Coculturing

1. International Journal of Biological Macromolecules 59 (2013) 377–383
Contents lists available at SciVerse ScienceDirect
International Journal of Biological Macromolecules
journal homepage: www.elsevier.com/locate/ijbiomac
Characterization of new exopolysaccharides produced by coculturing
of L. kefiranofaciens with yoghurt strains
Zaheer Ahmeda,∗
, Yanping Wangb
, Nomana Anjuma
, Hajra Ahmada
,
Asif Ahmadc
, Mohsin Razad
a
Department of Home & Health Sciences, Allama Iqbal Open University Islamabad, Pakistan
b
Key Laboratory of Food Nutrition and Safety, Ministry of Education, Tianjin University of Science & Technology, Tianjin 300457, China
c
Department of Food Technology, PMAS-Arid Agriculture University, Rawalpindi, Pakistan
d
Department of Chemistry, Allama Iqbal Open University Islamabad, Pakistan
a r t i c l e i n f o
Article history:
Received 29 March 2013
Received in revised form 21 April 2013
Accepted 27 April 2013
Available online 7 May 2013
Keywords:
Co-culturing
EPS
Characterization
L. kefiranofaciens
Yoghurt
a b s t r a c t
This project was designed to study the coculturing affect of exopolysaccharide (EPS) producing strains
Lactobacillus kefiranofaciens (L.k) ZW3, with non EPS producing strains L. bulgaricus (L.b) and Streptococ-
cus thermophilus (S.t) in three different combinations: L.k + L.b, L.k + S.t, and L.k + L.b + S.t. FTIR analysis
revealed presence of strong stretch in regions of 3400, 2900 and 1647 cm−1
which is characteristic of
a typical polysaccharide. Co-cultured EPSs were composed of glucose, galactose, arabinose and xylose;
and their sugar compositions were different from ZW3 polysaccharide that was mainly composed of
gluco-galactan. Peak temperature for L.k + L.b, L.k + S.t, L.k + S.t + L.b and ZW3 polymers were 90.59, 87.61,
95.18 and 97.38 ◦
C, respectively. Thermal analysis revealed degradation temperature of 326.44, 294.6,
296.7 and 299.62 ◦
C for L.k + L.b, L.k + S.t, L.k + S.t + L.b and ZW3 polymers, respectively. SEM and AFM
analysis divulged that three cocultured EPSs had different surface morphology than ZW3 polymer. Since
co-cultured polymers have different structure than the polymer produced exclusively by EPS producing
strain, it can be safely concluded from the study that co-culturing can be one way to change the structure
of polymers. Coculturing of L. kefiranofaciens with non-EPS producing strains resulted in yoghurt with
increased viscosity and delayed syneresis.
© 2013 Elsevier B.V. All rights reserved.
1. Introduction
Exopolysaccharide, the biological macromolecules produced by
the variety of microbes, has been studied extensively during the
last decade; firstly due to its numerous health benefits and sec-
ondly, owing to its various industrial applications [1–3]. Among
industrial applications, its action as viscosity enhancer, stabilizer
and emulsifier has been reported previously [4]. Long list of their
health benefits make them a potential candidate to be used in
nutraceutical and pharma industry that can utilize EPSs to produce
products having anti-inflammatory, antitumor, immunostimula-
tory, immunomodulatory, antiviral, and antioxidant characteristics
[3–5].
Lactic acid bacteria (LAB) are generally regarded as safe (GRAS)
and polymers produced by these microbes have received atten-
tion of many researchers [6] due to their potential application in
the improvement of the rheology, texture and mouth feel of fer-
mented milk products including yoghurt, cheese, viili, langfil and
∗ Corresponding author. Tel.: +92 519057265.
E-mail address: zaheer 863@yahoo.com (Z. Ahmed).
kefir [7]. Among LAB, Lactobacillus kefiranofaciens is unique for the
production of exopolysaccharide, kefiran, having numerous health
benefits [8–11].
Most of the biological and food industries are utilizing a sin-
gle culture for production of products. However, using coculturing
will not only improve the product quality but may improve the
nutritional status of the food products. To reap the full benefit of
coculturing techniques features like symbiosis, competition and
allelopathy are important that needs characterization [12]. Previ-
ously, coculturing is reported by various researchers; these include
co-culture of immobilized Zymomonas mobilis and free cells of
Pichia stipitis (reclassified as Scheffersomyces stipitis), co-culture of
ethanologenic Escherichia coli strain KO11 with Saccharomyces cere-
visiae, co-culture of Z. mobilis and Candida tropicalis for ethanol
production from hydrolyzed agricultural wastes, co-culture of S.
cerevisiae and Pachysolen tannophilus, and coculture of restricted
catabolite repressed mutant P. stipitis and respiratory-deficient
mutant S. cerevisiae [13]. However, very little or no work has been
done on the production and characterization of polysaccharide by
co-culturing. In our previous studies, we have isolated a new strain
ZW3 from Tibet Kefir, which was identified as L. kefiranofaciens ZW3
both by biochemical and molecular techniques and its complete
0141-8130/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ijbiomac.2013.04.075

2. 378 Z. Ahmed et al. / International Journal of Biological Macromolecules 59 (2013) 377–383
genome is available online [8,11,14]. Physicochemical properties
of the polymer produced L. kefiranofaciens ZW3 have been reported
in our previous research with some applicable functional attributes
[8,11]. The present study was aimed to coculture L. kefiranofaciens
ZW3 with non-EPS producing yoghurt strain, and the characteriza-
tion of resulted exopolysaccharides.
2. Materials and methods
2.1. Strains used
The strain L. kefiranofaciens ZW3 (L.k) was isolated from
Tibet Kefir; its chromosome and plasmid pWW1 and pWW2
sequences had been deposited in GenBank under accession num-
bers CP002764, CP002765, and CP002766 and had been used in
our previous studies [8,14]. The strains Streptococcus thermophilus
CICC6038 (S.t) and Lactobacillus bulgaricus CICC6032 (L.b) were
obtained from China Center of Industrial Culture Collection.
2.2. Media used
Milk whey used in liquid whey media was deproteinized by
adjusting skim milk to pH 4.6 with 2 N HCl, heating for 30 min at
100 ◦C, and filtering. The resulting supernatant was adjusted to pH
6.8 with 2 N NaOH, heated for 30 min at 100 ◦C, and filtered to obtain
deproteinized whey. Whey medium were prepared as described
by Yokoi et al. [15] with some modification. Supplemented whey
medium contained 100 ml of milk whey, 1 g lactose monohydrate,
0.5 g glucose, 0.5 g tryptone, 0.05 g 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 0.4 g/l of MgSO4·7H2O,
0.15 g/l of MnSO4·4H2O, 0.18 g/l of FeSO4·7H2O, and 0.1 g/l NaCl.
Skimmed milk was used to study coculturing behavior of L. kefi-
ranofaciens on viscosity of yoghurt when grown together with L.
bulgaricus and S. thermophilus.
2.3. Coculturing of L. kefiranofaciens ZW3 with yoghurt strains
and production of exopolysaccharide
To study coculturing behavior, L. kefiranofaciens ZW3 (EPS pro-
ducer) was cultured with traditional yoghurt starter culture i.e.
L. bulgaricus and S. thermophilus which were non EPS produc-
ing strains. EPS was produced by growing L. kefiranofaciens and
L. bulgaricus (L.k + L.b) (1:1), L. kefiranofaciens and S. thermophilus
(L.k + S.t) (1:1), and L. kefiranofaciens, L. bulgaricus and S. ther-
mophilus (L.k + L.b + S.t) (1:1:1). L.k + S.t, L.k + L.b and L.k + L.b + S.t
stand for cocultured EPSs produced by L. kefiranofaciens and S. ther-
mophilus, L. kefiranofaciens and L. bulgaricus, and L. kefiranofaciens,
L. bulgaricus and S. thermophilus, respectively.
The method used for isolation and purification of EPS was same
as described in our previous study [8].
2.4. Study of infrared (FT-IR) spectroscopy
The major structural groups of the purified EPS were
detected using Fourier-transformed infrared spectroscopy. For FTIR
spectrum of ZW3 EPS was obtained using KBr method. The polysac-
charide samples were pressed into KBr pellets at sample:KBr
ratio 1:100. The Fourier transform-infrared spectra were recorded
on a Bruker Vector 22 instrument (Germany) in the region of
4000–400 cm−1, at a resolution of 4 cm−1 and processed by Bruker
OPUS software.
2.5. Sugar composition analysis
For sugar composition determinations, polysaccharides were
hydrolyzed by treatment with 2MTFA (120 ◦C for 2 h). Analysis was
performed using a Varian GC/MS 4000 instrument (USA) equipped
with VF-5ms 30 m × 0.25 mm × 0.10 ␮m column. Sugar identifica-
tion was done by comparison with reference sugars (l-rhamnose,
l-fructose, l-arabinose, d-xylose, d-mannose, d-galactose and d-
glucose). Detailed procedure of sample preparation and analysis
was same as described in our previous study [8].
2.6. Differential scanning calorimeter (DSC)
The thermal properties of EPS were analyzed using a differential
scanning calorimeter (DSC Model 141 SETARAM Scientific & Indus-
trial Equipment Co Ltd., France). The 4.2 mg of dried EPS sample
was placed in an aluminum pan. Then it was sealed and analyzed,
using empty pan as a reference, for determining the melting point
and enthalpy change. The heating rate was 10 ◦C/min from 20 to
300 ◦C.
2.7. Thermogram analysis (TGA)
Pyrolysis and combustion were carried out in Mettler Toledo
TGA/SDTA 851e thermal analyzer operating at atmospheric pres-
sure. The system was controlled by a compatible PC, which registers
the temperature measured by a thermocouple placed in the cru-
cible. The crucible was made of Al2O3. 10 mg of the EPS was
placed in a platinum crucible and heated at a linear heating rate
of 10 ◦C/min over a temperature range 25–1000 ◦C. The experi-
ments were performed separately in air and nitrogen atmosphere
at a flow rate of 50 ml/min. Prior to the experiment, TGA/SDTA unit
was calibrated for temperature reading using indium as melting
standard.
2.8. Scanning electron microscopy (SEM)
The surface morphology of the copolymers was investigated by
scanning electron microscopy (SEM, JEOL/EO, and model JSM-6380,
Japan) at an accelerating voltage of 10 KV147. Samples for scanning
electron microscopy (SEM) analysis were glued to aluminum stubs
and gold-sputtered, before SEM examination.
2.9. Atomic force micrograph (AFM) of ZW EPS
EPS solution (1 mg/ml) was prepared by adding some puri-
fied ZW3 EPS into double distilled H2O. The aqueous solution was
stirred for about 1 h at 50 ◦C in a sealed bottle under N2 stream so
that ZW3 EPS dissolved completely. After cooling to room tem-
perature, the solution was diluted to the final concentration of
0.01 mg/ml. About 5 ␮l of diluted EPS solution was dropped on
the surface of a mica sample carrier, allowed to dry at room tem-
perature. Later, the AFM images were obtained by scanning probe
microscope (JEOL JSPM-5200, Japan) in tapping mode. The can-
tilever oscillated at its proper frequency (158 KHz) and the driven
amplitude was 0.430 V.
2.10. Yoghurt formation by coculturing L. kefiranofaciens with
yoghurt strains
Skimmed milk was used to study coculturing behavior of L.
kefiranofaciens on yoghurt viscosity, when grown together with L.
bulgaricus and S. thermophilus in equal proportions.

3. Z. Ahmed et al. / International Journal of Biological Macromolecules 59 (2013) 377–383 379
Fig. 1. Fourier-transformed infrared (FT-IR) spectrum of the cocultured exopolysac-
charides and the EPS produced by L. kefiranofaciens ZW3.
3. Results and discussion
3.1. FTIR analysis of co-cultured EPSs
After fermentation the cocultured EPSs was separated, purified
and characterized using FTIR. FTIR spectrum of resulted cocultured
EPSs is presented in comparative form in Fig. 1. In our previous
study, we have described the FTIR spectra of ZW3 polysaccharide
in detail [8]. It is depicted from the spectra (Fig. 1) that all the
cocultured EPSs had peaks ranging from 3395.07 to 589.13 cm−1.
The presence of relatively strong absorption peak at 1647.31 cm−1
is the characteristic IR absorption of polysaccharides [16] which
can be attributed to the bending vibration of O H [17]. The band
at 3395.07 cm−1 region was attributed to the stretching vibra-
tion of O H in the constituent sugar residues. The stretching at
2924.22 was associated with the stretching vibration of C H in
the sugar ring [3] while the broad stretch of C O C, C O at
1000–1200 cm−1 exhibited the presence of carbohydrates [21].
The signal at 1061 cm−1 was attributed to the stretch vibration
of C O and change angle vibration of O H [17]. The absorp-
tion at 1245 cm−1 revealed the presence of sulfate groups as S O
and C O S in ZW3, L.k and L.b, and L.k and S.t, while it was
absent in L.k, S.t and L.b polysaccharide similar to algal polysac-
charide. The stretching at 855.31 is indicating the presence of
primary and secondary sulfate groups. In addition, the absorption
at 890 cm−1 suggests the presence of an ␤-anomeric configuration
[18]. The presence of unique stretching at 1558.07 corresponds to
the N H bending (amide group). The extra peak such as 753.21
and 589.13 can be attributed to glycoside linkage. All of this FTIR
data substantiate that coculturing technique is effective in pro-
ducing EPS along with some proteinaceous and sulfur containing
substances. This discovery is important and shows the coculturing
potential of these microorganisms for production of nutraceuti-
cal food products. There are also some extra peaks present in
some co-cultured EPSs which were absent in ZW3 polysaccha-
ride.
3.2. Sugar composition
GCMS analysis of cocultured EPS (Fig. 2) indicated that polysac-
charide produced by L. kefiranofaciens is gluco-galactan in nature
and one example of that is ZW3 polysaccharide produced by L.
kefiranofaciens ZW3 [8]. By GCMs analysis, it was revealed that
cocultured EPSs contained some additional sugars such as arab-
inose and xylose which were not present in EPS produced by
L. kefiranofaciens ZW3. However, when ZW3 strain accompanied,
either of the yoghurt strain or by both, additional hexose were
added up in resulted EPS. It is important to note that both of con-
ventional yoghurt strains appeared as non EPS producer, only L.
kefiranofaciens have the ability to produce EPS in the tested cocul-
turing conditions. The gluco-galactan nature of EPS suggests that
the EPS is a heteropolysaccharide and confirm the previous stud-
ies of Kanmani et al. [1] who reported that Streptococcus phocae
PI80 produce heteropolysaccharide EPS that composed of arab-
inose, fructose and galactose; whereas Lactococcus lactis subsp.
lactis contains fructose and rhamnose as sugar unit [5]. In our
previous studies, we have reported the L. plantarum KF5 which pro-
duces the EPS composed of mannose, glucose and galactose [19].
Xylose is often one of the predominant sugars in plant biomass
and mostly not present in bacterial polysaccharide [20] and in our
case all the cocultured EPSs had xylose in their sugar composition
and so the produced EPSs can be claimed as the new polysaccha-
ride.
Fig. 2. Gas chromatogram of alditol acetate derivative of hydrolyzed cocultured exopolysaccharides: 1, L.k + L.b; 2, L.k. + L.b + S.t; 3, L.k + S.t EPS; 4, ZW3 EPS produced by L.
kefiranofaciens ZW3.

4. 380 Z. Ahmed et al. / International Journal of Biological Macromolecules 59 (2013) 377–383
Table 1
Thermal properties of cocultured L. kefiranofaciens ZW3 exopolysaccharide (EPS) by
differential scanning calorimetry (DSC).
Sample name Peak
temperature (◦
C)
Enthalpy
(J/g)
ZW3 EPS 97.38 249.7
Xanthan gum 153.4 93.2
Guar gum 490.1 192.9
Locust gum 109.11 87.1
L. kefiranofaciens ZW3 + S.
thermophilus EPS
87.61 243.6
L. kefiranofaciens ZW3 + L.
bulgaricus EPS
90.59 247.3
L. kefiranofaciens ZW3 + L.
bulgaricus + S.
thermophilus EPS
95.18 239.8
3.3. Differential scanning calorimeter (DSC)
Along with other attributes, industrial application and commer-
cial utilization of polysaccharide are largely dependent upon its
thermal properties [1,21]. Differential scanning calorimetric (DSC)
analysis was performed in order to investigate the energy levels
and changes in enthalpy ( H) values of cocultured EPSs with heat
flow from 25 to 300 ◦C. DSC results are depicted in Table 1.
The peak temperature for ZW3 EPS was 97.38 ◦C and the
enthalpy change needed to melt 1 g of EPS was about 249.7 J. How-
ever, when EPSs were produced by coculturing the L. kefiranofaciens
with yoghurt strains, the produced cocultured EPSs showed differ-
ent thermal behavior. L.k + S.t, L.k + L.b, and L.k + S.t + L.b have peak
temperature of 87.61, 90.59 and 95.18 ◦C; enthalpy change need
to melt 1 g of EPS was 243.6, 247.3 and 239.8, respectively. The
peak melting temperature for reference material such as locust
gum, xanthan gum and guar gum was 109.11, 153.4 and 490.1,
respectively; the enthalpy change needed to melt 1 g of EPS was
87.1, 93.2 and 192.9, respectively (Table 1). All the cocultured EPS
have lower peak temperature and enthalpy change, as compared to
ZW3 exopolysaccharide and reference material. The values for peak
temperature and enthalpy changes are slightly lower as reported
by Kanmani et al. [1] for the EPS produced by S. phocae PI80 with
melting point of 120.09 ◦C and the enthalpy change needed to melt
1 g of EPS was about 404.6 J.
3.4. Thermogram analysis
Thermogravimetric analysis (TGA) is a simple analytical tech-
nique that measures the weight loss of a material as a function
of temperature [22]. 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 [19]. The thermo
gravimetric analysis was carried out dynamically (weight loss
versus temperature) and the experimental results are presented
in Fig. 3. A degradation temperature (Td) 294.6 ◦C was deter-
mined for L.k + S.t EPS. An initial weight loss (10%) between 50
and 105 ◦C was attributed to moisture and alcohol content trapped
in the exopolysaccharide. The presence of the increased mois-
ture content and alcohol can be attributed to the presence of
high carboxyl groups. A dramatic weight loss (about 60%) occurs
between 273.38 and 294.96 ◦C. Complete weight loss of L.k + S.t
EPS occurs after 400 ◦C. In case L.k + L.b cocultured EPS, a degra-
dation temperature (Td) 326.44 ◦C was recorded. An initial weight
loss (8%) was between 30 and 95 ◦C and after that a dramatic loss
weight loss (45%) between 263.63 and 326.44 ◦C was observed;
however, the TGA curve was less steep sloped as compared to
L.k + S.t EPS at the same temperature. Complete weight loss of
L.k + L.b EPS occurs after 600 ◦C leading to conclusion that it is
more heat tolerable as compared to L.k + S.t EPS. In the third kind
Fig. 3. TG curves of cocultured exopolysaccharides. (A) L.k + S.t, (B) L.k + L.b, (C)
L.k + L.b + S.t EPS.
of cocultured EPS, i.e. L.k + S.t + L.b EPS, a degradation temperature
296.7 ◦C was observed. About 15% weight loss occurred when EPS
was exposed to temperature range of 30–92 ◦C. Like both of other
cocultured EPSs, major weight loss (50%) occurred between tem-
perature range of EPS 275.83–296.76 ◦C and TGA curve had steep
slope similar to L.k + S.t EPS. Complete weight loss of L.k + S.t + L.b
EPS occurs after 350 ◦C. In our previous study, we have reported
the TGA analysis of ZW3 and reference polysaccharide such as
xanthan gum and locust gum. In case of ZW3 EPS, a degradation

5. Z. Ahmed et al. / International Journal of Biological Macromolecules 59 (2013) 377–383 381
Fig. 4. Atomic force microscopy (AFM) of different cocultured EPSs. (A and B) L.k + S.t, (C and D) L.k + L.b, (E and F) L.k + L.b + S.t EPS.
temperature (Td) of 299.62 ◦C was determined from the TGA curve
for the polysaccharide ZW3 and about 18% weight loss occur
between temperature range of 40–90 ◦C. The onset of decomposi-
tion occurred at 261.4 ◦C and up to 299.62 ◦C the recorded mass loss
was 20%. ZW3 EPS was completely decomposed when temperature
approached beyond 600 ◦C. TG analysis of xanthan gum and locust
gum as reference material indicated a degradation temperature of
282.65 ◦C for xanthan gum, whereas for locust gum it was 278.46 ◦C.
From TGA analysis of cocultured EPSs, it is clear that all the
polysaccharides have different pattern of stability to the exposed
temperature.
3.5. SEM analysis
Scanning electron microscope is a useful tool to study the
surface morphology of polymer and also to predict its physical
properties [11,19,23]. There was significant difference in surface
morphology of the three cocultured polymer (Fig. 4). Surface of
L.k + S.t polymer at 600× is very smooth and is similar up to some
extent to surface of polysaccharide produced by L. kefiranofaciens
ZW3 [8]. At 1000×, it seems that polymer is made of long threads
which are very compact. A smooth surface is good prediction to use
the polymer for film making. At 1000×, the surface of the second
cocultured polymer, i.e. L.k + L.b, is significantly different from the
surface of L.k + S.t and seems to be made of thin sheets. At 6000×,
the surface L.k + L.b polymer is smooth with glittering properties.
Smoothness of the surface is less as compared to L.k + S.t poly-
mer. When examined at 1000×, the surface of the L.k + L.b + S.t EPS
is rough and is quite different from the L.k + S.t and L.k + L.b + S.t
polymer. The difference is even obvious when it is observed at
6000× which gives indication of very rough surface of the polymer.
From SEM scan it can be predicted that L.k + L.b and L.k + S.t poly-
mers were composed of homogeneous matrix, while L.k + L.b + S.t
was made of heterogeneous material. Homogenous consistency
of L.k + L.b and L.k + S.t polymers is indication of their structural
integrity which makes them a good choice to be used in polymer
film making [11]; whereas L.k + L.b + S.t may result in inferior film
formation due to its dull and rough appearance. KF5 EPS reported

6. 382 Z. Ahmed et al. / International Journal of Biological Macromolecules 59 (2013) 377–383
Fig. 5. SEM results of different cocultured EPSs. (A) and (B) at 1000× and 6000× of L.k + S.t; (C) and (D) at 1000× and 6000× of L.k + L.b; and (E) and (F) at 1000× and 6000×
of L.k + L.b + S.t EPS.
by Wang et al. [19] also had dull and porous surface, which made
it unfit for film making.
3.6. Atomic force micrograph (AFM) of ZW EPS
SEM has not sufficient vertical resolution to appreciate vari-
ations of the topography at the nanometer scale [24]. AFM
measurement was performed to analyze the surface roughness and
morphology of the polymer and a owing to its ability to measure
interaction forces in liquids at a pico- or nano-Newton level with
high vertical and lateral resolutions [11,25]. The AFM images of
cocultured EPSs are presented in Fig. 5. Size and arrangements of
the molecules of resulted three cocultured EPSs are significantly
different from each other. In case of L.k + S.t, maximum height of
the lump was 41.3 nm. The presence of long thread like lumps
make its quite different from other two cocultured EPSs, i.e. L.k + L.b
and L.k + L.b + S.t. If we ignore the thread like structure, the surface
of polymer looks like a film having uniform texture. The second
cocultured polymer L.k + L.b has lumps with maximum height of
65.5 nm. Most of the lumps are closely associated and have uni-
formly distribution with tight packaging. The third cocultured EPS,
L.k + L.b + S.t., has lump with maximum size 20 nm. Lumps with big
size are dispersed in patches; otherwise the polymer gives the look
of a uniform film with compact structure.

7. Z. Ahmed et al. / International Journal of Biological Macromolecules 59 (2013) 377–383 383
Fig. 6. The coculturing behavior of L. kefiranofaciens on viscosity of yoghurt when
grown together with L. bulgaricus and S. thermophilus.
3.7. Yoghurt formation by coculturing L. kefiranofaciens with
yoghurt strains
L. kefiranofaciens strain was grown together with L. bulgaricus
and S. thermophilus which were non EPS producing strain and effect
on viscosity was studied and depicted in Fig. 6. Controlled yoghurt
was also produced by traditional yoghurt strains i.e. L. bulgaricus
and S. thermophilus. It is clear from Fig. 6 that yoghurt produced
by coculturing with L. kefiranofaciens has higher viscosity as com-
pared to yoghurt made by only traditional yoghurt strains. So the
L. kefiranofaciens has good potential to be used in yoghurt indus-
try. Moreover, co-cultured yoghurt showed no syneresis at room
temperature kept up to 1 month. Preliminary sensory evaluation
of yoghurt was also done; it was liked by the most of the con-
sumer in its fresh mode or, when kept in refrigerator even up to
3 months. However, acceptability of yoghurt stored at room tem-
perature decreased with passage of time, mainly due to production
of off flavored (results not depicted here).
4. Conclusion
In this study, coculturing effect of L. kefiranofaciens ZW3 with
traditional yoghurt strain i.e. L. bulgaricus and S. thermophilus
was explored. Characterizations of cocultured polymers revealed
that these have different physiochemical properties than ZW3
exopolysaccharide produced exclusively by L. kefiranofaciens ZW3.
Gas chromatography revealed that cocultured EPS is a het-
eropolysaccharide and is quite different from ZW3 polysaccharide.
AFM and SEM also predicted that polymers have different sur-
face morphology and topography. Exopolysaccharide produced by
coculturing of L.k + L.b and L.k + S.t has a potential for making of
biopolymer films. Moreover, the coculturing can result in yoghurt
with enhanced viscosity and with delayed syneresis mechanism up
to 3 months.
Acknowledgment
Author is grateful to Higher Education Commission of Pakistan
for financial support.
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