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帯広畜産大学学術情報リポジトリOAK:Obihiro university Archives of Knowledge
' '
TitleStudies on Exopolysaccharide Produced by
Lactobacillus fermentum TDS030603
Author(s) Shi, Tala
Citation
Issue Date 2009-06
URL http://ir.obihiro.ac.jp/dspace/handle/10322/2451
Rights
Studies on Exopolysaccharide Produced by Lactobacillus fermentum TDS030603
Tala, Shi
June 2009
Master Course of Food and Animal Hygiene,
Graduate school of
Obihiro University of Agriculture and Veterinary Medicine
Contents
Abstract
1. Introduction
1-1. Lactic acid bacteria 1
1-2. Exopolysaccharides produced by microorganism 2
1-3. Classification of exopolysaccharide from LAB 4
1-4. Objectives of this study 5
2. Materials and methods
2-1. Bacterial strain and chemicals 7
2-2. Culture condition for EPS production 7
2-3. Production and isolation of EPS 7
2-4. Estimation of molecular mass distribution of EPS 8
2-5. Analyses of EPS monosaccharide composition 8
2-5-1. Thin layer chromatography 8
2-5-2. High performance liquid chromatography 9
2-6. 1H-NMR spectroscopy 9
2-7. Viscosity measurement 10
3. Results
3-1. Cell growth of L. fermentum TDS030603 in CDM 11
3-2. EPS production in CDM with various carbohydrate sources 11
3-3. Chemical structure of EPS 11
3-4. Molecular mass and monosaccharide composition of EPS 12
3-5. Viscosity of EPS 12
4. Discussion 14
5. Concluding remarks 17
Acknowledgements
Tables and Figures
References
Abstract
A lactic acid bacterium that was capable of producing a neutral hetero
exopolysaccharide (EPS), Lactobacillus fermentum TDS030603, was previously isolated from
a fermented milk product in our laboratory. Related to the cell growth in de
Man-Rogosa-Sharpe (MRS) broth, this strain produced about 100 mg/L of EPS in a purified
form. The EPS was composed of D-glucose and D-galactose as previously reported.
Furthermore, the EPS produced in MRS in this study exhibited the molar ratio of D-glucose to
D-galactose was ranging from 2.6 to 2.8, depending on the media, similar to the previously
reported value. The 1% (w/v) solution of the EPS was a non-Newtonian fluid, which
exhibited pseudoplastic behavior with an apparent viscosity ( app) of 0.88 Pa s at a shear rate
of 10/s. A chemically defined medium (CDM) is of great advantage to assess the effects of
medium components on EPS. In this study we investigated the effects of carbohydrates on the
production, chemical structures, and physicochemical properties of the EPS produced by L.
fermentum TDS030603. Among carbohydrate tested, glucose yielded the highest amount of
EPS (69mg/ml). Analyses using thin-layer chromatography, high performance liquid
chromatography, and 1H-NMR spectroscopy indicated that chemical structures of EPS
produced in MRS and in the CDM supplemented with glucose, galactose, lactose or sucrose
as a carbohydrate source, were substantially identical. All the EPS solutions at a concentration
of 1% (w/v) showed thread-forming properties, however, the viscosity was apparently
distinguishable, probably because of the difference of their molecular mass distributions. Our
results provide significant information on the applicable possibility of the EPS in the food
manufacture.
1
1. Introduction
1-1. Lactic acid bacteria
Lactic acid bacteria (LAB) constitute a group of gram-positive bacteria united by a
constellation of morphological, metabolic, and physiological characteristics.1) The general
description of the bacteria included in the group are gram-positive, nonsporing, nonrespiring
cocci or rods, which produce lactic acid as the major end product during the fermentation on
carbohydrate.2) The LAB term is initially associated with bacteria involved in food and feed
fermentation, including related bacteria normally associated with the (healthy) mucosal
surfaces of humans and animals.3,4) The classification of lactic acid bacteria into different
genera is largely based on morphology, mode of glucose fermentation growth at different
temperatures, configuration of the lactic acid produced, ability to grow at high salt
concentrations, and acid or alkaline tolerance.5) Chemotaxonomic markers such as fatty acid
composition and constituent of the cell wall are also used in classification. The term lactic
acid bacteria were then used to mean “milk-souring organisms”.6) Significantly, the first pure
culture of a bacterium was “bacterium lactis” (probably Lactobacillus lactis) obtained by J.
Liser in 1873.7) The lactic acid bacteria comprised four genera: Lactobacillus, Leuconostoc,
Pediococcus, and Streptococcus. But there has always been controversy as to boundaries of
the group. Proper classification of LAB partially rely on molecular biology methods, although
some of Orla-Jensen’s concepts are still viable.8) This is perhaps more true regarding
classification at the species than at the genus level. In some cases, only an analysis at the
nucleic acid level will resolve classification problems. Some characteristic used in the
phenotypic/biochemical characterization of strains are range of carbohydrates fermented,
arginine hydrolysis, acetain formation, bile tolerance, type of hemolysis, production of
extracellular polysaccharide, growth factor requirements, presence of certain
enzymes(β-galactosidase and β-glucuronidase), growth characteristic in milk, and serological
2
typing.9,10)
The physiology of LAB has been of interest ever since it was recognized that these
bacteria are involved in the acidification of the food and feed products.11) Increased
knowledge of LAB physiology, such as metabolism, nutrient utilization, etc., has been one
way to achieve more controlled processes. LAB are generally associated with habitats rich in
nutrients, such as various food products (milk, meat, vegetables, beverages), but some are also
members of the normal flora of the mouth, intestine, and vagina of mammals.4) All of these
properties are associated with certain LAB strains, and played an important roles for the
industrial and clinical level. Also it endowed LAB an enormous economical value and
scientific attention to this group of microbes. In addition to prophylactic uses, human
therapeutic bacterial preparations may be aimed at special groups, the many kinds of
antimicrobial substances produced by several species and strains of LAB could offer
alternatives to chemical food additives used to control pathogenic or otherwise harmful
contaminants in food, e.g., Listeria, Pseudomonas, Enterobacteriancae, etc.12) In fermented
foods the strains producing antimicrobials could be used themselves, while in other food
products isolated and purified antimicrobial substances could be added. (Table 1).13) Sugar
metabolism is central to the physiology of LAB, as carbohydrates probably represent their
most important source of energy. Therefore the perception of a direct connection between
sugar metabolism and host fitness is the importance of these organisma for human health and
well-being.
1-2. Exopolysaccharides produced by microorganism
Various polymers of plant, animal, and microbial origin are indispensable tools in food
formulations, among which microbial polysaccharide received increased interest in recent
years.14-17) Food polymers are long-chain, high-molecular mass (MM) that dissolve or
disperse in water to give texturizing properties to food products. Also food polymers that are
3
used for their secondary effects which include gel formation, stabilization, emulsification,
suspension of particulates, control of crystallization, inhibition of syneresis, encapsulation,
and film formation.15,17) The majority of food polymers that are nowadays used by the food
industry are polysaccharides from plants. (e.g., starch, cellulose, pectin, locust been gum, and
guar gum) or seaweeds (e.g., alginate, carrageenan, and agar) and animal proteins such as
caseinate and gelatin.18) The functional properties of these polymers are determined by quite
subtle structure characteristic and may not, as native molecules, always meet the required
rheological properties or quality standards. Therefore, these biothickeners are often
chemically modified, which restricts their use as a food polymer.14, 19) Alternative classes of
biothickeners are the microbial exopolysaccharides (EPSs). EPSs occur widely among
bacteria and microalgae and less among yeasts and fungi.16, 20, 21) In general, microbial EPSs
can be defined as extracellular polysaccharides that are either associated with, and often
covalently bound to the cell surface in the form of capsules, or secreted in the environment in
the form of slime.22) They are referred to as capsular polysaccharides (CPSs) or slime EPSs,
23) the terms ESPs, however, maybe used to describe either type of extracellular
polysaccharide. CPSs can be visualized microscopically, while EPSs often result to improve
viscosity of the medium. The roles of EPSs in the bacteria are not clearly defined. The
capsular structure may protect the cell against unfavorable environment conditions such as
desiccation, presence of toxic compounds, low temperature or high osmotic pressures and
may contribute to the uptake of metal ions.24) Moreover, the presence of EPS favors the
interaction between physical supports and bacteria, resulting in the appearance of biofilms.
Food grade bacteria that produce EPSs are an interesting alternative for food uses of
EPSs, in particular lactic acid bacteria,15,17) dairy propionobacteria,25) and bifidobacteria.26,27)
These EPS producing bacteria have a generally recognized as safe (GRAS) status, which is
definitely an advantage in today’s marketing of these polymers.15) Indeed, consumers have an
increasing awareness about the food products they consume, which is reflected by the
increasing demand for 100% natural, safe, and healthy food products that are free for
4
additives, have a low fat or sugar content, and have undergone minimal processing.
Enzymatic treatment of polysaccharide instead of chemical modifications contributes to
natural development of modified structures with enhanced functional properties.28) Another
advantage of EPSs produced by LAB is huge diversity of their structures and, therefore, they
have a broad application potential. The major disadvantage of EPSs produced by LAB is the
low production yield in comparison with xanthan.29) Genetic engineering strategies could
increase the yield and enable the design of novel polysaccharide structures with improved
properties, but the application of generally modified products is difficult for acceptance by
EPS particularly interesting for fermented dairy products to improve their viscosity and
rheology, texture and body, and month feel. 24, 30) Finally, several health claims are associated
with EPSs from LAB such as immunostimulatory and probiotic effects.1, 31) Therefore, EPSs
from LAB have potential for development and exploitation as food additives or functional
food ingredients with both health and economical benefits. Hence, novel microbial
biopolymers may fill gaps in the market available polymers or may replace traditional food
products in terms of improved rheological and stability characteristics. These developments,
however, require a thorough understanding of the structure function relationships and the
biosynthetic mechanisms (Fig. 1).32)
1-3. Classification of exopolysaccharides from LAB
EPSs from LAB can be subdivided into two groups: (i) the Homo polysaccharide
(HoPSs) that are composed of one type of monosaccharide, and (ii) the Hetero polysaccharide
(HePSs) composed of a repeating unit that contains two or more different monosaccharide.30)
This classification also takes into account the way of biosynthesis, that one is, extracellular,
enzymatic synthesis by the action of one enzyme (a glycansucrase) with sucrose as the sole
precursor in the case of HoPSs. And another is intracellular biosynthesis which through the
assembly of building blocks by the subsequent action of different types of glycosyltransferase
5
enzymes (with sugar nucleotides) as precursor molecules followed their polymerization in the
case of HePSs. HePSs from LAB are produced in a greater variety with regard to
monosaccharide composition, monosaccharide ratio, and molecular structure (monosaccharide
components, ring forms, anomeric configurations, and stereo–and region–specific linkages) of
the repeating units, as well as the conformation and MM of the polymer.4) HePSs are
produced by mesophilic (e.g., L. lactis, L. brevis, L.casei, L. paracasei, L. rhamnosus, L.
sakei) and thermophilic (e.g., L. acidophilus, L. delbrueckii subsp. bulgaricus, L. helveticus, S.
macedonicus, and S. thermophilus). Composition and structure of HePSs are composed of an
almost always branched backbone of repeating units that consist of monosaccharides,
derivatives of monosaccharides, or substituted monosaccharides.17,30) In 1990, the first
repeating units of an HePS produced by S. thermophilus was determined.33) To date, structures
of HePSs from more than 50 LAB strains have been analyzed, yielding 34 unique HePS
repeating units (Table 2).17) The monosaccharides can be present as the - or -anomer and
can be linked in many different ways. The MM of the HePSs produced by LAB varies from
1.0 104 to 6.0 106 as extrapolated from dextran calibration curve.4,12) Some LAB strains
produce high-MM HePSs, while others produce low-MM HePSs. Also variability in the MM
of structurally identical HePSs produced by different strains as well as strains that are capable
of simultaneously producing two HePSs with different MM has been reported.34-36)
1-4. Objectives of this study
A large number of EPS produced by LAB have been reported, but little is known about
the effects of medium components on chemical structures and rheological properties of
EPS.17,24,32,37) Since the complexity of media composition possibly lead to an incorrect
structural analysis of EPS, a chemically defined medium (CDM) is of great advantage to
assess the effects of medium components on EPS.17) Using CDM, to date, it has been shown
that adenine or orotic acid stimulated both the cell growth and the yield of EPS in
6
Lactobacilli.38, 39) Also the variation and the concentration of the carbohydrate source
exhibited significant effects on the EPS yield, but the preference of sugar for the maximum
EPS production was strain-dependent.17,42,43) On the other hand, effects of carbohydrate
source on monosaccharide composition of EPS are unclear: the constitutive monosaccharides
were found to be the same in L. helveticus by altering carbohydrate source, but the relative
proportion of the individual monosaccharide varied in L. delbrueckii subsp. bulgaricus.39,40)
The rheological properties of the EPS produced by LAB are attributed to its molecular mass,
molecular mass distribution, constituent sugar residues, linkage between the sugar monomers
and the presence of side groups.41) However, effects of altered medium composition on
rheological properties of EPS have been scarcely reported.
In the present work, we aimed to evaluate the effects of carbohydrate source on the
yield, chemical structure and viscosity of a neutral hetero EPS produced by L. fermentum
TDS030603. In this context, we use the new CDM for the strain modifying the previously
reported media.42) That has developed in our laboratory. From the CDM supplemented with
various carbohydrate sources, EPS was successfully produced, and it was easily purified by a
one-step batch method. The production of EPS by L. fermentum TDS030603 was growth
related as reported before for other thermophilic LAB.38,39) The chemical structure and
viscosity of the EPS produced in the CDM were evaluated, using the EPS in MRS as a
reference. We have found that the variation of carbohydrate source affected not the chemical
structure, but the viscosity of the EPS, especially at low shear rates. Possible determinant that
leads the rheological variation of EPS will also be discussed.
7
2. Materials and methods
2-1. Bacterial strain and chemicals
L. fermentum TDS030603 was obtained from the bacterial collection in our
laboratory.43) The strain was stored until used at -80 C in MRS broth containing 20% (v/v)
glycerol. MRS (deMan-Rogosa-Sharp) was from Oxoid (Cambridge, UK). DEAE-Sephadex
A-50 and Toyopearl HW-55F were from GE Healthcare (Buckinghamshire, UK) and Tosoh
(Tokyo, Japan), respectively. D2O (99.99% atom %D) was from Sigma-Aldrich (St. Louis,
USA). All the chemicals used were analytical grade.
2-2. Culture condition for EPS production
The composition of the chemically defined medium used in this study was summarized
in Table 3. After static culture in MRS for 24 h at 30 C under aerobic condition, cells were
harvested, washed thoroughly by sterilized phosphate buffered saline (PBS), and inoculated in
fresh MRS or CDM to be 0.2 of the optical density (OD) at 600 nm. Cell growth (OD600nm)
and pH of the static culture were monitored. Simultaneously, viable cell number was counted
on MRS-agar plates. At 12, 24, 48, and 72 h after inoculation, a 1-ml aliquot of culture media
was collected, diluted with MRS and spread on a MRS-agar plate. The plate was incubated at
30 C for 48 h under anaerobic condition. Colonies appeared were counted as viable cells.
2-3. Production and isolation of EPS
After 72 h cultivation in a 1-liter CDM at 30 C, cells were removed by centrifugation
(17,000 g, 1 h, 25 C). Crude EPS was precipitated by addition of equal volume of ice-cold
ethanol to the culture supernatant. The ethanol precipitant was collected by centrifugation
8
(17,000 g, 30 min, 4 C), dissolved thoroughly in 300 ml of ion-exchange water and
dialyzed against running tap water for 2 d and then lyophilized. In order to remove protein
contaminants, this lyophilized crude EPS was dissolved in 100 ml of 50 mM Tris-HCl (pH
8.7), and it was purified by a batch method using a 200-ml slurry of DEAE-Sephadex A-50
equilibrated with the same buffer. A non-adsorbed fraction was collected, and lyophilized. The
crude EPS produced in MRS was further purified by gel-filtration on a Toyopearl HW-55F
column (2.6 100 cm, 15 ml/h) equilibrated with water, in order to remove mannan derived
from yeast cell-wall in MRS. The purified EPSs were thoroughly dialyzed against water, and
lyophilized.
2-4. Estimation of molecular mass distribution of EPS
The molecular mass and distribution of EPS were estimated by a high performance
liquid chromatography (HPLC). The purified EPS was dissolved in water (1 mg/ml), and 100
l of this solution was loaded onto a TSK gel 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).
Shodex standard P-82 (Showa Denko, Tokyo, Japan), a series of pullulans with known
molecular weight ranging from 0.59 104 to 7.88 105 Da, were used as the standard.
2-5. Analyses of EPS monosaccharide composition
2-5-1. Thin layer chromatography
The purified EPS (2 mg) was hydrolyzed in 250 l of 2 M trifluoroacetic acid (TFA) at
100 C for 5 h. Excess TFA was removed by rotary evaporation, and the hydrolysate was
washed thoroughly by water, and lyophilized. This lyophilized powder was dissolved in 100
9
l of water, and a 5- l aliquot was used for thin-layer chromatography (TLC). Development
was done twice on a silica gel TLC plate (20 20 cm) with a developing solvent of
n-butanol:ethanol:water (2:1:1, v/v). Carbohydrates were visualized by heating the TLC plate
after sprayed with 5% (v/v) sulfuric acid in ethanol. Glucose, galactose, and mannose were
used as standard monosaccharides.
2-5-2. High performance liquid chromatography
Prior to the high performance liquid chromatography (HPLC) analysis, the EPS was
hydrolyzed and the hydrolysate was labeled with 2-aminobenzoic acid by a modified method
of Anumula and Dhume.44) For the labeling reaction, labeling reagent A (4% sodium acetate
trihydrate, 2% boric acid, in methanol, w/v) and labeling reagent B (0.32 M 2-aminobenzoic
acid, 1 M sodium cyanoborohydride, in labeling reagent A) were used. The hydrolysate (1.4
mg of EPS) in 70 l of water was mixed with 100 l of labeling reagent B, and the mixture
was heated at 80 C for 50 min. After cooling down to room temperature, an aqueous phase
was collected by centrifugation (900 g, 10 C, 10 min). The solvent was removed by rotary
evaporation, and then the dried sample was dissolved in 500 l of water. The sample was
diluted, and then separated on an ODS-100Z column (4.6 250 mm, Tosoh) at a flow rate of
0.75 ml/min, using 150 mM trisodiumcitrate (pH 4.5)/7.5 % (v/v) acetonitrile as a solvent.
Different concentrations of glucose and galactose, labeled by 2-aminobenzoic acid, were used
as standards. The molar ratio of glucose and galactose was calculated from the peak areas.
The experiment was performed in triplicate.
2-6. 1H-NMR spectroscopy
Exchangeable protons in the purified EPS (2 mg) were replaced by deuterium in
99.99% D2O. Using a 500 MHz FT-NMR spectrometer, Jeol ECP-500 (Jeol, Tokyo, Japan),
10
1H-NMR spectra have been recorded 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. The spectrum was measured by referring to an internal
acetone ( = 2.225), but chemical shifts (ppm) were represented by reference to an internal
sodium 2,2-dimethyl-2-silapentane-5-sulphonate.
2-7. Viscosity measurement
Using a Dynamic Analyzer RDA II (Rheometric Scientific, Piscataway, USA) equipped
with a cone-and-plate attachment (diameter, 25 mm; angle, 0.1 radian; gap, 65 m), the shear
stress of 1% (w/v) solution of the purified EPS was monitored for 300 sec at 22 C up to 300/s,
in a steady shear testing mode. The apparent viscosity, app, was calculated from the shear
stress at a certain point of shear rate.
11
3. Results
3-1. Cell growth of L. fermentum TDS030603 in CDM
In the developed CDM consisting of 42 chemicals, the cell growth, which was
monitored by optical density 600 nm at a stationary phase, was around one-fourth of that in
MRS (Fig. 2A) the viable cell count showed similar profile to the optical density at 600 nm of
the culture broth (Fig. 2B). After incubation, the final pH of MRS broth was around 4.0,
whereas those of CDMs were around 5.0 (Fig. 2C).
3-2. EPS production in MRS and CDM with various carbohydrate sources
EPS production was investigated using MRS and the CDM supplemented with either
glucose (CDMGlc), galactose (CDMGal), lactose (CDMLac), or sucrose (CDMSuc), each at a
concentration of 1% (w/v). Neither cell growth nor EPS production was observed, when
maltose and fructose were used as carbohydrate sources (result not shown). Associated with
cell growth, the EPS was produced in the culture supernatant (Fig. 2D). After 72 h cultivation,
the highest EPS production (97.1 mg/L) was found in MRS. Among the carbohydrates tested,
glucose gave the best EPS production (69.0 mg/L), while lactose, galactose or sucrose yielded
73%, 51% or 19%, respectively (Table 4). But when the strain was cultivated in MRS,
CDMGlc, or CDMGal, the EPS production reach to the highest at 24-48 h, with a slight
decrease of the EPS observed at 72 h (Fig. 2D).
3-3. Chemical structure of EPS
All the EPSs that were produced in MRS and the CDM supplemented with various
carbohydrate sources consisted of glucose and galactose, and no other monosaccharide was
12
detected on a TLC plate (Fig. 3). The 1H-NMR spectrum of each EPS exhibited a very similar
profile (Fig. 4). In all spectra, typical chemical shifts that represent an H-2 signal glucose,
which was substituted at OH-2 and OH-3 ( = 5.661), -anomeric configuration of the
glucose or galactose ( = 4.978 and 5.314) and -anomeric configuration of the glucose or
galactose ( = 4.510 and 4.725) were identified.
3-4. Molecular mass and monosaccharide composition of EPS
The high molecular mass of EPS prevented good estimations, however the molecular
masses of the major EPSs (peak I, Fig. 5) produced in MRS and CDM supplemented with
various carbohydrates showed similar values. Only the major EPS produced in CDMGal
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 at least 106 Da. The EPS produced in MRS contained a lower
molecular mass fraction (peak III, Fig. 5), of which molecular weight was 2.8 104 Da. An
apparent shoulder was observed in the vicinity of peak I (peak II, Fig. 5). The monosaccharide
composition of EPS was investigated by an HPLC experiment (Fig. 6). The molar ratio of
glucose to galactose was ranging from 2.6 to 2.8 (Table 5). This result was similar to the value
of 2.5, which had been previously determined for the EPS produced in MRS.27)
3-5. Viscosity of EPS
The 1% (w/v) solution of each EPS showed a strong ropy character (data not shown).
Analysis using a rheometer revealed that those EPS solutions exhibited pseudoplastic
behavior that was typical for aqueous solution of high molecular mass biopolymers. Among
the EPS solutions, however, the viscosities were obviously different, especially in the low
shear rate range (Fig. 7). At the shear rate of 10/s, the solution of EPS produced in MRS gave
13
the apparent viscosity, app = 0.88 Pa s. Compared to this value, only the EPS in CDMGlc gave
a higher viscosity, app = 1.27 Pa s. Other carbohydrates yielded EPSs whose apparent
viscosities at the same shear rate were much lower, by 43 to 65%, than that of the EPS
produced in MRS.
14
4. Discussion
L. fermentum TDS030603 was initially isolated from a fermented milk product was
found to secrete a relatively high amount of viscous EPS (100 mg/L, in purified form), when
compared to other EPS producing LAB.43) As was expected, the strain grew and produced
EPS in a cheese-whey based medium. Furthermore, the strain grew and maintained ability of
EPS production even in MRS. In order to obtain precise information on EPS production by
the strain, we used a CDM. In the prototype CDM, previously reported as a synthetic medium
of L. helveticus,42) L. fermentum TDS030603 was able to proliferate, and the maximum cell
growth at the stationary phase was about one-fourth of that in MRS (Fig. 2A). According to
the information provided by manufacturer, the MRS contained 2% (w/v) glucose. Since
increased concentration of carbohydrate amount in media often improve EPS production
relative to cell growth,45) the strain exhibited improved proliferation and EPS yield in MRS
comparing to the prototype CDM. A broad requirement of amino acids has been observed in L.
fermentum TDS030603, being coincident with other LAB reported.46-51) On the other hand,
the strain did not require six amino acids, L-alanine, L-cysteine, glycine, L-lysine, L-proline
and L-threonine, indicating that the biosynthetic pathways of these amino acids appeared to be
functionally active in the strain.42) Therefore we used a developed CDM, consisting from 42
chemical compounds (Table 3), in which L. fermentum TDS030603 could proliferated well
under experimental conditions. Using the CDM supplemented with various carbohydrate
sources, we observed a clear relationship between the varied carbohydrate source and the cell
growth. The most efficient carbohydrate for the strain was glucose, and no proliferation has
been observed on maltose and fructose. In our case, EPS production showed clear correlation
to the cell growth in accordance with other EPS producing LAB.38, 39) Thus the EPS yield of
the strain seems to depend, partially at least, on the concentration and the use efficiency of
carbohydrate. There is no consistency in the reported preference of carbohydrate source
regarding EPS production, and thus that seems to be depend on the strains and the medium
15
composition.17,39,40,43) Even the reason why the EPS production yields decreased at 72 h
cultivation is unclear.
The TLC analysis revealed that the constitutive monosaccharides of L. fermentum
TDS030603 EPS were not altered by carbohydrate source (Fig. 3), in agreement with other
Lactobacillus strains.38, 39, 46) This was further supported by 1H-NMR (Fig. 4), and we
concluded that carbohydrate source did not affect the chemical structure of EPS of L.
fermentum TDS030603. By contrast, the carbohydrate source had a significant influence on
the viscosity of EPS (Fig. 7). Generally, viscosity of EPS is affected by (i) electrostatic
interactions of charged residues, (ii) entanglement of long sugar chains, and (iii) effect of
branched residues. 1H-NMR and monosaccharide composition analysis demonstrated that the
chemical structures of the EPSs were substantially identical. Furthermore, no correlation has
been observed between the viscosity and the monosaccharide composition ratio of the EPSs;
therefore, the differences in EPS chain length are unlikely to cause variations in viscosity,
corresponding to the previous report.52) Among the EPSs in CDM, their molecular mass
distributions were clearly divided into two groups: one includes the EPSs in CDMGlc and
CDMLac and the other the EPSs in CDMGal and CDMSuc. The peak II fraction was higher in
the former group than in the later. However, the peak II fraction of the EPS in MRS was not
apparent, thus the fraction might not affect the viscosity variation. It was difficult to clarify
the relationship between molecular mass distribution and viscosity; however heterogeneity of
molecular mass distribution was the most probable cause for the variation of EPS viscosity.
The molar ratio of the repeating unit in the major polysaccharide of our strain producing EPS
was 3:1, whereas that in the minor polysaccharide was 1:1.43) The heterogeneity of the
molecular mass distribution of the EPS might reflect these major and minor components exist
in the EPS.
LAB is widely accepted as GRAS status, and thus the EPS produced by L. fermentum
TDS03603 is potentially useful as a food additive. The viscosity of the EPS is comparable to
that of the xanthan gum produced by Xanthomonas campestris, which is used widely as a
16
viscosifier.29) However the yield of the EPS produced by L. fermentum TDS030603 may
prevent it from becoming commercially available, for which at least 100 times higher
production (ca. 10 g/L) will be required. To achieve the EPS yields in acceptable amount for
its commercial use, optimization of the conditions for EPS production such as growth
temperature, pH control of the culture broth, examination of the appropriate carbon-nitrogen
ratio, exploring substances which enhance EPS production, and so on, is prerequisite.
17
Concluding remarks
A CDM containing different carbohydrate sources medium was used as a culture broth
for L. fermentum TDS030603 strain. The ability of the strain to produce highly viscous EPS
was observed in the CDM as well as in MRS. The production of the EPS was growth
associated, but degradation of the EPS was observed during 72 h cultivation. The variation of
the carbohydrate source is unlikely to influence the production yield per cell and the chemical
structure of the EPS. Moreover the viscosity of the EPS was also affected by carbohydrates,
possibly owing to the heterogeneity in the molecular mass distribution of the EPS.
18
Acknowledgements
This work was carried out at the Department of Food Hygiene, Obihiro
University of Agriculture and Veterinary Medicine; it was one of the Graduate School
Programme of Food and Animal hygiene. Sincerest thanks are due to the following for
their support, encouragement and constructive criticisms made this study possible.
Assist Prof. Kenji Fukuda, Prof. Tadasu Urashima, my supervisors and associate Prof.
Tadashi Nakamura for their excellent guidance. Their continuous encouragement and
kind supports during all stages of the study have been of great importance to the
accomplishment of this work. Also thanks for Prof. Hiroshi Koaze and Ms. Kumi
Yasuda in Division of Food Technology and Biotechnology, Department of Food
Science, Obihiro University of Agriculture and Veterinary Medicine for the technical
advice on the rheological analysis of the EPS produced by L. fermentum TDS030603.
All the students in the laboratory of Dairy Science of this university for their hospitality
and for the informative discussions.
Table 1. Foods and their associated lactic acid bacteriaa
Food types Lactic acid bacteria
Milk and dairy foods
Hard cheese without eye formation Lc. Lactis subsp. cremoris and subsp. lactis
Cottage cheese and cheeses with a few or
small eyes(Edam)
Lc. Lactis subsp. cremoris and subsp.lactis; and
Lc. mesenteroides subsp.cremoris
Cultures butter, buttermilk cheeses with
round eyes (Gouda)
Lc. Lactis subsp. cremoris and subsp. lactis and var.
diacetylactis; and Lc. mesenteroides subsp.cremoris; Lc.lactis
Swiss–type cheeses Lb. delbrueckll subsp. bulgaricus; lb. helvericus
Dairy products in general Lb. brevis; Lb. buchneri; Lb. casei; Lb. paracasei; Lb. fermentum;
Lb. plantarum; LC. subsp. cremoris; Lc.lactis
Fermented milks
–Yougurt Streptococcus thermophilus and Lb.delbrueckii subsp. bulgaricus;
Lc. lactis subsp. diacetylactis
–Acidophilus milk Lb. acidophilus
–Kefir Lb.kefir; Lb. kefiranofaciens
Meats
raw C. divergens; C.piscicola(maltaromicus)
semi–preserved Lb. viridescens (spoilage); Lc. carnosum and Lc. gelidum;
Fermented meat p. acidilactici and p. pentosaceus (inoculated into semi–dry
sausages); Lb. sake; Lb. curvatus; Lb. fraciminis;
fish
Marinated fish products Lb. alimentarius; C. piscicola
Fermented vegetables P. acidilactici and P. pentosaceus; Lb. plantarum; Lb. sake;
Lb. buchneri; Lb. fermentum
Cucumbers, sauerkraut Lc. mesenteroides; Lb. bavaricus; Lb. brevis; Lb. sake;
Lb. plantarum;
Olives Lc. mesenteroides; Lb. pentosus;
Soy sauce Tetragenococcus (Pediococcus) halophilus
Baked goods
Sourdough bread Lb. sanfranciso(wheat and rye sourdough)
Lb. fraciminis; Lb. plantarum
Lb. brevis; Lb. plantarum;
Lb. amylovorus; Lb. reuteri
Wine (malo–lactic fermented ) Lc. oenosc.
aSources of information: (Hammes & Tichaczek, 1994); (Wood&Holzapfel, 1995).
C, Carnobacterium; Lb, Lactobacillus; Lc, Lactococcus; Le, Leuconostoc; P, Pediococcus.
20
Table 2. Heteropolysaccharides produced by lactic acid bacteria
Strain Growth medium EPSpproduction (mg/L culture)
Molecular mass
EPS composition Gal Glc Rha
Repeating unit
Reference
L. casei CG11 BMM 160 1 17 3 Pentamer Robijin et al.1996c
L.caseiCRL87 skim milk 121 7.9×105 1 1.1 Mozzi et al.1992
L.bulgaricusCNRZ416 Skim milk 285 4.9×105 4 1 1 Cerning et al.1986
L.bulgaricusNCFB2772 CDM 37 1.5× 106 6.8 1 0.7 Grobben rt al.1995
L.Bulgaricus0ll1037R-1 skim milk 58 1.2 ×106 3 2 Uemma et al.1998
L. bulgaricusII SDM 354 5 1 1 heptamer Gruter et al.1993
L. helveticus TN 4 skim milk 180 1.8× 106 1 1 hexamer Yamamoto et al. 1995
L. helveticus Lb 59 skim milk 272 2.0× 106 1 1 hexamer Stingele et al.1997
L.helveticusTY1-2 skim milk 200 1.6× 106 2.8 3 heptamer Yamamoto et al. 1994
L.paracasei34-1 SDM 3 tetramer Robijin et al.1996a
L.lactis ssp. cremorisSBT0495
whey medium 150 1.7×106 2 2 1 pentamer Nakajima et al.1990
S.thermophilus CNCMI 733
skim milk 42 1.0× 106 2 1 tetramer Doco et al.1990
S.thermophilusSFi12 SkimKmilk+amino acid
105 2.0×106 3 1 2 hexamer Lemoine et al. 1997
S.thermophilusRs skim milk 135 2.6×106 5 2 heptamer Faber et al. 1998
BMM: basal minimum medium; CDM: chemically defined medium; SDM: semi-defined medium; Gal: galactose; Glc: glucose; Rha: rhamnose
Table 3. Chemical composition of the CDM for L. fermentum TDS030603
D-Glucose 10.0a
DL-Alanine 0.2b
L-Arginine 0.1a
L-Aspartic acid 0.1b
L-Glutamic acid 0.2a
L-Histidine 0.1a
L-Isoleucine 0.1a
L-Leucine 0.1a
L-Methionine 0.1a
L-Phenylalanine 0.1a
L-Serine 0.1b
L-Tryptophan 0.1a
L-Tyrosine 0.1a
L-Valine 0.1a
p-Aminobenzoic acid 0.002b
Biotin 0.00001a
Folic acid 0.0001b
Nicotinamide 0.001b
Nicotinic acid 0.001b
Pantotheic acid 0.002a
Pyridoxal 0.002a
Pyridoxol 0.001b
Riboflavin 0.0002b
Adenine 0.01a
Guanine 0.01b
Thymine 0.005a
Uracil 0.01a
Xanthine 0.01b
Adenylic acid 0.02a
Cytidylic acid 0.05a
2’-Deoxyguanosine 0.01a
Ammonium citrate 1.0a
Sodium acetate 6.0b
Sodium citrate 0.5b
Sodium thioglycolate 0.5b
FeSO4 7H2O 0.02b
K2HPO4 3.0a
KH2PO4 3.0a
MgSO4 7H2O 0.5b
MnSO4 5H2O 0.2a
Spermidine phosphate 0.005b
Tween80 1.0a
Components Concentration(g/L)
D-Glucose 10.0a
DL-Alanine 0.2b
L-Arginine 0.1a
L-Aspartic acid 0.1b
L-Glutamic acid 0.2a
L-Histidine 0.1a
L-Isoleucine 0.1a
L-Leucine 0.1a
L-Methionine 0.1a
L-Phenylalanine 0.1a
L-Serine 0.1b
L-Tryptophan 0.1a
L-Tyrosine 0.1a
L-Valine 0.1a
p-Aminobenzoic acid 0.002b
Biotin 0.00001a
Folic acid 0.0001b
Nicotinamide 0.001b
Nicotinic acid 0.001b
Pantotheic acid 0.002a
Pyridoxal 0.002a
Pyridoxol 0.001b
Riboflavin 0.0002b
Adenine 0.01a
Guanine 0.01b
Thymine 0.005a
Uracil 0.01a
Xanthine 0.01b
Adenylic acid 0.02a
Cytidylic acid 0.05a
2’-Deoxyguanosine 0.01a
Ammonium citrate 1.0a
Sodium acetate 6.0b
Sodium citrate 0.5b
Sodium thioglycolate 0.5b
FeSO4 7H2O 0.02b
K2HPO4 3.0a
KH2PO4 3.0a
MgSO4 7H2O 0.5b
MnSO4 5H2O 0.2a
Spermidine phosphate 0.005b
Tween80 1.0a
Components Concentration(g/L)
aEssential
bNot essential but important
Media OD600nm Viable cell count Crude EPS Purified EPS
(72 h cultivation) (log10cfu/ml) (mg/L)
MRS 3.96 9.6 462 97.1
CDMGlc 0.3 7.78 122 69
CDMGal 0.52 7.89 83.3 34.9
CDMLac 1.06 7.98 84.9 50.2
CDMSuc 1.19 8.7 43.2 13
Table 4. Cell growth and EPS production of L. fermentum TDS030603 in MRS
broth and CDM supplemented with various carbohydrate sources
MRS 2.6 0.05
CDMGlc 2.7 0
CDMGal 2.6 0.03
CDMLac 2.6 0.02
CDMSuc 2.8 0
Media Molar ratio
(Glucose:Galactose)
MRS 2.6 0.05
CDMGlc 2.7 0
CDMGal 2.6 0.03
CDMLac 2.6 0.02
CDMSuc 2.8 0
Media Molar ratio
(Glucose:Galactose)
Table 5. Monosaccharide composition of L. fermentum
TDS030603 EPS in MRS and CDM supplemented with
various carbohydrate sources
Fig. 1. Primary structure of EPS produced by LAB: (1) Homopolysaccharide: A, dextran; B,
Levan; C, polygalactan; (2) heteropolysaccharide from mesophilic LAB: D, L. lactis subsp.
cremoris SBT0495; E, Lb. sake 0-1; F, Lb. paracesei 34-1; and (3) hereopolysaccharide from
thermophilic LAB: G, Lb. acidophilus LMG9433; H, Lb. delbrueckii subsp. bulgaricus rr; I, Lb.
helveticus NCDD766; J, Lb. helveticus TY 1-2 and K, its mutant TN-4; L, S. thremophilus Sc6;
M, S. thermophilus OR901; N, S. thermophilus Sc32; O, S. thermopiles Ac12. Glc, glucose;
Gal, galactose; Rha, rhamnose; GlcNAc, N-acetyl-glucosamine; GalNAc,
N-acetyl-galactosamine; Ac, acetyl. TheD-(D) and L-(L) concguration, and pyranose(p) and
fruanose (f) structure are indicated.
(A) (B)
(C) (D)
Opt
ical
den
sity
at 6
00 n
m
Cul
tiva
ble
cell
s (l
og 1
0cfu
/ml)
pH
Cultivation time (h)Cultivation time (h)
7
8
9
10
11
0 20 40 60 80
3
4
5
6
7
0 20 40 60 80
EPS
in p
urif
ied
form
(m
g/L
)0
1
2
3
4
5
0 20 40 60 80
0
40
80
120
160
0 20 40 60 80
(A) (B)
(C) (D)
Opt
ical
den
sity
at 6
00 n
m
Cul
tiva
ble
cell
s (l
og 1
0cfu
/ml)
pH
Cultivation time (h)Cultivation time (h)
7
8
9
10
11
0 20 40 60 80
3
4
5
6
7
0 20 40 60 80
EPS
in p
urif
ied
form
(m
g/L
)0
1
2
3
4
5
0 20 40 60 80
0
40
80
120
160
0 20 40 60 80
Fig. 2. 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.
Standard EPS
CDMGlc CDMGal CDMLac CDMSuc MRSGlc Gal Man
Standard EPS
CDMGlc CDMGal CDMLac CDMSuc MRSGlc Gal Man
Fig. 3. 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 standard. MRS, EPS produced in
MRS; CDMGlc, EPS in glucose supplemented CDM; CDMGal, EPS in galactose
supplemented CDM; CDMLac, EPS in lactose supplemented CDM; CDMSuc, EPS in
sucrose supplemented CDM.
6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0
Chemical shift (ppm)
MRS
CDMGlc
CDMGal
CDMLac
CDMSuc
H-2 -anomer -anomer
D2O Acetone
6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0
Chemical shift (ppm)
MRS
CDMGlc
CDMGal
CDMLac
CDMSuc
6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.06.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0
Chemical shift (ppm)
MRS
CDMGlc
CDMGal
CDMLac
CDMSuc
H-2 -anomer -anomer
D2O Acetone
Fig. 4. NMR spectra of EPSs produced by L. fermentum TDS030603 in MRS and
the CDM supplemented with various carbohydrate sources. Chemical shifts
derived from H-2 substitution ( = 5.661), -anomer ( = 4.978 and 5.314) and
-anomer ( = 4.510 and 4.725) were observed. Chemical shift of D2O and
acetone was = 4.348 and 2.225, respectively.
0
2
4
6
8
1 0
1 2
1 4
0 5 1 0 1 5 2 0 2 5 3 0
- 2
0
2
4
6
8
1 0
1 2
0 5 1 0 1 5 2 0 2 5 3 0
0
2
4
6
8
1 0
1 2
0 5 1 0 1 5 2 0 2 5 3 0
- 2
0
2
4
6
8
1 0
0 5 1 0 1 5 2 0 2 5 3 0
- 2
0
2
4
6
8
0 5 1 0 1 5 2 0 2 5 3 00 5 10 15 20 25 30
Retention time (min)
141210
86420
121086420
-2
1086420
12
10
8
6
4
20
-28
6
4
2
0
-2
MRS
CDMGlc
CDMGal
CDMLac
CDMSuc
Vol
tage
(m
V)
I II III
0
2
4
6
8
1 0
1 2
1 4
0 5 1 0 1 5 2 0 2 5 3 0
- 2
0
2
4
6
8
1 0
1 2
0 5 1 0 1 5 2 0 2 5 3 0
0
2
4
6
8
1 0
1 2
0 5 1 0 1 5 2 0 2 5 3 0
- 2
0
2
4
6
8
1 0
0 5 1 0 1 5 2 0 2 5 3 0
- 2
0
2
4
6
8
0 5 1 0 1 5 2 0 2 5 3 00 5 10 15 20 25 30
Retention time (min)
141210
86420
121086420
-2
1086420
12
10
8
6
4
20
-28
6
4
2
0
-2
MRS
CDMGlc
CDMGal
CDMLac
CDMSuc
Vol
tage
(m
V)
I II III
Fig. 5. Molecular mass distribution of EPSs produced by L. fermentum TDS030603
in MRS and CDM supplemented with various carbohydrate sources. 100 g 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.
18.24, 3929
23.98, 84513
26.56, 412106
28.81, 359159
30.80, 73752
32.73, 110170
33.97, 5204
35.65, 53136387
41.99, 13020
49.61, 20255689
59.43, 48932
63.93, 3440
0 10 20 30 40 50 60
保持時間(min)
0
200
400
600
800
1000
1200
1400
信号強度(mV)
12.95, 5520
17.43, 31764
18.93, 4739
20.81, 29341
21.61, 53648
22.99, 29764
25.53, 250305
27.01, 564948
28.76, 631671
31.19, 100866
31.92, 57210
33.17, 58534060
35.68, 83200
38.43, 17641
41.76, 39420
49.61, 33571750
55.21, 11690
58.08, 83500
62.80, 144674
0 10 20 30 40 50 60
保持時間(min)
0
200
400
600
800
1000
1200
1400
信号強度(mV)
14.09, 8278
21.37, 12430
24.11, 6350
25.64, 222782
27.32, 1002084
33.17, 8343367
35.67, 28201126
41.93, 3552
49.61, 30069945
57.60, 14701
63.52, 120390
0 10 20 30 40 50 60
保持時間(min)
0
200
400
600
800
1000
1200
1400
信号強度(mV)
Fig. 6. Monosaccharide ratio of EPS produced by L. fermentum TDS030603 in MRS
and the CDM supplemented with various carbohydrate sources. (A), glucose; (B),
galactose; (C), EPS produced in CDM supplemented with glucose.
(A)
(B)
(C) Retention time (min)
Retention time (min)
Retention time (min)
Sig
nal i
nten
sity
(m
V)
Sig
nal i
nten
sity
(m
V)
Sig
nal i
nten
sity
(m
V)
Fig. 7. Viscosity of EPSs produced by L. fermentum TDS030603 in MRS and the
CDM supplemented with various carbohydrate sources. Symbols; open circle, EPS
produced in MRS; closed circle, EPS in CDMGlc; open triangle, EPS in CDMGal;
closed triangle, EPS in CDMLac; cross, EPS in CDMSuc.
0 50 100 150 200 250 3000
0.5
1.0
1.5
2.0
2.5
3.0
Shear rate (1/s)
Vis
cosi
ty (
Pa
s)
0 50 100 150 200 250 3000
0.5
1.0
1.5
2.0
2.5
3.0
Shear rate (1/s)
Vis
cosi
ty (
Pa
s)
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