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8/3/2019 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.
doi:10.1016/j.ijbiomac.2008.06.011
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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
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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
8/3/2019 2008 - Physicochemical Properties of Exopolysaccharide Produced by Lac to Bacillus Kefiranofaciens ZW3 Isolated From Tibet Kefir
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286 Y. Wang et al. / International Journal of Biological Macromolecules 43 (2008) 283–288
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
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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.
8/3/2019 2008 - Physicochemical Properties of Exopolysaccharide Produced by Lac to Bacillus Kefiranofaciens ZW3 Isolated From Tibet Kefir
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288 Y. Wang et al. / International Journal of Biological Macromolecules 43 (2008) 283–288
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).
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