Food Hydrocolloids xxx (2013) 1e7

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Food Hydrocolloids
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Exopolysaccharides produced by Rhizobium radiobacter S10 in whey

and their rheological properties
Fangfang Zhou, Zhengjun Wu*, Chen Chen, Jin Han, Lianzhong Ai, Benheng Guo
State Key Laboratory of Dairy Biotechnology, Institute of Bright Dairy & Food Co., Ltd., 1518 Jiangchangxi Road, Shanghai 200436, China

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

Article history: Exopolysaccharides (EPSs) produced by a number of bacteria genera are very attractive to food industries
Received 3 December 2012 because of their stabilizing and emulsifying capabilities. Rhizobium radiobacter S10, an EPS-producing
Accepted 14 August 2013 strain, was isolated from kefir. The chemical and rheological properties of the produced EPS were
investigated. The EPS yield of the strain can reach 2834.2 mg L
1. The flow and viscoelastic behavior of
Keywords: S10 EPS were investigated by steady-shear and small-amplitude oscillatory experiments, respectively.
Exopolysaccharide
The EPS solution (0.1%e1.0%, w/v) exhibited non-Newtonian and shear thinning behavior (pseudoplas-
Rhizobium radiobacter
ticity) at shear rates that ranged from 0.01 s
1 to 1000 s
1. Weak gels were obtained with EPS con-
Purification
Rheological properties
centrations as high as 0.75% at room temperature (25  C). The changes in the elastic (G0 ) and viscous (G00 )
moduli during the heating and cooling cycles indicated that S10 EPS can form a thermal reversible gel.
The predominant fraction of S10 EPS, PK2, was composed of galactose and glucose at a molar ratio of
1.00:4.92 and had an approximate molecular weight of 3.03  106 Da.
Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction stabilizing, suspending, and thickening agent, succinoglycan is

Microbial exopolysaccharides (EPSs) are mainly secreted by bac-
teria and microalgae into their surroundings during growth. EPSs,
such as xanthan (Queiroz & Fontes, 2008), gellan (Banik, Kanari, &
Upadhyay, 2000; Elboutachfaiti, Delattre, Petit, & Michaud, 2010),
and curdlan (Funami & Nishinari, 2007; Salah et al., 2011), have been
extensively and intensively studied for their gelling, stabilizing, and
texturizing properties. These properties make the polymers suitable
for a wide range of applications in the food, pharmaceutical, and
other industries (Nampoothiri, Singhania, Sabarinath, & Pandey,
2003). However, the physical properties of these polymers are un-
suitable for all applications. Thus, the discovery and development of
new microbial EPSs with improved rheological characteristics have
been considerably explored.
Rhizobium radiobacter, formerly classified as Agrobacterium tume-
faciens/Agrobacterium radiobacter, is versatile in the biosynthesis of a
range of EPSs (Edmond, Riddler, Baxter, Wicklund, & Pasculle, 1993;
Garfinkel & Nester, 1980; Rho, Kang, & Lee, 2001). Many members of
this species are important EPS producers. Succinoglycan, first isolated
from Alcaligenes faecalis, can also be synthesized by different strains of
R. radiobacter in high yields on large scale. Succinoglycan is used in
agrochemical formulations, oil and gas field chemicals, and personal
and home care products. Given its useful applications as an emulsion-
industrially produced by Shell (Zevenhuizen, 1997). Curdlan, another
commercially important EPS produced by R. radiobacter strains, is a
linear b-(1 / 3)-glycan used as a gelling agent to improve the textural
quality, water-holding capacity, and thermal stability of various foods
(Sutherland, 1999). The EPSs produced by R. radiobacter attract great
interest because of their good viscosity-enhancing capability (Hebbar
et al., 1992; Salah et al., 2011).
Kefir grains, the source of fermented milk kefir, are a combination
of bacteria and yeasts in a matrix of proteins, lipids, and poly-
saccharides. Kefiran, the EPS produced by kefir microflora, is
increasingly used in the food industry as a gelling agent or film-
forming material (Ghasemlou, Khodaiyan, Oromiehie, & Yarmand,
2011; Piermaria, Pinotti, Garcia, & Abraham, 2009). Various strains
with EPS-producing ability have been isolated from kefir grains,
including lactic acid bacteria, yeast, and other bacteria. This study
isolated R. radiobacter strain S10 from kefir grains. This strain can
produce highly viscous EPS in whey. The rheological properties of S10
EPS and its chemical properties, such as its molecular weight (Mw)
distribution and monosaccharide composition, were also investigated.
2. Materials and methods
2.1. Isolation, identification, and storage of R. radiobacter S10

* Corresponding author. Tel.: þ86 21 66553513; fax: þ86 21 66553536. Kefir grains obtained from northwest China were washed with
E-mail address: wuzhengjun@brightdairy.com (Z. Wu). sterile distilled water and homogenized in sterilized physiological

0268-005X/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodhyd.2013.08.016

Please cite this article in press as: Zhou, F., et al., Exopolysaccharides produced by Rhizobium radiobacter S10 in whey and their rheological
properties, Food Hydrocolloids (2013), http://dx.doi.org/10.1016/j.foodhyd.2013.08.016
2 F. Zhou et al. / Food Hydrocolloids xxx (2013) 1e7
saline using a blender. The homogenized sample was serially diluted
in sterilized physiological saline, and 1-mL aliquots were plated on
skim milk agar. After incubation at 30  C under aerobic conditions for
120 h, ropy colonies were further streaked on skim milk agar plates
and incubated as described above. The fresh culture was inoculated
separately into 250-mL flasks with 50 mL 10% (w/w) sterile recon-
stituted whey from 90% desalted whey powder (Valio Co., Finland)
and incubated at 30  C for 96 h on a shaker at 200 rpm. The broth was
then bathed in boiling water for 5 min and cooled to room temper-
ature. After trichloroacetic acid (TCA) was added to the broth at a final
concentration of 5% (w/w), the broth was stored in a refrigerator
overnight and then centrifuged (Beckman Coulter J-30I, CA, USA) at
12,000 g and 4  C for 20 min to remove the pellets. The supernatant
was dialyzed in membrane tubes with Mw cut-off of 14 kDa against
distilled water at 4  C for 72 h, with several water changes. EPS
content in the dialyzed supernatant was determined by the phenol-
sulfuric acid method using glucose as standard (Dubois, Gilles,
Hamilton, Rebers, & Smith, 1956). All tests were conducted in tripli-
cate, and results were expressed in mg equivalent to glucose per liter
of the growth medium. Among the tested strains, S10 exhibited the
highest EPS yields.
S10 was first identified by morphological shape, spore forming,
Gram staining, catalase, and oxidase tests and then by carbon
resource utilization tests. The identification result was confirmed
by partial sequencing the 16S rRNA gene of S10.
S10 was routinely propagated on skim milk agar and stored in
sterile 10% (w/w) reconstituted milk at
80  C.
2.2. Preparation of EPS
A 10% (w/w) solution of reconstituted whey was prepared as
mentioned above. Fresh culture of S10 was inoculated in 250-mL
flasks with 50 mL sterile reconstituted whey and incubated on a
shaker at 200 rpm and 30  C for 48 h. The culture was then trans-
ferred to a 7-L fermentor (BioTron Inc., Korea) with 4.5 L sterile
reconstituted whey at an inoculation ratio of 4.0% (v/v). Fermentation
was performed at 30  C for 96 h at a stirring speed of 200 rpm, and
sampling was conducted every 24 h to assay EPS yield as indicated in
Section 2.1. EPS yield was calculated as the average result of three
batches.
At the end of fermentation, the broth was heated at 100  C for
5 min to inactivate enzymes that might degrade the EPS. After the
broth was cooled to room temperature, a final concentration of 5%
(w/v) TCA was added to the broth with gentle stirring. The mixture
was kept at 4  C for 12 h. Cells and precipitated proteins were
removed by centrifugation (12,000 g, 4  C, 20 min). The EPS in the
supernatant was precipitated by adding three volumes of pre-
chilled 95% ethanol, and the mixture was kept at 4  C for 12 h. A
second centrifugation (12,000 g, 4  C, 20 min) was then performed.
The precipitated EPS was washed twice with pre-chilled absolute
ethanol. The obtained EPS pellet was redissolved in de-ionized
water and dialyzed in membrane tubes (with Mw cut-off of
14 kDa) against de-ionized water for 72 h, with the de-ionized
water changed four times each day. The pellet was dialyzed to
remove small carbohydrates and TCA until no monosugar could be
detected outside. The retentate was lyophilized (Labconco-12L,
Labconco, MO, USA) for 48 h, and S10 EPS was obtained and stored
in a dryer at room temperature. The carbohydrate content of the
S10 EPS was determined by the phenol-sulfuric acid method using
glucose as standard (Dubois et al., 1956), while the protein content
of the S10 EPS was determined by the Bradford assay using bovine
serum albumin (Sigma, St. Louise, MO, USA) as standard (Bradford,
1976). The moisture and ash contents of the S10 EPS were deter-
mined using AOAC Method (AOAC International, 2005). All the S10
EPS composition assays were carried out in triplicates.
Please cite this article in press as: Zhou, F., et al., Exopolysaccharides produced by Rhizobium radiobacter S10 in whey and their rheological
properties, Food Hydrocolloids (2013), http://dx.doi.org/10.1016/j.foodhyd.2013.08.016
2.3. Rheological properties of S10 EPS solutions
All rheological properties of the S10 EPS were determined with a
stress- and strain-controlled ARES-G2 rheometer (TA instruments,
New Castle, USA) using a parallel plate (50 mm in diameter, 1.0 mm
gap). Shear rates from 0.01 s
1 to 1000 s
1 were used in flow
behavior tests, and shear thinning behavior was fitted to the Wil-
liamson model to calculate the consistency and flow index: h ¼ h0/
[1þ(kg_ )n], where k is the consistency index, n is the flow behavior
index, and h0 is the zero-rate viscosity.
Prior to dynamic experiments, the linear viscoelastic region was
assessed at 1 Hz by strain sweep experiments, with 8% strain within
the linear region selected for the S10 EPS solutions. The viscoelastic
properties, storage modulus (G0 ), and loss modulus (G00 ) were deter-
mined through a small-amplitude oscillatory test at frequencies from
0.01 Hz to 10.00 Hz at 25  C. Dynamic temperature sweep tests were
performed at 0.1 Hz between 5 and 75  C. During the testing, EPS
solution was heated from 5  C to 75  C and then cooled down to 5  C.
The heating and cooling rate was 2  C min
1. A thin layer of low-
viscosity mineral oil was used to cover the sample to prevent sol-
vent evaporation during the measurements. All rheological mea-
surements were reported as the mean of at least two replicates.
2.4. Purification of EPS
S10 EPS was fractionated by anion-exchange chromatography on
a DEAE-Sepharose CL-6B column (V2.6 cm  30 cm, GE Health, USA),
equilibrated with 0.05 M TriseHCl buffer (pH 7.6), and eluted with
the buffer at a flow rate of 3 mL min
1, and fraction PK1 was obtained.
A linear gradient of NaCl solution (0 Me1 M) was subsequently
performed, and fraction PK2 was obtained. The elution was moni-
tored for proteins by an online ultraviolet detector at 280 nm. The
eluted fractions were assayed for carbohydrate content by the
phenol-sulfuric acid method described above. The two poly-
saccharide fractions PK1 and PK2 were obtained, dialyzed for 3 d,
and lyophilized. PK1 was less than 5% of the S10 EPS and abandoned
(data not shown). PK2 was further loaded onto a Sepharose 4B col-
umn (V1.6 cm  100 cm, GE Health, USA) and eluted with distilled
water at 0.3 mL min
1. Only one EPS peak during gel permeation was
detected by the phenol-sulfuric acid method. Elutes with EPS were
pooled and lyophilized, and purified PK2 was obtained.
2.5. Monosaccharide composition analysis
To determine the sugar composition, S10 EPS or PK2 was dissolved
in de-ionized water to prepare a solution of 5 mg mL
1, and 200 mL of
PK2 solution was hydrolyzed with 4 M trifluoroacetic acid (TFA) at
110  C for 2 h. After the mixture was cooled to room temperature,
400 mL of methanol was added to the mixture and evaporated to
dryness under an N2 stream. Residual TFA in the sample was removed
by repeating the above treatment thrice. The hydrolyzed sample was
then dissolved in de-ionized water, adjusted to its initial volume, and
filtered through a membrane (0.22 mm, Millipore, MA, USA). Monos-
accharides were identified and quantified with a Dionex high-perform
ance anion-exchange chromatography (HPAEC) system (DX-500) equi
pped with pulsed amperometric detection (Sunnyvale, CA, USA). The
monosaccharides were eluted by a linear gradient of NaOH solutions
(100 mMe300 mM) according to the procedure described by Wood
et al. (Wood, Weisz, & Blackwell, 1994). The percentage of the mono-
saccharides was calculated from the peak areas using a response factor.
2.6. Distribution of molecular weight
The distribution of the Mw of PK2 was determined by a high-
performance liquid chromatography (HPLC, Waters 2690, Waters,
 F. Zhou et al. / Food Hydrocolloids xxx (2013) 1e7 3

MA, USA) system equipped with a Waters 2410 refractive index

detector and Waters Ultrahydrogel TM linear column
(V7.8 mm  300 mm). The PK2 sample was dissolved in 100 mM
NaNO3 (50  C, 1 h), cooled, and filtered through a 0.45 mm filter
prior to injection. The mobile phase was 0.1 mol L
1 NaNO3, and the
separation was carried out at 0.9 mL min
1 at a constant column
temperature of 35  C. Pullulan (P20-P800, JM Science, NY, USA) was
used as reference compound.

3. Results and discussion

3.1. Screening and identification of S10 strain

Screening was performed on milk agar and EPS yields of

different strains in 10% (w/w) skim milk were assayed. Among the
tested strains, S10, which produced the highest amount of EPS, was
selected for further study. S10 is a Gram-negative, non-spore-
forming, catalase-positive, and oxidase-positive rod belonging to
R. radiobacter as indicated by carbon resource utilization tests. For
further confirmation, the 16S rDNA genes of S10 were sequenced.
Approximately 1500 base pairs of the 16S rDNA genes were
amplified and sequenced, and the sequence was deposited into the
GenBank database under accession no. JX498970. The nucleotide
sequence was used to analyze sequence similarity through BLAST
(http://www.ncbi.nlm.nih.gov/blast) and was subsequently found
to be 100% similar to those of Agrobacterium sp. BD-32,
A. tumefaciens strain 30D, A. tumefaciens strain W3, Agrobacterium
sp. WR57, and A. tumefaciens strain C115. According to these results,
S10 was identified as R. radiobacter.
3.2. EPS yield
EPS yields of R. radiobacter S10 in different media, including
reconstituted whey and skim milk, were carried out in a fermentor
at 30  C at a stirring speed of 200 rpm for 96 h (Fig. 1). R. radiobacter
S10 produced hardly any EPS in all tested media in the first 24 h of
cultivation. EPS yields increased slowly from 36 h to 48 h but
rapidly up to 72 h and were kept constant until the end of
fermentation. Reconstituted whey was more suitable than skim
milk for R. radiobacter S10 in biosynthesizing EPS. The highest EPS
yield was 2834.2  226 mg L
1 in 10% (w/w) reconstituted whey at
72 h (calibrated as glucose). R. radiobacter PTCC 1654 strain produce
EPSs of up to 20 g L
1 (Moosavi-Nasab, Taherian, Bakhtiyari,
Farahnaky, & Askari, 2012). Curdlan yield from R. radiobacter
ATCC 6466 achieved 16.5 g L
1 (Salah et al., 2011). EPS yields of
Fig. 2. Flow viscosity curves of S10 EPS solutions at 25  C. Samples were dissolved for
3 h and held at 25  C for 1 h before measurement. Symbols represent solution con-
centrations: (;) ¼ 1.00%; (-) ¼ 0.75%; (C) ¼ 0.50%; and (A) ¼ 0.25% (w/v).
Rhizobium strains are influenced by many factors, such as temper-
ature, pH, growth medium components, and fermentation time.
The optimization of culture conditions for EPS production by
R. radiobacter S10 is undertaken.
Given that the amount of EPS synthesized by R. radiobacter S10
in sterile reconstituted whey was much higher than that in skim
milk (Fig. 1), only S10 EPS synthesized in 10% (w/w) sterile recon-
stituted whey incubated at 30  C for 96 h was obtained for further
study. The obtained S10 EPS was composed of carbohydrate
(84.65  3.96%, calibrated as glucose)，moisture (8.73  0.64%),
ash (0.32  0.17%). At an S10 EPS solution of 0.1% (w/w), no protein
could be detected by the Bradford Assay.
3.3. Rheological measurements
3.3.1. Flow behavior
The effect of shear rate on apparent viscosity for 0.25%, 0.50%,
0.75%, and 1.00% (w/v) S10 EPS solutions at 25  C is shown in Fig. 2.
The apparent viscosity of all tested EPS solutions significantly
decreased with increasing shear rate (up to 1000 s
1), indicating
typical shear thinning behavior (pseudoplasticity). This property is
important for processing applications, indicating that the solution
containing the EPS flows easily when poured from a container or
during certain processing operations, such as pumping, spray dry-
ing, and stirring, despite its high viscosity (Nindo, Tang, Powers, &
Singh, 2005). The parameters obtained via numerically fitting the
data in Fig. 2 to this equation are listed in Table 1.
The viscosity of S10 EPS was much higher (about two orders of
magnitude) than that of EPS solution made from date syrup using
A. radiobacter PTCC1654 (Moosavi-Nasab et al., 2012) and also of

Table 1
Comparison of rheological parameters for S10 EPS solutions at different concen-
trations at 25  C.

Concentration Zero-rate Rate index (n) Consistency (k) R2

(w/v%) viscosity
(Pa s)

0.25 2.35 0.483 3.63 0.982

0.50 5.63 0.578 6.48 0.998
0.75 479.01 0.864 52.64 0.999
1.00 5557.84 0.873 338.85 0.998
Fig. 1. EPS production by R. radiobacter S10 grown in different media containing 8%
whey (-), 10% whey (C), or 10% skim milk (:) (w/w). Data were obtained by fitting the Williamson model (p < 0.05).

Please cite this article in press as: Zhou, F., et al., Exopolysaccharides produced by Rhizobium radiobacter S10 in whey and their rheological
properties, Food Hydrocolloids (2013), http://dx.doi.org/10.1016/j.foodhyd.2013.08.016
4 F. Zhou et al. / Food Hydrocolloids xxx (2013) 1e7

EPS produced by some other microorganisms in kefir grains

(Piermaria et al., 2009) at the same condition (1% w/w, without
plasticizer, 25  C, shear rate of 300 s
1). This higher viscosity may
have been caused by the high Mw and branch degree of S10 EPS, an
observation that warrants confirmation by further structural and
conformational analysis.

3.3.2. Viscoelastic behavior

The results of the dynamic frequency sweep tests of 0.10%e
1.00% (w/v) S10 EPS solutions are shown in Fig. 3. At low polymer
concentration (0.1%), the rheological behavior of S10 EPS solutions
exhibited typical viscous fluid properties with the viscous modulus
(G00 ) higher than the elastic modulus (G0 ) over the whole frequency
range (Fig. 3A). At higher concentrations (0.25% and 0.50% w/v),
both the G0 and G00 moduli increased with increasing frequency
(Fig. 3B and C), but the G0 modulus increased more rapidly than the
G00 modulus. As a result, the modulus curves intersected at the
crossover point (fc), and the G0 modulus was predominantly high at
frequencies higher than fc. The EPS solution transitioned from a Fig. 5. Elution profile of S10 EPS on DEAE-Sepharose CL-6B column. PK1, PK2: two
fluid-like to a gel-like structure. At concentrations above 0.75% polysaccharide fractions.
(Fig. 3D and E), the G0 values were greater than G00 over the entire
frequency range explored, and frequency dependence was strong gel, which has a G0 one or two orders of magnitude higher
observed, which indicated the presence of an apparent gel network than its G00 with frequency independence (Balaghi, Mohammadifar,
in the system. Frequency dependence is an important indication of Zargaraan, Gavlighi, & Mohammadi, 2011; Tzoumaki, Moschakis, &
a typical weak gel (Ikeda & Nishinari, 2001) but not of a typical Biliaderis, 2011). Compared with the 0.75% EPS solution, the 1.00%

Fig. 3. Mechanical spectra of S10 EPS solutions at different concentrations at 25  C: (A) 0.10% EPS solution, (B) 0.25% EPS solution, (C) 0.50% EPS solution, (D) 0.75% EPS solution, and
(E) 1.00% EPS solution (w/v).

Fig. 4. Changes in elastic G0 (-) and viscous G00 (,) moduli during initial heating from 5  C to 75  C and subsequent cooling to 5  C at 2  C min
1 for 1.0% w/v of EPS.

Please cite this article in press as: Zhou, F., et al., Exopolysaccharides produced by Rhizobium radiobacter S10 in whey and their rheological
properties, Food Hydrocolloids (2013), http://dx.doi.org/10.1016/j.foodhyd.2013.08.016
 F. Zhou et al. / Food Hydrocolloids xxx (2013) 1e7
Fig. 6. Chromatogram of PK2 obtained by gel permeation HPLC. Mw was
3.03  106 Da.
solution exhibited weakened frequency dependence (Fig. 3E). The
gap between G0 and G00 increased, indicating that increasing EPS
concentration strengthens intermolecular interactions, thereby
strengthening and stabilizing the gel system.
The most important effect of temperature on EPS, except for
degradation in some exceptional cases, is an orderedisorder
conformational change, usually followed by rotation measurement
(Hebbar et al., 1992). The heating or cooling modulus curve of the 1%
S10 EPS solutions was obtained at a heating or cooling rate of
2  C min
1 at a constant frequency of 0.1 Hz (Fig. 4). At the beginning
of heating that ranged from 5  C to 62  C, the moduli of the EPS
Fig. 7. HPAEC results of hydrolyzed S10 EPS and PK2. S10 EPS was composed mainly of galactose and glucose at a molar ratio of 1:4.52 (A) whereas PK2 was composed mainly of
galactose and glucose at a molar ratio of 1:4.92 (B).
Please cite this article in press as: Zhou, F., et al., Exopolysaccharides produced by Rhizobium radiobacter S10 in whey and their rheological
properties, Food Hydrocolloids (2013), http://dx.doi.org/10.1016/j.foodhyd.2013.08.016
5
solutions exhibited little temperature dependence and only slightly
decreased, and G0 dominated G00 (Fig. 4A). Both moduli sharply
decreased from 63  C to 65  C, and the two modulus curves inter-
sected, indicating that the gel system began to melt. Therefore, the
melting point of the gel was 64  C  1  C. The modulus curves were
almost constant from 66  C to 75  C, with G00 higher than G0 . During
cooling, the G0 and G00 curves were nearly reversible with the heating
curves, but the gelling point was approximately 59  C  1  C,
exhibiting thermal hysteresis compared with the melting point
(about 5  C lower). At the end of the procedure, both moduli almost
resumed to their original levels, indicating that heating and cooling
did not influence the gelling ability of the EPS and that the gel formed
by the S10 EPS was a thermal reversible gel. Compared with gels
formed by some other polysaccharides, such as carrageenan, gellan,
and flaxseed gum, the gel formed by S10 EPS has higher gelling and
melting temperatures, indicating that the gel system is rather stable.
3.4. Purification of EPS
S10 EPS was fractionated by ion-exchange chromatography into
two fractions (PK1 and PK2; Fig. 5). During the fractionation, no
absorbance at 280 nm was observed, suggesting the absence of
residual protein in the S10 EPS. PK1 was less than 5% of the S10 EPS
and abandoned (data not shown), indicating that S10 EPS was
composed almost entirely of PK2. PK2 was further isolated by gel
filtration chromatography, and only one peak of EPS was detected
by the phenol-sulfuric acid method, indicating that PK2 was a
6 F. Zhou et al. / Food Hydrocolloids xxx (2013) 1e7
homogeneous polysaccharide. The purity of PK2 was further
ascertained by HPLC assay, which indicated PK2 with one size
distribution (at 13.5 min) (Fig. 6).
3.5. Monosaccharide composition and distribution of Mw
The monosugar composition of S10 EPS as well as PK2 was
analyzed using HPAEC. The S10 EPS was composed of galactose
and glucose at an approximate ratio of 1.00:4.52 and trace
amounts of glucosamine and mannose (Fig. 7(A)), whereas PK2
was composed of galactose and glucose at an approximate ratio of
1.00: 4.92 and trace amounts of glucosamine and mannose
(Fig. 7(B)). The high similarity of the monosaccharide composition
between PK2 and S10 EPS provided further support to the point
that S10 EPS is mainly composed of PK2. The monosugar
composition of EPS produced by Rhizobium (formerly Agro-
bacterium) is controversial. Harada (Harada, 1965) isolated and
identified succinoglycan from A. radiobacter strain 10C3, which
contains 77.0% glucose, 6.7% galactose, 1.9% mannose, and 10.6%
succinic acid. Water-soluble EPS from A. radiobacter is composed
of 89.0% glucose, 10.0% galactose, and 0.7% rhamnose (Triveni,
Shamala, & Rastogi, 2001). Curdlan produced by R. radiobacter
contains glucose, galactose, and pyruvate at the ratio of 1:1:1
(Zevenhuizen, 1997). By contrast, curdlan produced by
R. radiobacter ATCC 6466 contains only glucose (Salah et al., 2011).
Considering its monosugar composition, S10 EPS may be neither
succinoglycan nor curdlan. Therefore, the toxicological assess-
ment should be done before the application of this new EPS in
food (Calliari et al., 2011).
The monosugar composition of bacterial EPS depends on the
genetic mechanisms involved in EPS biosynthesis and can be
influenced by various factors, such as the culture medium and
environmental conditions (McKellar, Van Geest, & Cui, 2003;
Triveni et al., 2001). Further studies should analyze the difference
of the monosaccharide composition of EPS produced by Rhizobium
from the perspectives of genetics and culture conditions.
The chromatogram of PK2 (Fig. 6) obtained by gel permeation
HPLC depicted a single distribution of Mw corresponding to
3.03  106 Da, consistent with the high viscosity of PK2. Although
molecular mass is critical for rheological property of the EPS, little
information is available about molecular weight of EPS produced
by R. radiobacter. EPS expressed by a strain of A. tumefaciens, now
classified as R. radiobacter, was with a molecular weight distri-
bution of 1.55e5.30  106 Da (Stredansky, Conti, Bertocchi,
Matulova, & Zanetti, 1998), which was comparable with the
value of our current result. On the contrary, low molecular weight
(approximate 3  105Da) had been reported about curdlan pro-
duced by Agrobacterium sp. ATCC 31750 (Kim, Ryu, Choi, Rhee, &
Lee, 2003).
4. Conclusion
R. radiobacter S10 isolated from kefir grains can biosynthesize
EPS with high viscosity in whey. The S10 EPS fraction PK2 was
composed of galactose and glucose at a molar ratio of 1.00:4.92,
differed in monosugar composition from either succinoglycan or
curdlan, and exhibited an approximate Mw of 3.03  106 Da. The
rheological behavior of S10 EPS solutions of different concentra-
tions exhibited typical shear thinning and thermal reversible weak-
gel properties. The rheological properties of S10 EPS give it great
potential as a thickener or stabilizer. Extensive research is needed
to fully explore this new material and its potential application in
the food and pharmaceutical industries.
Please cite this article in press as: Zhou, F., et al., Exopolysaccharides produced by Rhizobium radiobacter S10 in whey and their rheological
properties, Food Hydrocolloids (2013), http://dx.doi.org/10.1016/j.foodhyd.2013.08.016
Acknowledgments
This study was supported by the State Key Project for Basic
Research (Project 973, No. 2010CB735705).
References
AOAC. (2005). Official methods of analysis of the AOAC International (18th ed.).
Gaithersburg, MD: Association of Official Analytical Chemists.
Balaghi, S., Mohammadifar, M. A., Zargaraan, A., Gavlighi, H. A., & Mohammadi, M.
(2011). Compositional analysis and rheological characterization of gum traga-
canth exudates from six species of Iranian Astragalus. Food Hydrocolloids, 25(7),
1775e1784.
Banik, R., Kanari, B., & Upadhyay, S. (2000). Exopolysaccharide of the gellan family:
prospects and potential. World Journal of Microbiology and Biotechnology, 16(5),
407e414.
Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein-dye binding.
Analytical Biochemistry, 72(1), 248e254.
Calliari, C. M., Magnani, M., Saito, A. Y., Dionízio Filho, P. S., Malvezi, A. D., &
Gómez, R. H. C. (2011). In vitro and in vivo toxicity studies of Agrobacterium
radiobacter k84 biopolymer (ARB). Semina: Ciências Agrárias, 32, 1915e1920.
Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P., & Smith, F. (1956). Colorimetric
method for determination of sugars and related substances. Analytical Chem-
istry, 28(3), 350e356.
Edmond, M. B., Riddler, S. A., Baxter, C. M., Wicklund, B. M., & Pasculle, A. W. (1993).
Agrobacterium radiobacter: a recently recognized opportunistic pathogen.
Clinical Infectious Diseases, 16(3), 388e391.
Elboutachfaiti, R., Delattre, C., Petit, E., & Michaud, P. (2010). Polyglucuronic
acids: structures, functions and degrading enzymes. Carbohydrate Polymers,
84(1), 1e13.
Funami, T., & Nishinari, K. (2007). Gelling characteristics of curdlan aqueous dis-
persions in the presence of salts. Food Hydrocolloids, 21(1), 59e65.
Garfinkel, D. J., & Nester, E. W. (1980). Agrobacterium tumefaciens mutants affected
in crown gall tumorigenesis and octopine catabolism. Journal of Bacteriology,
144(2), 732e743.
Ghasemlou, M., Khodaiyan, F., Oromiehie, A., & Yarmand, M. S. (2011). Development
and characterisation of a new biodegradable edible film made from kefiran, an
exopolysaccharide obtained from kefir grains. Food Chemistry, 127(4), 1496e
1502.
Harada, T. (1965). Succinoglucan 10C3: a new acidic polysaccharide of Alcaligenes
faecalis var. myxogenes. Archives of Biochemistry and Biophysics, 112(1), 65e69.
Hebbar, K., Gueniot, B., Heyraud, A., Colin-Morel, P., Heulin, T., Balandreau, J., et al.
(1992). Characterization of exopolysaccharides produced by rhizobacteria.
Applied Microbiology and Biotechnology, 38(2), 248e253.
Ikeda, S., & Nishinari, K. (2001). “Weak Gel”-type rheological properties of aqueous
dispersions of nonaggregated k-carrageenan helices. Journal of Agricultural and
Food Chemistry, 49(9), 4436e4441.
Kim, M.-K., Ryu, K.-E., Choi, W.-A., Rhee, Y.-H., & Lee, I.-Y. (2003). Enhanced pro-
duction of (1/3)-b-d-glucan by a mutant strain of Agrobacterium species.
Biochemical Engineering Journal, 16(2), 163e168.
McKellar, R., Van Geest, J., & Cui, W. (2003). Influence of culture and environmental
conditions on the composition of exopolysaccharide produced by Agro-
bacterium radiobacter. Food Hydrocolloids, 17(4), 429e437.
Moosavi-Nasab, M., Taherian, A. R., Bakhtiyari, M., Farahnaky, A., & Askari, H. (2012).
Structural and rheological properties of succinoglycan biogums made from low-
quality date syrup or sucrose using Agrobacterium radiobacter inoculation. Food
and Bioprocess Technology, 5(2), 638e647.
Nampoothiri, K. M., Singhania, R. R., Sabarinath, C., & Pandey, A. (2003). Fermen-
tative production of gellan using Sphingomonas paucimobilis. Process Biochem-
istry, 38(11), 1513e1519.
Nindo, C., Tang, J., Powers, J., & Singh, P. (2005). Viscosity of blueberry and raspberry
juices for processing applications. Journal of Food Engineering, 69(3), 343e350.
Piermaria, J. A., Pinotti, A., Garcia, M. A., & Abraham, A. G. (2009). Films based on
kefiran, an exopolysaccharide obtained from kefir grain: development and
characterization. Food Hydrocolloids, 23(3), 684e690.
Queiroz, V. M. S., & Fontes, S. (2008). Experimental analysis of structural change and
rheological behavior of macromolecular solutions with guar and xanthan gums
in crossflow microfiltration processing. Food and Bioprocess Technology, 1(2),
180e186.
Rho, H. S., Kang, S., & Lee, Y. H. (2001). Agrobacterium tumefaciens-mediated
transformation of the plant pathogenic fungus, Magnaporthe grisea. Molecules
and Cells, 12(3), 407e411.
Salah, R. B., Jaouadi, B., Bouaziz, A., Chaari, K., Blecker, C., Derrouane, C., et al. (2011).
Fermentation of date palm juice by curdlan gum production from Rhizobium
radiobacter ATCC 6466Ô: purification, rheological and physico-chemical char-
acterization. Food Science and Technology, 44(4), 1026e1034.
Stredansky, M., Conti, E., Bertocchi, C., Matulova, M., & Zanetti, F. (1998). Succino-
glycan production by Agrobacterium tumefaciens. Journal of Fermentation and
Bioengineering, 85(4), 398e403.
Sutherland, I. W. (1999). Biofilm exopolysaccharides. In Microbial extracellular
polymeric substances (pp. 73e92). Springer.
 F. Zhou et al. / Food Hydrocolloids xxx (2013) 1e7 7

Triveni, R., Shamala, T., & Rastogi, N. (2001). Optimised production and utilisation of
exopolysaccharide from Agrobacterium radiobacter. Process Biochemistry, 36(8),
787e795.
Tzoumaki, M. V., Moschakis, T., & Biliaderis, C. G. (2011). Mixed aqueous chitin
nanocrystalewhey protein dispersions: microstructure and rheological behav-
iour. Food Hydrocolloids, 25(5), 935e942.
Wood, P. J., Weisz, J., & Blackwell, B. A. (1994). Structural studies of (l-3),(l-4)-/3-D-
glucans by 13C-nuclear magnetic resonance spectroscopy and by rapid analysis of
cellulose-like regions using high-performance anion-exchange chromatography
of oligosaccharides released by lichenase. Cereal Chemistry, 71(3), 301e307.
Zevenhuizen, L. (1997). Succinoglycan and galactoglucan. Carbohydrate Polymers,
33(2), 139e144.

Please cite this article in press as: Zhou, F., et al., Exopolysaccharides produced by Rhizobium radiobacter S10 in whey and their rheological
properties, Food Hydrocolloids (2013), http://dx.doi.org/10.1016/j.foodhyd.2013.08.016