrafiltration set-up and procedure
Crossflow ultrafiltration was conducted with a Vivaflow 200 cross
2


S. Eder et al.

Carbohydrate Polymers 260 (2021) 117830

flow device equipped with Hydrosart (HY) or polyethersulfone (PES)
ăttingen, Germany). The
crossflow membrane cassettes (Sartorius AG, Go
nominal molecular weight cut-offs (MWCO) provided by the manufac­
turer were 2, 5 and 10 kDa for HY and 3 and 10 kDa for PES membranes.
Further characteristics of the membrane can be found in the supple­

mentary information (Table S1, Fig. S1). Ultrafiltration was performed
in constant volume diafiltration operation mode at a constant pressure
of 2.5 bar set with a Masterflex L/S peristaltic pump (Cole-Parmer
GmbH, Wertheim, Germany) (Fig. 1). The resulting circulation flowrates
were between 20.9–24.5 L/h (Fig. S2). In each trial, 250 mL of poly­
saccharide feed solution (0.025%, w/v) were subjected to diafiltration
for 1 h at RT. MilliQ water, connected to the feed tank, was used as
exchange solution in order to maintain a constant volume of 250 mL.
The number of diavolumes exchanged during the crossflow diafiltration
were recorded for each membrane (Table S2). Crossflow ultrafiltrations
were conducted in triplicates for each membrane material with distinct
MWCO. After each ultrafiltration run, the ultrafiltration device was
washed with the corresponding washing solution to avoid carry-over.
Washing solutions were 0.5 M NaOH and 0.5 mM NaOCl in 0.5 M
NaOH for HY and PES membranes, respectively. The feed solutions and
retentates were analyzed in terms of molecular weight distribution and
conformational parameters using size-exclusion chromatography
coupled to light scattering and viscometer detectors (HPSEC-triple
detection) (see Section 2.4). The resulting yield of the respective poly­
saccharide in the retentate was obtained as mass-ratio according to the
following formula:
Yield (%) =

mretentate
∗100,
mfeed

intrinsic viscosity [η], and the Mark-Houwink plot of the polysaccharide
feed and retentate solutions were determined using high-performance
size exclusion chromatography (HPSEC) equipped with triple detec­

tion (OMNISEC, Malvern Panalytical Ltd., Malvern, United Kingdom)
according to the procedure described by Demuth, Betschart, and
ăm (2020). In short, the HPSEC-triple detection system consisted of
Nystro
a OMNISEC resolve unit (OMNISEC, Malvern Panalytical Ltd, Malvern,
United Kingdom) coupled to the multi-detector module OMNISEC reveal
(OMNISEC, Malvern Panalytical Ltd, Malvern, United Kingdom)
encompassing a refractive index (RI), right-angle light scattering (RALS)
at 90◦ , low-angle light scattering (LALS) at 7◦ , and a viscometer detec­
tor. Two A6000M columns with an exclusion limit of 20 000 000 Da
connected in series (Malvern Panalytical Ltd., Malvern, United
Kingdom) were maintained at 30 ◦ C. Polysaccharide solutions were
filtered through a 0.45 μm Nylon syringe filter prior to analysis. Sample
injections of 100 μL were eluted with 0.1 M aq. NaNO3 containing 0.02%
(w/v) NaN3 at a flow rate of 0.7 mL/min. The system was calibrated with
a one-point calibration using a Malvern PolyCAL™ polyethylene glycol
(PEO24 K) standard and verified with a dextran (DEX-T70 K) standard.
Data analysis was performed using the OMNISEC 10.30 software (Mal­
vern Panalytical Ltd., Malvern, United Kingdom) using a refractive index
increment value (dn/dc) of 0.145 mL/g for both polysaccharide stan­
dards. The Mark-Houwink equation was used to investigate the
conformation:
(3)

[η] = KM α ,

where M is the molecular weight at a given point within the mo­
lecular weight distribution; [η] is intrinsic viscosity; K is a constant, and
α is a scalar related to the conformation in solution. The value of α re­
sults from the slope of the Mark-Houwink plot. The data for M and [η]

were extracted from HPSEC measurements. In general, the value of α is
below 0.5 for spherical-like (theoretically 0 for a fully collapsed coil in a
poor solvent, as predicted by the Einstein equation), between 0.5–0.8 for
random coil, and larger than 0.8 for rigid rod conformation (Q. Guo
et al., 2013; He, Zhang, Wang, Qu, & Sun, 2017). Mark-Houwink plots
with two fractions were evaluated for the value of α using a MATLAB
script computing the optimal breakpoint on a given data set for two
linear fits by minimizing the overall sum of squared errors (SSE). The
SSE was calculated using:

(1)

where mretentate and mfeed are the masses of available polysaccharide
in the retentate and feed solution, respectively, determined by highperformance anion-exchange chromatography with pulsed ampero­
metric detection (HPAEC-PAD) after TFA hydrolysis (see Section 2.5)
(Cheryan, 1998). The standard deviation of Eq. (1) was calculated,
assuming independent variables, according to (Taylor, 1982):
√̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅
√((
)
) )
(
√
σmfeed 2
σmretentate 2
.
(2)
σ Yield = Yield√
+
mretentate

mfeed

2.4. Molecular weight determination and conformation analysis

SSE =

N
∑
(

)2

αcal − αexp i .

(3)

i=1

The weight average molecular weight (Mw), dispersity Đ (Mw/Mn),

2.5. Determination of purity and yield of polysaccharides
Purity of polysaccharides and membrane rejection in terms of poly­
saccharide yield was analyzed by quantifying monosaccharides using
high-performance anion-exchange chromatography with pulsed
amperometric detection (HPAEC-PAD) after TFA hydrolysis based on
the method described by Boual, Abdellah, Aminata, Michaud, and Hadj
(2012). In brief, 1 mL aqueous polysaccharide solution was incubated
with 1.5 mL 3.3 M TFA at 100 ◦ C for 4 h. After complete evaporation
under N2 gas stream at RT, the dried hydrolysate was dissolved in 10 mL
water. Hydrolysate solutions were filtered through a hydrophilic 0.45

μm PTFE syringe filter prior to analysis. For the polysaccharide analysis,
a Dionex ICS-5000+ System (Thermo Scientific, Sunnyvale, United
States) equipped with a Dionex CarboPac PA1 (4 × 250 mm) column and
a CarboPac PA1 (4 × 50 mm) guard column operating at 25 ◦ C was used.
Injection volume of the samples was 10 μL and eluted using a combi­
nation of the two mobile phases: (A) 200 mM NaOH and (B) purified
water at a flow rate of 1 mL/min. The applied gradient program was
adapted from the method described by Rohrer, Cooper, and Townsend
(1993) with slight modifications. The resulting gradient program was:
0–20 min, isocratic 8% A and 92% B; 20–30 min, isocratic 100% A; and
30–39 min, isocratic 8% A and 92% B. Eluents were kept under helium
atmosphere. Quantification of samples was performed with the internal

Fig. 1. Scheme of the cross-flow diafiltration set-up. Reprinted with permission
from Sartorius© (Directions for Use Vivaflow 50 | 50R | 200, 2016).
3


S. Eder et al.

Carbohydrate Polymers 260 (2021) 117830

calibration method using the Chromeleon Chromatography Data System
(CDS) Version 7 (Thermo Scientific, Sunnyvale, United States).
D-Glucose at seven concentration levels between 1.25–30 mg/L was used
as external standard and D-Sorbitol as internal standard. D-Glucose was
the only identified monomeric sugar for both polysaccharides in
HPEAC-PAD analysis. The purity of the polysaccharides on a w/w basis
was determined as follows:
Purity (%) =

mhydrolysate
∗100,
mpolysaccharide

flattening and without any further processing.
2.7. Dynamic light scattering
Correlation function and zeta potential (ζ) were measured by dy­
namic light scattering (DLS) using a Zetasizer Nano (Malvern Panalytical
Ltd., Malvern, United Kingdom). The experiments were performed at 25
◦
C and each sample was measured three times with 11 runs per mea­
surement. The results were processed using the Zetasizer software.

(5)

where mpolysaccharide and mhydrolysate represent the masses of crude poly­
saccharide powder and polysaccharide available after TFA hydrolysis
determined by HPAEC-PAD, respectively.

2.8. Statistical analysis
All experiments were performed at least in triplicates and the data
were expressed as mean values ± standard deviation. One-way analysis
of variance (ANOVA) with Tukey’s post-hoc test was performed to
compare mean group values. An alpha value of 0.05 was considered
significant. We analyzed the data using Origin, Version 2018 (OriginLab
Corporation, Northampton, United States).

2.6. Atomic force microscopy
Imaging of the polysaccharides was conducted by high-resolution

atomic force microscopy (AFM). For the sample preparation, 20 μL of
1 μg/mL filtered scleroglucan solution were deposited on freshly cleaved
mica, left to adsorb for 30 s and subsequently gently dried with pres­
surized air. AFM height images were then obtained using a Nanoscope
VIII Multimode Scanning Force Microscope (Bruker AXS, Karlsruhe,
Germany) equipped with commercial silicon nitride cantilevers in tap­
ping mode at ambient conditions. Images are presented after a 3rd order

3. Results & discussion
3.1. Characterization of polysaccharides
Pullulan and scleroglucan standards required comprehensive

Fig. 2. (A) DLS correlation functions showing the solubility of native pullulan and scleroglucan in aqueous solutions and respective purities determined with HPSECRI and HPAEC-PAD (pullulan, n = 15; scleroglucan, n = 17). (B) HPSEC-RI signal overlay for scleroglucan after dissolution at varying temperature and duration. The
dotted arrows indicate increased signal intensity upon prolonged dissolution time. (C) Representative HPSEC-LALS chromatograms for native pullulan and scle­
roglucan solutions with Mw and Đ indications of the fractions observed (pullulan and scleroglucan, n = 6). (D) Representative Mark-Houwink plot for pullulan and
scleroglucan solutions. The α values for the scleroglucan fractions were calculated using a MATLAB script computing the optimal breakpoint on a given data set for
two linear fits by minimizing overall SSE. Simplistic illustrations of the respective conformation given by the Mark-Houwink α value and dotted lines are included for
visualization purposes.
4


S. Eder et al.

Carbohydrate Polymers 260 (2021) 117830

characterization to ensure the molecular comparability of both poly­
saccharides and to constrain ultrafiltration separation variations exclu­
sively to the respective conformation in solution. The characterization
focused on the solubility, purity, molecular weight, and the conforma­
tion in aqueous solution of both polysaccharides under investigation

(Fig. 2). A fast decay in the correlation functions obtained by DLS
illustrated the complete solubility in aqueous solution for pullulan and
scleroglucan (Fig. 2A). The slower decay observed for the scleroglucan
correlation curve demonstrated the larger hydrodynamic radius of
scleroglucan compared to pullulan. The consistency of the poly­
saccharide purity in solution measured by HPSEC-RI along with the
polysaccharide powder purities obtained by HPAEC-PAD after TFA hy­
drolysis corroborated the complete solubility of pullulan and scle­
roglucan standards. The pullulan purity of 73 ± 2% observed with
HPSEC-RI matched the purity of 72 ± 4% determined by HPAEC-PAD
after dissolving pullulan for 1 h at RT under constant stirring
(Fig. 2A). This observation is in accordance with previous work on the
high solubility and stability of pullulan in aqueous solution (Adolphi &
Kulicke, 1997). Scleroglucan dissolution trials at various combinations
of temperature and incubation duration provided the optimal dissolu­
tion procedure and ensured the complete solubility as monitored by
HPSEC-RI (Fig. 2B). Incubation for 24 h at 80 ◦ C resulted in a purity in
solution of 32 ± 1% with HPSEC-RI, which agreed with the purity
determined of 36 ± 4% by HPAEC-PAD (Fig. 2A). Hence, a dissolution
procedure of 1 h at RT for pullulan and 24 h at 80 ◦ C for scleroglucan
were adopted (see Section 2.2).
HPSEC-triple detection revealed a uniform pullulan population with
a Mw of 270 ± 7 kDa and moderate dispersity (Đ) of 1.52 ± 0.02,
whereas scleroglucan exhibited distinct high-Mw and low-Mw fractions,
with 3730 ± 60 kDa and 1510 ± 50 kDa, respectively (Fig. 2C). Both
scleroglucan fractions showed uniform Đ of 1.017 ± 0.002 and 1.092 ±
0.024, respectively. The Mark-Houwink plot derived from HPSEC-triple
detection analysis provides a valuable measure for polysaccharide
conformational elucidation. Pullulan exhibited a random coil confor­
mation across the total polysaccharide population indicated by α = 0.68

(Fig. 2D). The two fractions present in scleroglucan showed two
distinctly different conformations in solution. The Mark-Houwink plot
indicated a spherical conformation in the high-Mw fraction and a rigid
rod-like conformation in the low-Mw fraction, reflected by α = 0.03 and
α = 2.5, respectively. Literature suggests that the low-Mw fraction might
be composed of several scleroglucan strands coordinated to rigid rodlike entities in solution (Sletmoen & Stokke, 2008; Zhang, Zhang, &
Xu, 2004). The low α value of the high-Mw scleroglucan fraction indi­
cated the presence of aggregates (Q. Guo et al., 2013). Yanaki and

Norisuye (1983) confirmed the presence of two fractions of scleroglucan
in aqueous solution. Furthermore, their study proposed that the high-Mw
fraction consists of two or more linear rigid rod entities, in line with our
observation of high-Mw scleroglucan aggregates with uniform Đ. Ultra­
filtration separation evaluation based on conformation requires the
breakdown of aggregates and the presence of the total scleroglucan
population in a rigid rod-like conformation beside an adjustment of the
molecular weight.
3.2. Treatment of scleroglucan solution
3.2.1. Aggregate breakdown with alkaline treatment and subsequent
neutralization
Alkaline treatment with subsequent neutralization of native scle­
roglucan solution (AN-scleroglucan) was evaluated for its suitability to
break down high-Mw aggregates and to induce a rigid rod-like confor­
mation across the total scleroglucan population. The successive break­
down of aggregates upon increasing NaOH concentration up to 0.2 M
prior to neutralization resulted in a distinct shift of the molecular weight
distribution of the total scleroglucan population towards lower Mw
(Fig. 3A). Treatment with 0.2 M NaOH followed by neutralization suc­
cessfully induced the transition to a rigid rod-like conformation over the
entire scleroglucan population. Previous work showed that scleroglucan

strands in rigid rod-like entities undergo a conformational transition
from rigid rod-like structures to random coil at 0.1– 0.2 M NaOH
induced by electrostatic repulsion owing to high ionic strength (Slet­
moen & Stokke, 2008; Zhang et al., 2004). Furthermore, the introduced
charges destabilize hydrogen bonds and lead to the breakdown of ag­
gregates. The transition to a rigid rod-like conformation observed is
consistent with the described ability of alkaline-treated and denaturated
random coil scleroglucan strands to spontaneously renaturate and form
rigid rod-like structures after subsequent neutralization (Sletmoen &
Stokke, 2008; Zhang et al., 2004). The overlay of the Mark-Houwink
plots of the alkaline treatment at various NaOH concentration after
neutralization revealed increasing slopes and hence increasing α values
with higher NaOH concentration (Fig.3 B). Consequently, more scle­
roglucan strands were separated and structures with higher rigidity
renaturated after neutralization as the NaOH concentration in the
treatment increased. The treatment with 0.2 M NaOH showed an
increased uniformity in the resulting molecular weight distribution of
scleroglucan with high rigidity, displayed by a Đ value of 1.31 ± 0.02,
and a Mark-Houwink α of 2.03 ± 0.04, compared to treatments with
0.01 M and 0.1 M NaOH (Fig. 3A, B). At concentrations equal to or lower
than 0.1 M NaOH, scleroglucan exhibited distinct fractions composed of

Fig. 3. (A) HPSEC-RI monitoring for molecular weight distribution and Đ alteration (n = 6) of scleroglucan after alkaline treatment for 10 min at RT with varying
NaOH concentration followed by neutralization and (B) corresponding Mark-Houwink plot illustrating conformational transitions. Colored areas depict corre­
sponding molecular weight fractions in panel (A) and (B) for enhanced visualization guiding.
5


S. Eder et al.

Carbohydrate Polymers 260 (2021) 117830

aggregates and rigid rod-like entities. Interestingly, aggregate formation
first increased during treatment with low NaOH concentration, as
observed after alkaline treatment with 0.01 M NaOH and subsequent
neutralization in comparison to the untreated control (Fig.3 A). This
observation was consistent with the expected equilibrium of co-existing
aggregates and linear rigid rod-like structures in solution below 0.1–0.2
M NaOH (Sletmoen & Stokke, 2008). Ding, Jiang, Zhang, and Wu
(1998)) observed a similar phenomenon for pachyman, a (1,
3)-β-D-glucan, in aqueous NaOH solution and concluded that large ag­
gregates are formed.
Alkaline treatment with 0.2 M NaOH for 10 min and subsequent
neutralization facilitated the breakdown of the majority of aggregates
and the conformational transition to rigid rod-like structures across the
entire scleroglucan population. The optimal treatment conditions
enabled the preparation of AN-scleroglucan solutions with uniform
molecular weight distribution and conformation.

(Fig. 4A). The shoulder of the elution profile around retention volume =
12.25 mL at t = 0 vanishes after 5 min sonication, thus illustrating the
complete breakdown of aggregates (Fig. 4B). Furthermore, the unaltered
Đ observed for ANS-scleroglucan indicates the absence of any structural
selectivity of the applied treatment. The change of α values might
originate from an altered higher-order structure within the rigid rod-like
entities owing to the polysaccharide degradation (Sletmoen & Stokke,
2008), yet with no observed effect on the resulting conformation in
solution (Fig. 4A). The simultaneous decrease in α values and Mw
observed matches the controversially discussed presence of rigid
rod-like structure of scleroglucan below a critical Mw (Li, Xu, & Zhang,

2010; Wang et al., 2017). Denaturation of scleroglucan polymer chain
ends or incomplete strand breakage might hinder the sterical alignment
and contribute to the abated rigidity through sonication treatment.
The findings on sonication-induced scleroglucan degradation ob­
tained pushes further studies on the biological activities of native (1,3)β-D-glucans, as controlled sonication is a promising approach to reduce
their viscosity (Sletmoen & Stokke, 2008) and was already successfully
applied for cellulose (Arcari et al., 2020). Overall, preparation of
ANS-scleroglucan enabled a controlled and reproducible molecular
weight adjustment with unaltered Đ and remaining rigid rod-like
conformation of scleroglucan in solution over the total treatment
period investigated. Furthermore, ANS-scleroglucan showed structural
stability over the entire ultrafiltration and analysis period, facilitating a
meaningful ultrafiltration evaluation and subsequent chromatographic
investigation (Fig. S3). For more information on the structural stability
of ANS-scleroglucan solution, see supplementary information.

3.2.2. Molecular weight adjustment by sonication
The assessment of the effect of polysaccharide conformation on ul­
trafiltration performance requires comparable molecular weights with
distinct conformations in solution. For this purpose, the adjustment of
molecular weight by sonication and its effect on the Đ and conformation
were investigated. Sonication of AN-scleroglucan (see Section 3.2.1) for
180 min resulted in a significant decrease of Mw from 1860 ± 130 kDa to
387 ± 14 kDa and yielded alkaline-treated, neutralized and sonicated
scleroglucan (ANS-scleroglucan) (Fig. 4A). The gradual shift of the
respective molecular weight distribution to higher retention volume
(Fig. 4B), which is inversely proportional to the molecular weight,
illustrated the successful Mw reduction with increasing sonication time
(Fig. 4A). The uniform Đ of AN-scleroglucan solution remained un­
changed, without any significant variation, in the ANS-scleroglucan

solution, as shown by the Đ values of 1.31 ± 0.02 and 1.36 ± 0.07,
respectively (Fig. 4A). Simultaneously, the value of Mark-Houwink α
decreased significantly from 2.03 ± 0.04 to 1.1 ± 0.1 during the soni­
cation process. However, α values above 0.8 are ascribed to a rigid rodlike conformation, indicating that the conformation of scleroglucan was
maintained (Q. Guo et al., 2013; He et al., 2017).
The cleavage of glycosidic linkages owing to shear forces in the fluid
caused by imploding cavitation bubbles presumably governs the
sonication-driven polysaccharide degradation (Cizova, Bystricky, &
Bystricky, 2015). Consequently, aggregates formed by hydrogen bonds
are more susceptible to sonication degradation and readily disentangled.
Hence, the breakdown of remaining aggregates accounted for the
considerable decrease of Mw within the first 5 min of sonication

3.3. Visualization of scleroglucan treatment by AFM
AFM imaging was used to visualize the morphology and changes
thereof during the preliminary preparation of the ANS-scleroglucan feed
solution. Scleroglucan in the native state covered the complete surface
revealing a mesh of branched, overlapping and intertwined poly­
saccharides chains with varying heights (Fig. 5A), resembling observa­
tions made in previous studies (McIntire & Brant, 1997; Vuppu, Garcia,
& Vernia, 1997). After the alkaline treatment and neutralization, the
majority of aggregates were separated with individual aggregates still
being observable, supporting the observation from HPSEC-RI of a
decreasing effect on the molecular weight (Fig. 5B). As fuzzy ends and
branching points were observable, the interaction of multiple poly­
saccharide chains was confirmed, as already reported in other linear
polysaccharides such as the carrageenans and gellan gum (Diener et al.,
2019, 2020). The effect of sonication was visually confirmed as the

Fig. 4. (A) Alteration of Mw, Đ, and Mark-Houwink α in AN-scleroglucan solution during sonication treatment over 180 min. Different letters denote significant

differences (one-way ANOVA + Tukey’s post hoc test, p < 0.05, n = 3). (B) HPSEC-RI overlay of sonicated AN-scleroglucan illustrating gradual reduction of Mw with
prolonged sonication time.
6


S. Eder et al.

Carbohydrate Polymers 260 (2021) 117830

Fig. 5. Representative AFM height images of aqueous scleroglucan deposited on mica: (A) In its native state; (B) after alkaline treatment and neutralization (ANscleroglucan); (C) after subsequent sonication (ANS-scleroglucan); (D) retentate after ultrafiltration. Colored squares highlight the location of the enlarged AFM
images displayed right below. Height applies to all images.

remaining aggregated structures, and thus the detected molecular
weight, were further disintegrated (Fig. 5C). The sonication treatment of
the AN-scleroglucan solution resulted in the liberation of rigid rod-like,
linear polysaccharide chains, confirming the expected conformation of
scleroglucan. Similarly to the ANS-scleroglucan solution, mainly rigid
rod-like, linear polysaccharide chains were observed in the retentate
solution after ultrafiltration (Fig. 5D). The rigidity of the polysaccharide
chains may explain the indifference of the ANS-scleroglucan solution
before and after application of ultrafiltration and points again at the
importance of the conformation of the polysaccharide conformation for
the assessment of a membrane. The apparent alignment of the polymers
and aggregates is presumably caused by the drying step in the sample
preparation and emphasizes the rigidity of the scleroglucan polymers
(Stokke & Brant, 1990). Interestingly, a small number of the single
polysaccharides were ring-like shaped, also observed in ι-carrageenan
owing to their chiral secondary structure (Fig. 5C, D) (Schefer, Usov, &
Mezzenga, 2015). In our observations, AFM imaging provided a simple
characterization pathway to explore conformational changes and verify

the effect of the pretreatments.

3.4.1. Characterization of pullulan and ANS-scleroglucan feed solutions
The scleroglucan solution pretreatment described (see Section 3.2)
facilitated the elimination of potential influencing factors on the ultra­
filtration separation beside the polysaccharide conformation in solution
and ensured the comparability with pullulan. The feed solutions of
pullulan and ANS-scleroglucan showed a Mw of 271 ± 9 and 383 ± 22
kDa, respectively, fulfilling supposed molecular weight comparability
with ΔMw ≈ 100 kDa (Fig. 6A, B). Moreover, the α values of 0.68 ± 0.05
and 1.08 ± 0.04 depict the random coil and rigid rod-like conformation
of pullulan and ANS-scleroglucan in solution, respectively. The Đ of 1.59
± 0.08 for pullulan and 1.34 ± 0.05 for ANS-scleroglucan reflect the
narrow to moderate Đ of the desired conformation considering the
respective polysaccharide. Additionally, the intrinsic viscosity [η] of
pullulan, 0.84 ± 0.27 dL/g, and ANS-scleroglucan, 1.13 ± 0.13 dL/g,
corroborate the elucidated conformation (Fig. 6A, B). The [η] might be
considered as “inverse density“, with higher values indicating a more
extended and less dense polymer in solution. Hence, the low [η] value of
pullulan illustrates the compact random coil conformation in compari­
son to scleroglucan. The high [η] of ANS-scleroglucan substantiates the
extended polymer arrangement corresponding to the linear conforma­
tion. The ζ-potential measurement of the feed solutions reflected the
neutral character of pullulan and ANS-scleroglucan and excluded any
charge-induced differences among them (Fig. 6A, B). Furthermore,
density and viscosity values of both polysaccharide solutions were
comparable to water (Table S3, Fig. S4). Therefore, influences on the
diafiltration process due to physical properties of the polysaccharide
solutions could be excluded.

3.4. Evaluation of pullulan and ANS-scleroglucan ultrafiltration
Ultrafiltration investigations of polysaccharides with identical
monomeric units, comparable molecular weight, and distinct confor­
mation in aqueous solution reveal insight into the effect of conformation
on the membrane filtration process. Pullulan and ANS-scleroglucan so­
lutions were subjected to ultrafiltration using Hydrosart (HY) and pol­
yethersulfone (PES) membranes with distinct molecular weight cut-offs
of 2, 5 and 10 kDa, and 3, and 10 kDa, respectively. Ultrafiltration
separation performance was evaluated considering percentage differ­
ence in Mw of retentate and feed solution (Δ%Mw), revealing the impact
on the molecular weight distribution, and the corresponding recovery
yield of pullulan and ANS-scleroglucan achieved with the membranes
studied. The comparison of pullulan and ANS-scleroglucan for each
membrane and the separate consideration of pullulan and ANSscleroglucan ultrafiltration performance across all membranes studied
provides comprehensive inferences on the separation processes
observed.

3.4.2. Separation efficiency of pullulan and ANS-scleroglucan by
ultrafiltration
The comparison of pullulan and ANS-scleroglucan for each mem­
brane provided insight into the separation efficiency for both poly­
saccharides. Pullulan and ANS-scleroglucan recoveries after
ultrafiltration mostly revealed no statistically significant differences,
irrespective of the membrane used or MWCO selected (Fig. 7), apart
from the 10 kDa PES membrane. The filtration process with this
particular membrane resulted in a substantial difference between ANSscleroglucan and pullulan, with a yield of 71% and a marginal recovery
yield of 1%, respectively. The rejection coefficients and permeability
7

S. Eder et al.

Carbohydrate Polymers 260 (2021) 117830

Fig. 6. Characteristics of prepared (A) pullulan and (B) ANS-scleroglucan feed solutions used to investigate the conformational effect of polysaccharides on ul­
trafiltration separation (pullulan, n = 10; scleroglucan, n = 15). Simplistic illustrations of the respective conformation given by the Mark-Houwink α value are
included for visualization purposes. Mark-Houwink α values were derived from areas indicated by the dotted lines, corresponding to the major weight fraction of the
respective polysaccharide, and excluding software extrapolation at the border areas within the molecular weight distribution. Mw, Đ, and [η] values were derived
from areas within the molecular weight distribution indicated by the brackets.
Fig. 7. Percentage difference in Mw between
the pullulan and ANS-scleroglucan retentate
and feed solutions (Δ%Mw) and corresponding
recovery yields of the respective Hydrosart
(HY) and polyethersulfone (PES) membranes
observed. The statistical evaluation allows for
comparison between pullulan and ANSscleroglucan for each membrane studied.
Different letters denote significant differences
of recovery yields (oneway ANOVA + Tukey’s
post hoc test, p < 0.05, n = 3). Asterisks indi­
cate significant differences of Δ%Mw (oneway
ANOVA + Tukey’s post hoc test, *p < 0.05, n =
3).

values for pullulan and ANS-scleroglucan considering each membrane
were in line with the yields presented (Table S2, Fig. 7). This striking
difference in remaining yield demonstrates the fundamental effect of
polysaccharide conformation on ultrafiltration separation. Moreover,
the significantly higher values for Δ%Mw of ANS-scleroglucan after ul­
trafiltration with 5 kDa HY, 10 kDa HY and 3 kDa PES indicate the
conformational effect on membrane separation. Interestingly, Δ%Mw of

pullulan and ANS-scleroglucan did not differ significantly for filtrations
with 2 kDa HY and 10 kDa PES membranes (Fig. 7). It appears that the
10 kDa PES membrane offers potential merits to selectively separate
pullulan and scleroglucan and provides great potential for other poly­
saccharide applications with similar conformational differences.
The variations observed can be ascribed to the distinct conformations
in solution. The higher chain flexibility of pullulan permits a more
compact spatial alignment in solution. Considering the applied trans­
membrane pressure during ultrafiltration, the hydrodynamic volume of
flexible polymers can be additionally decreased by shear-induced
deformation at the membrane interface (Fried, 1997). Ultimately,
these circumstances enhance the transport across the membrane and
reduce the pullulan yield. On the contrary, ANS-scleroglucan possesses a
linear rigid rod-like conformation, which entails an increased

hydrodynamic volume relative to the molecular weight in case of a
spatial consideration along the polysaccharide chain. The results ob­
tained are consistent with the statistical model proposed by Vinther
et al. (2012), claiming that linear shapes have a lower probability of
entering a membrane pore compared to spherical shapes. Our observa­
tions affirm the importance to consider conformation in ultrafiltration
separation, since the physical separation directly relies on the hydro­
dynamic volume under ultrafiltration conditions of the particles to be
retained.
3.4.3. Membrane performance for pullulan and ANS-scleroglucan yields
The evaluation of pullulan and ANS-scleroglucan ultrafiltration
across all membranes studied permits comprehensive inferences on
membrane performance for the considered polysaccharide. The com­
parison within a respective polysaccharide for all membranes studied
showed no significant effect of membrane selection on pullulan yield,

except for the aforementioned 10 kDa PES membrane (Fig. 8). ANSscleroglucan exhibited a trend of decreasing yield with increasing
MWCO, although solely the difference between 3 kDa PES, showing 94%
yield, and 10 kDa PES, showing 71% yield, was statistically significant.
Moreover, the yield of 98% achieved with 2 kDa HY was significantly
8


S. Eder et al.

Carbohydrate Polymers 260 (2021) 117830

Fig. 8. Percentage difference in Mw between
the pullulan and ANS-scleroglucan retentate
and feed solutions (Δ%Mw) and corresponding
recovery yields of the respective Hydrosart
(HY) and polyethersulfone (PES) membranes
observed. The statistical evaluation allows for
comparison within a respective polysaccharide
across the membranes studied. Different letters
denote significant differences of recovery yields
(oneway ANOVA + Tukey’s post hoc test, p <
0.05, n = 3). Asterisks indicate significant dif­
ferences of Δ%Mw (oneway ANOVA + Tukey’s
post hoc test, *p < 0.05, n = 3).

higher than the ANS-scleroglucan yield of 71% obtained with 10 kDa
PES. Furthermore, the extrapolation of the yield remaining after
assuming the highest ND observed (ND = 20 for pullulan diafiltration
with 10 kDa PES, see Table S2), corroborated the trends of poly­
saccharide yields observed (Table S2, Fig. 8). Pullulan ultrafiltration

revealed significant differences in Δ%Mw between distinct MWCO within
a given membrane material, except for 5 kDa HY (Fig. 8). However, 2
kDa HY showed a higher Δ%Mw value than 10 kDa HY, whereas the
opposite effect was observed for PES membranes. Moreover, Δ%Mw of 10
kDa PES membrane exhibited a significant difference to all other pul­
lulan ultrafiltrations. ANS-scleroglucan showed significant differences
in Δ%Mw between HY and PES membranes after ultrafiltration, but no
differences within the same membrane material were observed.
Overall, the recovery yields revealed remarkable deviations between
the nominal MWCO and the actual Mw of the respective feed solution.
The MWCO represents the lowest molecular weight of a considered
molecule that is 90% rejected by the membrane (Koros et al., 1996). A
yield of 90% for pullulan and ANS-scleroglucan with PES membranes
were achieved with a 3 kDa MWCO only, which implies a 90–fold and
128–fold deviation between nominal MWCO and actual Mw, respec­
tively. Generally, selection of a membrane with a MWCO 3 to 6 times
smaller than the molecular weight of the molecule to be retained is
recommended in order to assure complete retention (Schwartz, 2003).
Since the Mw of the prepared pullulan and ANS-scleroglucan feed solu­
tions were 27–fold and 38–fold greater than the highest MWCO chosen
(Figs. 7 and 8), respectively, a recovery yield of 90% was expected for all
membranes studied. These observations demonstrate that MWCOs based
on globular proteins are not applicable to polysaccharides. Pullulan and
ANS-scleroglucan ultrafiltration with HY membranes achieved over
90% or insignificant lower yields irrespective of the MWCO selected.
The reduction in Δ%Mw with increasing MWCO of HY membranes
observed for pullulan contradicts the principle of MWCO rating, indi­
cating an effect of membrane material on separation performance.
Higher MWCO are expected to result in the rejection of larger molecules
with a concomitant increase in Mw of the retentate. Pullulan ultrafil­

tration with 10 kDa PES demonstrated clearly the rejection of larger
molecules with higher MWCO corresponding to the expected effect and
to the conformational effect discussed above. However, the Δ%Mw re­
sults obtained for ANS-scleroglucan, showing that differences occurred
solely among membrane materials, corroborate the considered effect of
membrane material on the molecular weight distribution.

weight and hydrodynamic volume. In this case, ultrafiltration perfor­
mance evaluation based on resulting yields is an adequate measure to
reflect the membranes suitability for a considered application. However,
polysaccharide often present broader molecular weight distribution and
thus specific molecular weight fractions might get lost despite a satis­
factory yield. Ideally, polysaccharide filtration achieves the highest
yield whilst maintaining an unchanged molecular weight distribution of
the desired polysaccharide. The combined assessment of Δ%Mw and
yield elucidates the effect of membrane selection on the molecular
weight distribution and separation efficiency of the considered
polysaccharide.
Pullulan ultrafiltration with yields of at least 90% without adverse
alteration of the molecular weight distribution was achieved with 2 kDa
HY or 3 kDa PES (Fig. 8). This observation corresponds to a 136-fold and
90-fold deviation between nominal MWCO and pullulan Mw, respec­
tively. ANS-scleroglucan ultrafiltration with yields of at least 90% and
smallest alteration of molecular weight distribution was observed for 2
and 5 kDa HY membranes (Fig. 8), corresponding to a 192–fold and
77–fold deviation between nominal MWCO and ANS-scleroglucan Mw,
respectively. The significant difference in ANS-scleroglucan yield after
ultrafiltration with 3 kDa and 10 kDa PES was not accompanied with any
significant change in Δ%Mw (Fig. 8). Since ANS-scleroglucan possesses a
linear rigid rod-like structure, the ability to pass the membrane may also

depend on the spatial orientation. Under ultrafiltration conditions with
elevated transmembrane pressure, linear structures might be forced
through the membrane pore irrespective of their molecular weight,
whereas spherical structures with high Mw are retained. Such a phe­
nomenon would result in an unchanged Δ%Mw of the rigid rod-like
polymer with simultaneously decreasing yield upon an increasing
MWCO, as observed. Interestingly, pullulan ultrafiltration with HY
membranes displayed no significant differences in terms of yield, but the
significant reduction in Δ%Mw with increasing MWCO indicated the loss
of higher-Mw pullulan fractions (Fig. 8). Furthermore, the reverse
pattern of significant differences between pullulan Δ%Mw and yield with
10 kDa HY and PES reinforce the effect of membrane material on Mw
alteration and recovery yield. Moreover, the ultrafiltration of ANSscleroglucan with HY membranes, where neither significant difference
in Δ%Mw nor in the yield were observed, substantiates a greater effect of
membrane material rather than the nominal MWCO on resulting
retentate properties. In addition, the distinct observations within Pul­
lulan and ANS-scleroglucan filtration emphasize the fundamental effect
of polysaccharide conformation on the resulting separation process. The
smallest MWCOs of HY and PES membranes provided the desired
membranes performance regarding yield and Mw for pullulan. In case of
ANS-scleroglucan, ultrafiltration with HY membranes yielded the

3.4.4. Combined Mw and yield evaluation for optimal pullulan and ANSscleroglucan rejection
Globular proteins usually exhibit a narrow relation of molecular
9


S. Eder et al.

Carbohydrate Polymers 260 (2021) 117830

highest recovery yields while maintaining the closest weight distribu­
tion to the initial feed solution observed, irrespective of the selected
MWCO.
The impact of membrane material on ultrafiltration performance
within a considered polysaccharide is of particular interest since both
membrane materials have been extensively used in filtration of poly­
saccharides (Kothari et al., 2014; Susanto, Arafat, Janssen, & Ulbricht,
2008). Saha, Balakrishnan, and Ulbricht (2007) found that
cellulose-based membranes are more prone to fouling than PES mem­
branes when filtering a high molecular weight fraction of 130 kDa
containing arabinogalactan. For this study, the monitoring of the
permeate flow obviously indicated that fouling didn`t occur during the
time of the diafiltrations, irrespective the membrane utilized (Fig. S5).
Many factors possibly contribute to fouling, such as the concentration of
solutes or the interaction of solutes and membrane e.g. electrostatic in­
teractions, hydrophobic interactions, and hydrogen bonding. Given the
diluted concentration of the polysaccharide feed solutions (0.025%
(w/v)) utilized in the diafiltration, any adverse fouling effect due to
solute concentration can be neglected. In particular, charges on the
membrane and solute surface are an important factor to consider in
ultrafiltration since electrostatic interactions can influence the separa­
tion process (Hu et al., 2018). Electrostatic attraction owing to oppo­
sitely charged surfaces of membrane and solute might induce fouling,
whereas same charges suppress fouling by repulsive effects (Breite,
Went, Thomas, Prager, & Schulze, 2016). The isoelectric point (IEP) of
the PES membranes in this study is reported to be around 5.5 (Salgin,
Salgin, & Soyer, 2013). Membranes based on regenerated cellulose, such
as the HY membranes used, have IEP`s between 3–5 (Pontie, Chasseray,
Lemordant, & Laine, 1997; Pontie, Durand-Bourlier, Lemordant, &

Laine, 1998). Since pullulan and ANS-scleroglucan feed solutions were
neutral, both membrane materials are operated above their IEP, hence
exhibiting slightly negative surfaces charges during ultrafiltration.
Furthermore, it could be assumed that the ionic strength of μ = 0.2 in the
ANS-scleroglucan solution resulting from NaCl after alkaline treatment
and neutralization had no effect on the IEP (Salgin et al., 2013).
Consequently, potential constraints due to adverse charge-charge in­
teractions at the membrane surface, such as adsorptive effects, could be
neglected owing to the ζ-potential measurements and the membrane
IEPs reported. Hence, fouling cannot explain the differences observed
between e.g. 10 kDa HY and 10 kDa PES in the ultrafiltration of pullulan.
However, further investigations are needed to ascertain the underlying
mechanistic cause for the observed difference between membrane ma­
terials for a given polysaccharide.
The assembled data suggest that polysaccharide conformation sub­
stantially affects ultrafiltration performance when separating distinct
polysaccharide geometries. Considering a respective polysaccharide
conformation, the membrane material seems to influence largely the
rejection behavior. However, conformation might play a decisive role as
the separation variations in terms of membrane material differ for both
glucose-based polysaccharides. Considering polysaccharide purifica­
tions, we recommend choosing the smallest MWCO applicable for a
desired application. In case of pullulan, HY and PES membranes proved
to be suitable selections, whereas HY showed superior performance in
terms of yields without Mw alteration for ANS-scleroglucan. Based on
our results, ultrafiltration with 10 kDa PES membrane might be an asset
for the selective separation of pullulan and ANS-scleroglucan in future
studies.

between apparent and nominal MWCO were observed for certain

membranes. The conformation as crucial factor was evidenced by a
higher molecular weight and yield in the retentate of rigid rod-like ANSscleroglucan compared to randomly coiled pullulan. Furthermore, the
effect of spatial orientation of linear molecules on the transport across
the membrane was illustrated with ANS-scleroglucan. While the mo­
lecular weight remained unchanged after ultrafiltration, the yield
significantly decreased, indicating membrane transport irrespective of
molecular weight for linear polysaccharides. Eventually, Hydrosart
membranes may be recommended for purification purposes of glucosebased polysaccharides with comparable conformation and molecular
weight as in this study, to ensure high polysaccharide yield and smallest
possible effects on the molecular weight distribution. Moreover, the
smallest MWCO feasible for the considered application should be cho­
sen. We anticipate that polyethersulfone membranes with elevated
MWCO will facilitate the selective separation of pullulan and ANSscleroglucan and offer great potential for polysaccharides with similar
structural feature and conformation. This work provides the empirical
framework for the development of an improved membrane selection for
polysaccharide filtration, paving the way to revised membrane guide­
lines in general and high-performance separation of polysaccharides in
particular.
CRediT authorship contribution statement
Severin Eder: Conceptualization, Methodology, Formal analysis,
Investigation, Writing - original draft, Writing - review & editing,
Visualization, Supervision. Patrick Zueblin: Methodology, Formal
analysis, Investigation, Writing - original draft. Michael Diener:
Conceptualization, Formal analysis, Investigation, Writing - original
draft, Visualization. Mohammad Peydayesh: Conceptualization,
Writing - review & editing. Samy Boulos: Conceptualization, Methodư
ology, Writing - review & editing. Raffaele Mezzenga: Resources,
ă m: Resources, Writing - review
Writing - review & editing. Laura Nystro
& editing, Supervision, Project administration, Funding acquisition.

Declaration of Competing Interest
The authors reported no declarations of interest.
Acknowledgment
The authors gratefully thank Dr. Pascal Bertsch from the Laboratory
of Food Process Engineering, ETH Zürich for his assistance in the
ultrasonication setup and sharing his expertise. The authors acknowlư
edge Dr. Joăel Zink from the Laboratory of Food Process Engineering,
ETH Zürich for supporting the viscosity and density measurements and
his help. This work was supported by European Research Council ERC,
under the European Union’s Horizon 2020 research and innovation
programme (Grant agreement No. 679037), and ETH Zurich.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi: />References

4. Conclusions

Adolphi, U., & Kulicke, W. M. (1997). Coil dimensions and conformation of
macromolecules in aqueous media from flow field-flow fractionation/multi-angle
laser light scattering illustrated by studies on pullulan. Polymer, 38(7), 1513–1519.
Arcari, M., Axelrod, R., Adamcik, J., Handschin, S., S´
anchez-Ferrer, A., Mezzenga, R., &
Nystră
om, G. (2020). Structureproperty relationships of cellulose nanofibril hydroand aerogels and their building blocks. Nanoscale, 12(21), 11638–11646.
Atkins, E. (1985). Conformations in polysaccharides and complex carbohydrates. Journal
of Biosciences, 8(1–2), 375–387.
Boual, Z., Abdellah, K., Aminata, K., Michaud, P., & Hadj, M. (2012). Partial
characterization and hydrolysis procedure of water soluble polysaccharides

Polysaccharide conformation in aqueous solution showed a

remarkable effect on ultrafiltration retentates in terms of recovery yield
and molecular weight distribution. Overall, the impact of poly­
saccharide conformation and membrane material, considering a distinct
conformation, were more decisive on the membrane separation than the
manufacturer’s declared MWCO. Consequently, large deviations
10


S. Eder et al.

Carbohydrate Polymers 260 (2021) 117830
McIntire, T. M., & Brant, D. A. (1997). Imaging of individual biopolymers and
supramolecular assemblies using noncontact atomic force microscopy. Biopolymers,
42(2), 133–146.
Montesdeoca, V. A., Bakker, J., Boom, R. M., Janssen, A. E. M., & Van der Padt, A.
(2019). Ultrafiltration of non-spherical molecules. Journal of Membrane Science, 570,
322–332.
Muzzarelli, R. A. A. (2012). Frontmatter. In Y. Habibi, & L. A. Lucia (Eds.), Polysaccharide
building blocks (pp. i–xiii).
Navard, J., & Navard, P. (2012). Introduction: Challenges and opportunities in building a
multinational, interdisciplinary research and education network on polysaccharides.
In P. Navard (Ed.), Polysaccharide research the European polysaccharide network of
excellence (EPNOE) research initiatives and results (pp. 1–12). Wien: Springer.
Nishinari, K., Kohyama, K., Williams, P. A., Phillips, G. O., Burchard, W., & Ogino, K.
(1991). Solution properties of pullulan. Macromolecules, 24(20), 5590–5593.
Pinelo, M., Jonsson, G., & Meyer, A. S. (2009). Membrane technology for purification of
enzymatically produced oligosaccharides: Molecular and operational features
affecting performance. Separation and Purification Technology, 70(1), 1–11.
Platt, S., Mauramo, M., Butylina, S., & Nystrom, M. (2002). Retention of pegs in crossflow ultrafiltration through membranes. Desalination, 149(1-3), 417–422.
Pontie, M., Chasseray, X., Lemordant, D., & Laine, J. M. (1997). The streaming potential

method for the characterization of ultrafiltration organic membranes and the control
of cleaning treatments. Journal of Membrane Science, 129(1), 125–133.
Pontie, M., Durand-Bourlier, L., Lemordant, D., & Laine, J. M. (1998). Control fouling
and cleaning procedures of UF membranes by a streaming potential method.
Separation and Purification Technology, 14(1-3), 1–11.
Rohrer, J. S., Cooper, G. A., & Townsend, R. R. (1993). Identification, quantification, and
characterization of glycopeptides in reversed-phase hplc separations of glycoprotein
proteolytic digests. Analytical Biochemistry, 212(1), 7–16.
Saha, N. K., Balakrishnan, M., & Ulbricht, M. (2007). Sugarcane juice ultrafiltration: FTIR
and SEM analysis of polysaccharide fouling. Journal of Membrane Science, 306(1-2),
287–297.
Salgin, S., Salgin, U., & Soyer, N. (2013). Streaming potential measurements of
polyethersulfone ultrafiltration membranes to determine salt effects on membrane
zeta potential. International Journal of Electrochemical Science, 8(3), 4073–4084.
Schefer, L., Usov, I., & Mezzenga, R. (2015). Anomalous stiffening and ion-induced
coil–Helix transition of carrageenans under monovalent salt conditions.
Biomacromolecules, 16(3), 985–991.
Schwartz, L. (2003). Diafiltration for desalting or buffer Exchange. BioProcess International
(Vol. 5, pp. 43–49). Boston, MA: BioProcess International.
Scott, K. (1995). Introduction to membrane separation. Handbook of industrial membranes
(pp. 3–185). Amsterdam, NL: Elsevier Science.
Sletmoen, M., & Stokke, B. T. (2008). Review : Higher order structure of (1,3)-beta-Dglucans and its influence on their biological activities and complexation abilities.
Biopolymers, 89(4), 310–321.
Stokke, B. T., & Brant, D. A. (1990). The reliability of wormlike polysaccharide chain
dimensions estimated from electron-micrographs. Biopolymers, 30(13–14),
1161–1181.
Sun, H. J., Qi, D., Xu, J. Y., Juan, S., & Zhe, C. (2011). Fractionation of polysaccharides
from rapeseed by ultrafiltration: Effect of molecular pore size and operation
conditions on the membrane performance. Separation and Purification Technology, 80
(3), 670–676.

Susanto, H., Arafat, H., Janssen, E. M. L., & Ulbricht, M. (2008). Ultrafiltration of
polysaccharide-protein mixtures: Elucidation of fouling mechanisms and fouling
control by membrane surface modification. Separation and Purification Technology, 63
(3), 558–565.
Taylor, J. R. (1982). 3.3 sums and differences; Products and quotient. In J. R. Taylor
(Ed.), An introduction to error analysis: The study of uncertainties in physical
measurements. Sausalito, California: University Science Books.
Vinther, F., Pinelo, M., Brons, M., Jonsson, G., & Meyer, A. S. (2012). Statistical
modelling of the interplay between solute shape and rejection in porous membranes.
Separation and Purification Technology, 89, 261–269.
Vuppu, A. K., Garcia, A. A., & Vernia, C. (1997). Tapping mode atomic force microscopy
of scleroglucan networks. Biopolymers, 42(1), 89–100.
Wang, Q., Sheng, X. J., Shi, A. M., Hu, H., Yang, Y., Liu, L., … Liu, H. Z. (2017). Betaglucans: Relationships between modification, conformation and functional activities.
Molecules, 22(2).
Yanaki, T., & Norisuye, T. (1983). Triple Helix and random coil of scleroglucan in dilutesolution. Polymer Journal, 15(5), 389–396.
Zhang, X. F., Zhang, L. N., & Xu, X. J. (2004). Morphologies and conformation transition
of Lentinan in aqueous NaOH solution. Biopolymers, 75(2), 187–195.

extracted from onesaharian medicinal plant: Malvaaegyptiaca l. International Journal
of Bioscience Biochemistry and Bioinformatics, 2, 100–103.
Breite, D., Went, M., Thomas, I., Prager, A., & Schulze, A. (2016). Particle adsorption on a
polyether sulfone membrane: How electrostatic interactions dominate membrane
fouling. RSC Advances, 6(70), 65383–65391.
Cano, A., & Palet, C. (2007). Xylooligosaccharide recovery from agricultural biomass
waste treatment with enzymatic polymeric membranes and characterization of
products with MALDI-TOF-MS. Journal of Membrane Science, 291(1-2), 96–105.
Castillo, N. A., Valdez, A. L., & Farina, J. I. (2015). Microbial production of scleroglucan
and downstream processing. Frontiers in Microbiology, 6.
Chen, X., Qi, T., Zhang, Y., Wang, T., Qiu, M., Cui, Z., & Fan, Y. (2020). Facile pore size
tuning and characterization of nanoporous ceramic membranes for the purification

of polysaccharide. Journal of Membrane Science, 597, Article 117631.
Cheryan, M. (1998). Process design. In M. Cheryan (Ed.), Ultrafiltration and microfiltration
handbook (pp. 293–344). CRC Press.
Cizova, A., Bystricky, P., & Bystricky, S. (2015). Ultrasonic and free-radical degradation
of mannan from Candida albicans. International Journal of Biological Macromolecules,
75, 32–36.
Coviello, T., Palleschi, A., Grassi, M., Matricardi, P., Bocchinfuso, G., & Alhaique, F.
(2005). Scleroglucan: A versatile polysaccharide for modified drug delivery.
Molecules, 10(1), 633.
Demuth, T., Betschart, J., & Nystră
om, L. (2020). Structural modifications to watersoluble wheat bran arabinoxylan through milling and extrusion. Carbohydrate
Polymers, 240, Article 116328.
Diener, M., Adamcik, J., Bergfreund, J., Catalini, S., Fischer, P., & Mezzenga, R. (2020).
Rigid, fibrillar quaternary structures induced by divalent ions in a carboxylated
linear polysaccharide. ACS Macro Letters, 9(1), 115–121.
Diener, M., Adamcik, J., S´
anchez-Ferrer, A., Jaedig, F., Schefer, L., & Mezzenga, R.
(2019). Primary, secondary, tertiary and quaternary structure levels in linear
polysaccharides: From random coil, to single Helix to supramolecular assembly.
Biomacromolecules, 20(4), 1731–1739.
Ding, Q., Jiang, S. H., Zhang, L. N., & Wu, C. (1998). Laser light-scattering studies of
pachyman. Carbohydrate Research, 308(3-4), 339–343.
Directions for Use Vivaflow 50 | 50R | 200. (2016). Goettingen. Germany: Sartorius Lab
Instruments.
Fried, J. R. (1997). Basic Principles of Membrane Technology By Marcel Mulder
(University of Twente, The Netherlands). Kluwer Academic: Dordrecht. 1996. 564
pp. $255.00. ISBN 0-7823-4247-X. Journal of the American Chemical Society, 119(36),
8582-8582.
Gidley, M. J., & Nishinari, K. (2009). Physico-chemistry of (1,3)-β-glucans. In A. Bacic,
G. B. Fincher, & B. A. Stone (Eds.), Chemistry, biochemistry, and biology of 1-3 Beta

glucans and related polysaccharides (pp. 47–118). San Diego: Academic Press.
Guo, M. Q., Hu, X., Wang, C., & Ai, L. (2017). Polysaccharides: Structure and solubility.
In Z. Xu (Ed.), Solubility of polysaccharides (pp. 7–21). London: IntechOpen Limited.
Guo, Q., Wang, Q., Cui, S. W., Kang, J., Hu, X., Xing, X., & Yada, R. Y. (2013).
Conformational properties of high molecular weight heteropolysaccharide isolated
from seeds of Artemisia sphaerocephala Krasch. Food Hydrocolloids, 32(1), 155–161.
Harding, S. E., Abdelhameed, A. S., & Morris, G. A. (2011). On the hydrodynamic
analysis of conformation in mixed biopolymer systems. Polymer International, 60(1),
2–8.
He, P. F., He, L., Zhang, A. Q., Wang, X. L., Qu, L., & Sun, P. L. (2017). Structure and
chain conformation of a neutral polysaccharide from sclerotia of Polyporus
umbellatus. Carbohydrate Polymers, 155, 61–67.
Hu, C. Z., Liu, Z. T., Lu, X. L., Sun, J. Q., Liu, H. J., & Qu, J. H. (2018). Enhancement of
the Donnan effect through capacitive ion increase using an electroconductive rGOCNT nanofiltration membrane. Journal of Materials Chemistry A, 6(11), 4737–4745.
Kim, K. J., Fane, A. G., Benaim, R., Liu, M. G., Jonsson, G., Tessaro, I. C., & Bargeman, D.
(1994). A Comparative-Study of Techniques Used for Porous Membrane
Characterization - Pore Characterization. Journal of Membrane Science, 87(1-2),
35–46.
Koros, W. J., Ma, Y. H., & Shimidzu, T. (1996). Terminology for membranes and
membrane processes. Pure and Applied Chemistry, 68(7), 1479–1489.
Kothari, S., Kim, J. A., Kothari, N., Jones, C., Choe, W. S., & Carbis, R. (2014).
Purification of O-specific polysaccharide from lipopolysaccharide produced by
Salmonella enterica serovar Paratyphi A. Vaccine, 32(21), 2457–2462.
Li, S., Xu, S. Q., & Zhang, L. N. (2010). Advances in conformations and characterizations
of Fungi polysaccharides. Acta Polymerica Sinica, 12, 1359–1375.
Liu, J. H. Y., Brameld, K. A., Brant, D. A., & Goddard, W. A. (2002). Conformational
analysis of aqueous pullulan oligomers: An effective computational approach.
Polymer, 43(2), 509–516.

11