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International Journal of Biological Macromolecules 43 (2008) 283–288

Journal of Biological Macromolecules 43 (2008) 283–288 Contents lists available at ScienceDirect International

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

journal homepage: www.elsevier.com/locate/ijbiomac

journal homepage: www.elsevier.com/locate/ijbiomac Physicochemical properties of exopolysaccharide produced by

Physicochemical properties of exopolysaccharide produced by Lactobacillus kefiranofaciens ZW3 isolated from Tibet kefir

Yanping Wang , Zaheer Ahmed, Wu Feng, Chao Li, Shiying Song

Tianjin Key Laboratory of Food Nutrition and Safety, Faculty of Food Engineering and Biotechnology, Tianjin University of Science and Technology, No. 29, 13th street at TEAD, Tianjin 300457, PR China

article

info

Article history:

Received 26 April 2008 Received in revised form 21 June 2008 Accepted 23 June 2008 Available online 9 July 2008

Keywords:

Physicochemical Properties Exopolysaccharide Lactobacillus kefiranofaciens ZW3

abstract

An exopolysaccharide (EPS) producing strain, ZW3, was isolated from Tibet kefir grain and was identi- fied as Lactobacillus kefiranofaciens. FT-IR spectroscopy revealed the presence of carboxyl, hydroxyl, and amide groups, which correspond to a typical heteropolymeric polysaccharide. The GC analysis of ZW3 EPS revealed that it was glucogalactan in nature. Exopolymer showed similar flocculation stability like xanthan gum but better than guar gum with a melting point of 93.38 C which is lower than xanthan gum (153.4 C) and guar gum (490.11 C). Compared with other commercially available hydrocolloids like xanthan gum, guar gum and locust gum ZW3 EPS showed much better emulsifying capability. © 2008 Elsevier B.V. All rights reserved.

1. Introduction

The increased demand for natural polymers for various indus- trial applications in recent years has led to a renewed interest in exopolysaccharide (EPS) production by microorganisms. Many microorganisms including lactic acid bacteria, algae, fungi and plants have an ability to synthesize extracellular polysaccha- rides and excrete them out of cell either as soluble or insoluble polymers [1–3]. The exopolysaccharides produced by microorgan- isms are widely used in the food, pharmaceutical and chemical industries, and function as bioflocculants, bioabsorbents, heavy metal removal agents, drug delivery agents, etc. [4,2,5]. Exam- ples of industrially important microbial exopolysaccharides are dextrans, xanthan, gellan, pullulan, yeast glucans and bacte- rial alginates [6]. Polysaccharides of microbial origin have been developed as food additive including xanthan from Xanthomonas campestris [7] and gellan from Pseudomona elodea [8]. How- ever physical properties of these polymers are such that they are not suited for all applications and there is a demand for novel materials that gives improved rheological characteristic

[9].

Lactic acid group of bacteria (LAB) which excretes polysac- charide of elevated molecular weight (MW) has been studied extensively during the last decade [6,1012]. Their particular phys-

Corresponding author. Tel.: +86 22 60601400; fax: +86 22 60601478. E-mail address: ypwang40@yahoo.com (Y. Wang).

0141-8130/$ – see front matter © 2008 Elsevier B.V. All rights reserved.

doi:10.1016/j.ijbiomac.2008.06.011

ical and rheological properties, which make them suitable as viscosifying, stabilizing, gelling, or emulsifying agents, in com- bination with the GRAS (generally recognized as safe) status of EPS-producing lactic acid bacteria, make EPSs promising as a new generation of food thickeners [13,14,5]. Lactobacillus kefiranofaciens, which was isolated from kefir grains and used as the starter Caucasian cultured milk, produces an exopolysaccharide called kefiran [15]. Various isolates have been reported and described as Lactobacillus kefir [14], L. kefiranofaciens [16], Lactobacillus sp. KPB-167B [17], L. kefirgranum and L. parakefir [18]. This non-exhaustive listing indicates that the complex taxo- nomic relationships among the bacterial species of kefir have not been completely explored. In addition, the influence of the geo- graphical origin of kefir grains is also to be taken into account [19–21]. Kefiran, is a water-soluble glucogalactan, which has been reported to have antibacterial and antitumour activity, modulates gut immune system and protects epithelial cells against Bacillus cereus exocelullar factors [22,17,23–26]. Kefiran also can be used as a food grade additive for fermented product since it enhances the rheological properties of chemically acidified skim milk gels increasing their apparent viscosity and the storage and loss mod- ulus of these gels. This phenomenon was strengthened by the previous heat treatment usually applied for yogurts manufacture [27]. However, the physicochemical properties of the exopolysac- charide from this strain have not been completely studied yet. In this study we reported some physicochemical properties of exopolysaccharide produced by L. kefiranofaciens ZW3 isolated from Tibet kefir such as thermal stability, emulsifying capability, floc-

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culating activity and FT-IR spectra which previously had not been reported yet. The ZW3 EPS proved to be having good emulsion sta- bility and flocculating activity as compared to other commercially available gums.

2. Experimental

2.1. Kefir grain

Kefir grain was taken from Tibet, China and was preserved by our laboratory and propagated at 25 C.

2.2. Media

Media used were supplemented MRS, supplemented M17 and supplemented whey. Whey medium were prepared as described by Yokoi et al. [28], with some modification. Supplemented whey medium contained 100 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 MgSO 4 ·7H 2 O, 0.15 g/l of MnSO 4 ·4H 2 O, 0.18 g/l of FeSO 4 ·7H 2 O, and 0.1 g/l NaCl. Milk whey used in the agar media was prepared by the filtration of skim milk, which was adjusted to pH 5.5 with lactic acid and heated for 30 min at 100 C. Milk whey used in liquid whey media was deproteinized by adjusting skim milk to pH 4.6 with 2N HCl, heating for 30 min at 100 C, and filtering. The resulting supernatant was adjusted to pH 6.8 with 2N NaOH, heated for 30 min at 100 C, and filtered to obtain deproteinized whey. Supplemented MRS broth medium was prepared by addition of following components in commercial MRS broth (Oxoid) medium: 5 mM CaCl 2 , 0.04% MnSO 4 ·4H 2 O, 0.07% MgSO 4 and % lactose monohydrate, where as M17 (Oxoid) broth media was supplemented with 0.5% glucose and 1% lactose only. The purpose of using agar wheymedia was to isolate ropy strains, where as supplemented liquid whey, supplemented MRS and supplemented M17 media were aimed at exopolysaccha- ride production. The final pH of each medium was adjusted to 6.2, and was subsequently autoclaved at 115 C for 20 min.

2.3. Screening of the isolates for EPS production

The kefir grains washed with sterile distilled water were homog- enized with a Waring blender. For isolation, 1 ml of homogenized and serially diluted in salt solution kefir grain was plated on whey agar medium. After incubation at 30 C for 7–9 days in an anaero- bic atmosphere with a GasPack filled with a gas mixture consisting of 80% N 2 , 10% CO 2 and 10% H 2 (v/v), ropy bacteria were isolated. EPS-producing LAB strains were first screened according to the stickiness and ropiness characteristics of their colonies. Follow- ing primitively screened isolates were inoculated into 50 ml liquid whey medium in the screw-capped bottles with an inoculation per- centage of 2%. The tightly capped bottles were incubated at 30 C for 24–72 h in anaerobic conditions. After the broth was centrifuged at 12,000 × g for 30 min, a certain volume of supernatant was taken to dialysis through 10 kDa membrane against distilled water at 4 C for 72 h with 2–4 changes per day. Exopolysaccharide productions of different strains were then determined by phenol–sulphuric acid method [30] until no single sugar was detected in distilled water.

fermentation which included: l-arabinose, ribose, d-xylose, galac- tose, d-fructose, mannitol, sorbitol, cellobiose, maltose, lactose, mélibiose, saccharose, tréhalose, and d-raffinose. The strain identi- fication was also confirmed by partially sequencing 16S rRNA genes analysis. A primer pair, P1 (5 -GAGTTTGATCCTGG CTCAG-3 ) and P2 (5 -TACCGCGGCTGCTGGCAC-3 ), corresponding to positions 8–28 and 542–549 of the 16S rDNA, respectively was used to clone 16S rRNA genes of target isolate. Total chromosomal DNA from MRS (Oxoid) 48 h broth culture was extracted as described by Forsman and Alatossava [29]. DNA fragments were amplified as follows:

initial denaturation at 94 C for 10 min, followed by 30 cycles con- sisting of denaturation at 94 C for 30 s, annealing at 58 C for 30 s, extension at 72 C for 1 min and a 10-min final extension step at 72 C. PCR product was checked with agarose gel electrophoresis. Amplified products of about 550 bp in length after verification were sequenced by using DNA sequencing kit (Shanghai Sangon Biolog- ical Engineering Technology & Services Co. Ltd.). The nucleotide sequences were used for the analysis of sequence similarity through BLAST (http://www.ncbi.nlm.nih.gov/blast).

2.5. Isolation and purification of EPS

The EPS was purified by using method of García-Garibay and Marshall [7], with some modification. The strain ZW3 was grown in 500 ml liquid whey medium in Erlenmeyer flaks at 30 C for 72 h in anaerobic conditions. The flasks were taken out and heat at 100 C for 30 min to dissolve cell attached EPS and subsequently centrifuged at 12,000 rpm for 15 min. Crude EPS was precipitated by the addition of an equal volume of chilled absolute ethanol to the supernatant fluid. After overnight precipitation at 4 C, the sam- ple was centrifuged, at above given conditions, and the pellet was retained. The sample was redissolved in distilled water (100 ml) with gentle heating (less than 50 C) and the EPS was recovered by precipitation on the addition of an equal volume of chilled abso- lute ethanol. The sample was centrifuged at 25,000 × g for 25 min at 4 C. The resulting EPS pellet was redissolved in not more than 20 ml of distilled water with gentle heating (less than 50 C) and then small neutral sugars were removed by dialysis, for 72 h at 4 C, against three changes of distilled water per day. The contents of the dialysis bag were freeze-dried to provide EPS. This EPS was named as partially purified EPS and was later on used to study physical characteristic. Partially purified EPS was further purified by dissolv- ing it in 14% trichloroacetic acid (TCA) and stirred over night. The precipitated protein was removed by centrifugation at 12,000 × g for 15 min. The resulted supernatant was adjusted to pH 7 and EPS was precipitated by putting an equal volume of chilled ethanol at 21 C. The pellet was dissolved in double distilled water and was lyphophilized.

2.6. Study of infrared (FT-IR) spectroscopy

The major structural groups of the purified EPS were detected using Fourier-transformed infrared spectroscopy. For FT- IR spectrum of ZW3 EPS was obtained using KBr method. The polysaccharide samples were pressed into KBr pellets at sam- ple: KBr ratio 1:100. The Fourier transform-infrared spectra were 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.

2.4. Identification of strain ZW3

The strain ZW3 was identified by using Gram stain reac- tion, catalase reaction; ability to grow at 15, 37 and 45 C, gas production from glucose, arginine hydrolysis and by sugar

2.7. Sugar composition

For sugar composition determinations, polysaccharides were hydrolyzed by treatment with 2 M TFA (120 C for 2 h); the released sugars were converted to their alditol acetates and analyzed by

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GC–MS. The standard alditol acetates were generated by sub- jecting an intimate mixture of equal proportions of rhamnose, glucose, ribose, arabinose, xylose, mannose, glucose and galac- tose to the same experimental conditions that were applied to the polysaccharide The composition of exopolysaccharide was deter- mined by comparison of retention time of different peaks of alditol acetates with mixture standard alditol acetates. Analysis was per- formed by using a Varian GC/MS 4000 instrument (USA) equipped with VF-5ms 30 m × 0.25 mm × 0.10 m column under following conditions—injector temperature: 320 C, split ratio: 10, column flow: 1 ml/min, carrier gas: He 99.999%, column oven temperature:

150 C (hold time 2 min) to 300 C (hold time 2 min) reached via a rising gradient of 10 C min 1 and ionization: EI scan type full.

2.8. Analysis of thermal properties

The thermal properties of EPS were analyzed by using a differ- ential scanning calorimeter (DSC Model 141 SETARAM Scientific & Industrial Equipment Co Limited, France). After placing 4.2 mg of dried EPS sample in an aluminium pan, it was sealed and analyzed, using empty pan as a reference, for determining the melting point and enthalpy change. The heating rate was 10 C min 1 from 20 to 300 C.

2.9. Emulsion stability

The emulsifying activity of EPS was assayed as described by Bramhachari et al. [31]. Lyophilized EPS (0.5 mg) was dissolved in 0.5 ml deionized water by heating at 100 C for about 15–20 min and allowed to cool to room temperature (25 C). The volume was then made up to 2 ml using phosphate-buffered saline (PBS). The sample was vortexed for 1 min after the addition of 0.5 ml hex- adecane. The absorbance at 540 nm was read immediately before and after vortexing (A 0 ). The fall in absorbance was recorded after incubation at room temperature for 30 and 60 min (A t ). A control was run simultaneously with 2 ml PBS and 0.5 ml hexadecane. The emulsification activity was expressed as the percentage retention of emulsion during incubation for time t: A t /A 0 × 100.

2.10. Flocculating activity

The flocculating activity was measured by using the method as described by Lim et al. [32]. Charcoal-activated carbon that was used as a testing material was suspended in deionized water at a concentration of 5 g/l. In a test tube, 10 ml of a charcoal-activated carbon suspension was added and mixed with 0.1 ml of CaCl 2 solu- tion (6.8 mM). To this mixture, various amounts of EPS were added and vortexed for 30 s and allowed to stand for 10 min at room tem- perature. The turbidities of the upper 1 ml phase were measured at 550 nm. A control experiment without the EPS was also pursued in the same manner. The flocculating activity (%) was defined and calculated according to the following equation:

flocculating activity = B A × 100 × dilution rate

B

A: turbidity of EPS-containing suspension; B: turbidity of control.

3.

Results and discussion

3.1.

Screening and identification of ZW3 strain

Kefir samples were taken from Tibet, China. Different media such as, supplementedMRS, supplementedM17 and supplemented whey media was used for screening of EPS-producing strains. But we found the supplemented whey media as the best, both for

we found the supplemented whey media as the best, both for Fig. 1. Ropy behaviour of

Fig. 1. Ropy behaviour of colony of L. kefiranofaciens ZW3 strain.

screening and exopolysaccharide production. Initially the strains were screened on the basis of the morphology and colonies which have mucoid, slimy or ropy appearance, were selected for next step. In final step capability of strains to produce EPS were tested by phe- nol sulphuric acid method. The ropy strain of ZW3 which produced the highest amount of EPS among screened strains, was selected for present study. Strain ZW3 was, Gram positive, catalase negative and rod shaped bacteria. It did not grow at 15 C. This strain did not produce gas from arginine. This homofermentative profile along with the combination of sugar fermentation pattern suggests that strain ZW3 might belong to the L. kefiranofaciens species [33]. To confirm the biochemical results, partial sequencing of variable regions of 16S rRNA genes was also performed. About 550 base pair (bp) variable regions of 16S rRNA genes was amplified and 500 bp were sequenced. The nucleotide sequences were used for the analysis of sequence similarity through BLAST (http://www.ncbi.nlm.nih.gov/blast) and it gave 100% similarity with L. kefiranofaciens subsp. kefirgranum and L. kefiranofaciens subsp. Kefiranofaciens. But differentiate physical test between two subspecies such as growth at 15 C, its convex colony with extremely sliminess appearance (Fig. 1), large amount of exopolysaccharide production and negative aesculin hydrolysis proved that strain L. kefiranofaciens ZW3 belonged to subsp. kefi- ranofaciens [18]. So strain ZW3 was identified as L. kefiranofaciens subsp. kefiranofaciens and named as L. kefiranofaciens subsp. Kefira- nofaciens ZW3.

3.2. EPS production, Isolation and quantification

Initially L. kefiranofaciens ZW3 and other strains were grown in 50 ml liquid whey medium to screen the strains for EPS quantifi- cation. After incubation for 48–72 h, the broth was centrifuged at 12,000 rpm at 4 C for 15 min. After removing cell, the supernatant was dialysed and EPS amount was determined by phenol sulphuric acid method using glucose as standard. Strain ZW3 produced a very high amount of EPS up to 1215 mg/l. And it produced even high amount up to 1675 mg/l if the incubated broth was heated at 100 C for 30 min and followed by centrifugation and EPS quantification. Heat treatment of the samples as a first step in the polysaccha- ride isolation procedure is critical for complete recovery of the EPS. Samples without this step gave lower polysaccharide concen- tration than those including this treatment but it should be used only where the exopolysaccharide is thermally stable [34,35]. Other drastic methods include boiling the cell suspension for 15 min in water, heating at 60 C in saline solution, heating in a mixture of

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Journal of Biological Macromolecules 43 (2008) 283–288 Fig. 2. Fourier-transformed infrared (FT-IR) spectrum of

Fig. 2. Fourier-transformed infrared (FT-IR) spectrum of the exopolysaccharide pro- duced by L. kefiranofaciens ZW3.

phenol water at 65 C or sonicating the cell suspension. Autoclav- ing is the most frequently used treatment for releasing capsular polysaccharides from cells [35]. Also the amount of EPS produced by L. kefiranofaciens ZW3 was high as compared to previously reported EPS production in the same medium which was up to 405 mg [28]. So the strain L. kefiranofaciens may be has better capability of EPS production than previously reported strains.

3.3. Sugar analysis and infrared (FT-IR) spectroscopy

Fourier transform infrared spectroscopy has been a useful tool in monitoring structural changes in biopolymers [36]. Carbohy- drates such as xanthan components are recognized by peaks at wave numbers of 1040 cm 1 (C–O bond from the alcohol group), 2940 cm 1 (C–H stretch) and 3400 cm 1 (–OH stretch) [37]. The IR spectra of purified exopolysaccharide ZW3 is given in Fig. 2, which shows more complex pattern of peaks from 2950 to 1200 cm 1 . Polysaccharides contain a significant number of hydroxyl groups, which exhibit a broad rounded absorption band above wave num- ber 3000 cm 1 . The absorption in that region (Fig. 2) has the rounded trait typical of hydroxyl groups [38] which suggests that the substance is polysaccharide. The IR spectra of L. kefiranofaciens ZW3 exopolysaccharide revealed characteristic functional such as a broad-stretching hydroxyl group at 3405 cm 1 and a weak C–H stretching peak of methyl group at 2924 cm 1 [31]. A broad stretch of C–O–C, C–O at 1000–1200 cm 1 corresponds to the presence of carbohydrates [39], so in the fingerprint region (region below 1500 cm 1 where bands characterise the molecule as a whole), the strongest absorption band at 1067 cm 1 is attributed to that substance is polysaccharide [40]. Strong absorption at 1643 cm 1 which corresponds to amide I > C O str and C–N bending of protein and peptide amines, and a peak at 1378 cm 1 could be assigned to C O str of the COO and C–O bond from COO [41,42]. The FT-IR spectra of the polymer evidenced the presence of carboxyl groups, which may serve as binding sites for divalent cations [31]. Further, the spectrum showed the presence of carboxyl, hydroxyl, and amine groups, which are the preferred groups for the flocculation pro- cess similar to that observed in polyelectrolyte [43]. Noticeably the exopolysaccharide differ from the algal polysaccharide by having an additional peak at around 1240 cm 1 region due to the presence of o-acetyl ester [44]. The sugar composition of the EPS, analyzed using MS–gas chro- matography (Fig. 3). Here only qualitative results are given which revealed that ZW3 exopolysaccharide is composed of glucose and

that ZW3 exopolysaccharide is composed of glucose and Fig. 3. Gas chromatogram of alditol acetate derivative

Fig. 3. Gas chromatogram of alditol acetate derivative of hydrolyzed exopolysac- charide from L. kefiranofaciens ZW3.

galactose only. The presence of different sugar moieties suggests that the exopolymer is a heteropolysaccharide. Similar biochemi- cal compositions were observed in previous studies of the EPS from L. kefiranofaciens species isolated from kefir [23]. In this contrast it did not differ from previously reported results.

3.4. Analysis of thermal properties

Besides chemical properties, applicability of polysaccharide is largely dependent on its thermal and rheological behaviour [45]. As for the thermal characteristics of exopolysaccharides, heat absorp- tion and emission are accompanied with the physical change by deformation of polymer structure or melting of crystalline polysac- charides. Energy level of the polysaccharide was scanned from 25 to 350 C using a differential scanning calorimeter and was compared with xanthan gum and guar gum used as standard. The melting of ZW3 EPS, xanthan gum and guar gum started at about 93.38, 153.4, and 490.1 C, respectively, and the endothermic enthalpy change

( H) required to melt 1 g of ZW3 EPS, xanthan gum and guar gum

were 249.7, 93.2 and 192.9, respectively (Table 1). Thus the ZW3 polysaccharide showed a different thermal behaviour than xanthan gum and guar gum. As for the exopolysaccharides obtained from a mutant of Bacillus polymyxa, the melting point was 183.25 C, and enthalpy was 100.3 cal/g [46]. In an earlier report, the mea- surement of the thermal characteristics of levan synthesized with levansucrase showed the highest melting point to be 178.4 C with an enthalpy of 1.66 cal/g, similar to the thermal characteristics of the exopolysaccharides derived from legacy microorganisms [47].

3.5. Emulsion stability

Microbial and plant gums as well as some plant and animal proteins have been known to possess lipid emulsifying effects. Especially, xanthan gum with microorganism origin has been widely used in the food industry because of its high emulsifying

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

 

Peak temperature ( C)

Enthalpy (J g 1 )

ZW3 EPS

97.38

249.7

Xanthan gum

153.4

93.2

Guar gum

490.1

192.9

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287

Table 2 Emulsifying activity of ZW3 exopolysaccharide (EPS)

Emulsifier

Incubation time (min)

Sample OD at A 540 nm

Emulsifying activity (%)

Standard

0

0.173 ± 0.0.009

100 ± 0.00 38.15 ± 2.31 17.91 ± 1.74

30

0.066 ± 0.004

60

0.031 ± 0.003

Xanthan gum

0

0.518 ± 0.011

100 ± 0.00 92.66 ± 2.44 81.10 ± 2.68

30

0.480 ± 0.015

60

0.421 ± 0.013

Guar gum

0

0.571 ± 0.015

100 ± 0.00 72.67 ± 2.43 37.47 ± 2.98

30

0.415 ± 0.014

60

0.214 ± 0.017

Locust gum

0

0.249 ± 0.004

100 ± 0.00 86.34 ± 3.22 65.46 ± 2.41

30

0.215 ± 0.008

60

0.163 ± 0.006

ZW3 EPS puried

0

0.343 ± 0.009

100 ± 0.00 91.25 ± 2.05 88.04 ± 2.63

30

0.313 ± 0.007

60

0.302 ± 0.009

ZW3 EPS partially purified

0

1.26 ± 0.032

100 ± 0.00 88.09 ± 2.30 84.12 ± 1.83

30

1.11 ± 0.029

60

1.06 ± 0.023

Hexadecane, 0.5 ml, was added to 0.5 ml EPS (1 mg/ml), diluted to 2 ml with phosphate buffer saline (PBS), vortexed for 1 min and the absorbance monitored at 540 nm. A control was run with 2 ml PBS without EPS.

activity [48]. The emulsifying activity of EPS is determined by its strength in retaining the emulsion of the hydrocarbon in water. Generally the emulsion breaks rapidly within an initial incubation of 30 min. The absorbance reading after 30 and 60 min gives a fairly good indication of the stability of the emulsion [49]. The emulsion stabilities of L. kefiranofaciens ZW3 exopolysaccharide were com- pared with various commercial polysaccharides including xanthan gum, guar gum and locust gum and results are listed in Table 2. The purified fraction of the exopolymer produced by L. kefiranofaciens ZW3 retained 91.25% and 88.04% of the emulsification activity after 30 and 60 min, respectively. Results of partially purified fraction of the exopolymer, which did not differ much from purified fraction, produced 88.09% and 84.12% after 30 and 60 min, respectively. The other polysaccharides such as locust bean and guar gum showed relatively poor emulsifying activities as compared to L. kefiranofa- ciens ZW3 exopolysaccharide. The guar gum retained 72.67% and 37.47%, locust gum, 86.34% and 65.46%, where as xanthan gum pro- duced 92.66% and 81.10% emulsion activity after 30 and 60 min, respectively. So the purified and partially purified L. kefiranofaciens ZW3 exopolysaccharide showed almost similar activity, where as purified exopolysaccharide showed better activity when compared with xanthan gum. From these results, the polysaccharide produced by L. kefiranofaciens ZW3 is expected to have a great potential for use as an emulsifier.

3.6. Flocculating capability

A variety of flocculants, such as inorganic flocculants (polya- luminium chloride and aluminium sulphate), organic flocculants (polyacrylamide, polyethyleneimine) and natural flocculants or bio flocculants (gelatin, chitosan guar gum) have been widely used in chemical and mineral industrial fields such as tap water producing, wastewater treatment, dredging, downstream processing, fermen- tation and food industries [50–52]. Although chemical flocculants have been used widely due to their effective flocculating activity and low cost, they have neurotoxic and carcinogenic monomers and their usage is restricted [1,53]. On the contrary, biofloccu- lants produced by microorganisms during their growth are safe and biodegradable polymers [54]. Recently, many studies have been reported on the flocculating effect of microbial polysaccha-

rides to replace synthetic flocculants, which are industrially used

[55–57].

Flocculating capability test was performed at EPS concentration ranging from 0.1 to 0.6 mg in 5 mg/l dispersion of charcoal-activated carbon containing 6.8 mM CaCl 2 ·H 2 O (Fig. 4). The flocculating capa- bility of isolated exopolysaccharide was compared with that of xanthan gum and guar gum used as control. This capability ini- tially increased with increasing the concentration of EPS, and gave the greatest flocculating activity between concentration range of 0.3–0.5 mg/l and on word it had a decreasing trend as the EPS (flocculant) concentration increased. The optimal flocculant con- centration in test solution was determined to be 0.4 mg/ml. Where as the optimal flocculant concentration for xanthan gum and guar gum was 0.3 and 0.5 mg/l, respectively. As shown in Fig. 4 that the flocculating capability initially increased with increasing con- centration and then started to decrease after attaining a highest and purified point and this may be due to that the adsorption of excess flocculants destabilized the particles. Because of incomplete dispersion of excess flocculants, only particles around flocculants participated in the flocculating reaction at that moment. A large molecular weight flocculant is usually long enough and has a suf- ficient number of free functional groups that can act as bridges to bring many suspended particles together, and hence causes a larger floc size in the flocculation reaction [32]. L. kefiranofaciens ZW3 EPS

flocculation reaction [32] . L. kefiranofaciens ZW3 EPS Fig. 4. Flocculating capacity of L. kefiranofaciens ZW3

Fig. 4. Flocculating capacity of L. kefiranofaciens ZW3 EPS, xanthan gum and guar gum.

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showed a better flocculating activity than guar gum and almost sim- ilar to xanthan gum. Moreover the flocculating behaviour of ZW3 is also supported by its IR analysis. So ZW3 EPS is expected to be useful flocculating agents in the areas of wastewater treatment, drinking water processing, and downstream processing in the food indus- try because of their biodegradability and harmlessness towards humans and the environment.

Acknowledgments

This work was supported by grant from China the Fifteenth National Scientific Support Grant (No. 2006BAD 04A 06) and by Tianjin Municipal Science and Technology Commission Grant (No.

08JCYBJC01900).

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