2008 - Physicochemical Properties of Exopolysaccharide Produced by Lac to Bacillus Kefiranofaciens ZW3 Isolated From Tibet Kefir

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International Journal of Biological Macromolecules 43 (2008) 283–288

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / i j b i o m a c

Physicochemical properties of exopolysaccharide produced by Lactobacillus

kefiranofaciens ZW3 isolated from Tibet kefir

Yanping Wang∗, Zaheer Ahmed, Wu Feng, Chao Li, Shiying Song

Tianjin Key Laboratory of Food Nutrition and Safety, Faculty of Food Engineering and Biotechnology, Tianjin University of Science and Technology,

No. 29, 13th street at TEAD, Tianjin 300457, PR China

a r t i c l e i n f o

Article history:

Received 26 April 2008

Received in revised form 21 June 2008

Accepted 23 June 2008

Available online 9 July 2008

Keywords:

Physicochemical

Properties

Exopolysaccharide

Lactobacillus kefiranofaciens ZW3

a b s t r a c t

An exopolysaccharide (EPS) producing strain, ZW3, was isolated from Tibet kefir grain and was identi-

fied as Lactobacillus kefiranofaciens. FT-IR spectroscopy revealed the presence of carboxyl, hydroxyl, and

amide groups, which correspond to a typical heteropolymeric polysaccharide. The GC analysis of ZW3

EPS revealed that it was glucogalactan in nature. Exopolymer showed similar flocculation stability like

xanthan gum but better than guar gum with a melting point of 93.38 ◦C which is lower than xanthan

gum (153.4◦C) and guar gum (490.11 ◦C). Compared with other commercially available hydrocolloids like

xanthan gum, guar gum and locust gum ZW3 EPS showed much better emulsifying capability.

© 2008 Elsevier B.V. All rights reserved.

1. Introduction

The increased demand for natural polymers for various indus-

trial applications in recent years has led to a renewed interest

in exopolysaccharide (EPS) production by microorganisms. Many

microorganisms including lactic acid bacteria, algae, fungi and

plants have an ability to synthesize extracellular polysaccha-

rides and excrete them out of cell either as soluble or insoluble

polymers [1–3]. The exopolysaccharides produced by microorgan-

isms are widely used in the food, pharmaceutical and chemical

industries, and function as bioflocculants, bioabsorbents, heavy

metal removal agents, drug delivery agents, etc. [4,2,5]. Exam-

ples of industrially important microbial exopolysaccharides are

dextrans, xanthan, gellan, pullulan, yeast glucans and bacte-

rial alginates [6]. Polysaccharides of microbial origin have been

developed as food additive including xanthan from Xanthomonas

campestris [7] and gellan from Pseudomona elodea [8]. How-ever physical properties of these polymers are such that they

are not suited for all applications and there is a demand for

novel materials that gives improved rheological characteristic

[9].

Lactic acid group of bacteria (LAB) which excretes polysac-

charide of elevated molecular weight (MW) has been studied

extensively during the last decade [6,10–12]. Their particular phys-

∗ Corresponding author. Tel.: +86 22 60601400; fax: +86 22 60601478.

E-mail address: [email protected] (Y. Wang).

ical and rheological properties, which make them suitable as

viscosifying, stabilizing, gelling, or emulsifying agents, in com-

bination with the GRAS (generally recognized as safe) status of

EPS-producing lactic acid bacteria, make EPSs promising as a new

generation of food thickeners [13,14,5].

Lactobacillus kefiranofaciens, which was isolated from kefir

grains and used as the starter Caucasian cultured milk, produces

an exopolysaccharidecalled kefiran [15]. Various isolates have been

reported and described as Lactobacillus kefir [14], L. kefiranofaciens

[16], Lactobacillus sp. KPB-167B [17], L. kefirgranum and L. parakefir

[18]. This non-exhaustive listing indicates that the complex taxo-

nomic relationships among the bacterial species of kefir have not

been completely explored. In addition, the influence of the geo-

graphical origin of kefir grains is also to be taken into account

[19–21]. Kefiran, is a water-soluble glucogalactan, which has been

reported to have antibacterial and antitumour activity, modulates

gut immune system and protects epithelial cells against Bacilluscereus exocelullar factors [22,17,23–26]. Kefiran also can be used

as a food grade additive for fermented product since it enhances

the rheological properties of chemically acidified skim milk gels

increasing their apparent viscosity and the storage and loss mod-

ulus of these gels. This phenomenon was strengthened by the

previous heat treatment usually applied for yogurts manufacture

[27]. However, the physicochemical properties of the exopolysac-

charide from this strain have not been completely studied yet.

In this study we reported some physicochemical properties of

exopolysaccharideproduced by L. kefiranofaciensZW3isolatedfrom

Tibet kefir such as thermal stability, emulsifying capability, floc-

0141-8130/$ – see front matter © 2008 Elsevier B.V. All rights reserved.

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284 Y. Wang et al. / International Journal of Biological Macromolecules 43 (2008) 283–288

culating activity and FT-IR spectra which previously had not been

reported yet. The ZW3 EPS proved to be having good emulsion sta-

bility and flocculating activity as compared to other commercially

available gums.

2. Experimental

2.1. Kefir grain

Kefir grain was taken from Tibet, China and was preserved by

our laboratory and propagated at 25 ◦C.

2.2. Media

Media used were supplemented MRS, supplemented M17 and

supplemented whey. Whey medium were prepared as described

by Yokoi et al. [28], with some modification. Supplemented whey

medium contained100 of milkwhey,1 g lactose monohydrate, 0.5g

glucose, 0.5 g tryptone, 0.05g cysteine monohydrochloride, 0.5 g

sodium acetate, 0.1 ml Tween 80, 1 ml mineral solution, and 2 g

agar. The mineral solution was composed of 0.4g/l of MgSO4·7H2O,

0.15g/l of MnSO4·4H2O, 0.18 g/l of FeSO4·7H2O, and 0.1g/l NaCl.Milk whey used in the agar media was prepared by the filtration

of skim milk, which was adjusted to pH 5.5 with lactic acid and

heated for 30min at 100 ◦C. Milk whey used in liquid whey media

was deproteinized by adjusting skim milk to pH 4.6 with 2N HCl,

heatingfor30minat100 ◦C, andfiltering. The resulting supernatant

was adjusted to pH 6.8 with 2N NaOH, heated for 30 min at 100 ◦C,

and filtered to obtain deproteinized whey. Supplemented MRS

broth medium was prepared by addition of following components

in commercial MRS broth (Oxoid) medium: 5 mM CaCl2, 0.04%

MnSO4·4H2O, 0.07% MgSO4 and % lactose monohydrate, where as

M17 (Oxoid) broth mediawas supplemented with0.5% glucose and

1%lactoseonly.Thepurposeofusingagarwheymediawastoisolate

ropy strains, where as supplemented liquid whey, supplemented

MRS and supplemented M17 media were aimed at exopolysaccha-ride production. The final pH of each medium was adjusted to 6.2,

and was subsequently autoclaved at 115 ◦C for 20min.

2.3. Screening of the isolates for EPS production

The kefirgrainswashedwith sterile distilledwaterwere homog-

enized with a Waring blender. For isolation, 1 ml of homogenized

and serially diluted in salt solution kefir grain was plated on whey

agar medium. After incubation at 30 ◦C for 7–9 days in an anaero-

bic atmosphere with a GasPack filled with a gas mixture consisting

of 80% N2, 10% CO2 and 10% H2 (v/v), ropy bacteria were isolated.

EPS-producing LAB strains were first screened according to the

stickiness and ropiness characteristics of their colonies. Follow-

ing primitively screened isolates were inoculated into 50 ml liquidwhey medium in the screw-cappedbottleswith an inoculation per-

centage of 2%. The tightly capped bottles were incubated at 30 ◦C

for 24–72h in anaerobic conditions. Afterthe broth wascentrifuged

at 12,000× g for30 min, a certain volume of supernatant was taken

to dialysis through 10 kDamembrane against distilled water at 4 ◦C

for 72 h with 2–4 changes per day. Exopolysaccharide productions

of differentstrainswere thendetermined by phenol–sulphuric acid

method [30] until no single sugar was detected in distilled water.

2.4. Identification of strain ZW3

The strain ZW3 was identified by using Gram stain reac-

tion, catalase reaction; ability to grow at 15, 37 and 45 ◦C,

gas production from glucose, arginine hydrolysis and by sugar

fermentation which included: l-arabinose, ribose, d-xylose, galac-

tose, d-fructose, mannitol, sorbitol, cellobiose, maltose, lactose,

mélibiose, saccharose, tréhalose, and d-raffinose. The strain identi-

fication wasalso confirmed by partially sequencing 16S rRNA genes

analysis. A primer pair, P1 (5-GAGTTTGATCCTGG CTCAG-3) and P2

(5-TACCGCGGCTGCTGGCAC-3), corresponding to positions 8–28

and 542–549 of the 16S rDNA, respectively was used to clone 16S

rRNA genes of target isolate. Total chromosomal DNA from MRS(Oxoid) 48h broth culture was extracted as described by Forsman

and Alatossava [29]. DNA fragments were amplified as follows:

initial denaturation at 94 ◦C for 10 min, followed by 30 cycles con-

sisting of denaturation at 94 ◦C for 30 s, annealing at 58◦C for 30s,

extension at 72 ◦C for 1 min and a 10-min final extension step at

72 ◦C. PCR product was checked with agarose gel electrophoresis.

Amplified productsof about 550bp in length afterverificationwere

sequenced by using DNA sequencing kit (Shanghai Sangon Biolog-

ical Engineering Technology & Services Co. Ltd.). The nucleotide

sequences were usedfor theanalysis of sequencesimilarity through

BLAST (http://www.ncbi.nlm.nih.gov/blast ).

2.5. Isolation and purification of EPS

The EPS was purified by using method of García-Garibay and

Marshall [7], with some modification. The strain ZW3 was grown

in 500ml liquid whey medium in Erlenmeyer flaks at 30 ◦C for

72h in anaerobic conditions. The flasks were taken out and heat

at 100 ◦C for 30 min to dissolve cell attached EPS and subsequently

centrifuged at 12,000 rpm for 15min. Crude EPS was precipitated

by the addition of an equal volume of chilled absolute ethanol to

the supernatant fluid.After overnight precipitationat 4 ◦C,thesam-

ple was centrifuged, at above given conditions, and the pellet was

retained. The sample was redissolved in distilled water (100 ml)

with gentle heating (less than 50 ◦C) and the EPS was recovered by

precipitation on the addition of an equal volume of chilled abso-

lute ethanol. The sample was centrifuged at 25,000× g for 25 min

at 4 ◦C. The resulting EPS pellet was redissolved in not more than

20 ml of distilled water with gentle heating (less than 50◦C) andthen small neutral sugars were removed by dialysis, for 72h at 4 ◦C,

against threechangesof distilled water perday. Thecontents of the

dialysis bag were freeze-dried to provide EPS. This EPS was named

as partially purified EPS and was later on used to study physical

characteristic.Partially purified EPS wasfurther purified by dissolv-

ing it in 14% trichloroacetic acid (TCA) and stirred over night. The

precipitated protein was removed by centrifugation at 12,000 × g

for 15min. The resulted supernatant was adjusted to pH 7 and EPS

was precipitated by putting an equal volume of chilled ethanol at

−21◦C. The pellet was dissolved in double distilled water and was

lyphophilized.

2.6. Study of infrared (FT-IR) spectroscopy

The major structural groups of the purified EPS were

detected using Fourier-transformed infrared spectroscopy. For FT-

IR spectrum of ZW3 EPS was obtained using KBr method. The

polysaccharide samples were pressed into KBr pellets at sam-

ple: KBr ratio 1:100. The Fourier transform-infrared spectra were

recordedon a BrukerVector 22 instrument (Germany)in the region

of 4000–400cm−1, at a resolution of 4cm−1 and processed by

Bruker OPUS software.

2.7. Sugar composition

For sugar composition determinations, polysaccharides were

hydrolyzedby treatment with 2 M TFA (120◦C for 2 h); the released

sugars were converted to their alditol acetates and analyzed by

http://www.ncbi.nlm.nih.gov/blast




Y. Wang et al. / International Journal of Biological Macromolecules 43 (2008) 283–288 285

GC–MS. The standard alditol acetates were generated by sub-

jecting an intimate mixture of equal proportions of rhamnose,

glucose, ribose, arabinose, xylose, mannose, glucose and galac-

tose to the same experimental conditions that were applied to the

polysaccharide The composition of exopolysaccharide was deter-

mined by comparison of retention time of different peaks of alditol

acetates with mixture standard alditol acetates. Analysis was per-

formed by using a Varian GC/MS 4000 instrument (USA) equippedwith VF-5ms 30m ×0.25mm×0.10m column under following

conditions—injector temperature: 320 ◦C, split ratio: 10, column

flow: 1 ml/min, carrier gas: He 99.999%, column oven temperature:

150◦C (hold time 2 min) to 300◦C (hold time 2 min) reached via a

rising gradient of 10◦Cmin−1 and ionization: EI scan type full.

2.8. Analysis of thermal properties

The thermal properties of EPS were analyzed by using a differ-

ential scanning calorimeter (DSC Model 141 SETARAM Scientific &

Industrial Equipment Co Limited, France). After placing 4.2 mg of

dried EPS sample in an aluminium pan, it was sealed and analyzed,

using empty pan as a reference, for determining the melting point

and enthalpy change. The heating rate was 10◦Cmin−1 from 20 to

300 ◦C.

2.9. Emulsion stability

The emulsifying activity of EPS was assayed as described by

Bramhachari et al. [31]. Lyophilized EPS (0.5 mg) was dissolved in

0.5 ml deionized water by heating at 100◦C for about 15–20min

and allowed to cool to room temperature (25 ◦C). The volume was

then made up to 2 ml using phosphate-buffered saline (PBS). The

sample was vortexed for 1 min after the addition of 0.5 ml hex-

adecane. The absorbance at 540 nm was read immediately before

and after vortexing ( A0). The fall in absorbance was recorded after

incubation at room temperature for 30 and 60 min ( At ). A control

was run simultaneously with 2 ml PBS and 0.5 ml hexadecane. The

emulsification activity was expressed as the percentage retentionof emulsion during incubation for time t : At / A0 ×100.

2.10. Flocculating activity

The flocculating activity was measured by using the method as

described by Lim et al. [32]. Charcoal-activated carbon that was

used as a testing material was suspended in deionized water at a

concentration of 5 g/l. In a test tube, 10 ml of a charcoal-activated

carbonsuspension was added and mixed with 0.1 ml of CaCl2 solu-

tion (6.8 mM). To this mixture, various amounts of EPS were added

and vortexed for 30s and allowed to stand for 10 min at room tem-

perature. The turbidities of the upper 1 ml phase were measured

at 550nm. A control experiment without the EPS was also pursued

in the same manner. The flocculating activity (%) was defined andcalculated according to the following equation:

flocculating activity =

B− A

B

× 100× dilution rate

A: turbidity of EPS-containing suspension; B: turbidity of control.

3. Results and discussion

3.1. Screening and identification of ZW3 strain

Kefir samples were taken from Tibet, China. Different media

suchas, supplementedMRS, supplemented M17 andsupplemented

whey media was used for screening of EPS-producing strains. But

we found the supplemented whey media as the best, both for

Fig. 1. Ropy behaviour of colony of L. kefiranofaciens ZW3 strain.

screening and exopolysaccharide production. Initially the strains

were screened on the basis of the morphology and colonies which

have mucoid, slimy or ropy appearance, were selected for nextstep.

In final step capability of strains to produce EPSweretested by phe-nol sulphuric acid method. The ropy strain of ZW3 which produced

the highest amount of EPS among screened strains, was selected

for present study.

Strain ZW3 was, Gram positive, catalase negative and rod

shaped bacteria. It did not grow at 15 ◦C. This strain did not

produce gas from arginine. This homofermentative profile along

with the combination of sugar fermentation pattern suggests

that strain ZW3 might belong to the L. kefiranofaciens species

[33]. To confirm the biochemical results, partial sequencing of

variable regions of 16S rRNA genes was also performed. About

550 base pair (bp) variable regions of 16S rRNA genes was

amplified and 500 bp were sequenced. The nucleotide sequences

were used for the analysis of sequence similarity through BLAST

(http://www.ncbi.nlm.nih.gov/blast ) and it gave 100% similaritywith L. kefiranofaciens subsp. kefirgranum and L. kefiranofaciens

subsp. Kefiranofaciens . But differentiate physical test between

two subspecies such as growth at 15 ◦C, its convex colony

with extremely sliminess appearance (Fig. 1), large amount of

exopolysaccharide production and negative aesculin hydrolysis

proved that strain L. kefiranofaciens ZW3 belonged to subsp. kefi-

ranofaciens [18]. So strain ZW3 was identified as L. kefiranofaciens

subsp. kefiranofaciens and named as L. kefiranofaciens subsp. Kefira-

nofaciens ZW3.

3.2. EPS production, Isolation and quantification

Initially L. kefiranofaciens ZW3 and other strains were grown in

50 ml liquid whey medium to screen the strains for EPS quantifi-cation. After incubation for 48–72h, the broth was centrifuged at

12,000 rpm at 4 ◦C for 15 min. After removing cell, the supernatant

was dialysed and EPS amount was determined by phenol sulphuric

acid method usingglucose as standard.Strain ZW3produced a very

high amount of EPS up to 1215 mg/l. And it produced even high

amountup to 1675 mg/l if the incubatedbroth was heatedat 100 ◦C

for 30 min and followed by centrifugation and EPS quantification.

Heat treatment of the samples as a first step in the polysaccha-

ride isolation procedure is critical for complete recovery of the

EPS. Samples without this step gave lower polysaccharide concen-

tration than those including this treatment but it should be used

onlywhere the exopolysaccharide is thermally stable [34,35]. Other

drastic methods include boiling the cell suspension for 15 min in

water, heating at 60◦

C in saline solution, heating in a mixture of






Fig.2. Fourier-transformedinfrared (FT-IR)spectrum of the exopolysaccharide pro-

duced by L. kefiranofaciens ZW3.

phenol water at 65 ◦C or sonicating the cell suspension. Autoclav-

ing is the most frequently used treatment for releasing capsularpolysaccharides from cells [35]. Also the amount ofEPS produced by

L. kefiranofaciens ZW3 washigh as comparedto previously reported

EPS production in the same medium which was up to 405 mg [28].

So the strain L. kefiranofaciens may be has better capability of EPS

production than previously reported strains.

3.3. Sugar analysis and infrared (FT-IR) spectroscopy

Fourier transform infrared spectroscopy has been a useful tool

in monitoring structural changes in biopolymers [36]. Carbohy-

drates such as xanthan components are recognized by peaks at

wave numbers of 1040 cm−1 (C–O bond from the alcohol group),

2940cm−1 (C–H stretch) and 3400 cm−1 (–OH stretch) [37]. The IR

spectra of purified exopolysaccharide ZW3 is given in Fig. 2, whichshows more complex pattern of peaks from 2950 to 1200 cm−1.

Polysaccharides contain a significant number of hydroxyl groups,

which exhibit a broad rounded absorption band above wave num-

ber 3000cm−1. The absorption in that region (Fig. 2) has the

rounded trait typical of hydroxyl groups [38] which suggests that

the substance is polysaccharide. The IR spectra of L. kefiranofaciens

ZW3 exopolysaccharide revealed characteristic functional such as

a broad-stretching hydroxyl group at 3405 cm−1 and a weak C–H

stretching peak of methyl group at 2924 cm−1 [31]. A broad stretch

of C–O–C, C–O at 1000–1200 cm−1 corresponds to the presence

of carbohydrates [39], so in the fingerprint region (region below

1500 cm−1 where bands characterise the molecule as a whole),

the strongest absorption band at 1067 cm−1 is attributed to that

substance is polysaccharide [40]. Strong absorption at 1643 cm−1

which corresponds toamideI > C O str and C–N bending of protein

and peptide amines, and a peak at 1378 cm−1 could be assigned to

C O str of the COO− and C–O bond from COO− [41,42]. The FT-IR

spectra of the polymer evidenced the presence of carboxyl groups,

which may serve as binding sites for divalent cations [31]. Further,

thespectrumshowed thepresenceof carboxyl, hydroxyl,and amine

groups, which are the preferred groups for the flocculation pro-

cess similar to that observed in polyelectrolyte [43]. Noticeably the

exopolysaccharide differ from the algal polysaccharide by having

an additional peak at around1240 cm−1 region due to the presence

of o-acetyl ester [44].

The sugar composition of the EPS, analyzed using MS–gas chro-

matography (Fig. 3). Here only qualitative results are given which

revealed that ZW3 exopolysaccharide is composed of glucose and

Fig. 3. Gas chromatogram of alditol acetate derivative of hydrolyzed exopolysac-

charide from L. kefiranofaciens ZW3.

galactose only. The presence of different sugar moieties suggeststhat the exopolymer is a heteropolysaccharide. Similar biochemi-

cal compositionswereobservedin previous studies of the EPSfrom

L. kefiranofaciens species isolated from kefir [23]. In this contrast it

did not differ from previously reported results.

3.4. Analysis of thermal properties

Besides chemical properties, applicability of polysaccharide is

largely dependenton its thermal and rheological behaviour[45]. As

for the thermal characteristics of exopolysaccharides, heat absorp-

tion and emission are accompanied with the physical change by

deformation of polymer structure or melting of crystalline polysac-

charides. Energy level of the polysaccharide wasscannedfrom 25 to

350

◦

C using a differential scanningcalorimeter and was comparedwith xanthan gum and guar gum used as standard. The melting of

ZW3EPS, xanthan gumand guar gumstarted at about 93.38, 153.4,

and 490.1 ◦C, respectively, and the endothermic enthalpy change

(H) required to melt 1 g of ZW3 EPS, xanthan gum and guar gum

were 249.7, 93.2 and 192.9, respectively (Table 1). Thus the ZW3

polysaccharide showed a differentthermalbehaviour thanxanthan

gum and guar gum. As for the exopolysaccharides obtained from

a mutant of Bacillus polymyxa, the melting point was 183.25 ◦C,

and enthalpy was 100.3 cal/g [46]. In an earlier report, the mea-

surement of the thermal characteristics of levan synthesized with

levansucrase showed the highest melting point to be 178.4 ◦C with

an enthalpy of 1.66cal/g, similar to the thermal characteristics of

the exopolysaccharides derived from legacy microorganisms [47].

3.5. Emulsion stability

Microbial and plant gums as well as some plant and animal

proteins have been known to possess lipid emulsifying effects.

Especially, xanthan gum with microorganism origin has been

widely used in the food industry because of its high emulsifying

Table 1

Thermal propertiesof L. kefiranofaciens ZW3 exopolysaccharide(EPS) by differential

scanning calorimetry (DSC)

Peak temperature (◦C) Enthalpy (J g−1)

ZW3 EPS 97.38 249.7

Xanthan gum 153.4 93.2

Guar gum 490.1 192.9



Y. Wang et al. / International Journal of Biological Macromolecules 43 (2008) 283–288 287

Table 2

Emulsifying activity of ZW3 exopolysaccharide (EPS)

Emulsifier Incubation time (min) Sample OD at A540nm Emulsifying activity (%)

Standard 0 0.173 ± 0.0.009 100 ± 0.00

30 0.066 ± 0.004 38.15 ± 2.31

60 0.031 ± 0.003 17.91 ± 1.74

Xanthan gum 0 0.518 ± 0.011 100 ± 0.00

30 0.480 ± 0.015 92.66 ± 2.4460 0.421 ± 0.013 81.10 ± 2.68

Guar gum 0 0.571 ± 0.015 100 ± 0.00

30 0.415 ± 0.014 72.67 ± 2.43

60 0.214 ± 0.017 37.47 ± 2.98

Locust gum 0 0.249 ± 0.004 100 ± 0.00

30 0.215 ± 0.008 86.34 ± 3.22

60 0.163 ± 0.006 65.46 ± 2.41

ZW3 EPS purified 0 0.343 ± 0.009 100 ± 0.00

30 0.313 ± 0.007 91.25 ± 2.05

60 0.302 ± 0.009 88.04 ± 2.63

ZW3 EPS partially purified 0 1.26 ± 0.032 100 ± 0.00

30 1.11 ± 0.029 88.09 ± 2.30

60 1.06 ± 0.023 84.12 ± 1.83

Hexadecane, 0.5ml, was added to 0.5 ml EPS (1 mg/ml), diluted to 2 ml with phosphate buffer saline (PBS), vortexed for 1 min and the absorbance monitored at 540 nm. A

control was run with 2ml PBS without EPS.

activity [48]. The emulsifying activity of EPS is determined by its

strength in retaining the emulsion of the hydrocarbon in water.

Generally the emulsion breaks rapidly within an initial incubation

of 30min. The absorbance reading after 30 and 60min gives a fairly

good indication of the stability of the emulsion [49]. The emulsion

stabilities of L. kefiranofaciens ZW3 exopolysaccharide were com-

pared with various commercial polysaccharides including xanthan

gum, guar gum and locust gum and results are listed in Table 2. The

purified fraction of the exopolymer produced by L. kefiranofaciens

ZW3 retained 91.25% and 88.04% of the emulsification activity after

30 and 60 min, respectively. Results of partially purified fraction of

the exopolymer, which did not differ much from purified fraction,

produced 88.09% and 84.12% after 30 and 60 min, respectively. The

other polysaccharides such as locust bean and guar gum showed

relatively poor emulsifying activities as compared to L. kefiranofa-

ciens ZW3 exopolysaccharide. The guar gum retained 72.67% and

37.47%, locustgum, 86.34%and 65.46%, whereas xanthan gumpro-

duced 92.66% and 81.10% emulsion activity after 30 and 60 min,

respectively. So the purified and partially purified L. kefiranofaciens

ZW3 exopolysaccharide showed almost similar activity, where as

purified exopolysaccharide showed better activity when compared

withxanthan gum.Fromthese results,the polysaccharide produced

by L. kefiranofaciens ZW3 is expected to have a great potential for

use as an emulsifier.

3.6. Flocculating capability

A variety of flocculants, such as inorganic flocculants (polya-

luminium chloride and aluminium sulphate), organic flocculants

(polyacrylamide, polyethyleneimine) and natural flocculants or bio

flocculants (gelatin, chitosan guar gum) have been widely used in

chemical and mineral industrial fields suchas tap water producing,

wastewater treatment, dredging, downstream processing, fermen-

tation and food industries [50–52]. Although chemical flocculants

have been used widely due to their effective flocculating activity

and low cost, they have neurotoxic and carcinogenic monomers

and their usage is restricted [1,53]. On the contrary, biofloccu-

lants produced by microorganisms during their growth are safe

and biodegradable polymers [54]. Recently, many studies have

been reported on the flocculating effect of microbial polysaccha-

rides to replace synthetic flocculants, which are industrially used

[55–57].

Flocculating capability test was performed at EPS concentration

rangingfrom 0.1 to 0.6mg in 5 mg/ldispersion of charcoal-activated

carbon containing 6.8 mM CaCl2·H2O (Fig.4). The flocculating capa-

bility of isolated exopolysaccharide was compared with that of

xanthan gum and guar gum used as control. This capability ini-

tially increased with increasing the concentration of EPS, and gave

the greatest flocculating activity between concentration range of

0.3–0.5mg/l and on word it had a decreasing trend as the EPS

(flocculant) concentration increased. The optimal flocculant con-

centration in test solution was determined to be 0.4mg/ml. Where

as the optimal flocculant concentration for xanthan gum and guar

gum was 0.3 and 0.5 mg/l, respectively. As shown in Fig. 4 that

the flocculating capability initially increased with increasing con-

centration and then started to decrease after attaining a highest

and purified point and this may be due to that the adsorption of

excess flocculants destabilized the particles. Because of incomplete

dispersion of excess flocculants, only particles around flocculants

participated in the flocculating reaction at that moment. A large

molecular weight flocculant is usually long enough and has a suf-

ficient number of free functional groups that can act as bridges to

bring many suspended particlestogether, and hence causes a larger

flocsize in the flocculation reaction [32]. L. kefiranofaciens ZW3EPS

Fig. 4. Flocculating capacity of L. kefiranofaciens ZW3 EPS, xanthan gum and guar

gum.




showeda better flocculating activitythanguar gumand almost sim-

ilar to xanthan gum. Moreover theflocculatingbehaviour of ZW3is

also supportedby itsIR analysis. SoZW3 EPSis expected tobe useful

flocculating agents in the areas of wastewater treatment, drinking

water processing, and downstream processing in the food indus-

try because of their biodegradability and harmlessness towards

humans and the environment.

Acknowledgments

This work was supported by grant from China the Fifteenth

National Scientific Support Grant (No. 2006BAD 04A 06) and by

Tianjin Municipal Science and Technology Commission Grant (No.

08JCYBJC01900).

References

[1] C. Arezoo, Nephrol. Dial. Transplant. 16 (2002) 17–20.[2] I.Y. Lee, W.T. Seo, G.J. Kim, M.K. Kim, S.G. Ahn, G.S. Kwon, Bioprocess Biosyst.

Eng. 16 (1997) 71–75.[3] L. De Vuyst, F. Vanderveken, S. van den Ven, B. Degeest, J. Appl. Microbiol. 84

(1998) 1059–1068.[4] J. Bender,S.R. Eaton, U.M.Ekanemesang,P. Phillips,Appl. Environ.Microbiol. 60

(1994) 2311–2315.[5] I.W. Sutherland, Trends Biotechnol. 16 (1998) 41–46.[6] L. De Vuyst, B. Degeest, FEMS Microbiol. Rev. 23 (1999) 153–177.[7] M. Garciagaribay, V.M.E. Marshall, J. Appl. Bacteriol. 70 (1991) 325–328.[8] S. Matsukawa, T. Watanabe, Food Hydrocolloids 21 (2007) 1355–1361.[9] A. Laws, Y. Gu, V. Marshall, Biotechnol. Adv. 19 (2001) 597–625.

[10] L. De Vuyst, F.De Vin,F. Vaningelgem, B.Degeest,Int.Dairy J. 11 (2001)687–708.[11] A.D. Welman, I.S. Maddox, Trends Biotechnol. 21 (2003) 269–274.[12] A. Laws, V. Marshall, Int. Dairy J. 11 (2001) 709–721.[13] L. Jolly, S.J.F. Vincent, P. Duboc, J.R. Neeser, Antonie Leeuwenhoek 82 (2002)

367–374.[14] O. Kandler, P. Kunath, Syst. Appl. Microbiol. 4 (1983) 286–294.[15] P. Kooiman, Carbohyd. Res. 7 (1968) 220–221.[16] T. Fujisawa, S. Adachi, T. Toba, K. Arimara, T. Mitsuoka, Int. J. Syst. Bacteriol. 38

(1988) 12–14.[17] H. Yokoi, Y. Fujii, T. Mukai, T. Toba, S. Adachi, Int. J. Food Microbiol. 13 (1991)

257–264.[18] S. Takizawa, S. Kojima, S. Tamura, S. Fujinaga, Y. Benno, T. Nakase, Int. J. Syst.

Bacteriol. 44 (1994) 435–438.[19] G. Ottogalli, A. Galli, P. Resmini, G. Volonterio, Annu. Microbiol. 23 (1973)

109–121.[20] L. Angulo, E. Lopez, C. Lema, J. Dairy Res. 60 (1993) 263–267.[21] M.E. Pintado, J.A. Lopes Da Silva, P.B. Fernandes, F. Xavier Malcata, T.A. Hogg,

Int. J. Food Sci. Technol. 31 (1996) 15–26.[22] M. Murofushi, M. Shiomi, K. Aibara, Jpn. J. Med. Sci. Biol. 36 (1983) 49–53.

[23] H. Maeda, X. Zhu, K. Omura, S. Suzuki, S. Kitamura, J. Agric. Food Chem. 52(2004) 5533–5538.

[24] K.L. Rodrigues, L.R. Gaudino Caputo, J.C. Tavares Carvalho, J. Evangelista, J.M.Schneedorf, Int. J. Antimicrob. Agents 25 (2005) 404–408.

[25] G. Vinderola, G. Perdigón, J. Duarte, E. Farnworth, C. Matar, Cytokine 36 (2006)254–260.

[26] M. Medrano, P.F. Pérez, A.G. Abraham, Int. J. Food Microbiol. 122 (2008) 1–7.[27] P.S. Rimada, A.G. Abraham, Int. Dairy J. 16 (2006) 33–39.[28] H. Yokoi, T. Watanabe, Y. Fujii, T. Toba, S. Adachi, J. Dairy Sci. 73 (1990)

1684–1689.

[29] P. Forsman, T. Alatossava, Arch. Virol. 137 (1994) 43–54.[30] M. Dubois, K.A.Gilles, J.K.Hamilton, P.A. Rebers, F.Smith,Anal. Chem.28 (1956)

350–356.[31] P.V. Bramhachari, P.B. Kavi kishor, R. Ramadevi, R. Kumar, B.R. Rao, S.K. Dub ey,

J. Microbiol. Biotechnol. 17 (2007) 14–51.[32] D.J.Lim, J.D.Kim, M.Y. Kim,S.H. Yoo, J.U.Kong,J. Microbiol.Biotechnol. 17 (2007)

979–984.[33] I. Mainville, N. Robert, B. Lee, E.R. Farnworth, Syst. Appl. Microbiol. 29 (2006)

59–68.[34] P.S. Rimada, A.G. Abraham, Lait 83 (2003) 79–87.[35] A.S. Kumar, K. Mody, B. Jha, J. Basic Microbiol. 47 (2007) 103–117.[36] R.H. Wilson, B.J. Goodfellow, P. Belton, Food Hydrocolloids 2 (1988) 169–178.[37] S. Cetin, A. Erdincler, Water Sci. Technol. 5 (2004) 49–56.[38] K.J. Howe, K.P. Ishida, M.M. Clark, Desalination 147 (2002) 251–255.[39] P.J. Bremer, G.G. Geesey, Biofouling 3 (1991) 89–100.[40] S. Nataraj, R. Schomacker, M. Kraume, M.I. Mishra, A. Drews, J. Membr. Sci. 308

(2008) 152–161.[41] D. Helm, D. Naumann, FEMS Microbiol. Lett. 126 (1995) 75–80.

[42] K. Haxaire, Y. Marechal, M. Milas, M. Rinaudo, Biopolymers 72 (2003) 10–20.[43] J.E. Zajic, E. Knetting, Development in Industrial Microbiology, American Insti-tute of Biological Science, Washington, D.C., 1970, pp. 87–98.

[44] S.K.Kazy, P. Sar, S.P. Singh, A.K. Sen,S.F. D’Souza, World J. Microbiol.Biotechnol.18 (2002) 583–588.

[45] E. Marinho-Soriano, E. Bourret, Bioresour. Technol. 96 (2005) 379–382.[46] G.S. Kwon, H.K. Joo, T.K. Oh, Kor. J. Appl. Microbiol. Biotechnol. 20 (1992) 34–

39.[47] S.J. Jung, K.B. Song, B.Y. Kim, U.H. Chun, S.K. Rhee, Food Eng. Prog. 3 (1999)

176–180.[48] M.C. Cirigliano, G.M. Carman, Appl. Environ. Microbiol. 51 (1985) 846–850.[49] S. Royan, C. Parulekar, S. Mavinkurve, Lett. Appl. Microbiol. 29 (1999) 342–

346.[50] H. Salehizadeh, M. Vossoughi, I. Alemzadeh, Biochem. Eng. J. 5 (2000)

39–44.[51] H. Salehizadeh, S.A. Shojaosadati, Biotechnol. Adv. 19 (2001) 371–385.[52] Z.Q. Zhang,B. Lin, S.Q. Xia, X.J. Wang,A.M. Yang, J. Environ.Sci. China 19 (2007)

660–666.[53] C. Ruden, Food Chem. Toxicol. 42 (2004) 335–349.

[54] S.B. Deng, R.B. Bai, X.M. Hu, Q. Luo, Appl. Microbiol. Biotechnol. 60 (2003)588–593.

[55] K. Toeda, R. Kurane, Agric. Biol. Chem. 55 (1991) 2793–2799.[56] J. Zhang, Z. Liu, S. Wang, P. Jiang, Appl. Microbiol. Biotechnol. 59 (2002) 517–

522.[57] X.W. Huang, W. Cheng, Y.Y. Hu, J. Environ. Sci. China 17 (2005) 494–498.

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2008 - Physicochemical Properties of Exopolysaccharide Produced by Lac to Bacillus Kefiranofaciens ZW3 Isolated From Tibet Kefir