International Journal of Biological Macromolecules 109 (2018) 810–818

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

journal homepage: www.elsevier.com/locate/ijbiomac

Physicochemical properties of a high molecular weight levan from

Brenneria sp. EniD312
Wei Xu a , Qian Liu a , Yuxiang Bai a , Shuhuai Yu a , Tao Zhang a , Bo Jiang a,b ,
Wanmeng Mu a,b,∗
a
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, 214122, People’s Republic of China
b
International Joint Laboratory on Food Safety, Jiangnan University, Wuxi 214122, People’s Republic of China

a r t i c l e i n f o a b s t r a c t

Article history: A high molecular weight levan was produced by a novel levansucrase and some properties of this polymer
Received 1 September 2017 were investigated. The levan exhibited a poroid microstructure as well as series of individual ellipsoidal
Received in revised form 24 October 2017 or spheroidal particles. The weight-average molecular weight (M̄w ) of the levan was determined to be
Accepted 9 November 2017
1.41 × 108 Da. In a 0.1% solution, the levan showed a mean diameter of 176 nm, while in a 1% solution
Available online 11 November 2017
the diameter was 182 nm. The decomposition temperature was determined to be 216.67 ◦ C, with an
endothermic peak at 147.41 ◦ C and a melting enthalpy of 76.9 J/g. The small angle X-ray diffraction pattern
Keywords:
showed a distinctive peak pattern between 15◦ and 40◦ (2q). The levan solution showed a shear-thinning
Levan
Microstructure
behaviour. These results suggest this levan could be a good additive in the food processing industry, as
Thermal behaviour well as an important bio-based material in the medicinal or chemical industry.
Particle size © 2017 Elsevier B.V. All rights reserved.
Shear-thinning

1. Introduction
Levan is a type of fructan that is composed of fructose with b-2,6
linkage. In addition to the levan-type fructan, two additional types
of fructan include inulin-type fructan, containing sole b-2,1 link-
ages, and graminan-type fructan, containing both b-2,6 and b-2,1
linkages [1,2]. In general, levan can be produced from microor-
ganisms, and a few plant species that possess levansucrase which
synthesize levan using sucrose as a sole substrate [3].
Unlike other polysaccharides, levan has a unique combination
of several important properties that facilitate its broad applica-
tion in many fields. High molecular weight levan is considered to
be a good fat-substitute to improve the flavour of dairy products
[4]. A patent using levan as an additive was obtained by a confec-
tionery company [5]. Recently, levan-type fructans have attracted
much attention of the bread making industry due to its significant
physiological effects. Because of its ability to form a hydrocolloid
microgel, levan can be used as an ingredient in wheat bread to
extend the shelf life of the product [6]. In addition to its application
in the food industry, levan is also widely used in the chemical and
medicinal industries due to its outstanding physicochemical prop-
∗ Corresponding author at: State Key Laboratory of Food Science and Technology,
Jiangnan University, Wuxi, Jiangsu, 214122, People’s Republic of China.
E-mail address: wmmu@jiangnan.edu.cn (W. Mu).
erties. For example, in the medicinal industry, levan can help heal
burned or mechanically damaged tissue through metalloproteinase
activation [7]. It was reported that levan had a significant antiox-
idant effect and protected against oxidative stress that is linked
to atherosclerosis [8]. Other studies have also shown the ability of
levan to have anti-obesity [9], anti-inflammatory, weight-loss [10],
lower cholesterol and anti-tumour effects [11,12]. In the chemical
industry, levan exhibits excellent tensile properties, with a tensile
strength on bare aluminum of 500–1500 psi, and can be used in
food packaging as a levan-based film [13]. In addition, levan can
also be potentially used as cosmeceutical agent due to its anti-
inflammatory and cell-proliferative effects [14].
The objective of the work is to characterize a high molecular
weight levan produced by levansucrase from a new Gram-negative
species Brenneria sp. EniD312. It is well known the factual appli-
cation of levan is largely influenced by the molecular weight and
its unique physicochemical properties [15]. For example, the pure
levan films were reported to possess worse mechanical proper-
ties than the levan-based composite material because of the high
degree of branch of original levan [16]. Apart from branching condi-
tion, the X-ray diffraction pattern and degree of crystallinity as well
as the thermal transition properties are also essential to the applica-
tion of bioactives. The study focusing on the low-digestible starch
conducted by Kim et al. found that the modified starch not only
change its crystallinity from A-form to B-form, but also increase

https://doi.org/10.1016/j.ijbiomac.2017.11.056
0141-8130/© 2017 Elsevier B.V. All rights reserved.
 W. Xu et al. / International Journal of Biological Macromolecules 109 (2018) 810–818 811

Fig. 1. (A) Scanning electron micrograph of the levan produced by the levansucrase from Brenneria sp. EniD312 (Left: 160×, Right: 300×). (B) Atomic force microcopy images
of the levan produced by the levansucrase from Brenneria sp. EniD312 (Left: planar graph, Right: vertical graph).

the content of slowly digestible starch (SDS) and resistant starch
(RS), expanding its application to a deeper extent [17].
In this study, a high molecular weight levan was obtained using a
levansucrase from a new bacterial strain Brenneria sp. EniD312. The
polysaccharide product was structurally determined to be levan
with a weight-average molecular weight (M̄w ) of 1.41 × 108 Da and
number-average molecular weight (M̄n ) of 1.27 × 108 Da, as deter-
mined by multi-angle laser light scattering, and represented the
largest levan ever reported. In addition, some basic investigations
about the structural, thermal behaviour, and solution rheology
properties of this polymer were also carried out.
2. Materials and methods
tryptone) (W/V) containing 100 mg/mL ampicillin was used as
the culture medium. As levansucrase is an intracellular protein,
the cells were harvested by centrifugation and disruption by a
Vibra-Cell72405 Sonicator (Bioblock, Illkirch, France), then after
the cell fragments were removed, the levansucrase purification
steps reported in our previous study were followed to obtain the
purified Brsp-LS [18]. Sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE) was used to check the protein purity,
and the enzyme activity was validated using sucrose as a sole
substrate at pH 6.5 and 35 ◦ C. The production of levan was detected
by a HPLC (Waters Corporation, MA, USA) that was equipped with a
Sugar-Pak I column (6.5 mm × 300 mm, Waters, USA) and a Waters
2414 RI detector.

2.1. Bioproduction of levan by Brenneria sp. EniD312 2.2. Microscopic analysis

levansucrase
An expression vector containing the full length levansucrase-
encoding gene from Brenneria sp. EniD312 (GeneBank:
BrE312 3941, 1311 bp) with a 3¢-terminal 6 × histidine-tag
was commercially synthesized by Shanghai Generay Biotech
Co., Ltd. (Shanghai, China). The plasmid pET-22b(+) was used
as an expression vector, with NdeI and XhoI restriction sites at
the 5¢- and 3¢-termini of the fusion gene used to construct the
recombinant plasmid (pET-Brsp-LS). E. coli BL21(DE3) was used
as expression vector. LB broth (1% NaCl, 0.05% yeast extract, 1%
The surface morphology of the levan was viewed by scanning
electron microscopy (SEM). Samples were dried and fractured,
mounted on aluminium stubs, coated with a thin gold film
(∼10 nm) with a sputtering apparatus (Bal-Tec SCD 050, Balzers
Technique) and were examined with a field effect SEM (Rexdale,
Ont., Canada) at an acceleration voltage of 5 kV and a magnification
ratio of 80–300.
To investigate the fine morphological characteristics of the
levan, atomic force microscopy (AFM) was also used. An aque-
ous solution of the levan (1 mg/mL) was prepared, by stirring the
812 W. Xu et al. / International Journal of Biological Macromolecules 109 (2018) 810–818
Fig. 2. TGA thermogram of the levan produced by the levansucrase from Brenneria sp. EniD312.
solution for 1 h at 75 ◦ C in a hermetic bottle under a stream of
nitrogen. After cooling down to room temperature, successive dilu-
tions were made to a final levan concentration of 5 mg/mL. Using
a micropipette, approximately 2 mL of the sample solution was
placed on a freshly cleaved mica slide (SPIChem TM Mica, West
Chester, PA), then the samples were dried in a desiccator before
scanning. The samples were photographed at room temperature in
air. The atomic force microcope (Bruker Dimension® IconÔ, Bruker
Corporation, Germany), was operated in tapping-mode applied.
The images were collected at a resolution of 256 × 256 pixels,
and for each sample, various scan areas were chosen to probe
(10 mm × 10 mm, 5 mm × 5 mm, 2 mm × 2 mm).
2.3. Molecular weight determination
The molecular weight of the levan was determined using a
high-performance size-exclusion chromatography system (Agi-
lent Technologies, Santa Clara, CA) supplemented with a Dawn
HeleosII multi-angle laser-light scattering detector, an Optilab T-
rEX refractive-index detector and a Shodex OHpak SB-806 HQ
column (8 × 300 mm). The solvent delivery system consisted of a
vacuum degasser, an auto sampler and a pump. The polysaccharide
sample was dissolved in a 0.1 mmol/L NaNO3 solution and filtered
through a 0.45 mm millipore filter (Merck, Newark, NJ) before injec-
tion. The mobile phase was a 0.1 mmol/L NaNO3 solution containing
0.02% NaN3 and the column was eluted at a flow rate of 0.5 mL/min
at 45 ◦ C.
2.4. Thermal analysis
To investigate the thermal behaviour of the levan, differential
scanning calorimetry (DSC) (Rigaku Co. Ltd., Tokyo, Japan) was car-
ried out. Samples (∼4 mg) were sealed in an aluminium pan. The
energy profile of the sample was recorded from 60 to 460 ◦ C at a
heating rate of 10 ◦ C/min under a nitrogen atmosphere. A thermo-
gravimeter analyser (TA Instruments model SDT600) was used for
the thermal gravimetric analysis (TGA) of the levan. The thermo-
gram was obtained in a temperature range from 50 to 600 ◦ C with
a ramp rate of 10 ◦ C/min under a nitrogen atmosphere. The DGT
trace was obtained by taking the first derivative of the TGA line.
2.5. X-ray diffraction analysis
X-ray diffraction patterns of the levan were determined by a
Rigaku MiniFlex diffractometer (Rigaku Inc., Tokyo, Japan). The
operating conditions of the diffractometer were: Cu Ka radiation,
40 kV, 200 mA, with the scattering angles (2q) of 5–50◦ . The scatter-
ing intensity data were recorded at a rate of 0.5◦ /min, with a step
resolution of 0.02◦ .
2.6. Solution properties
The levan sample was dissolved in deionized water at a concen-
tration of 3, 6, 9, and 12% (W/V) at room temperature. The solution
was stirred and dissolved for 0.5 h and then was transferred to a
glass tube. The appearance aqueous solutions of the levan were
photographedd by a digital camera (Sony, Shanghai, China).
2.7. Particle size distribution
The particle size of the levan in aqueous solution was
determined with a commercial dynamic light scattering and micro-
electrophoresis device (Nano-ZS, Malvern Instruments, UK) using
fresh samples of levan in aqueous solution that were diluted to 0.1%
and 1% (W/V) with deionized water at room temperature before
measurement. The particle size data are reported as the intensity-
weighted (“Z-average”) mean particle diameter.
2.8. Rheological behaviour
The apparent viscosities of the levan (at the concentration of
3, 6, 9, and 12% W/V) were determined. The shear rate was varied
from 0.01 to 100 1/s and the resulting flow curves were analysed
using Origin 8.0 Pro software package. The dynamic shear rheo-
logical properties were also investigated using a stress-controlled
rheometer (AR-G2, TA Instrument, DE, USA) at room tempera-
ture. Oscillatory measurements were implemented for the different
 W. Xu et al. / International Journal of Biological Macromolecules 109 (2018) 810–818
Fig. 3. Small-angle X-ray diffraction patterns of pure levan produced by the levansucrase from Brenneria sp. EniD312. Patterns for the angular range of 0–40 ◦ C.
aqueous solutions of levan, with the frequency ranging from 0.1 to
10 Hz. The storage (G’) and loss (G”) moduli were then determined.
2.9. Storage stability
To test the storage stability of levan solution, the 10% (W/V)
aqueous solution of levan was stored at room temperature in a
10 mL inverted centrifuge tube for 4 weeks, and the absorbance
of aqueous levan solutions was detected by spectrophotometer at
l=515 nm. All the data were the means of triplicate independent
experiments.
3. Results and discussions
3.1. Production of levan the Brenneria sp. EniD312 levansucrase
In this study, a gene (GeneBank: BrE312 3941) encoding a
putative levansucrase (protein ID: EHD23269.1) was cloned from
Brenneria sp. EniD312. Up to now, the levan-producing levan-
sucrases have been characterized from various microorganisms,
including Acetobacter xylinum NCI 1005 [19], Bacillus amyloliquefa-
ciens [20], Bacillus licheniformis 8-37-0-1 [21], Bacillus megaterium
DSM319 [22], Lactobacillus reuteri 121 [23], Leuconostoc mesen-
teroides B-512 FMC [24], Lactobacillus sanfranciscensis TMW 1.392
[25], Pseudomonas aurantiaca S-4380 [26], Pseudomonas syringae
pv. tomato [27], and Zymomonas mobilis [28].
Different from microbial fermentation to produce polysaccha-
rides [29], levan biosynthesis in this study was carried out in a
solution containing 480 g/L of sucrose at pH 6.0 and 35 ◦ C using
6 U/g (sucrose hydrolytic activity) of the recombinant Brenneria
sp. EniD312 levansucrase. The highest levan yield (175 g/L) was
obtained after 6 h reaction, after which the levan was harvested
by alcohol precipitation, and no protein was detected in the pre-
ciptated levan by the Lowry method using bovine serum albumin
as a standard [30].
Upon mild acid drolysis of the levan, only fructose was detected
by HPLC analysis, revealing that the polysaccharides generated by
813
the Brenneria sp. EniD312 levansucrase was a homopolymer of fruc-
tose. In order to determining the glycosidic linkages comprising
the levan, methylation analysis to distinguish bewteen 6¢-Fru and
1¢-Fru was introduced [31]. The mole fraction of the branching
fructose monomers was not detected in this high molecular weight
levan, suggesting that it was composed of b-(2,6)-d-fructofuranosyl
repeating units, similar to the linear levan produced by Halomonas
sp. AAD6 [32]. Compared to the levan produced by other species,
the branching degree of levan from S. salivarius was 10%, B. polymixa
12%, and A. levanicum 9% [33].
3.2. Microscope observations
According to Qin et al. [34], the physical properties of an
unknown polymer could be efficiently predicted through eluci-
dating its surface morphology by scanning electron microscopy.
As shown in Fig. 1(A), the granules of this high molecular weight
levan showed a tendency to be closely aggregated, had a cemen-
titious material appearance and exhibited a porous networks. The
levan fraction obtained from S. salivarius showed two different mor-
phologies depending on the molecular weight, the M̄w > l05 fraction
behaved as compact spheres, whereas praticles in the M̄w < l05
fraction were primarily characterized as linear random coils [35].
According to E. Newbrun et al. [36] reported that the morphologies
of levan and glucan simultaneously showed a tendency to aggre-
gate, and the difference was that the glucans (dextran) frequently
formed clumped clusters of numerous molecules, while individual
molecules in the levan appeared as ellipsoidal particles. A similar
poroid structure was observed by Majumder and Goyal [37] for
the glucan from L. dextranicum NRRL B-1146. This kind of meshed
structure with a small pore size distribution of polysaccharide from
would be applied as a water-binding agent in the food industry
[38,39].
The topographical atomic force microcope images of this pure,
high molecular weight levan using a 5 mg/mL aqueous solution
were shown in Fig. 1(B). A series of individual ellipsoidal or
spheroidal particles were observed in the 1.5 mm × 1.5 mm scan
814 W. Xu et al. / International Journal of Biological Macromolecules 109 (2018) 810–818

areas, with a peak-to-valley value exceeding 10 nm. The average Table 1

Chemical and thermal characteristics of levan produced by levansucrase from Bren-
vertical distance of these particles was 9.292 nm, and the mean
neria sp. EniD312.
roughness (Ra ) was 4.045 nm (values have 95% confidence inter-
vals). A similar result was reported for a levan from S. salivarius Levan Status
[36]. In addition, these large individual particles of levan aqueous TGA degradation Stage I (lost weight) 8.0%
solution demonstrated the excellent water solubility, unlike most Temperature 50–135 ◦ C
exopolysaccharides, such as cellulose, and curdlan, which would Stage II (lost weight) 50.8%
Tempearture 205–300 ◦ C
often swell in water [38].
Stage III (lost weight) 21.1%
Tempearture 300–500 ◦ C
Stage III (remained weight) 20.1%
3.3. Molecular weight distribution Temperature >500 ◦ C
DSC Melting enthalpy 76.9 J/g
Fig. S1 showed the HPSEC-MALLS-RI chromatogram of the levan. Peak temperature 147.41 ◦ C
A single distribution peak was observed with a weight-average Yield 175 g/L
Total protein ND
molecular weight (M̄w ) corresponding to 1.41 × 108 Da, indicating
Total carbohydrate 93.57%
the presence of a homopolymer of fructose. The radius of gyration Water 6.44%
(Rg ) and polydispersity (M̄w M̄n ) obtained for the polysaccharide
ND: Not Detected.
was 30.0 nm and 1.28, respectively. Based on the studies of E. New-
brun et al. [36], the M̄w of levan produced by Streptococcus salivarius
depended on the pH of the growth media. A molecular weight
of levan as low as l06 Da was obtained at pH 5.7. Compared to
the initial weight was lost. According to Stivila et al. [42], the main
the reported M̄w (106 to 109 Da) of engineering polysaccharides,
chain b(2 → 6) linkages and the breaking of pyranose rings might
including xanthan, gellan, cellulose, dextran, curdlan, hyaluronan,
happen during this phase, occasionally with other char-forming
succinoglycan, galactopol, frucopol and levan [3], the 1.41 × 108 Da
reactions. In the end, only 20.1% of the solid residue remained above
molecular weight M̄w of the levan in this study was much larger,
550 ◦ C. Previously, little attention has been paid to the thermal
except for the levan produced from B. goodwinii identified by Liu
behaviour of a fructosan such as levan. Compared to the sequential
et al. [40], which has a M̄w of 1.3 × 108 Da.
decomposition of a-d-glucan produced from Leuconostoc citreum
SK24.002 [28], three phases were also observed, and a degradation
3.4. Thermal behaviour temperature 292.6 ◦ C was recorded. However, in case of dextran,
only two main degradation phases were observed. Drastic weight
To assess the sequential decomposition, the decomposition loss of the levan occurred when temperature was above 300 ◦ C and
temperature and the thermal kinetics of the levan from Brenneria. loss of weight was complete above 500 ◦ C [43]. The difference in
sp. EniD312, the thermal gravimetric analysis (TGA) and differen- thermal behaviour may be attributed to the different composition
tial scanning calorimetric analysis (DSC) were performed and the and molecular structure.
result was showed in Fig. 2. In summary, the degradation stages of A differential scanning calorimetric analysis showed one
this levan were similar to the previous reports for polysaccharides endothermic peak at 147.41 ◦ C associated with a melting enthalpy
[41]. At the first stage of degradation, approximately 8.0% of the ini- value of 76.9 J/g (Table 1). The highest melting temperature
tial weight loss occurred between 50 and 135 ◦ C, owing to the loss (178.4 ◦ C), and melting enthalpy value 1.66 cal/g of a levan was
of moisture content. The levan broke down above 200 ◦ C, and the reported in 1999 by Jung et al. [44]. According to Miao et al. [45], the
weight dramatically decreased (approximately 50.8%) during the water-soluble extracellular homopolysaccharide from Lactobacillus
second degradation stage from 205 to 300 ◦ C, with a decomposition reuteri SK24.003 showed a similar endothermic peak at 147.7 ◦ C.
temperature of 216.67 ◦ C. This stage was probably caused by the However, compared to the exopolysaccharide composed of man-
gradual breaking of levan b(2 → 1) branch linkages, as well as the nose, glucose and galactose produced by Lactobacillus plantarum
breaking of bonds in the ring units. The third stage of degradation KF5, a much lower melting temperature of 86.35 ◦ C and a much
happened between 300 and 500 ◦ C, where approximately 21.1% of larger melting enthalpy of 133.5 J/g were observed. It was reason-

Fig. 4. (A) Appearance of several aqueous solution of concentrations of levan at concentration of 3, 6, 9, and 12%. (B) The appearance of aqueous solution of levan (10%) stored
at room temperature for 4 weeks.
 W. Xu et al. / International Journal of Biological Macromolecules 109 (2018) 810–818
Fig. 5. Particle size distributions of the levan in aqueous solution (0.1% and 1%).
able to believe the molecular configuration of polymers resulted in
the differences in thermal behaviour [57].
3.5. X-ray diffraction
Cellulose, which is composed of b(1 → 4) glucose, is an extra-
cellular polysaccharide that has a high crystallinity property [38],
and starch, another glucan composed of glucose a(1 → 4) linkages,
is also capable of generating A-, B-, and C-type X-ray diffraction
spectra [46,56]. However, little information is available regarding
the crystallinity of levan. The X-RD pattern of the purified levan in
this study showed a distinctive peak pattern between 15◦ and 40◦
(2q), mainly at 15.3◦ (2q), 17.1◦ (2q), and 20.0◦ (2q) (Fig. 3), but no
peaks in the 3–15◦ range, similar as the result reported by Chen et al.
[13]. To get an understanding of levan crystallinity, another fructan
(inulin) was introduced to make a contrast. It was noticeable that
the diffraction spectrum of inulin also showed strong peaks approx-
imately at 15◦ and 40◦ (2q), but some new peaks approximately at
7◦ and 35◦ (2q) arose in the diffraction spectrum. That is, inulin dis-
played a similar crystal pattern with levan, and also showed more
crystallinity than levan. The polythopic crystallinity of inulin has
been confirmed by Cooper in a study showed that inulin isoform
were noticeably distinguishable obviously by repeated additions
of one crystal unit, but that all isoforms showed indistinguishable
X-ray diffraction patterns [47,48]. Correlating the detailed crystal-
lization mechanism with observed crystal patterns is beyond the
scope of this study, but the presence of the inulin and levan pattern
spectrum as well as some discussions might help us to learn more
815
about the complexities of the levan structure, although there was
little information regarding this.
3.6. Solution properties analysis
Different concentrations of polysaccharides could affect the
depth of solution colour to some extent [49–51]. Therefore, a com-
parison of the appearance of aqueous solution of levan at different
concentrations was performed in this study. The result showed that
the levan solution at a 3.0% (W/V) concentration has an opales-
cent colour, and the colour became deeper as the concentration
increased from 3.0% to 12.0% (W/V), with the 12.0% aqueous solu-
tion of the levan showing a completely milky-white color (Fig. 4A).
According to Benigar et al. [55], different microbial levan solution
might show different opalescence, even at the same concentra-
tion. The levan from E. herbicola exhibited the most turbid solution,
followed by the Z. mobilis levan solution, and the most transpar-
ent was the solution of a B. subtilis levan. Before this study, little
attention has been focused on the levan aqueous solution from
Gram-negative bacterium Brenneria sp. EniD312, and little informa-
tion is available about the relationship bewteen the concentration
of levan aqueous solution and its related color. In this study, the
size and refractive index of the scattering particles were the main
causes of different sample turbidities. Besides, a reversed phe-
nomenon that at a higher concentration, a more opalescent glucan
solution was observed in the case of a-d-glucan was reported by
Miao et al. [46], but further discussion about the phenomenon was
missing.
816 W. Xu et al. / International Journal of Biological Macromolecules 109 (2018) 810–818
Fig. 6. Rheological properties of the levan produced by the levansucrase from Bren-
neria sp. EniD312. (shear rate − apparent viscosity curves).
3.7. Particle size distribution
The size distribution of the levan molecule was determined
using a commercial dynamic light scattering (DLS) instrument, and
the results were shown in Fig. 5. Two aqueous solutions of levan
(0.1% and 1%) (W/V) were each filtered by 0.45 mm Millipore fil-
ter. As can be seen, the 0.1% levan solution showed a mean particle
diameter of 176 nm and the 1% solution showed a mean particle
diameter as 182 nm. Two levan solutions showed similar normal
distributions of particle size. However, some subtle differences at
the beginning and the end of the distributions were observed. At
the high concentration (1%), the mean particle diameter increased
by 6 nm, which could be attributed to the excellent water solu-
ble of levan, such that more water molecules were absorbed by
intermolecular affinity and formed large aggregates. By compari-
son, cellulose acetate filter could retain some large levan molecules,
resulting in a different size distribution in the case of a Bacillus sp.
5% levan solution [13].
3.8. Rheological behaviour
Levan has been characterized as one of a polysaccharde with
an extremely low intrinsic viscosity (approximately 0.14 dl/g) and
film-forming ability [40]. The appearance of individual spherical
microscopic levan particles observed in this study (Fig. 1B) con-
firmed the low intrinsic viscosity of levan.
According to Arvidson and Han [52,53], some levans of bacte-
rial origin might show a rather surprising nongelling behaviour in
aqueous solutions. In this study, our efforts were focused on the
apparent viscosity of levan and its change during the shearing pro-
cess. The apparent viscosity vs shear rate of 3, 6, 9 and 12% (W/V)
aqeuous solutions of levan were determined and the results were
presented in Fig. 6.
The 6, 9 and 12% levan solution showed a shear thinning
behaviour (non-Newtonian pseudoplastic fluid) at shear rates from
0.01 to 100 1/s. Meanwhile, the viscosity decreased with an increas-
ing shear rate, and the change was finally weakened at higher shear
rates. However, the 3% levan solution showed an essentially New-
tonian behaviour at the shear rates between 1 and 100 1/s. A similar
result was reported in the case of Z. mobilis and E. herbicola levan in
aqueous solution that it could display Newtonian behaviour at a low
concentration and non-Newtonian at a high concentration, while a
B. subtilis levan showed a totally Newtonian behaviour at concen-
trations ranging from 1% to 8%. The shear thinning phenomenon
often occurred when macromolecules in solution orient themselves
in the same shear direction, facilitating the disengagement of the
polymer [33]. The differences in levan branching, polymer molec-
ular weight or interparticle interactions could result in different
rheological behaviour. Additionally, the shear thinning behaviour
of levan is favourable in the food industry that produce dairy prod-
ucts, edible syrups, and sauce salad [54].
The storage G’ and loss G” moduli of levan in this study were
determined through dynamic oscillatory tests, with the frequency
ranging from 0.1 to 10 Hz (Fig. 7). When the levan concentration
was 3%, the storage G’ and loss G” moduli showed a transition of
dominancy at a low frequency, but the storage G’ moduli remained
higher than the loss moduli (G”) over the whole frequency range,
with frequency dependence occurring at higher concentrations of
levan, and the frequency dependence trend weakening at concen-
trations between 9% and 12%. In other words, a weak gel-structure
could be formed at a high concentration. However, in the case
of levan from Bacillus subtilis, the storage G’ and loss G” moduli
were equal at an 8% concentration of levan, and the loss G” moduli
became dominant at higher levan concentrations. A similar result
of loss G” moduli remaining higher than storage G’ at high concen-
tration was reported for the Z. mobilis and E. herbicola levan [55].
Furthermore, it was previously observed that the storage modulus
G’ was higher than the loss modulus G” in all levan samples at a low
concentration [36]. The microbial strain or the method of obtaining
the levan most likely led to these different oscillatory test results.
Compared to other polysaccharides, gellan, alginate and curdlan
are known to have a high gelling capacity [3]. For example, the
exopolysaccharide from R. radiobacter S10 was reported to form
an apparent weak gel at a low concentration at room temperature,
and subsequent heating and cooling proceudures did not influence
the gelling ability of the polysaccharides. In summary, the levan
obtained in this study displayed a similar rheological behaviour to
other reported elastic gells such as a-glucan [56], and might be used
as an important bio-based film, however, due to our limited knowl-
edge of the 3D structure of the levan, a detailed interpretation of
its behaviour in such an application was insufficient.
3.9. Storage stability
It is well known that storage stability is an important criteria to
assess the properties of food additives and might cause a strongly
affect on the product shelf life [58], so storage tests were also done
in this study. Fig. 4B was taken from a 10 mL inverted centrifuge
tube, in which was the 10% (W/V) aqueous solution of levan after
stored at room temperature for nearly 4 weeks. As we can see from
the result, although the color is slightly lighter than that of the fresh
levan solution, there was still no stratified phenomenon, and what
was notably, the solution won not drop down, which strongly indi-
cated the well adhesion and homogeneity of levan solution during
storage.
In addition, the particle size distributions of levan solution
during the storage were also determined, and the results were pre-
sented in Fig. S2. It was the fact that the mean particle size of both
0.1% and 1% (W/V) aqueous solution of levan showed a tendency to
increase during the storage. However, the change was very slight,
because the particle size of 0.1% levan solution just changed from
176 to 228 nm during the 4 weeks storgae, and there was only a
61 nm rise in the particle size of 1% levan solution, which indicates
the excellent physical stability of levan in this study.
According to Chen et al. [13], the UV absorbance profile of levan
aqueous solution exhibited a strong absorption peak at 515 nm,
which means the amount of levan could be quantified by the
absroption intensity. 6% (W/V) aqueous solution of levan was pre-
treated by different buffers (pH 3.5, 5.5, 7.5 and 9.5) and stored at
room tempearture for different days. Fig. S3 showed the results, in
 W. Xu et al. / International Journal of Biological Macromolecules 109 (2018) 810–818
Fig. 7. Double logarithmic plot of storage G’ and loss G” moduli vs shear rate for aqueous levan solutions (3, 6, 9 and 12%) measured at room temperature and at an angular
frequency of 10/s.
which there was lilltle change in the absorbance OD515 , indicating
an excellent pH stability of levan during the storage. Also, the same
titer of levan solution was incubated at different temperatures (20,
40, 60 and 80 ◦ C) for spell, and the results were shown in Fig. S4.
Similarily, the OD515 exhibited very little fluctuation with a good
stability, implying there was no degradation in the levan solution,
which was much better than commerical starch.
4. Conclusions
In this study, a high molecular weight levan was produced by
a Brenneria sp. EniD312 levansucrase. A poroid structure and a
series of individual ellipsoidal or spheroidal particles of the levan
were observed by scanning electron microscopy and atomic force
microscopy. The weight-average molecular weight (M̄w ) was deter-
mined to be 1.41 × 108 Da, much larger than other levans. A 0.1%
levan solution showed a mean particle diameter of 176 nm and
the 1% solution showed the mean particle diameter of 182 nm.
The decomposition temperature of the levan was determined to be
216.67 ◦ C, and the endothermic peak appeared at 147.41 ◦ C, with
a melting enthalpy of 76.9 J/g. The small angle X-ray diffraction
pattern of the purified levan showed a distinctive peak pattern
between 15◦ and 40◦ (2q), primarily at 15.3◦ (2q), 17.1◦ (2q), and
817
20.0◦ (2q). A 3% levan solution showed an essentially Newtonian
behaviour at the shear rates between 1 and 100 1/s, whereas the 6,
9 and 12% levan solution showed a non-Newtonian pseudoplastic
behaviour (shear thinning) at shear rates from 0.01 to 100 1/s, and
the viscosity decreased with increasing shear rate. Taken toghther,
the characteristics of the levan identified in this study suggested
that it could be a good additive in the food processing industry,
as well as an important bio-based material in the medicinal and
chemical industries.
Acknowledgements
This work was supported by the NSFC Project (No. 21276001),
the 863 Project (No. 2013AA102102), the Fundamental Research
Funds for the Central Universities (No. JUSRP51304A), and the Sup-
port Project of Jiangsu Province (No. BK20130001).
Appendix A. Supplementary data
Supplementary data associated with this article can be found,
in the online version, at https://doi.org/10.1016/j.ijbiomac.2017.11.
056.
818 W. Xu et al. / International Journal of Biological Macromolecules 109 (2018) 810–818
References
[1] W. Van den Ende, Multifunctional fructans and raffinose family
oligosaccharides, Front. Plant Sci. 4 (4) (2013) 247–258.
[2] S.A. Kang, K. Hong, K.H. Jang, S. Kim, K.H. Lee, B.I. Chang, C.H. Kim, R. Choue,
Anti-obesity and hypolipidemic effects of dietary levan in high fat
diet-induced obese rats, J. Microbiol. Biotechnol. 14 (1) (2004) 796–804.
[3] F. Freitas, V.D. Alves, M.A. Reis, Advances in bacterial exopolysaccharides:
from production to biotechnological applications, Trends Biotechnol. 29 (8)
(2011) 388–398.
[4] F. Anne, B. Karl, D.S. Johan, Fructan – and/or polydextrose – containing dairy
powders, Process for preparing same and their use. EPEP0821885. (2006).
[5] L. Marcelo, Bravo, Cordero. (1991). Confectionery composition. EP, EP0410506
A2. (1991).
[6] F. Jakob, A. Pfaff, R. Novoa-Carballal, H. Rübsam, T. Becker, R.F. Vogel,
Structural analysis of fructans produced by acetic acid bacteria reveals a
relation to hydrocolloid function, Carbohydr. Polym. 92 (2) (2013) 1234–1242.
[7] C.P. Strube, A. Homann, M. Gamer, D. Jahn, J. Seibel, D.W. Heinz,
Polysaccharide synthesis of the levansucrase SacB from Bacillus megaterium is
controlled by distinct surface motifs, J. Biol. Chem. 286 (20) (2011)
17593–17600.
[8] I. Dahech, B. Harrabi, K. Hamden, A. Feki, H. Mejdoub, H. Belghith, K.S.
Belghith, Antioxidant effect of nondigestible levan and its impact on
cardiovascular disease and atherosclerosis, Int. J. Biol. Macromol. 58 (2013)
281–286.
[9] J. Oh, S.R. Lee, K.T. Hwang, G.E. Ji, The anti-obesity effects of the dietary
combination of fermented red ginseng with levan in high fat diet mouse
model, Phytother. Res. 28 (4) (2014) 617–622.
[10] R. Srikanth, G. Siddartha, C.H. Sundhar Reddy, B.S. Harish, R.M. Janaki, K.B.
Uppuluri, Antioxidant and anti-inflammatory levan produced from
Acetobacter xylinum NCIM2526 and its statistical optimization, Carbohydr.
Polym. 123 (2015) 8–16.
[11] H. Brend, T.M. Hans-Ulrich, H. Stephan, Cholesterol-reducing agent made of
dietary fibre and cholesterol-reducing substances. EPEP1526857 A1. (2005).
[12] S.-H. Yoo, E.J. Yoon, J. Cha, H.G. Lee, Antitumor activity of levan
polysaccharides from selected microorganisms, Int. J. Biol. Macromol. 34 (1)
(2004) 37–41.
[13] X. Chen, H. Gao, H.J. Ploehn, Montmorillonite–levan nanocomposites with
improved thermal and mechanical properties, Carbohydr. Polym. 101 (1)
(2014) 565–573.
[14] K.H. Kim, B.C. Chung, Y.H. Kim, K.S. Kim, C.S. Han, C.H. Kim, Cosmeceutical
properties of levan produced by Zymomonas mobilis, J. Cosmet. Sci. 56 (3)
(2005) 395–406.
[15] R. Dedonder, C. Peaud-Lenoel, Studies on the levansucrase of Bacillus subtilis. I.
Production of levans and levansucrase (levan-succharotransfructosidase) by
cultures of Bacillus subtilis, Bull. Soc. Chim. Biol. (Paris) 39 (5–6) (1957)
483–497.
[16] K. Majeed, M. Jawaid, A. Hassan, A.A. Bakar, H.P.S.A. Khalil, A.A. Salema,
Potential materials for food packaging from nanoclay/natural fibres filled
hybrid composites, Mater. Des. 46 (2013) 391–410.
[17] H.K. Ji, R.K. Ha, J.C. Seung, P. Cheon-Seok, W.M. Tae, Production of an in vitro
low-digestible starch via hydrothermal treatment of amylosucrase-Modified
normal and waxy rice starches and its structural properties, J. Agric. Food
Chem. 119 (2016) 5045–5052.
[18] R. Li, T. Zhang, B. Jiang, W. Mu, M. Miao, Purification and characterization of
an intracellular levansucrase derived from Bacillus methylotrophicus SK
21.002, Biotechnol. Appl. Biochem. 62 (6) (2014) 815–822.
[19] K. Tajima, T. Tanio, Y. Kobayashi, H. Kohno, M. Fujiwara, T. Shiba, T. Erata, M.
Munekata, M. Takai, Cloning and sequencing of the levansucrase gene from
Acetobacter xylinum NCI 1005, DNA Res. 7 (4) (2000) 237–242.
[20] D. Rairakhwada, J.W. Seo, M.Y. Seo, O. Kwon, S.K. Rhee, C.H. Kim, Gene
cloning, characterization, and heterologous expression of levansucrase from
Bacillus amyloliquefaciens, J. Int. Microbiol. Biotechnol. 37 (2) (2010) 195–204.
[21] L. Lu, F. Fu, R. Zhao, L. Jin, C. He, L. Xu, M. Xiao, A recombinant levansucrase
from Bacillus licheniformis 8–37-0-1 catalyzes versatile transfructosylation
reactions, Process Biochem. 49 (9) (2014) 1503–1510.
[22] A. Homann, R. Biedendieck, S. Gotze, D. Jahn, J. Seibel, Insights into polymer
versus oligosaccharide synthesis: mutagenesis and mechanistic studies of a
novel levansucrase from Bacillus megaterium, Biochem. J. 407 (2) (2007)
189–198.
[23] S.A. Van Hijum, E. Szalowska, M.J. Van der Maarel, L. Dijkhuizen, Biochemical
and molecular characterization of a levansucrase from Lactobacillus reuteri,
Microbiology 150 (3) (2004) 621–630.
[24] H.K. Kang, M.Y. Seo, E.S. Seo, D. Kim, S.Y. Chung, A. Kimura, D.F. Day, J.F. Robyt,
Cloning and expression of levansucrase from Leuconostoc mesenteroides B-512
FMC in Escherichia coli, Biochim. Biophys. Acta 1727 (1) (2005) 5–15.
[25] M. Tieking, M.A. Ehrmann, R.F. Vogel, M.G. Ganzle, Molecular and functional
characterization of a levansucrase from the sourdough isolate Lactobacillus
sanfranciscensis TMW 1.392, Appl. Microbiol. Biotechnol. 66 (6) (2005)
655–663.
[26] E.K. Jang, K.H. Jang, I. Koh, I.H. Kim, S.H. Kim, S.A. Kang, C.H. Kim, S.D. Ha, S.K.
Rhee, Molecular characterization of the levansucrase gene from Pseudomonas
aurantiacia S-4380 and its expression in Escherichia coli, J. Microbiol.
Biotechnol. 12 (2002) 603–609.
[27] T. Visnapuu, K. Mardo, C. Mosoarca, A.D. Zamfir, A. Vigants, T. Alamäe,
Levansucrases from Pseudomonas syringae pv. tomato and P. chlororaphis
subsp. aurantiaca: substrate specificity, polymerizing properties and usage of
different acceptors for fructosylation, J. Biotechnol. 155 (3) (2011) 338–349.
[28] K. Kyono, H. Yanase, K. Tonomura, H. Kawasaki, T. Sakai, Cloning and
characterization of Zymomonas mobilis genes encoding extracellular
levansucrase and invertase, Biosci. Biotechnol. Biochem. 59 (2) (1995)
289–293.
[29] M. Miao, C. Huang, X. Jia, S.W. Cui, B. Jiang, T. Zhang, Physicochemical
characteristics of a high molecular weight bioengineered ˛-D-glucan from
Leuconostoc citreum SK24.002, Food Hydrocoll. 50 (4) (2015) 37–43.
[30] O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein measurement with
the Folin phenol reagent, J. Biol. Chem. 193 (1) (1951) 265–275.
[31] N.K. Kochetkov, O.S. Chizhov, Mass spectrometry of carbohydrates, Gen.
Carbohydr. Methods 150 (1972) 540–554.
[32] A. Poli, H. Kazak, B. Gürleyendağ, G. Tommonaro, G. Pieretti, E.T. Öner, B.
Nicolaus, High level synthesis of levan by a novel Halomonas species growing
on defined media, Carbohydr. Polym. 78 (4) (2009) 651–657.
[33] E. Benigar, I. Dogsa, D. Stopar, A. Jamnik, I.K. Cigic, M. Tomsic, Structure and
dynamics of a polysaccharide matrix: aqueous solutions of bacterial levan,
Langmuir 30 (14) (2014) 4172–4182.
[34] Q. Qin, B. Xia, Y. Xiong, S. Zhang, Y. Luo, Y. Hao, Structural characterization of
the exopolysaccharide produced by Streptococcus thermophilus 05-34 and its
in situ application in yogurt, J. Food Sci. 76 (9) (2011) 1226–1230.
[35] S.S. Stivala, J.E. Zwegi, Physicochemical parameters of partially hydrolyzed S
salivarius levan fractions, Bioploymers 20 (3) (1981) 605–619.
[36] E. Newbrun, R. Lacy, T.M. Christie, The Morphology and size of the
extracellular polysacchairdes from oral streptococci, Arch. Oral Biol. 16 (8)
(1971) 863–872.
[37] A. Majumder, A. Goyal, Rheological and gelling properties of a novel glucan
from Leuconostoc dextranicum NRRL B-1146, Food Res. Int. 42 (4) (2009)
525–528.
[38] S. Badel, T. Bernardi, P. Michaud, New perspectives for Lactobacilli
exopolysaccharides, Biotechnol. Adv. 29 (1) (2011) 54–66.
[39] A. Xiu, M. Zhou, B. Zhu, S. Wang, J. Zhang, Rheological properties of Salecan as
a new source of thickening agent, Food Hydrocoll. 25 (7) (2011) 1719–1725.
[40] Q. Liu, S. Yu, T. Zhang, B. Jiang, W. Mu, Efficient biosynthesis of levan from
sucrose by a novel levansucrase from Brenneria goodwinii, Carbohydr. Polym.
157 (2017) 1732–1740.
[41] G.P. Pandey, T. Liu, C. Hancock, Y. Li, X.S. Sun, J. Li, Thermostable gel polymer
electrolyte based on succinonitrile and ionic liquid for high-performance
solid-state supercapacitors, J. Power Sources 328 (2016) 510–519.
[42] S.S. Stivala, J. Kimura, L. Reich, Thermal degradation of levan, Thermochim.
Acta 50 (1) (1981) 111–122.
[43] S.K. Bajpai, N. Chand, S. Tiwari, S. Soni, Swelling behavior of cross-linked
dextran hydrogels and preliminary Gliclazide release behavior, J. Int.
Microbiol. Biotechnol. 93 (2016) 978–987.
[44] S.J. Jung, B.S. Kim, B.Y. Kim, U.H. Chun, S.K. Rhee, Viscosity and thermal
characterization of levan, Food Eng. Progress. 3 (1991) 176–180.
[45] M. Miao, Y. Ma, C. Huang, B. Jiang, S.W. Cui, T. Zhang, Physicochemical
properties of a water soluble extracellular homopolysaccharide from
Lactobacillus reuteri SK24.003, Carbohydr. Polym. 131 (2015) 377–383.
[46] R.F. Tester, J. Karkalas, X. Qi, Starch—composition, fine structure and
architecture, J. Cereal Sci. 39 (2) (2004) 151–165.
[47] P.D. Cooper, T.G. Barclay, M. Ginic-Markovic, A.R. Gerson, N. Petrovsky, Inulin
isoforms differ by repeated additions of one crystal unit cell, Carbohydr.
Polym. 103 (4) (2014) 392–397.
[48] P.D. Cooper, K.H. Rajapaksha, T.G. Barclay, M. Ginic-Markovic, A.R. Gerson, N.
Petrovsky, Inulin crystal initiation via a glucose-fructose cross-link of
adjacent polymer chains: atomic force microscopy and static molecular
modelling, Carbohydr. Polym. 117 (2015) 964–972.
[49] I. Andr, J.L. Putauxa, H. Chanzy, F.R. Taravela, J.W. Timmermansb, D.D. Witb,
Single crystals of inulin, Int. J. Biol. Macromol. 18 (3) (1991) 195–204.
[50] G.L. Cote, Low-viscosity ˛-d-glucan fractions derived from sucrose which are
resistant to enzymatic digestion, Carbohydr. Polym. 19 (4) (1992) 249–252.
[51] S.A. Arvidson, B.T. Rinehart, F. Gadala-Maria, Concentration regimes of
solutions of levan polysaccharide
from Bacillus sp, Carbohydr. Polym. 65 (2) (2006) 144–149.
[52] Y.W. Hangita, M.A. Clarke, Production and characterization of microbial levan,
J. Agric. Food Chem. 38 (1990) 393–396.
[53] Z. Pang, H. Deeth, S. Prakash, N. Bansal, Development of rheological and
sensory properties of combinations of milk proteins and gelling
polysaccharides as potential gelatin replacements in the manufacture of
stirred acid milk gels and yogurt, J. Food Eng. 169 (5) (2016) 27–37.
[54] E. Benigar, I. Dogsa, D. Stopar, A. Jamnik, Structure and dynamics of a
polysaccharide matrix: aqueous solutions of bacterial levan, Langmuir 30 (14)
(2014) 4172–4182.
[55] C. Huang, M. Miao, Z. Jin, B. Jiang, Physicochemical properties of a novel
water-soluble(1 → 3)(1 → 6)-␣-D-glucan, J. Food Sci. Biotechnol. 11 (2015)
1129–1134.
[56] C. Meram, E. Yussef, T. Feral, W. Jian, Physicochemical and functional
properties of livetins fraction from hen egg yolk, Food Biosci. 18 (2017) 38–45.
[57] D. Zhang, L. Cheng, Z. Gu, Y. Hong, Z. Li, Preparation and properities of
microcrystalline cellulose from corn stalk, J. Food Sci. Biotechnol. 35 (2015)
1113–1119.