Exopolysaccharides, characterization

1. Characterization of exopolysaccharide produced by Lactobacillus kefiranofaciens
ZW3 isolated from Tibet kefir e Part II
Zaheer Ahmed a,b,*, Yanping Wang b
, Nomana Anjum a
, Asif Ahmad c
, Salman Tariq Khan d
a
Department of Home & Health Sciences, Allama Iqbal Open University, Islamabad, Pakistan
b
Tianjin Key Laboratory of Food Nutrition and Safety, Faculty of Food Engineering and Biotechnology, Tianjin University of Science and Technology, People’s Republic of China
c
Department of Food Technology, Pir Mehr Ali Shah Arid Agriculture University Rawalpindi, Pakistan
d
Pharmaceutical Research Centre, PCSIR Labs Complex, Karachi 75280, Pakistan
a r t i c l e i n f o
Article history:
Received 16 October 2011
Accepted 6 June 2012
Keywords:
Lactobacillus kefiranofaciens ZW3
Rheological
Tibet kefir
Characterization
a b s t r a c t
ZW3 is a newly discovered exopolysaccharide (EPS) produced by Lactobacillus kefiranofaciens ZW3,
isolated from Tibet kefir. Some of its properties have been characterized in our previous paper. Present
research demonstrates some other important aspects of this EPS. The molecular weight obtained by gel
permeation HPLC was 5.5 Â 104
Da. Solubility, water holding and oil binding capacity of ZW3 EPS were
14.2%, 496.0%, and 884.74% respectively. Scanning electron microscopy (SEM) of ZW3 EPS demonstrated
a smooth surface with compact structures. A topographical examination of EPS by atomic force
microscopy (AFM) revealed that ZW3 EPS is composed of almost uniform net of molecules. Rheological
study indicated that common salt did not affect the viscous behavior of ZW3 EPS and acidic pH may
enhance its viscosity. Exopolymer showed a melting point of 93.38
C. A degradation temperature (Td) of
299.62
C was observed from the TGA curve for the polysaccharide ZW3.
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
In recent years, polysaccharides have attained the considerable
attention of researchers because of their wide distribution in nature
and documented health benefits. These macromolecules are either
homopolymers or heteropolymers of neutral sugars (Badel,
Bernardi, Michaud, 2011). The thickening properties of poly-
saccharides make them ideal as food additives and can be extracted
through various sources including plant, fungi or seaweeds (Saija,
Welman, Bennett, 2010). Extraction and purification processes
may affect the physiochemical and structural properties of poly-
saccharides and hence characterization of polysaccharides is
essential to determine suitable properties for the purpose of their
utilization as food additives. Often the structure of these polymers
is modified to increase their rheological properties and to make
them suitable for various food applications (De Vuyst Degeest,
1999; Roller Dea, 1992). Another reason for acceptance of lactic
acid bacterial polysaccharides such as ZW3 EPS is that, addition of
certain polysaccharides from plant sources is not acceptable in
certain dairy products due to problem of “all dairy” label to dairy
foods; and also the use of such plant polymers in dairy products is
prohibited in many European countries (Saija et al., 2010; Wang,
Zaheer, Feng, Li, Song, 2008). These restrictions force the agro-
food industries to look for other possible sources of polymers
from dairy sources and for that the best option is exopolysaccharide
produced by lactic acid bacteria on dairy based source with a GRAS
(generally regarded as safe) status (Badel et al., 2011; Maeda, Zhu,
Suzuki, Suzuki, Kitamura, 2004; Wang et al., 2008, 2010).
Numerous strains of lactobacillus genera have a potential to
produce exopolysaccharide under specific growth conditions with
a wide range and diversity of structure and have a potential to be
used as nutraceuticals (Badel et al., 2011; Gorska et al., 2010; Wang
et al., 2008, 2010). Due to their characteristic functional properties,
LAB exopolysaccharides are used as stabilizing, viscosity modifying,
and gelling agents (Pan Mei, 2010). However the physiological
function of these polymers is still unknown and few are used in food
industries (Suresh Kumar, Mody, Jha, 2007; Sutherland, 2007).
Lactobacillus kefiranofaciens, an isolate from kefir is famous for its
polymer named as kefiran; and has attained the attention of many
researchers in recent years (Piermaria, de la Canal, Abraham,
2008, Piermaria, Pinotti, García, Abraham, 2009; Wang Bi,
2008; Wang et al., 2008). Deproteinized whey medium which is
waste product of cheese industry can be used as substrate which
ends in the production of a valuable product i.e. kefiran (Wang et al.,
2008). These carbohydrates have several health beneficial effects,
including the decrease of blood pressure induced by hypertension
* Corresponding author. Department of Home Health Sciences, Allama Iqbal
Open University, Islamabad, Pakistan. Tel.: þ92 519057265; fax: þ92 51 9250063.
E-mail address: zaheer_863@yahoo.com (Z. Ahmed).
Contents lists available at SciVerse ScienceDirect
Food Hydrocolloids
journal homepage: www.elsevier.com/locate/foodhyd
0268-005X/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodhyd.2012.06.009
Food Hydrocolloids 30 (2013) 343e350

2. (Maeda et al., 2004), immunomodulation, epithelium protection
and antagonic activity against Bacillus cereus on Caco-2 cells
(Piermaria et al., 2010), increased phagocytic activity of peritoneal
and lung macrophages (Vinderola, Perdigon, Duarte, Farnworth,
Matar, 2006) and increased IgA cells in these sites (Duarte,
Vinderola, Ritz, Perdigon, Matar, 2006), antitumor activity (Liu,
Wang, Lin, Lin, 2002), antimicrobial activity (Rodrigues, Caputo,
Carvalho, Evangelista, Schneedorf, 2005), and anti-
inflammatory activity (Moreira et al., 2008). Kefiran also improves
the rheological properties and better viscoelasticity can be achieved
through addition of kefiran up to a level of 300 mg/L (Badel et al.,
2011; Piermaria et al., 2010). Moreover, the kefiran can form
brittle and transparent films with good water vapor barrier char-
acteristics (Piermaria et al., 2009). Due to beneficial attributes of
this polymer the present research was conducted. In our previous
paper (Wang et al., 2008) we have characterized some of properties
of polymer produced by L. kefiranofaciens ZW3 isolated from Tibet
kefir. The strain produces a high amount of polymer having desir-
able physiochemical properties (Wang et al., 2008). However, to
explore its potential for application in food industry more charac-
terization on physiochemical, structural and rheological parameters
is required. Keeping in view all of this, the current project was
planned to discover industrially important physiochemical, struc-
tural and rheological parameters for this exopolysaccharide.
2. Materials and methods
2.1. Isolation and purification of EPS
The indigenous strain L. kefiranofaciens ZW3 was isolated and
further purified from Tibet kefir as described in our previous study
(Wang et al., 2008). Liquid whey media was used for its propagation
and media was incubated under anaerobic conditions at tempera-
ture of 30 C and for a time period of 72 h. Maximum recovery of
EPS was obtained when degrading enzymes were inactivated by
heating the media at a temperature of 100 C for 30 min. This was
followed by ultracentrifugation at 12,000 Â g for 15 min at refrig-
erated temperature. EPS was precipitated by using the chilled
absolute ethanol and was kept in refrigerator at temperature of 4 C
for 12 h, followed by a second centrifugation by maintaining the
above parameters. For further purification, EPS obtained by above
treatment was redissolved in distilled water (100 ml) with gentle
heating below 50 C and precipitated again with equal volume of
chilled absolute ethanol. Again an ultracentrifuge treatment was
applied (25,000 Â g) for 25 min at refrigeration temperature. To
achieve further purification, EPS pellets were once again redis-
solved in 20 ml of distilled water with gentle heating (below 50 C).
Dialysis technique was applied to remove small molecular weight
simple sugars at 4 C for 72 h with three changes of distilled water
in a day. Dialyzed EPS was recovered through freeze dryer. Recov-
ered EPS was named as partially purified EPS and was further
characterized for some important parameters. Trichloroacetic acid
(TCA 14%) was used for further purification by overnight stirring.
This technique is valuable to remove protein impurities from EPS.
Precipitated protein was separated through centrifugation at
12,000 Â g for 15 min. The resultant material was neutralized up to
pH level of 7.0 and was again precipitated by adding chilled ethanol
in equal volumes. Finally pellets of EPS were redissolved in double
distilled water and were lyophilized.
2.2. Study of common physical properties
Solubility of ZW3 EPS in water and oil was determined by
following the procedure of Chang and Cho (1997). A separate
suspension of EPS was made by dissolving EPS at rate 50 mg/ml in
water and oil with continuous agitation at 25 C for 24 h. This was
followed by centrifugation 5000 Â g for 15 min and collected
supernatant (0.2 ml) was precipitated with 3 volume of ethanol.
Again EPS in form of precipitate was recovered by centrifugation at
10,000 Â g for 5 min. Resultant material was vacuum dried at 50 C
and difference in weight was recorded .The solubility was calcu-
lated as follows:
Solubilityð%Þ ¼ ½Total carbohydrate concentration in
supernatantðaÞŠ=½Weight of sample
ðdry weight basisÞŠ Â 100
Sample of EPS was characterized for water holding capacity
(WHC) by suspending 0.2 g sample in 10 ml of deionized water on
a vortex mixer. Dispersed material was centrifuged at 16,000 Â g for
25 min. Unbound water that was not held by EPS material was
discarded. All EPS material was dropped on pre weight filter paper
for complete drainage of water. Weight of EPS precipitated was
recorded. The percentage of WHC was calculated through following
expression:
WHCð%Þ ¼ ½total sample weight after water absorptionŠ
=½total dry sample weightŠ Â 100
The oil binding capacity was also calculated for this EPS in
a similar manner by adopting method of Kato, Okamoto, Tokuya,
and Takahashi (1982). For that purpose soya bean oil was used as
dispersing media. The other steps were identical to the analysis
procedure of WHC.
2.3. Measurement of molecular weight
Extracted and refined EPS pellet was characterized for molecular
weight using Agilent 1100series HPLC system (Agilent technologies
Palo AHO, CA, USA). Equipment was equipped with refractive index
detector TOSOH TSK-G4000 PWxl column (7.8 mm  30 cm, 10 mm)
(TOSOH Corp., Tokyo, Japan). A sample of 20 mL was injected in the
system by maintaining a flow rate of 0.5 ml/min and column
temperature of 35 C. Separation was carried out by using 0.71%
sodium sulfate as mobile phase. Dextran D2000 with molecular
weight of 2 Â 106
, D8 with molecular weight of 1.338 Â 105
, D7 with
molecularweightof 41,100, D5 with molecular weight 21,400, D4 with
molecular weight 10,000, D0 with molecular weight 180 (glucose)
was used as reference compound. These reference standards were
added into the mobile phase at rate of 10 mg per 1 ml solution.
2.4. Measurement of rheological properties of ZW3 EPS
Brookfield Digital Rheometer Model DV III (Brookfield Engi-
neering Laboratories Inc., Stoughton, Massachusetts, USA) was used
to determine rheological properties. An RV type ULA spindle that
rotated in chamber equipped with temperature control system
(Thermomixs; B. Braun Biotech International) was attached with
rheometer. Brookfield Rheocalc software (Brookfield Engineering
Laboratories Inc.) was used to control the instrument.
For preparation of sample EPS was well dissolved at rate of
2 mg/ml and 4 mg/ml. Rheological behavior for test solution was
measured against time with increasing shear rate. Further rheo-
logical characteristic of EPS was performed at variable pH levels
and with addition of different salts. pH of EPS solution was adjusted
at level of 4.0, 5.0 and 6.5 by using lactic acid. Two salts solutions;
NaCl (0.1 M) and CaCl2 (0.1 M) separately were used to dissolve EPS
for characterization of rheological properties. Further character-
ization was carried out by dissolving EPS in skim milk and water
and their rheological properties were compared with each other.
Z. Ahmed et al. / Food Hydrocolloids 30 (2013) 343e350344

3. The data for all of the parameters mentioned above were recorded
in triplicate.
2.5. Atomic force micrograph (AFM) of ZW EPS
Purified ZW3 EPS was dissolved completely at rate of 1 mg/ml in
double distilled water under nitrogen stream and with continuous
stirring for 1 h. After attaining room temperature; mixture was
diluted to achieve a concentration of 0.01 mg/ml. Mica sheet was
used as carrier media. A sample size of 5 mL was uniformly
distributed on mica sheet and was dried at room temperature. AFM
images were taken by scanning Probe microscope (JEOL JSPM-
5200, JAPAN) in tapping mode. The cantilever oscillation was set
at frequency of 158 kHz with driven amplitude of 0.430 v.
2.6. Scanning electron microscopy (SEM) analysis of ZW3 EPS
Scanning Electron Microscope technique was used for charac-
terization of EPS. Exopolysaccharide was fixed on aluminum stub
and gold sputtered and examined through SEM by maintaining an
accelerated voltage of 10 kv.
2.7. Thermogram analysis (TGA)
For TGA study Mettler Toledo TGA/SDTA 851e thermal system
was employed. This system operates at atmospheric pressure.
Compatible system software was used to control various parame-
ters including temperature. Variable system software was also used
to record temperature, over period of time through installed
thermo couples that have connecting ends in crucibles. The crucible
was used made up of Al2O3. Sample of EPS (10 mg) was placed in
crucible. System was programmed for linear heating at rate of
10 C/min rise in temperature in 1 min over a temperature range of
25e100 C. Separate experiments were performed in air and
nitrogen atmosphere. The flow rate for air and nitrogen was
maintained at 50 ml/min. System was initially calibrated for
temperature reading using indium as melting standard.
3. Results discussion
3.1. Physical properties of ZW3 EPS
The chromatogram (Fig.1) obtained by gel permeation HPLC
depicted a single distribution of MW corresponding to 5.5 Â 104
Da.
Different authors have reported EPS with different molecular
weight by L. kefiranofaciens. Wang and Bi (2008) reported kefiran
with molecular weight of 1.5 Â 105
Da, whereas Piermaria and
coworkers and other researchers have reported molecular weight
of 107
Da (Piermaria et al., 2008; Sutherland, 1998; Wang Bi,
2008). Some of the most common physical properties of ZW3 EPS
are shown in Table 1. ZW3 EPS is water soluble with good water
holding capacity and oil binding capacity. These properties are
attributed to the permeable structure of polymer chains which can
hold large amounts of water through hydrogen bonds (Zhu, Huang,
Peng, Qian, Zhou, 2010). According to Kethireddipalli, Hung,
Phillips, and McWatters (2002) when fibrous material is ground
to form powder, it not only brings the changes in it size, but may
also adversely affects swelling and water holding capacity of
polymer and change in the fiber matrix structure. Due to good
water holding capacity the L. kefiranofaciens ZW3 EPS producing
strain has good potential to be used in fermented products along
with non-EPS producing strain. Yang et al. (2010) have reported
that water holding capacity of yoghurt increases when its starter
culture is co-cultured with EPS producing strain.
3.2. Measurement of rheological properties of ZW3 EPS
Study of rheological properties is important for better machin-
ability of the product. In recent years, whey separation or accu-
mulation of liquid (whey) on the surface of a milk gel is common
problem in fermented milk products with and superfluous sight in
these products. This problem often appears as a result of shrinkage
of a gel causing an expulsion of liquid (Harwalkar Kalab, 1986;
Mistry Hassan, 1992). Conventionally different types of thick-
eners, stabilizers and synthetic chemicals are being used to avoid
this problem (Ramaswamy Basak, 1992; Xu, Stanley, Goff,
Davidson, 1992). Often these synthetic chemicals are not allowed
in some parts of the world (Wang et al., 2008). EPS will offer a new
substitute for these chemicals as a natural counterpart.
The viscous behavior of exopolysaccharide is dependent on its
structure and mass (Freitas et al., 2009) that are affected by various
factors such as salts, ionic strength, pH and temperature. To use the
EPS in for different products, the knowledge of its rheological
behavior at different pH and ionic may provide some useful
application for various food products. The rheological behavior of
this newly discovered EPS in water, milk, salt and also at different
pH, against time and increasing shear rate is shown in Fig. 2. EPS
showed a thinning behavior of viscosity i.e. high initial value of
viscosity which decreases with time and later on becomes stable.
Whereas viscosity was increased with increasing the concentration
of EPS from 2 mg/ml to 4 mg/ml (Fig. 2A). The effect of salts on the
viscosity of the solutions of the EPS produced by strain ZW3 as
a function of shear rates is shown in Fig. 2B. At 30 C the viscosity of
EPS in 0.1 M CaCl2 0.1 M NaCl solutions was almost similar over
the whole shear rate range (Fig. 2B). The effect of salts on the
viscosity of the solutions of the EPS produced by strain ZW3 as
a function of time is shown in Fig. 2C. Comparatively higher
viscosity of EPS in CaCl2 was observed than NaCl solution and this
behavior of EPS was evident in initial stages as well as after 10 min
of elapse time. This viscous behavior may be attributed to different
intermolecular arrangement of charged polymers in these solutions
that cause different degree of electrostatic repulsion or contracted
by electrostatic attraction between the polymer chains and is in line
with the study of Kanmani et al. (2011) who reported a diversified
viscosity behavior of EPS in different salt solutions.
Fig. 1. The chromatogram of ZW3 EPS obtained by gel permeation HPLC.
Table 1
Physical properties of ZW3 EPS.
Sample Solubility (%) Water holding
capacity (%)
Oil binding
capacity (%)
ZW3 14.2 496.00 884.74
Z. Ahmed et al. / Food Hydrocolloids 30 (2013) 343e350 345

4. Fig. 2D shows the behaviors of EPS at different pH as a function
of time. It is depicted from Fig. 2D that neutral pH tends to lower
the viscosity of EPS. Viscosity of EPS increased as a function of
decrease in pH. ZW3 EPS viscosity was also checked in skim milk
and water as an increased function of shear rate and time (Fig. 2E
and F). Initially the viscosity was high both in water and skim milk
and it decreased with passage of time whether it was treated with
a fixed sear rate; or with an increasing shear rate. Similarly, while
studying the impact of pH on the viscosity of polysaccharide, Gauri,
Mandal, Mondal, Dey, and Pati (2009) have reported on the
increased viscosity of EPS in acidic pHs relative to those alkaline
pHs. Similar results are also reported by Kanmani et al. (2011) who
also observed a decrease in the viscosity once he lowered the pH
from 6 (208 mPa) to acidic pH 3 (226 mPa).
These results are very significant as final pH of fermented milk
product is always at acidic side and exopolysaccharide has a favor-
able behavior at acidic pHs at acidic pH. It appears thus that a good
choice would be to select this pH for the use of the EPS ZW3 as
a biothickener or a stabilizer.
3.3. Atomic force micrograph (AFM) of ZW EPS
In recent years exopolysaccharide has been studied extensively
by using atomic force microscopy (Abu-Lail Camesano, 2003;
Wang et al., 2010) that provides a powerful tool to characterize the
morphological features of polymers. Owing to its ability to measure
interaction forces in liquids at a pico- or nano-Newton level with
high vertical and lateral resolutions. This technique enables us to
characterize and inference properties of the EPS by observing the
conformation of individual macromolecules along with molecular
structure of exopolysaccharide and its dynamics (Abu-Lail
Camesano, 2003; Giannotti, Rinaudo, Vancso, 2007; Giannotti
Vancso, 2007; Wang et al., 2010). This technique can also be used
in absence of water as a dispersing medium, thus enables the
researchers to study the conformation of polymers under diversi-
fied controlled conditions, such as electrochemical potential,
temperature, salt and solvent (Haxaire, Marechal, Milas, Rinaudo,
2003). The topographical AFM images of ZW3 EPS are shown in
Fig. 3. ZW3 EPS deposited from 10 mg/ml aqueous solution have
Fig. 2. Rheological behavior ZW3 EPS (A) 2 and 4 mg/ml concentration against time, (B C) in 0.1 M NaCl CaCl2 against time and shear rate, (D) at different pH against time, and
(E F) in water and skim milk against time and shear rate.
Z. Ahmed et al. / Food Hydrocolloids 30 (2013) 343e350346

5. rounded to spherical lumps with almost similar size and formation
of chains is also visible. However the molecules are tightly packed
and have reticulated shape suggesting that they have strong affinity
for water molecules and have pseudoplastic behavior. Similar result
was reported by Wang et al. (2010) for KF5 EPS, however in the case
of ZW3 EPS the concentration was lower (10 mg/ml) as compared to
KF5 EPS concentration (100 mg/ml). Different EPS have different
shape and structure. Wang et al. (2010) reported roundness lumps
irregularly reticulation shape for KF5 EPS, whereas Feng, Gu, Jin,
and Zhuang (2008) reported different shapes, spherical lumps and
worms, respectively in low and high concentration. Our findings for
the ZW3 EPS showed rounded lumps with maximum height of
lump by of 31.1 nm.
In a previous research, Wang et al. (2010) reported the KF5 EPS
with a maximal size of 13 nm.
3.4. Scanning electron microscopy (SEM) analysis of ZW3 EPS
Along with AFM another tool which is mostly used for imaging
of exopolysaccharide is SEM and has been reported by many
researchers (Goh, Haisman, Singh, 2005) and as a very useful tool
to study surface topography of polymers (Wang et al., 2010). SEM
results of ZW3 EPS and a reference material i.e. xanthan gum are
shown in Fig. 4.
As observed by SEM, ZW3 EPS look like thin film with smooth
and glittering surface; exhibiting compact structure which is
characteristic of a material used to make the plasticized films. So it’s
a good choice for making such kind of films. Moreover the SEM scan
showed that ZW3 EPS was made of homogeneous matrix which is
an indicator of the structural integrity especially important in film
making. Much of the SEM properties of ZW3 EPS are similar to the
properties of polymer reported by Piermaria et al. (2008) and
(2010) but was different from KF5 EPS reported by Wang et al.
(2010) whose surface was dull and had pores.
3.5. Thermogram analysis (TGA)
The timeetemperature integral is the singularly most effective
stimulus on the polysaccharide disperse system, from the mildest
process that insures safety and elementary dissolution to the
severest process that initiates chemical decomposition. On the
lower response scale, gelatinization and swelling are primary
occurrences; on the upper response scale, chemical dehydration,
pyrolysis, and resynthesis generate higher M species in the volatile
phase (Fagerson, 1969) and flavors, aromas, colorants (Vercellotti,
Crippen, Lovegren, Sanders, 1992), and a host of other small
organic molecules terminally. Along with other physiochemical
characteristics; applicability of exopolymer is largely dependent on
its rheological and thermal behavior (Marinho-Soriano Bourret,
2005). In thermal analysis of EPS heat is emitted and absorbed
which is accompanied by change in structure of polymer and in
melting of crystalline polymer (Wang et al., 2010).
The thermogravimetric (TGA) analysis for ZW3 EPS was carried
out dynamically (weight loss versus temperature). Xanthan gum
and locust gum were used as reference material and the experi-
mental results are depicted in Fig. 5. A degradation temperature
(Td) of 299.62 C was determined from the TGA curve for the
Fig. 3. Atomic force microscopy (AFM) images of ZW3 EPS.
Z. Ahmed et al. / Food Hydrocolloids 30 (2013) 343e350 347

6. polysaccharide ZW3. The initial weight loss of polymer between 40
and 90 C may be attributed to its moisture contents. EPS with high
carboxyl groups is always rich in moisture contents and initial
weight loss in ZW3 EPS suggests that ZW3 polymer is rich in
carboxyl contents. This is because the higher the carboxyl content
the greater the affinity of the polysaccharide for interaction with
water molecules (Parikh Madamwar, 2006). The decline in
weights above 90 C is attributed to the degradation of the sample.
The onset of decomposition occurred at 261.4 C and the recorded
mass loss was 10%. The polymer weight loss decreased dramatically
around 300 C, Fig. 5B and C show the TG analysis of xanthan gum
and locust gum respectively as reference material. Degradation
temperature for xanthan gum is 282.65 C, where for locust gum it
is 278.46 C. ZW3 EPS is glucogalactan in nature (Wang et al., 2008)
whereas Locust gum is a linear polysaccharides, which is composed
of mannose and galactose (Dakia, Blecker, Robert, Wathelet,
Paquot, 2008); while Xanthan gum is a hetero-polysaccharide
containing D-glucose, D-mannose and D-glucoronic acid (Baird,
Fig. 4. SEM results of ZW3 EPS and of a reference material xanthan gum. Fig (A) and (B) at 1000Â and 2000Â of xanthan gum, whereas fig (C), (D), (E) (F) showing SEM results of
ZW3 EPS at 1000Â, 2000Â, 10,000Â and 10,000Â respectively.
Z. Ahmed et al. / Food Hydrocolloids 30 (2013) 343e350348

7. 1989). The different behavior of different polymers in thermogra-
vimetric analysis may be attributed to their structure (Wang et al.,
2010). So ZW3 showed a relatively higher degradation temperature
than both xanthan and locust gum which makes it safe to be used in
dairy industry where in most of processes temperature seldom
overpasses 150 C.
4. Conclusion
L. kefiranofaciens (ZW3) isolated from Tibet kefir depicted good
exoplolysaccharide (EPS) producing capacity. A new modified
extraction procedure was investigated, that maximized the
recovery of EPS. In our previous study we have characterized the
polymer for its chemical structure composition by using the FTIR
GCMS analysis. Polymer was also studied for its emulsion
stability, thermal properties flocculating activity. In present study
we have characterized the remaining aspects of polysaccharide
which were not explored in the prior study. Extracted EPS was
characterized as medium molecular weight and possess good
solubility, water binding capacity and oil binding capacity. Rheo-
logical properties showed a good potential of this EPS and has
compatibility with water, milk, salts at different pH and shear rates.
Viscous behavior in fermented milk product indicated its potential
to be used as biothickner or biostabilizer. Characterization data
through SEM and AFM showed rounded lumps with maximum
height of 31.1 nm with indication of better structural stability that
can be utilized for film formation and generation of edible nano-
structures for encapsulation of food additives. These findings were
also confirmed through thermogram analysis. ZW3 EPS has
demonstrated excellent properties and the knowledge of physical,
surface morphology, rheological and thermal analysis; along with
previous explored parameters will enable the food scientist to use
the polymer in food industry in an efficient way.
Acknowledgment
This study was supported by the National Natural Science
Foundation of China (grant no. 31171629) and grant from the
Twelfth Five National Scientific Support grant (863) (No.
2011AA100904 ). We are also thankful to Higher Education
Commission of Pakistan for its financial support.
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