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Food Hydrocolloids 30 (2013) 343e350

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Food Hydrocolloids

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Characterization of exopolysaccharide produced by Lactobacillus kefiranofaciensZW3 isolated from Tibet kefir e Part II

Zaheer Ahmed a,b,*, Yanping Wang b, Nomana Anjum a, Asif Ahmad c, Salman Tariq Khan d

aDepartment of Home & Health Sciences, Allama Iqbal Open University, Islamabad, Pakistanb Tianjin Key Laboratory of Food Nutrition and Safety, Faculty of Food Engineering and Biotechnology, Tianjin University of Science and Technology, People’s Republic of ChinacDepartment of Food Technology, Pir Mehr Ali Shah Arid Agriculture University Rawalpindi, Pakistand 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 2011Accepted 6 June 2012

Keywords:Lactobacillus kefiranofaciens ZW3RheologicalTibet kefirCharacterization

* Corresponding author. Department of Home & HOpen University, Islamabad, Pakistan. Tel.: þ92 51905

E-mail address: zaheer_863@yahoo.com (Z. Ahme

0268-005X/$ e see front matter � 2012 Elsevier Ltd.http://dx.doi.org/10.1016/j.foodhyd.2012.06.009

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. Presentresearch demonstrates some other important aspects of this EPS. The molecular weight obtained by gelpermeation HPLC was 5.5 � 104 Da. Solubility, water holding and oil binding capacity of ZW3 EPS were14.2%, 496.0%, and 884.74% respectively. Scanning electron microscopy (SEM) of ZW3 EPS demonstrateda smooth surface with compact structures. A topographical examination of EPS by atomic forcemicroscopy (AFM) revealed that ZW3 EPS is composed of almost uniform net of molecules. Rheologicalstudy indicated that common salt did not affect the viscous behavior of ZW3 EPS and acidic pH mayenhance its viscosity. Exopolymer showed a melting point of 93.38 �C. A degradation temperature (Td) of299.62 �C was observed from the TGA curve for the polysaccharide ZW3.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

In recent years, polysaccharides have attained the considerableattention of researchers because of their wide distribution in natureand documented health benefits. These macromolecules are eitherhomopolymers or heteropolymers of neutral sugars (Badel,Bernardi, & Michaud, 2011). The thickening properties of poly-saccharides make them ideal as food additives and can be extractedthrough various sources including plant, fungi or seaweeds (Saija,Welman, & Bennett, 2010). Extraction and purification processesmay affect the physiochemical and structural properties of poly-saccharides and hence characterization of polysaccharides isessential to determine suitable properties for the purpose of theirutilization as food additives. Often the structure of these polymersis modified to increase their rheological properties and to makethem suitable for various food applications (De Vuyst & Degeest,1999; Roller & Dea, 1992). Another reason for acceptance of lacticacid bacterial polysaccharides such as ZW3 EPS is that, addition ofcertain polysaccharides from plant sources is not acceptable incertain dairy products due to problem of “all dairy” label to dairyfoods; and also the use of such plant polymers in dairy products is

ealth Sciences, Allama Iqbal7265; fax: þ92 51 9250063.d).

All rights reserved.

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 polymersfrom dairy sources and for that the best option is exopolysaccharideproduced 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 toproduce exopolysaccharide under specific growth conditions witha wide range and diversity of structure and have a potential to beused as nutraceuticals (Badel et al., 2011; Gorska et al., 2010; Wanget 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 physiologicalfunction of these polymers is still unknown and feware used in foodindustries (Suresh Kumar, Mody, & Jha, 2007; Sutherland, 2007).

Lactobacillus kefiranofaciens, an isolate fromkefir is famous for itspolymer named as kefiran; and has attained the attention of manyresearchers 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 iswaste product of cheese industry can be used as substrate whichends 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

Z. Ahmed et al. / Food Hydrocolloids 30 (2013) 343e350344

(Maeda et al., 2004), immunomodulation, epithelium protectionand antagonic activity against Bacillus cereus on Caco-2 cells(Piermaria et al., 2010), increased phagocytic activity of peritonealand 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 improvesthe rheological properties and better viscoelasticity can be achievedthrough addition of kefiran up to a level of 300 mg/L (Badel et al.,2011; Piermaria et al., 2010). Moreover, the kefiran can formbrittle and transparent films with good water vapor barrier char-acteristics (Piermaria et al., 2009). Due to beneficial attributes ofthis polymer the present research was conducted. In our previouspaper (Wang et al., 2008) we have characterized some of propertiesof polymer produced by L. kefiranofaciens ZW3 isolated from Tibetkefir. The strain produces a high amount of polymer having desir-able physiochemical properties (Wang et al., 2008). However, toexplore its potential for application in food industry more charac-terization onphysiochemical, structural and rheological parametersis required. Keeping in view all of this, the current project wasplanned 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 andfurther purified from Tibet kefir as described in our previous study(Wang et al., 2008). Liquid wheymediawas used for its propagationand media was incubated under anaerobic conditions at tempera-ture of 30 �C and for a time period of 72 h. Maximum recovery ofEPS was obtained when degrading enzymes were inactivated byheating the media at a temperature of 100 �C for 30 min. This wasfollowed by ultracentrifugation at 12,000 � g for 15 min at refrig-erated temperature. EPS was precipitated by using the chilledabsolute ethanol and was kept in refrigerator at temperature of 4 �Cfor 12 h, followed by a second centrifugation by maintaining theabove parameters. For further purification, EPS obtained by abovetreatment was redissolved in distilled water (100 ml) with gentleheating below 50 �C and precipitated again with equal volume ofchilled absolute ethanol. Again an ultracentrifuge treatment wasapplied (25,000 � g) for 25 min at refrigeration temperature. Toachieve further purification, EPS pellets were once again redis-solved in 20ml of distilled water with gentle heating (below 50 �C).Dialysis technique was applied to remove small molecular weightsimple sugars at 4 �C for 72 h with three changes of distilled waterin a day. Dialyzed EPS was recovered through freeze dryer. Recov-ered EPS was named as partially purified EPS and was furthercharacterized 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 at12,000 � g for 15 min. The resultant material was neutralized up topH level of 7.0 and was again precipitated by adding chilled ethanolin equal volumes. Finally pellets of EPS were redissolved in doubledistilled water and were lyophilized.

2.2. Study of common physical properties

Solubility of ZW3 EPS in water and oil was determined byfollowing the procedure of Chang and Cho (1997). A separatesuspension 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 wasfollowed by centrifugation 5000 � g for 15 min and collectedsupernatant (0.2 ml) was precipitated with 3 volume of ethanol.Again EPS in form of precipitate was recovered by centrifugation at10,000 � g for 5 min. Resultant material was vacuum dried at 50 �Cand difference in weight was recorded .The solubility was calcu-lated as follows:

Solubilityð%Þ ¼ ½Total carbohydrate concentration insupernatantðaÞ�=½Weight of sample

ðdry weight basisÞ� � 100

Sample of EPS was characterized for water holding capacity(WHC) by suspending 0.2 g sample in 10 ml of deionized water ona vortexmixer. Dispersedmaterial was centrifuged at 16,000� g for25 min. Unbound water that was not held by EPS material wasdiscarded. All EPS material was dropped on pre weight filter paperfor complete drainage of water. Weight of EPS precipitated wasrecorded. The percentage ofWHCwas calculated through followingexpression:

WHCð%Þ ¼ ½total sample weight after water absorption�=½total dry sample weight� � 100

The oil binding capacity was also calculated for this EPS ina similar manner by adopting method of Kato, Okamoto, Tokuya,and Takahashi (1982). For that purpose soya bean oil was used asdispersing media. The other steps were identical to the analysisprocedure of WHC.

2.3. Measurement of molecular weight

Extracted and refined EPS pellet was characterized for molecularweight using Agilent 1100series HPLC system (Agilent technologiesPalo AHO, CA, USA). Equipment was equipped with refractive indexdetector TOSOH TSK-G4000 PWxl column (7.8 mm � 30 cm, 10 mm)(TOSOH Corp., Tokyo, Japan). A sample of 20 mL was injected in thesystem by maintaining a flow rate of 0.5 ml/min and columntemperature of 35 �C. Separation was carried out by using 0.71%sodium sulfate as mobile phase. Dextran D2000 with molecularweight of 2 � 106, D8 with molecular weight of 1.338 � 105, D7 withmolecularweightof 41,100,D5withmolecularweight 21,400,D4withmolecular weight 10,000, D0 with molecular weight 180 (glucose)was used as reference compound. These reference standards wereadded 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 usedto determine rheological properties. An RV type ULA spindle thatrotated in chamber equipped with temperature control system(Thermomixs; B. Braun Biotech International) was attached withrheometer. Brookfield Rheocalc software (Brookfield EngineeringLaboratories Inc.) was used to control the instrument.

For preparation of sample EPS was well dissolved at rate of2 mg/ml and 4 mg/ml. Rheological behavior for test solution wasmeasured against time with increasing shear rate. Further rheo-logical characteristic of EPS was performed at variable pH levelsandwith addition of different salts. pH of EPS solutionwas adjustedat 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 EPSfor characterization of rheological properties. Further character-ization was carried out by dissolving EPS in skim milk and waterand their rheological properties were compared with each other.

Z. Ahmed et al. / Food Hydrocolloids 30 (2013) 343e350 345

The data for all of the parameters mentioned above were recordedin triplicate.

2.5. Atomic force micrograph (AFM) of ZW EPS

Purified ZW3 EPS was dissolved completely at rate of 1 mg/ml indouble distilled water under nitrogen stream and with continuousstirring for 1 h. After attaining room temperature; mixture wasdiluted to achieve a concentration of 0.01 mg/ml. Mica sheet wasused as carrier media. A sample size of 5 mL was uniformlydistributed on mica sheet and was dried at room temperature. AFMimages were taken by scanning Probe microscope (JEOL JSPM-5200, JAPAN) in tapping mode. The cantilever oscillation was setat 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 stuband gold sputtered and examined through SEM by maintaining anaccelerated voltage of 10 kv.

2.7. Thermogram analysis (TGA)

For TGA study Mettler Toledo TGA/SDTA 851e thermal systemwas employed. This system operates at atmospheric pressure.Compatible system software was used to control various parame-ters including temperature. Variable system software was also usedto record temperature, over period of time through installedthermo couples that have connecting ends in crucibles. The cruciblewas used made up of Al2O3. Sample of EPS (10 mg) was placed incrucible. System was programmed for linear heating at rate of10 �C/min rise in temperature in 1 min over a temperature range of25e100 �C. Separate experiments were performed in air andnitrogen atmosphere. The flow rate for air and nitrogen wasmaintained at 50 ml/min. System was initially calibrated fortemperature reading using indium as melting standard.

3. Results & discussion

3.1. Physical properties of ZW3 EPS

The chromatogram (Fig.1) obtained by gel permeation HPLCdepicted a single distribution of MW corresponding to 5.5�104 Da.Different authors have reported EPS with different molecularweight by L. kefiranofaciens. Wang and Bi (2008) reported kefiranwith molecular weight of 1.5 � 105 Da, whereas Piermaria and

Fig. 1. The chromatogram of ZW3 EPS obtained by gel permeation HPLC.

coworkers and other researchers have reported molecular weightof 107 Da (Piermaria et al., 2008; Sutherland, 1998; Wang & Bi,2008). Some of the most common physical properties of ZW3 EPSare shown in Table 1. ZW3 EPS is water soluble with good waterholding capacity and oil binding capacity. These properties areattributed to the permeable structure of polymer chains which canhold 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 groundto form powder, it not only brings the changes in it size, but mayalso adversely affects swelling and water holding capacity ofpolymer and change in the fiber matrix structure. Due to goodwater holding capacity the L. kefiranofaciens ZW3 EPS producingstrain has good potential to be used in fermented products alongwith non-EPS producing strain. Yang et al. (2010) have reportedthat water holding capacity of yoghurt increases when its starterculture 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 commonproblem in fermented milk products with and superfluous sight inthese products. This problem often appears as a result of shrinkageof 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 avoidthis problem (Ramaswamy & Basak, 1992; Xu, Stanley, Goff, &Davidson, 1992). Often these synthetic chemicals are not allowedin some parts of the world (Wang et al., 2008). EPS will offer a newsubstitute for these chemicals as a natural counterpart.

The viscous behavior of exopolysaccharide is dependent on itsstructure and mass (Freitas et al., 2009) that are affected by variousfactors such as salts, ionic strength, pH and temperature. To use theEPS in for different products, the knowledge of its rheologicalbehavior at different pH and ionic may provide some usefulapplication for various food products. The rheological behavior ofthis newly discovered EPS in water, milk, salt and also at differentpH, against time and increasing shear rate is shown in Fig. 2. EPSshowed a thinning behavior of viscosity i.e. high initial value ofviscosity which decreases with time and later on becomes stable.Whereas viscosity was increased with increasing the concentrationof EPS from 2 mg/ml to 4 mg/ml (Fig. 2A). The effect of salts on theviscosity of the solutions of the EPS produced by strain ZW3 asa function of shear rates is shown in Fig. 2B. At 30 �C the viscosity ofEPS in 0.1 M CaCl2 & 0.1 M NaCl solutions was almost similar overthe whole shear rate range (Fig. 2B). The effect of salts on theviscosity of the solutions of the EPS produced by strain ZW3 asa function of time is shown in Fig. 2C. Comparatively higherviscosity of EPS in CaCl2 was observed than NaCl solution and thisbehavior of EPS was evident in initial stages as well as after 10 minof elapse time. This viscous behavior may be attributed to differentintermolecular arrangement of charged polymers in these solutionsthat cause different degree of electrostatic repulsion or contractedby electrostatic attraction between the polymer chains and is in linewith the study of Kanmani et al. (2011) who reported a diversifiedviscosity behavior of EPS in different salt solutions.

Table 1Physical properties of ZW3 EPS.

Sample Solubility (%) Water holdingcapacity (%)

Oil bindingcapacity (%)

ZW3 14.2 496.00 884.74

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

Fig. 2D shows the behaviors of EPS at different pH as a functionof time. It is depicted from Fig. 2D that neutral pH tends to lowerthe viscosity of EPS. Viscosity of EPS increased as a function ofdecrease in pH. ZW3 EPS viscosity was also checked in skim milkand water as an increased function of shear rate and time (Fig. 2Eand F). Initially the viscosity was high both in water and skim milkand it decreased with passage of time whether it was treated witha fixed sear rate; or with an increasing shear rate. Similarly, whilestudying the impact of pH on the viscosity of polysaccharide, Gauri,Mandal, Mondal, Dey, and Pati (2009) have reported on theincreased viscosity of EPS in acidic pHs relative to those alkalinepHs. Similar results are also reported by Kanmani et al. (2011) whoalso observed a decrease in the viscosity once he lowered the pHfrom 6 (208 mPa) to acidic pH 3 (226 mPa).

These results are very significant as final pH of fermented milkproduct is always at acidic side and exopolysaccharide has a favor-able behavior at acidic pHs at acidic pH. It appears thus that a goodchoice would be to select this pH for the use of the EPS ZW3 asa biothickener or a stabilizer.

3.3. Atomic force micrograph (AFM) of ZW EPS

In recent years exopolysaccharide has been studied extensivelyby using atomic force microscopy (Abu-Lail & Camesano, 2003;Wang et al., 2010) that provides a powerful tool to characterize themorphological features of polymers. Owing to its ability tomeasureinteraction forces in liquids at a pico- or nano-Newton level withhigh vertical and lateral resolutions. This technique enables us tocharacterize and inference properties of the EPS by observing theconformation of individual macromolecules along with molecularstructure 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 usedin absence of water as a dispersing medium, thus enables theresearchers 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 inFig. 3. ZW3 EPS deposited from 10 mg/ml aqueous solution have

Fig. 3. Atomic force microscopy (AFM) images of ZW3 EPS.

Z. Ahmed et al. / Food Hydrocolloids 30 (2013) 343e350 347

rounded to spherical lumps with almost similar size and formationof chains is also visible. However the molecules are tightly packedand have reticulated shape suggesting that they have strong affinityfor water molecules and have pseudoplastic behavior. Similar resultwas reported byWang et al. (2010) for KF5 EPS, however in the caseof ZW3 EPS the concentrationwas lower (10 mg/ml) as compared toKF5 EPS concentration (100 mg/ml). Different EPS have differentshape 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 andworms, respectively in low and high concentration. Our findings forthe ZW3 EPS showed rounded lumps with maximum height oflump by of 31.1 nm.

In a previous research, Wang et al. (2010) reported the KF5 EPSwith 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 imagingof exopolysaccharide is SEM and has been reported by manyresearchers (Goh, Haisman, & Singh, 2005) and as a very useful toolto study surface topography of polymers (Wang et al., 2010). SEMresults of ZW3 EPS and a reference material i.e. xanthan gum areshown in Fig. 4.

As observed by SEM, ZW3 EPS look like thin film with smoothand glittering surface; exhibiting compact structure which ischaracteristic of a material used tomake the plasticized films. So it’sa good choice for making such kind of films. Moreover the SEM scan

showed that ZW3 EPS was made of homogeneous matrix which isan indicator of the structural integrity especially important in filmmaking. Much of the SEM properties of ZW3 EPS are similar to theproperties 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 mildestprocess that insures safety and elementary dissolution to theseverest process that initiates chemical decomposition. On thelower response scale, gelatinization and swelling are primaryoccurrences; on the upper response scale, chemical dehydration,pyrolysis, and resynthesis generate higher M species in the volatilephase (Fagerson, 1969) and flavors, aromas, colorants (Vercellotti,Crippen, Lovegren, & Sanders, 1992), and a host of other smallorganic molecules terminally. Along with other physiochemicalcharacteristics; applicability of exopolymer is largely dependent onits rheological and thermal behavior (Marinho-Soriano & Bourret,2005). In thermal analysis of EPS heat is emitted and absorbedwhich is accompanied by change in structure of polymer and inmelting of crystalline polymer (Wang et al., 2010).

The thermogravimetric (TGA) analysis for ZW3 EPS was carriedout dynamically (weight loss versus temperature). Xanthan gumand 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. 4. SEM results of ZW3 EPS and of a reference material xanthan gum. Fig (A) and (B) at 1000� and 2000� of xanthan gum, whereas fig (C), (D), (E) & (F) showing SEM results ofZW3 EPS at 1000�, 2000�, 10,000� and 10,000� respectively.

Z. Ahmed et al. / Food Hydrocolloids 30 (2013) 343e350348

polysaccharide ZW3. The initial weight loss of polymer between 40and 90 �C may be attributed to its moisture contents. EPS with highcarboxyl groups is always rich in moisture contents and initialweight loss in ZW3 EPS suggests that ZW3 polymer is rich incarboxyl contents. This is because the higher the carboxyl contentthe greater the affinity of the polysaccharide for interaction withwater molecules (Parikh & Madamwar, 2006). The decline inweights 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 dramaticallyaround 300 �C, Fig. 5B and C show the TG analysis of xanthan gumand locust gum respectively as reference material. Degradationtemperature for xanthan gum is 282.65 �C, where for locust gum itis 278.46 �C. ZW3 EPS is glucogalactan in nature (Wang et al., 2008)whereas Locust gum is a linear polysaccharides, which is composedof mannose and galactose (Dakia, Blecker, Robert, Wathelet, &Paquot, 2008); while Xanthan gum is a hetero-polysaccharidecontaining D-glucose, D-mannose and D-glucoronic acid (Baird,

Fig. 5. TG curves of ZW3 EPS, xanthan gum, locust gum. (A) ZW3 EPS, (B) xanthangum, (C) locust gum.

Z. Ahmed et al. / Food Hydrocolloids 30 (2013) 343e350 349

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 temperaturethan both xanthan and locust gumwhichmakes it safe to be used indairy industry where in most of processes temperature seldomoverpasses 150 �C.

4. Conclusion

L. kefiranofaciens (ZW3) isolated from Tibet kefir depicted goodexoplolysaccharide (EPS) producing capacity. A new modified

extraction procedure was investigated, that maximized therecovery of EPS. In our previous study we have characterized thepolymer for its chemical structure & composition by using the FTIR& GCMS analysis. Polymer was also studied for its emulsionstability, thermal properties & flocculating activity. In present studywe have characterized the remaining aspects of polysaccharidewhich were not explored in the prior study. Extracted EPS wascharacterized as medium molecular weight and possess goodsolubility, water binding capacity and oil binding capacity. Rheo-logical properties showed a good potential of this EPS and hascompatibility with water, milk, salts at different pH and shear rates.Viscous behavior in fermented milk product indicated its potentialto be used as biothickner or biostabilizer. Characterization datathrough SEM and AFM showed rounded lumps with maximumheight of 31.1 nm with indication of better structural stability thatcan be utilized for film formation and generation of edible nano-structures for encapsulation of food additives. These findings werealso confirmed through thermogram analysis. ZW3 EPS hasdemonstrated excellent properties and the knowledge of physical,surface morphology, rheological and thermal analysis; along withprevious explored parameters will enable the food scientist to usethe polymer in food industry in an efficient way.

Acknowledgment

This study was supported by the National Natural ScienceFoundation of China (grant no. 31171629) and grant from theTwelfth Five National Scientific Support grant (863) (No.2011AA100904 ). We are also thankful to Higher EducationCommission of Pakistan for its financial support.

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