帯広畜産大学学術情報リポジトリOAK:Obihiro university Archives of Knowledge

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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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