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International Journal of Biological Macromolecules 59 (2013) 377– 383

Contents lists available at SciVerse ScienceDirect

International Journal of Biological Macromolecules

jo ur nal homep age: www.elsev ier .com/ locate / i jb iomac

haracterization of new exopolysaccharides produced by coculturingf L. kefiranofaciens with yoghurt strains

aheer Ahmeda,∗, Yanping Wangb, Nomana Anjuma, Hajra Ahmada,sif Ahmadc, Mohsin Razad

Department of Home & Health Sciences, Allama Iqbal Open University Islamabad, PakistanKey Laboratory of Food Nutrition and Safety, Ministry of Education, Tianjin University of Science & Technology, Tianjin 300457, ChinaDepartment of Food Technology, PMAS-Arid Agriculture University, Rawalpindi, PakistanDepartment of Chemistry, Allama Iqbal Open University Islamabad, Pakistan

a r t i c l e i n f o

rticle history:eceived 29 March 2013eceived in revised form 21 April 2013ccepted 27 April 2013vailable online 7 May 2013

eywords:o-culturingPS

a b s t r a c t

This project was designed to study the coculturing affect of exopolysaccharide (EPS) producing strainsLactobacillus 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 analysisrevealed presence of strong stretch in regions of 3400, 2900 and 1647 cm−1 which is characteristic ofa 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 ofgluco-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,

◦

haracterization. kefiranofaciensoghurt

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 AFManalysis divulged that three cocultured EPSs had different surface morphology than ZW3 polymer. Sinceco-cultured polymers have different structure than the polymer produced exclusively by EPS producingstrain, it can be safely concluded from the study that co-culturing can be one way to change the structureof polymers. Coculturing of L. kefiranofaciens with non-EPS producing strains resulted in yoghurt withincreased viscosity and delayed syneresis.

. Introduction

Exopolysaccharide, the biological macromolecules produced byhe variety of microbes, has been studied extensively during theast decade; firstly due to its numerous health benefits and sec-ndly, owing to its various industrial applications [1–3]. Amongndustrial applications, its action as viscosity enhancer, stabilizernd emulsifier has been reported previously [4]. Long list of theirealth benefits make them a potential candidate to be used inutraceutical and pharma industry that can utilize EPSs to produceroducts having anti-inflammatory, antitumor, immunostimula-ory, immunomodulatory, antiviral, and antioxidant characteristics3–5].

Lactic acid bacteria (LAB) are generally regarded as safe (GRAS)nd polymers produced by these microbes have received atten-

ion of many researchers [6] due to their potential application inhe improvement of the rheology, texture and mouth feel of fer-

ented milk products including yoghurt, cheese, viili, langfil and

∗ Corresponding author. Tel.: +92 519057265.E-mail address: zaheer 863@yahoo.com (Z. Ahmed).

141-8130/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.ijbiomac.2013.04.075

© 2013 Elsevier B.V. All rights reserved.

kefir [7]. Among LAB, Lactobacillus kefiranofaciens is unique for theproduction of exopolysaccharide, kefiran, having numerous healthbenefits [8–11].

Most of the biological and food industries are utilizing a sin-gle culture for production of products. However, using coculturingwill not only improve the product quality but may improve thenutritional status of the food products. To reap the full benefit ofcoculturing techniques features like symbiosis, competition andallelopathy are important that needs characterization [12]. Previ-ously, coculturing is reported by various researchers; these includeco-culture of immobilized Zymomonas mobilis and free cells ofPichia stipitis (reclassified as Scheffersomyces stipitis), co-culture ofethanologenic Escherichia coli strain KO11 with Saccharomyces cere-visiae, co-culture of Z. mobilis and Candida tropicalis for ethanolproduction from hydrolyzed agricultural wastes, co-culture of S.cerevisiae and Pachysolen tannophilus, and coculture of restrictedcatabolite repressed mutant P. stipitis and respiratory-deficientmutant S. cerevisiae [13]. However, very little or no work has been

done on the production and characterization of polysaccharide byco-culturing. In our previous studies, we have isolated a new strainZW3 from Tibet Kefir, which was identified as L. kefiranofaciens ZW3both by biochemical and molecular techniques and its complete

3 Biolog

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2.10. Yoghurt formation by coculturing L. kefiranofaciens withyoghurt strains

78 Z. Ahmed et al. / International Journal of

enome is available online [8,11,14]. Physicochemical propertiesf the polymer produced L. kefiranofaciens ZW3 have been reportedn our previous research with some applicable functional attributes8,11]. The present study was aimed to coculture L. kefiranofaciensW3 with non-EPS producing yoghurt strain, and the characteriza-ion of resulted exopolysaccharides.

. Materials and methods

.1. Strains used

The strain L. kefiranofaciens ZW3 (L.k) was isolated fromibet Kefir; its chromosome and plasmid pWW1 and pWW2equences had been deposited in GenBank under accession num-ers CP002764, CP002765, and CP002766 and had been used inur previous studies [8,14]. The strains Streptococcus thermophilusICC6038 (S.t) and Lactobacillus bulgaricus CICC6032 (L.b) werebtained from China Center of Industrial Culture Collection.

.2. Media used

Milk whey used in liquid whey media was deproteinized bydjusting skim milk to pH 4.6 with 2 N HCl, heating for 30 min at00 ◦C, and filtering. The resulting supernatant was adjusted to pH.8 with 2 N NaOH, heated for 30 min at 100 ◦C, and filtered to obtaineproteinized whey. Whey medium were prepared as describedy Yokoi et al. [15] with some modification. Supplemented wheyedium contained 100 ml of milk whey, 1 g lactose monohydrate,

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

.5 g sodium acetate, 0.1 ml Tween 80, 1 ml mineral solution, and 2 ggar. The mineral solution was composed of 0.4 g/l of MgSO4·7H2O,.15 g/l of MnSO4·4H2O, 0.18 g/l of FeSO4·7H2O, and 0.1 g/l NaCl.kimmed milk was used to study coculturing behavior of L. kefi-anofaciens on viscosity of yoghurt when grown together with L.ulgaricus and S. thermophilus.

.3. Coculturing of L. kefiranofaciens ZW3 with yoghurt strainsnd production of exopolysaccharide

To study coculturing behavior, L. kefiranofaciens ZW3 (EPS pro-ucer) was cultured with traditional yoghurt starter culture i.e.. bulgaricus and S. thermophilus which were non EPS produc-ng strains. EPS was produced by growing L. kefiranofaciens and. bulgaricus (L.k + L.b) (1:1), L. kefiranofaciens and S. thermophilusL.k + S.t) (1:1), and L. kefiranofaciens, L. bulgaricus and S. ther-ophilus (L.k + L.b + S.t) (1:1:1). L.k + S.t, L.k + L.b and L.k + L.b + S.t

tand for cocultured EPSs produced by L. kefiranofaciens and S. ther-ophilus, L. kefiranofaciens and L. bulgaricus, and L. kefiranofaciens,

. bulgaricus and S. thermophilus, respectively.The method used for isolation and purification of EPS was same

s described in our previous study [8].

.4. Study of infrared (FT-IR) spectroscopy

The major structural groups of the purified EPS wereetected using Fourier-transformed infrared spectroscopy. For FTIRpectrum of ZW3 EPS was obtained using KBr method. The polysac-haride samples were pressed into KBr pellets at sample:KBr

atio 1:100. The Fourier transform-infrared spectra were recordedn a Bruker Vector 22 instrument (Germany) in the region of000–400 cm−1, at a resolution of 4 cm−1 and processed by BrukerPUS software.

ical Macromolecules 59 (2013) 377– 383

2.5. Sugar composition analysis

For sugar composition determinations, polysaccharides werehydrolyzed by treatment with 2MTFA (120 ◦C for 2 h). Analysis wasperformed using a Varian GC/MS 4000 instrument (USA) equippedwith 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 analysiswas same as described in our previous study [8].

2.6. Differential scanning calorimeter (DSC)

The thermal properties of EPS were analyzed using a differentialscanning calorimeter (DSC Model 141 SETARAM Scientific & Indus-trial Equipment Co Ltd., France). The 4.2 mg of dried EPS samplewas placed in an aluminum pan. Then it was sealed and analyzed,using empty pan as a reference, for determining the melting pointand enthalpy change. The heating rate was 10 ◦C/min from 20 to300 ◦C.

2.7. Thermogram analysis (TGA)

Pyrolysis and combustion were carried out in Mettler ToledoTGA/SDTA 851e thermal analyzer operating at atmospheric pres-sure. The system was controlled by a compatible PC, which registersthe temperature measured by a thermocouple placed in the cru-cible. The crucible was made of Al2O3. 10 mg of the EPS wasplaced in a platinum crucible and heated at a linear heating rateof 10 ◦C/min over a temperature range 25–1000 ◦C. The experi-ments were performed separately in air and nitrogen atmosphereat a flow rate of 50 ml/min. Prior to the experiment, TGA/SDTA unitwas calibrated for temperature reading using indium as meltingstandard.

2.8. Scanning electron microscopy (SEM)

The surface morphology of the copolymers was investigated byscanning electron microscopy (SEM, JEOL/EO, and model JSM-6380,Japan) at an accelerating voltage of 10 KV147. Samples for scanningelectron microscopy (SEM) analysis were glued to aluminum stubsand 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 wasstirred for about 1 h at 50 ◦C in a sealed bottle under N2 stream sothat ZW3 EPS dissolved completely. After cooling to room tem-perature, the solution was diluted to the final concentration of0.01 mg/ml. About 5 �l of diluted EPS solution was dropped onthe surface of a mica sample carrier, allowed to dry at room tem-perature. Later, the AFM images were obtained by scanning probemicroscope (JEOL JSPM-5200, Japan) in tapping mode. The can-tilever oscillated at its proper frequency (158 KHz) and the drivenamplitude was 0.430 V.

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.

Z. Ahmed et al. / International Journal of Biolog

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ig. 1. Fourier-transformed infrared (FT-IR) spectrum of the cocultured exopolysac-harides and the EPS produced by L. kefiranofaciens ZW3.

. Results and discussion

.1. FTIR analysis of co-cultured EPSs

After fermentation the cocultured EPSs was separated, purifiednd characterized using FTIR. FTIR spectrum of resulted coculturedPSs is presented in comparative form in Fig. 1. In our previoustudy, we have described the FTIR spectra of ZW3 polysacchariden detail [8]. It is depicted from the spectra (Fig. 1) that all theocultured EPSs had peaks ranging from 3395.07 to 589.13 cm−1.he presence of relatively strong absorption peak at 1647.31 cm−1

s the characteristic IR absorption of polysaccharides [16] whichan be attributed to the bending vibration of O H [17]. The bandt 3395.07 cm−1 region was attributed to the stretching vibra-ion of O H in the constituent sugar residues. The stretching at924.22 was associated with the stretching vibration of C H inhe sugar ring [3] while the broad stretch of C O C, C O at000–1200 cm−1 exhibited the presence of carbohydrates [21].

he signal at 1061 cm−1 was attributed to the stretch vibrationf C O and change angle vibration of O H [17]. The absorp-ion at 1245 cm−1 revealed the presence of sulfate groups as S Ond C O S in ZW3, L.k and L.b, and L.k and S.t, while it was

ig. 2. Gas chromatogram of alditol acetate derivative of hydrolyzed cocultured exopolyefiranofaciens ZW3.

ical Macromolecules 59 (2013) 377– 383 379

absent in L.k, S.t and L.b polysaccharide similar to algal polysac-charide. The stretching at 855.31 is indicating the presence ofprimary and secondary sulfate groups. In addition, the absorptionat 890 cm−1 suggests the presence of an �-anomeric configuration[18]. The presence of unique stretching at 1558.07 corresponds tothe N H bending (amide group). The extra peak such as 753.21and 589.13 can be attributed to glycoside linkage. All of this FTIRdata substantiate that coculturing technique is effective in pro-ducing EPS along with some proteinaceous and sulfur containingsubstances. This discovery is important and shows the coculturingpotential of these microorganisms for production of nutraceuti-cal food products. There are also some extra peaks present insome 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 natureand one example of that is ZW3 polysaccharide produced by L.kefiranofaciens ZW3 [8]. By GCMs analysis, it was revealed thatcocultured EPSs contained some additional sugars such as arab-inose and xylose which were not present in EPS produced byL. kefiranofaciens ZW3. However, when ZW3 strain accompanied,either of the yoghurt strain or by both, additional hexose wereadded 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 thatthe EPS is a heteropolysaccharide and confirm the previous stud-ies of Kanmani et al. [1] who reported that Streptococcus phocaePI80 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 ourprevious 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 ourcase all the cocultured EPSs had xylose in their sugar compositionand so the produced EPSs can be claimed as the new polysaccha-ride.

saccharides: 1, L.k + L.b; 2, L.k. + L.b + S.t; 3, L.k + S.t EPS; 4, ZW3 EPS produced by L.

380 Z. Ahmed et al. / International Journal of Biological Macromolecules 59 (2013) 377– 383

Table 1Thermal properties of cocultured L. kefiranofaciens ZW3 exopolysaccharide (EPS) bydifferential scanning calorimetry (DSC).

Sample name Peaktemperature (◦C)

Enthalpy(J/g)

ZW3 EPS 97.38 249.7Xanthan gum 153.4 93.2Guar gum 490.1 192.9Locust gum 109.11 87.1L. kefiranofaciens ZW3 + S.

thermophilus EPS87.61 243.6

L. kefiranofaciens ZW3 + L.bulgaricus EPS

90.59 247.3

L. kefiranofaciens ZW3 + L. 95.18 239.8

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bulgaricus + S.thermophilus EPS

.3. Differential scanning calorimeter (DSC)

Along with other attributes, industrial application and commer-ial utilization of polysaccharide are largely dependent upon itshermal properties [1,21]. Differential scanning calorimetric (DSC)nalysis was performed in order to investigate the energy levelsnd changes in enthalpy (�H) values of cocultured EPSs with heatow from 25 to 300 ◦C. DSC results are depicted in Table 1.

The peak temperature for ZW3 EPS was 97.38 ◦C and thenthalpy change needed to melt 1 g of EPS was about 249.7 J. How-ver, when EPSs were produced by coculturing the L. kefiranofaciensith yoghurt strains, the produced cocultured EPSs showed differ-

nt thermal behavior. L.k + S.t, L.k + L.b, and L.k + S.t + L.b have peakemperature of 87.61, 90.59 and 95.18 ◦C; enthalpy change needo melt 1 g of EPS was 243.6, 247.3 and 239.8, respectively. Theeak melting temperature for reference material such as locustum, xanthan gum and guar gum was 109.11, 153.4 and 490.1,espectively; the enthalpy change needed to melt 1 g of EPS was7.1, 93.2 and 192.9, respectively (Table 1). All the cocultured EPSave lower peak temperature and enthalpy change, as compared toW3 exopolysaccharide and reference material. The values for peakemperature and enthalpy changes are slightly lower as reportedy Kanmani et al. [1] for the EPS produced by S. phocae PI80 withelting point of 120.09 ◦C and the enthalpy change needed to melt

g of EPS was about 404.6 J.

.4. Thermogram analysis

Thermogravimetric analysis (TGA) is a simple analytical tech-ique that measures the weight loss of a material as a functionf temperature [22]. In thermal analysis of EPS, heat is emittednd absorbed which is accompanied by change in structure ofolymer and in melting of crystalline polymer [19]. The thermoravimetric analysis was carried out dynamically (weight lossersus temperature) and the experimental results are presentedn Fig. 3. A degradation temperature (Td) 294.6 ◦C was deter-

ined for L.k + S.t EPS. An initial weight loss (10%) between 50nd 105 ◦C was attributed to moisture and alcohol content trappedn the exopolysaccharide. The presence of the increased mois-ure content and alcohol can be attributed to the presence ofigh carboxyl groups. A dramatic weight loss (about 60%) occursetween 273.38 and 294.96 ◦C. Complete weight loss of L.k + S.tPS occurs after 400 ◦C. In case L.k + L.b cocultured EPS, a degra-ation temperature (Td) 326.44 ◦C was recorded. An initial weight

oss (8%) was between 30 and 95 ◦C and after that a dramatic losseight loss (45%) between 263.63 and 326.44 ◦C was observed;

owever, the TGA curve was less steep sloped as compared to.k + S.t EPS at the same temperature. Complete weight loss of.k + L.b EPS occurs after 600 ◦C leading to conclusion that it isore 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 temperature296.7 ◦C was observed. About 15% weight loss occurred when EPSwas exposed to temperature range of 30–92 ◦C. Like both of othercocultured EPSs, major weight loss (50%) occurred between tem-perature range of EPS 275.83–296.76 ◦C and TGA curve had steepslope 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 reportedthe TGA analysis of ZW3 and reference polysaccharide such asxanthan gum and locust gum. In case of ZW3 EPS, a degradation

Z. Ahmed et al. / International Journal of Biological Macromolecules 59 (2013) 377– 383 381

EPSs. (

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Fig. 4. Atomic force microscopy (AFM) of different cocultured

emperature (Td) of 299.62 ◦C was determined from the TGA curveor the polysaccharide ZW3 and about 18% weight loss occuretween temperature range of 40–90 ◦C. The onset of decomposi-ion occurred at 261.4 ◦C and up to 299.62 ◦C the recorded mass lossas 20%. ZW3 EPS was completely decomposed when temperature

pproached beyond 600 ◦C. TG analysis of xanthan gum and locustum as reference material indicated a degradation temperature of82.65 ◦C for xanthan gum, whereas for locust gum it was 278.46 ◦C.rom TGA analysis of cocultured EPSs, it is clear that all theolysaccharides have different pattern of stability to the exposedemperature.

.5. SEM analysis

Scanning electron microscope is a useful tool to study the

urface morphology of polymer and also to predict its physicalroperties [11,19,23]. There was significant difference in surfaceorphology of the three cocultured polymer (Fig. 4). Surface of

.k + S.t polymer at 600× is very smooth and is similar up to some

A and B) L.k + S.t, (C and D) L.k + L.b, (E and F) L.k + L.b + S.t EPS.

extent to surface of polysaccharide produced by L. kefiranofaciensZW3 [8]. At 1000×, it seems that polymer is made of long threadswhich are very compact. A smooth surface is good prediction to usethe polymer for film making. At 1000×, the surface of the secondcocultured polymer, i.e. L.k + L.b, is significantly different from thesurface 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 EPSis rough and is quite different from the L.k + S.t and L.k + L.b + S.tpolymer. The difference is even obvious when it is observed at6000× 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.twas made of heterogeneous material. Homogenous consistency

of L.k + L.b and L.k + S.t polymers is indication of their structuralintegrity which makes them a good choice to be used in polymerfilm making [11]; whereas L.k + L.b + S.t may result in inferior filmformation due to its dull and rough appearance. KF5 EPS reported

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×o

bi

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y Wang et al. [19] also had dull and porous surface, which madet unfit for film making.

.6. Atomic force micrograph (AFM) of ZW EPS

SEM has not sufficient vertical resolution to appreciate vari-tions of the topography at the nanometer scale [24]. AFMeasurement was performed to analyze the surface roughness and

orphology of the polymer and a owing to its ability to measure

nteraction forces in liquids at a pico- or nano-Newton level withigh vertical and lateral resolutions [11,25]. The AFM images ofocultured EPSs are presented in Fig. 5. Size and arrangements of

the molecules of resulted three cocultured EPSs are significantlydifferent from each other. In case of L.k + S.t, maximum height ofthe lump was 41.3 nm. The presence of long thread like lumpsmake its quite different from other two cocultured EPSs, i.e. L.k + L.band L.k + L.b + S.t. If we ignore the thread like structure, the surfaceof polymer looks like a film having uniform texture. The secondcocultured polymer L.k + L.b has lumps with maximum height of65.5 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 bigsize are dispersed in patches; otherwise the polymer gives the lookof a uniform film with compact structure.

Z. Ahmed et al. / International Journal of Biolog

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ig. 6. The coculturing behavior of L. kefiranofaciens on viscosity of yoghurt whenrown together with L. bulgaricus and S. thermophilus.

.7. Yoghurt formation by coculturing L. kefiranofaciens withoghurt strains

L. kefiranofaciens strain was grown together with L. bulgaricusnd S. thermophilus which were non EPS producing strain and effectn viscosity was studied and depicted in Fig. 6. Controlled yoghurtas also produced by traditional yoghurt strains i.e. L. bulgaricus

nd S. thermophilus. It is clear from Fig. 6 that yoghurt producedy coculturing with L. kefiranofaciens has higher viscosity as com-ared to yoghurt made by only traditional yoghurt strains. So the. kefiranofaciens has good potential to be used in yoghurt indus-ry. Moreover, co-cultured yoghurt showed no syneresis at roomemperature kept up to 1 month. Preliminary sensory evaluationf yoghurt was also done; it was liked by the most of the con-umer in its fresh mode or, when kept in refrigerator even up to

months. However, acceptability of yoghurt stored at room tem-erature decreased with passage of time, mainly due to productionf off flavored (results not depicted here).

. Conclusion

In this study, coculturing effect of L. kefiranofaciens ZW3 withraditional yoghurt strain i.e. L. bulgaricus and S. thermophilusas explored. Characterizations of cocultured polymers revealed

hat these have different physiochemical properties than ZW3xopolysaccharide produced exclusively by L. kefiranofaciens ZW3.

as chromatography revealed that cocultured EPS is a het-ropolysaccharide and is quite different from ZW3 polysaccharide.FM and SEM also predicted that polymers have different sur-

ace morphology and topography. Exopolysaccharide produced by

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ical Macromolecules 59 (2013) 377– 383 383

coculturing of L.k + L.b and L.k + S.t has a potential for making ofbiopolymer films. Moreover, the coculturing can result in yoghurtwith enhanced viscosity and with delayed syneresis mechanism upto 3 months.

Acknowledgment

Author is grateful to Higher Education Commission of Pakistanfor financial support.

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