Effects of carbohydrate source on physicochemical properties of the exopolysaccharide produced by Lactobacillus fermentum TDS030603 in a chemically defined medium

Article abstract of DOI:10.1016/j.carbpol.2009.10.037

A thermophilic lactic acid bacterium, Lactobacillus fermentum TDS030603, produced about 100 mg/L of EPS in purified form when grew in de Man-Rogosa-Sharpe (MRS) broth. The 1% (w/v) solution of the purified EPS was highly viscous, exhibiting an apparent vi

Full text of DOI:10.1016/j.carbpol.2009.10.037

Carbohydrate Polymers 79 (2010) 1040–1045
Contents lists available at ScienceDirect
Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
Effects of carbohydrate source on physicochemical properties of the
exopolysaccharide produced by Lactobacillus fermentum TDS030603 in a
chemically defined medium
a
b
a
b
b
Kenji Fukuda a, , Tala Shi , Kentaro Nagami , Fiame Leo , Tadashi Nakamura , Kumi Yasuda ,
*
Akitsugu Senda a, Hidemasa Motoshima c, Tadasu Urashima a
a Department of Animal and Food Hygiene, Obihiro University of Agriculture and Veterinary Medicine, 2-11 Inada-cho, Obihiro, Hokkaido 080-8555, Japan
b Department of Food Science, Obihiro University of Agriculture and Veterinary Medicine, 2-11 Inada-cho, Obihiro, Hokkaido 080-8555, Japan
c Research Center, Yotsuba Milk Products Co. Ltd., 465-1 Wako, Kitahiroshima, Hokkaido 061-1264, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A thermophilic lactic acid bacterium, Lactobacillus fermentum TDS030603, produced about 100 mg/L of
Received 27 April 2009
Received in revised form 25 September
2009
Accepted 15 October 2009
Available online 24 October 2009
EPS in purified form when grew in de Man–Rogosa–Sharpe (MRS) broth. The 1% (w/v) solution of the
purified EPS was highly viscous, exhibiting an apparent viscosity (gapp) of 0.88 Pa s at a shear rate of
10/s. To investigate the impact of carbohydrate source on the production yield and chemical structure
of EPS and the viscosity of EPS solution, a chemically defined medium (CDM) has been developed. Results
of TLC, HPLC, and 1H NMR spectroscopy indicated that the chemical structures of EPS released in MRS and
in the CDM supplemented with glucose, galactose, lactose or sucrose were very similar. All the 1% solu-
tions of EPSs released in CDMs were highly viscous similar to the EPS released in MRS, but their viscos-
ities appeared to differ, presumably because of the differences in their molecular mass distributions.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Chemical structure
Chemically defined medium
Exopolysaccharide
Lactic acid bacteria
Viscosity
1. Introduction
A large number of EPSs produced by LAB have been described
(Cerning, 1990; De Vuyst & Degeest, 1999; Jolly, Vincent, Duboc,
An exopolysaccharide (EPS) is a sugar polymer that is produced
mainly by bacteria and microalgae, either in a form bound to the
cell-wall, a so-called capsular polysaccharide (CPS), or in a free
form liberated into the culture medium, known as a slime EPS
(Sutherland, 1972). It is believed that the physiological function
of EPS is as the first line of biological defense against phagocytosis,
phage attack, antibiotics, toxic metal ions and physical stresses
such as desiccation and osmotic stress (Looijesteijn, Trapet, de
Vries, Abee, & Hugenholtz, 2001; Roberts, 1996; Weiner, Langille,
& Quintero, 1995; Whitfield, 1988). EPS produced by lactic acid
bacteria (LAB) is very useful in the food industry, because it pro-
vides consistency to the resulting fermented milk products, such
as Scandinavian ropy milks, viili and långfil (Duboc & Mollet,
2001). Furthermore, some EPSs have been claimed to show bioac-
tivities beneficial to health, including prebiotic or anti-inflamma-
tory effects (Salazar, Gueimonde, Hernández-Barranco, Ruas-
Madiedo, & de los Reyes-Gavilán, 2008; Vinderola, Perdigón,
Duarte, Farnworth, & Matar, 2007).
& Neeser, 2002; Laws, Gu, & Marshall, 2001; Welman & Maddox,
2003), but little is known about the effects of medium components
on the chemical structure of the EPS and its rheological properties
(Vaningelgem et al., 2004). Since the complexity of media compo-
sition can lead to an incorrect structural analysis of EPS (De Vuyst
& Degeest, 1999), a chemically defined medium (CDM) is of great
advantage when assessing the effects of medium components on
the chemical structure and the physicochemical properties of EPS
(Grobben et al., 1998). It has been shown, using CDM, that adenine
or orotic acid stimulates both the cell growth and the yield of EPS
in Lactobacilli (Petry, Furlan, Crepeau, Cerning, & Desmazeaud,
2000; Torino, Hébert, Mozzi, & de Valdez, 2005). Composition of
carbohydrate source also exhibited significant effects on the EPS
yield, but the preferences for sugar for the maximum EPS produc-
tion were strain-dependent (Cerning et al., 1994; Tallon, Bressol-
lier, & Urdaci, 2003; Torino et al., 2005). On the other hand, the
effects of carbohydrate source on the monosaccharide composition
of EPS are still unclear: the constitutive monosaccharides were
found to be the same in Lactobacillus helveticus following altera-
tions in the carbohydrate source (Torino et al., 2005), but the rela-
tive proportions of the individual monosaccharides varied in L.
delbrueckii subsp. bulgaricus (Petry et al., 2000). The rheological
* Corresponding author. Tel.: +81 155 49 5564; fax: +81 155 49 5577.
E-mail address: fuku@obihiro.ac.jp (K. Fukuda).
0144-8617/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carbpol.2009.10.037

K. Fukuda et al. / Carbohydrate Polymers 79 (2010) 1040–1045
1041
Table 1
properties of the EPS produced by LAB are attributed to its molec-
Chemical composition of the CDM for L. fermentum TDS030603.
ular mass, molecular mass distribution, constituent sugar residues,
linkages between the sugar monomers and the presence of side
groups (Shene, Canquil, Bravo, & Rubilar, 2008). However, effects
of altered medium composition on the rheological properties of
EPS have been scarcely reported.
In the present paper we aimed to evaluate the effects of carbo-
hydrate source on the yield, chemical structure and viscosity of a
neutral hetero-EPS produced by L. fermentum TDS030603. In this
context, we have developed a CDM for this strain, having modified
the previously reported media (Morishita, Deguchi, Yajima, Saku-
rai, & Yura, 1981). The chemical structure and viscosity of EPS re-
leased into CDM were assessed using the EPS released into MRS
as the reference. Possible determinants leading to rheological vari-
ations in the EPS will be discussed.
Components Concentration (g/L)
10.0a
0.2b
0.1a
0.1b
0.2a
0.1a
0.1a
0.1a
0.1a
0.1a
0.1b
0.1a
0.1a
0.1a
D
-Glucose
DL-Alanine
L
L
L
L
L
L
L
L
L
L
L
L
-Arginine
-Aspartic acid
-Glutamic acid
-Histidine
-Isoleucine
-Leucine
-Methionine
-Phenylalanine
-Serine
-Tryptophan
-Tyrosine
2. Materials and methods
-Valine
p-Aminobenzoic acid
Biotin
Folic acid
Nicotinamide
Nicotinic acid
Pantotheic acid
Pyridoxal
Pyridoxol
Riboflavin
Adenine
Guanine
0.002b
0.00001a
0.0001b
0.001b
0.001b
0.002a
0.002a
0.001b
0.0002b
0.01a
0.01b
0.005a
0.01a
0.01b
0.02a
0.05a
0.01a
1.0a
2.1. Bacterial strain and chemicals
Lactobacillus fermentum TDS030603 was obtained from the bac-
terial collection of our own laboratory (Leo et al., 2007). MRS was
from Oxoid (Cambridge, UK). DEAE-Sephadex A-50 and Toyopearl
HW-55F was from GE Healthcare (Uppsala, Sweden) and Tosoh (To-
kyo, Japan), respectively. D2O (99.99%atom % D) was from Sigma–Al-
drich (St. Louis, USA). All the chemicals used were analytical grade.
Thymine
Uracil
Xanthine
2.2. Development of CDM and culture condition
Adenylic acid
Cytidylic acid
20-Deoxyguanosine
Ammonium citrate
Sodium acetate
Sodium citrate
Sodium thioglycolate
FeSO4ꢁ7H2O
K2HPO4
Following the previous report (Morishita et al., 1981), we firstly
tested a prototype CDM consisting of 48 constituents, of which six
were non-essential amino acids (see below) and the other 42 are
listed in Table 1. Essential or important chemical compounds for
cell growth were determined by checking the cell density of cul-
ture medium from which one of the above constituents had been
omitted. After static culture in MRS for 24 h at 30 °C under aerobic
conditions, the cells were harvested, washed thoroughly with ster-
ilized phosphate buffered saline, and inoculated into 1 L of either
MRS or CDM to yield an optical density (OD) of 0.2 at 600 nm. Cell
growth (OD600 nm) and pH of the static culture were monitored.
Simultaneously, cultivable cell numbers were counted on MRS-
agar plates; after a given time period, a 1-ml aliquot of culture
medium was collected, diluted with MRS and spread on a MRS-
agar plate, which was incubated at 30 °C for 24 h under anaerobic
condition. Colonies appearing on the plate were counted as cultiva-
ble cells. To measure the amount of EPS produced, a 100-ml aliquot
of the culture medium was collected, and the EPS was purified fol-
lowing the procedure described below.
6.0b
0.5b
0.5b
0.02b
3.0a
KH2PO4
3.0a
MgSO4ꢁ7H2O
MnSO4ꢁ5H2O
Spermidine phosphate
Tween 80
0.5b
0.2a
0.005b
1.0a
a
Essential.
Important but not essential.
b
ever, it required further purification using a Toyopearl HW-55F col-
umn (2.6 ꢀ 100 cm, 15 ml/h) equilibrated with water. The polymer
dry mass of purified EPS was determined by measuring the weight.
The lyophilized EPS was stored in a desiccator until used.
2.4. Estimation of EPS molecular mass
2.3. Production and isolation of EPS
The molecular mass distribution of EPS was estimated using
high performance liquid chromatography (HPLC). The purified
Glucose, galactose, lactose, or sucrose was used in the CDM as
carbohydrate source at the final concentration of 1% (w/v). After
cultivation as described above, the cells were removed by centrifu-
gation (17,000g, 1 h, 4 °C). Crude EPS released in CDM was precip-
itated by addition of an equal volume of ice-cold ethanol to the
supernatant. The ethanol precipitate was collected by centrifuga-
tion (17,000g, 30 min, 4 °C), dissolved in 30 ml of water, dialyzed
overnight with water at 4 °C, and lyophilized. The lyophilized
crude EPS (from 100 ml of the culture medium) was dissolved in
10 ml of 50 mM Tris–HCl (pH 8.7), and purified by a batch method
using a 20-ml slurry of DEAE-Sephadex A-50 equilibrated with the
same buffer. The non-adsorbed fraction was collected, thoroughly
dialyzed against water, and lyophilized. Crude EPS released in
MRS was subjected to the same procedure described above, how-
EPS was dissolved in water (1 mg/ml), and 100
ll of this solution
was loaded onto a TSKgel G6000PWXL column (7.8 ꢀ 300 mm,
Tosoh). Elution was done with water at 40 °C at a flow rate of
1 ml/min. The EPS was detected by measuring the refractive index
of the eluate using a refractive index monitor RI-8020 (Tosoh). Sho-
dex Standard P-82 (Showa Denko, Tokyo, Japan), a series of pullu-
lans with known molecular masses ranging from 0.59 ꢀ 104 to
7.88 ꢀ 105 Da, was used as the standard.
2.5. Monosaccharide composition of EPS
The purified EPS (2 mg) was hydrolyzed in 250
ll of 2 M triflu-
oroacetic acid (TFA) at 100 °C for 5 h. Excess TFA was removed by

1042
K. Fukuda et al. / Carbohydrate Polymers 79 (2010) 1040–1045
rotary evaporation, and the hydrolysate was washed thoroughly
with water and lyophilized. The lyophilized powder was dissolved
in 100 ll of water, and a 5-ll aliquot was used for thin-layer chro-
The spectrum was measured by reference to internal acetone
(d = 2.225), but chemical shifts (ppm) were represented by refer-
ence to internal sodium 2,2-dimethyl-2-silapentane-5-sulphonate.
matography (TLC). Development was done twice on a silica gel TLC
plate (20 ꢀ 20 cm) using a developing solvent of n-butanol:etha-
nol:water (2:1:1, v/v). Carbohydrates were visualized by heating
the TLC plate after spraying with 5% (v/v) sulfuric acid in ethanol.
Glucose, galactose, and mannose were used as standard
monosaccharides.
The molar ratio of the monosaccharides of the hydrolysate was
analyzed using an HPLC. Prior to the analysis, the hydrolysate was
labeled with 2-aminobenzoic acid using a modified method of
Anumula and Dhume (1998). For the labeling reaction, labeling re-
agent A (4% sodium acetate trihydrate, 2% boric acid, in methanol,
w/v) and labeling reagent B (0.32 M 2-aminobenzoic acid, 1 M so-
dium cyanoborohydride, in the labeling reagent A) were used. The
2.7. Viscosity measurement
Using a Dynamic Analyzer RDA II (Rheometric Scientific, Piscat-
away, USA) equipped with a cone-and-plate attachment (diameter,
25 mm; angle, 0.1 radian; gap, 65 lm), the shear stress of a 1% (w/
v) solution of the purified EPS was monitored for 300 s at 22 °C up
to 300/s, in a steady shear testing mode. The apparent viscosity,
gapp, was calculated from the shear stress at a certain point of shear
rate.
3. Results and discussion
hydrolysate (1.4 mg of EPS) in 10 ll of water was mixed with 50 ll
of the labeling reagent B, and the mixture was heated at 80 °C for
50 min. After cooling to room temperature, 1 ml of chloroform and
0.5 ml of water were added, mixed well, and then an aqueous
phase was collected by centrifugation (900g, 10 °C, 10 min). The
solvent was removed by rotary evaporation, and the dried sample
3.1. Development of CDM and the growth of L. fermentum TDS030603
in CDM
By subtracting each component from the prototype CDM, out of
48 chemicals, we have determined 25 essential and 17 important
but not essential compounds for the cell growth of L. fermentum
TDS030603 (Table 1). When the strain was cultivated in the proto-
type CDM which did not contain one of the essential components,
no cell growth was observed (results not shown). Optimum cell
growth was not obtained even in a medium that contained the
25 essential compounds, so we added other components individu-
ally and thereby determined 17 compounds that were important
was dissolved in 500 ll of water. The sample was diluted 50 times
with 150 mM trisodium citrate (pH 4.5)/7.5% (v/v) acetonitrile, and
passed through an ODS-100Z column (4.6 ꢀ 250 mm, Tosoh) at a
flow rate of 0.75 ml/min, using the same buffer as eluant. Glucose
and galactose, labeled with 2-aminobenzoic acid, were used as
standards. The molar ratio of glucose and galactose was calculated
from the peak areas. Each analysis was performed in triplicate.
for optimum cell growth. Six amino acids,
L-alanine, L-cysteine, gly-
2.6. 1H NMR spectroscopy
cine, -lysine, -proline and -threonine, were not required for the
L
L
L
growth of L. fermentum TDS030603, indicating that the biosyn-
thetic pathways of these 6 amino acids were functionally active
(Morishita et al., 1981). In the developed CDM consisting of 42
chemicals, the bacterial population only reached an OD600nm of
1.5 units whereas 4.5 units were detected after the bacterial
growth in MRS (Fig. 1A). At the end of the exponential phase, the
great difference of bacterial population between CDM and MRS
Exchangeable protons in the purified EPS (2 mg) were replaced
by deuterium in 99.99% D2O. Using a 500 MHz FT-NMR spectrom-
eter, Jeol ECP-500 (Jeol, Tokyo, Japan), 1H NMR spectra were re-
corded at a probe temperature of 343 K that allowed us to
observe the chemical shifts in the range of 4.75–4.35 ppm, which
were overlapped by a large signal of HDO at ambient temperature.
5
1
(B)
(A)
4
3
2
1
0
1
9
8
7
0
20
40
60
80
0
20
40
60
800
160
120
80
40
0
7
6
5
4
3
(C)
(D)
0
20
40
60
80
0
20
40
60
80
Cultivation time (h)
Cultivation time (h)
Fig. 1. Cell growth and EPS production of L. fermentum TDS030603 and pH profile of the culture broth. The cell growth was monitored by OD600nm (A) and cultivable cell count
(B). (C) pH profile of the culture broth. EPS was purified and its dry mass was weighed (D). Symbols; open circle, EPS released in MRS; closed circle, EPS released in CDM
supplemented with glucose; open triangle, EPS released in CDM supplemented with galactose; closed triangle, EPS released in CDM supplemented with lactose; cross, EPS
released in CDM supplemented with sucrose.

K. Fukuda et al. / Carbohydrate Polymers 79 (2010) 1040–1045
1043
Glc
Gal
Man
CDMGlc CDMGal CDMLac CDMSuc MRS
EPSs
Standards
Fig. 2. Monosaccharide composition of EPSs produced by L. fermentum TDS030603 in MRS and CDM supplemented with various carbohydrate sources. Glucose (Glc),
galactose (Gal), and mannose (Man) were used as standards. MRS, EPS released in MRS broth; CDMGlc, EPS released in CDM supplemented with glucose; CDMGal, EPS released
in CDM supplemented with galactose; CDMLac, EPS released in CDM supplemented with lactose; CDMSuc, EPS released in CDM supplemented with sucrose.
cultures was confirmed by cultivable cell counts and pH values
reflecting acidification of the culture medium (Fig. 1B and C).
of L. fermentum TDS030603 grown in MRS and CDM supplemented
with various carbohydrate sources reaches the maximal concentra-
tion at the beginning of the stationary phase, in accordance with
other EPS producing LAB (Petry et al., 2000; Torino et al., 2005).
Of the carbohydrates tested, glucose gave the second highest EPS
production (69.0 mg/L), while lactose, galactose or sucrose yielded
73%, 51% or 19%, respectively, of the EPS released in CDMGlc. There
is no consistency in the reported preference for carbohydrates
regarding EPS production (Cerning et al., 1994; Tallon et al.,
2003; Torino et al., 2005), and thus this seems to be strain-depen-
3.2. EPS production in MRS and CDM supplemented with various
carbohydrate sources
EPS production was investigated using MRS and the CDM sup-
plemented with either glucose (CDMGlc), galactose (CDMGal), lac-
tose (CDMLac) or sucrose (CDMSuc), each at a concentration of 1%
(w/v). Neither cell growth nor EPS production was observed when
maltose and/or fructose were used as carbohydrate sources (re-
sults not shown). After 72 h cultivation, the best EPS production
(97.1 mg/L) in purified form was found in MRS. The EPS production
dent. When the strain was cultivated in MRS, CDMGlc, or CDMGal
,
the EPS production reached to the highest at 24–48 h, but a decline
of the EPS yield was observed at 72 h (Fig. 1D). The decline of EPS
production during prolonged fermentation has been observed in L.
rhamnosus R being attributed to the enzymatic degradation of EPS
(Pham, Dupont, Roy, Lapointe, & Cerning, 2000). Therefore, EPS
degrading enzymes might be expressed in L. fermentum
TDS030603 in 72 h cultivation.
Table 2
Monosaccharide composition of L. fermentum TDS030603 releasing EPS in MRS and
CDM supplemented with various carbohydrate sources.
Media
Molar ratioa (Glucose/Galactose)
3.3. Chemical structure of EPS
MRS
2.6 0.05
CDMGlc
CDMGal
CDMLac
CDMSuc
2.7
2.6 0.03
2.6 0.02
0
All the EPSs that were released in MRS and the CDM supple-
mented with various carbohydrates consisted of glucose and gal-
actose; no other monosaccharide was detected on TLC (Fig. 2).
The monosaccharide composition of EPS was also investigated by
2.8
0
HDO
acetone
H-2
α-anomer β-anomer
CDMSuc
CDMLac
CDMGal
CDMGlc
MRS
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
Chemical shift (ppm)
Fig. 3. NMR spectra of EPSs produced by L. fermentum TDS030603 in MRS and CDM supplemented with various carbohydrate sources. Chemical shifts derived from a H-2
signal of glucose, which was substituted at OH-2 and OH-3 (d = 5.661), -anomer (d = 4.978 and 5.314), and b-anomer (d = 4.510 and 4.725) were observed. Chemical shifts of
heavy water (HDO) and acetone were d = 4.348 and 2.225, respectively.
a

1044
K. Fukuda et al. / Carbohydrate Polymers 79 (2010) 1040–1045
an HPLC experiment. As the result, the molar ratio of glucose to
galactose ranged from 2.6 to 2.8 (Table 2). This result was similar
to the value of 2.5 which had been previously determined for the
EPS produced by L. fermentum TDS030603 in MRS (Leo et al.,
2007). The 1H NMR spectra of the EPSs released in MRS and CDMs
were very similar (Fig. 3). In all spectra, typical chemical shifts that
represent (i) an H-2 signal of glucose, which was substituted at
yielded a higher molecular mass than the others. Referring to the
standard pullulan and considering the size exclusion limit of the
column (approx. 5 ꢀ 107 Da), the molecular mass of peak I was
estimated to be more than 106 Da. The EPS produced in MRS con-
tained a lower molecular mass fraction (peak III, Fig 4), whose
molecular mass was 2.8 ꢀ 104 Da. An apparent shoulder was ob-
served in the vicinity of peak I in the EPSs released in CDMs supple-
mented with different carbohydrate sources, although it was not
apparent in the EPS released in MRS (peak II, Fig. 4). The molecular
mass distributions of peak II were clearly divided into two groups:
one includes the EPSs released in CDMGlc and CDMLac and the other
the EPSs released in CDMGal and CDMSuc (Fig. 4). The peak II frac-
tion was higher in the former group than in the latter.
OH-2 and OH-3 (d = 5.661), (ii)
a-anomeric configuration of the
glucose (d = 4.978 and 5.314), and (iii) b-anomeric configuration
of the glucose (d = 4.510 and 4.725) were identified. All these data
indicated that composition in monosaccharides of L. fermentum
TDS030603 EPS were not dependent on the nature of carbohy-
drates supplemented in the culture medium as was the case for
other Lactobacillus strains (Petry et al., 2000; Torino et al., 2005;
van den Berg et al., 1995).
3.5. Viscosity of EPS
Rheological analysis revealed that the EPS solutions exhibited
pseudoplastic behavior that was typical of aqueous solutions of
high molecular mass biopolymers. Among the EPS solutions, how-
ever, the viscosities were obviously different, especially in the low
shear rate range (Fig. 5). At a shear rate of 10/s, the solution of EPS
released in MRS yielded an apparent viscosity, gapp of 0.88 Pa s.
Compared with this value, only the EPS in CDMGlc had a higher vis-
cosity, gapp of 1.27 Pa s. Other carbohydrates yielded EPSs whose
apparent viscosities at the same shear rate were much lower,
namely 43–65% of that of the EPS produced in MRS.
3.4. Molecular mass of EPS
The molecular masses of the major EPSs (peak I, Fig. 4) released
either in MRS or CDM supplemented with various carbohydrates
showed similar values. Only the major EPS produced in CDMGal
I II
III
1 4
14
1 2
12
1 0
10
8
8
6
6
3.6. Possible determinant of the viscosity of EPS solution
4
4
MRS
2
2
In contrast to the effect on chemical structure of EPS, carbohy-
drates had a significant influence on its viscosity (Fig. 5). Generally,
the viscosity of EPS is affected by (i) electrostatic interactions be-
tween charged residues, (ii) entanglement of long sugar chains,
and (iii) the effect of branching. The electrostatic interaction is
not the case for L. fermentum TDS030603 producing EPS, since it
was revealed to be a neutral polysaccharide (Leo et al., 2007). 1H
NMR and monosaccharide composition analysis demonstrated that
the chemical structures of the EPSs were very similar (Figs. 2 and
3). Furthermore, no correlation was observed between the viscos-
ity of EPS solution and the monosaccharide composition ratios of
EPS; therefore variations in monosaccharide composition are not
a cause of the variations in viscosity. Although no significant vari-
ations could be found in the chemical structures of the EPSs, some
0
0
0
5
1 0
1 5
2 0
2 5
3 0
1 2
12
1 0
10
8
8
6
6
4
4
2
2
CDMGlc
0
0
0
5
1 0
1 5
2 0
2 5
3 0
- 2
-2
1 2
12
1 0
10
8
8
6
6
4
4
2
2
CDMGal
0
0
0
5
1 0
1 5
2 0
2 5
3 0
1 0
10
10
8
8
6
6
4
4
2
2
CDMLac
1
0
0
0
5
1 0
1 5
2 0
2 5
3 0
- 2
-2
8
8
6
6
4
0.1
0.01
4
2
2
CDMSuc
0
0
- 2
-2
0
0
5
5
1 0
10
1 5
15
2 0
20
2 5
25
3 0
30
1
10
100
1000
Retention time (min)
Shear rate(1/s)
Fig. 4. Molecular mass distribution of EPSs produced by L. fermentum TDS030603 in
MRS and CDM supplemented with various carbohydrate sources. One hundred
micrograms of the each purified EPS were used. I, II, and III indicate corresponding
peaks in the chromatograms. The elution of the EPS was monitored by refractive
index of the eluent.
Fig. 5. Viscosity of EPSs produced by L. fermentum TDS030603 in MRS and CDM
supplemented with various carbohydrate sources. Symbols; open circle, EPS
released in MRS; closed circle, EPS released in CDMGlc; open triangle, EPS released
in CDMGal; closed triangle, EPS released in CDMLac; cross, EPS released in CDMSuc
.

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1045
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(Fig. 4). Even though the major EPS fraction (peak I) in CDMGal had
the highest molecular mass, EPS in CDMGal showed the lowest vis-
cosity. A low molecular mass fraction (peak III) could be found only
in the EPS from MRS indicating that differences in EPS chain length
are unlikely to cause variations in viscosity. In Lactobacillus
rhamnosus R lowering molecular mass of EPS caused a decline of
the viscosity of EPS solution (Pham et al., 2000). Therefore, the vis-
cosity of EPS solution might be affected by the ratio of low molec-
ular mass EPS. A certain relationship between viscosity and
molecular mass distribution of EPS has been observed in peak II
(Fig. 4). Degradation of peak II fraction may cause the lower viscos-
ity of EPS released in CDMGal and CDMSuc. It was difficult to find a
clear relationship between molecular mass distribution and viscos-
ity; nevertheless, heterogeneity of molecular mass distribution
was the most probable cause for the variation of EPS viscosity.
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To conclude, a CDM for L. fermentum TDS030603 has been
developed. The ability of the strain to produce a highly viscous
EPS was observed in the CDM as well as in MRS. The production
of the EPS reaches the maximal concentration at the beginning of
the stationary phase, but degradation of the EPS was observed dur-
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structure of the EPS, but did affect the production yields. Moreover,
the viscosity of the EPS was also affected by carbohydrates, possi-
bly owing to the heterogeneity in the molecular mass distribution
of the EPS.
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We thank Associate Professor H. Koaze and Associate Professor
K. Hironaka of Obihiro University of Agriculture and Veterinary
Medicine, Japan, for giving us much useful technical advice on
the rheological analysis of the EPS produced by L. fermentum
TDS030603. We gratefully acknowledge Professor M. Messer of
The University of Sydney, Australia, for reviewing the manuscript.
This study was supported by research grants from Morinaga-
Housikai Foundation, Yotsuba Milk Product Co., and Global COE
Program ‘‘Frontier Program for Animal Global Health and Hygiene”,
Ministry of Education, Culture, Sports, Science, and Technology,
Japan.
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