International Journal of Biological Macromolecules 43 (2008) 283288

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

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

Physicochemical properties of exopolysaccharide produced by Lactobacillus keranofaciens ZW3 isolated from Tibet ker
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

a r t i c l e

i n f o

a b s t r a c t
An exopolysaccharide (EPS) producing strain, ZW3, was isolated from Tibet ker grain and was identied as Lactobacillus keranofaciens. 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 occulation 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.

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 keranofaciens ZW3

1. Introduction The increased demand for natural polymers for various industrial 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 polysaccharides and excrete them out of cell either as soluble or insoluble polymers [13]. The exopolysaccharides produced by microorganisms are widely used in the food, pharmaceutical and chemical industries, and function as bioocculants, bioabsorbents, heavy metal removal agents, drug delivery agents, etc. [4,2,5]. Examples of industrially important microbial exopolysaccharides are dextrans, xanthan, gellan, pullulan, yeast glucans and bacterial alginates [6]. Polysaccharides of microbial origin have been developed as food additive including xanthan from Xanthomonas campestris [7] and gellan from Pseudomona elodea [8]. However 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 polysaccharide 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 combination 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 keranofaciens, which was isolated from ker grains and used as the starter Caucasian cultured milk, produces an exopolysaccharide called keran [15]. Various isolates have been reported and described as Lactobacillus ker [14], L. keranofaciens [16], Lactobacillus sp. KPB-167B [17], L. kergranum and L. paraker [18]. This non-exhaustive listing indicates that the complex taxonomic relationships among the bacterial species of ker have not been completely explored. In addition, the inuence of the geographical origin of ker grains is also to be taken into account [1921]. Keran, 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 [22,17,2326]. Keran also can be used as a food grade additive for fermented product since it enhances the rheological properties of chemically acidied skim milk gels increasing their apparent viscosity and the storage and loss modulus of these gels. This phenomenon was strengthened by the previous heat treatment usually applied for yogurts manufacture [27]. However, the physicochemical properties of the exopolysaccharide from this strain have not been completely studied yet. In this study we reported some physicochemical properties of exopolysaccharide produced by L. keranofaciens ZW3 isolated from Tibet ker such as thermal stability, emulsifying capability, oc-

284

Y. Wang et al. / International Journal of Biological Macromolecules 43 (2008) 283288

culating activity and FT-IR spectra which previously had not been reported yet. The ZW3 EPS proved to be having good emulsion stability and occulating activity as compared to other commercially available gums. 2. Experimental 2.1. Ker grain Ker 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 modication. 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 MgSO4 7H2 O, 0.15 g/l of MnSO4 4H2 O, 0.18 g/l of FeSO4 7H2 O, and 0.1 g/l NaCl. Milk whey used in the agar media was prepared by the ltration 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 ltering. The resulting supernatant was adjusted to pH 6.8 with 2N NaOH, heated for 30 min at 100 C, and ltered to obtain deproteinized whey. Supplemented MRS broth medium was prepared by addition of following components in commercial MRS broth (Oxoid) medium: 5 mM CaCl2 , 0.04% MnSO4 4H2 O, 0.07% MgSO4 and % lactose monohydrate, where as M17 (Oxoid) broth media was supplemented with 0.5% glucose and 1% lactose only. The purpose of using agar whey media was to isolate ropy strains, where as supplemented liquid whey, supplemented MRS and supplemented M17 media were aimed at exopolysaccharide production. The nal 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 ker grains washed with sterile distilled water were homogenized with a Waring blender. For isolation, 1 ml of homogenized and serially diluted in salt solution ker grain was plated on whey agar medium. After incubation at 30 C for 79 days in an anaerobic atmosphere with a GasPack lled with a gas mixture consisting of 80% N2 , 10% CO2 and 10% H2 (v/v), ropy bacteria were isolated. EPS-producing LAB strains were rst screened according to the stickiness and ropiness characteristics of their colonies. Following primitively screened isolates were inoculated into 50 ml liquid whey medium in the screw-capped bottles with an inoculation percentage of 2%. The tightly capped bottles were incubated at 30 C for 2472 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 24 changes per day. Exopolysaccharide productions of different strains were then determined by phenolsulphuric acid method [30] until no single sugar was detected in distilled water. 2.4. Identication of strain ZW3 The strain ZW3 was identied by using Gram stain reaction, catalase reaction; ability to grow at 15, 37 and 45 C, gas production from glucose, arginine hydrolysis and by sugar

fermentation which included: l-arabinose, ribose, d-xylose, galactose, d-fructose, mannitol, sorbitol, cellobiose, maltose, lactose, mlibiose, saccharose, trhalose, and d-rafnose. The strain identication was also conrmed by partially sequencing 16S rRNA genes analysis. A primer pair, P1 (5 -GAGTTTGATCCTGG CTCAG-3 ) and P2 (5 -TACCGCGGCTGCTGGCAC-3 ), corresponding to positions 828 and 542549 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 amplied as follows: initial denaturation at 94 C for 10 min, followed by 30 cycles consisting 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 nal extension step at 72 C. PCR product was checked with agarose gel electrophoresis. Amplied products of about 550 bp in length after verication were sequenced by using DNA sequencing kit (Shanghai Sangon Biological 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 purication of EPS The EPS was puried by using method of Garca-Garibay and Marshall [7], with some modication. The strain ZW3 was grown in 500 ml liquid whey medium in Erlenmeyer aks at 30 C for 72 h in anaerobic conditions. The asks 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 uid. After overnight precipitation at 4 C, the sample 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 absolute 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 puried EPS and was later on used to study physical characteristic. Partially puried EPS was further puried by dissolving 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 puried EPS were detected using Fourier-transformed infrared spectroscopy. For FTIR spectrum of ZW3 EPS was obtained using KBr method. The polysaccharide 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 4000400 cm1 , at a resolution of 4 cm1 and processed by Bruker OPUS software. 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

Y. Wang et al. / International Journal of Biological Macromolecules 43 (2008) 283288

285

GCMS. The standard alditol acetates were generated by subjecting an intimate mixture of equal proportions of rhamnose, glucose, ribose, arabinose, xylose, mannose, glucose and galactose to the same experimental conditions that were applied to the polysaccharide The composition of exopolysaccharide was determined by comparison of retention time of different peaks of alditol acetates with mixture standard alditol acetates. Analysis was performed by using a Varian GC/MS 4000 instrument (USA) equipped with VF-5ms 30 m 0.25 mm 0.10 m column under following conditionsinjector temperature: 320 C, split ratio: 10, column ow: 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 min1 and ionization: EI scan type full. 2.8. Analysis of thermal properties The thermal properties of EPS were analyzed by using a differential scanning calorimeter (DSC Model 141 SETARAM Scientic & 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 min1 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 1520 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 hexadecane. The absorbance at 540 nm was read immediately before and after vortexing (A0 ). The fall in absorbance was recorded after incubation at room temperature for 30 and 60 min (At ). A control was run simultaneously with 2 ml PBS and 0.5 ml hexadecane. The emulsication activity was expressed as the percentage retention of emulsion during incubation for time t: At /A0 100. 2.10. Flocculating activity The occulating 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 CaCl2 solution (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 temperature. 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 occulating activity (%) was dened and calculated according to the following equation: occulating activity = BA 100 dilution rate B
Fig. 1. Ropy behaviour of colony of L. keranofaciens 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 nal step capability of strains to produce EPS were tested by phenol 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 prole along with the combination of sugar fermentation pattern suggests that strain ZW3 might belong to the L. keranofaciens species [33]. To conrm 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 amplied 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. keranofaciens subsp. kergranum and L. keranofaciens subsp. Keranofaciens. 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. keranofaciens ZW3 belonged to subsp. keranofaciens [18]. So strain ZW3 was identied as L. keranofaciens subsp. keranofaciens and named as L. keranofaciens subsp. Keranofaciens ZW3. 3.2. EPS production, Isolation and quantication Initially L. keranofaciens ZW3 and other strains were grown in 50 ml liquid whey medium to screen the strains for EPS quantication. After incubation for 4872 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 quantication. Heat treatment of the samples as a rst step in the polysaccharide isolation procedure is critical for complete recovery of the EPS. Samples without this step gave lower polysaccharide concentration 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

A: turbidity of EPS-containing suspension; B: turbidity of control. 3. Results and discussion 3.1. Screening and identication of ZW3 strain Ker samples were taken from Tibet, China. Different media such as, supplemented MRS, supplemented M17 and supplemented whey media was used for screening of EPS-producing strains. But we found the supplemented whey media as the best, both for

286

Y. Wang et al. / International Journal of Biological Macromolecules 43 (2008) 283288

Fig. 2. Fourier-transformed infrared (FT-IR) spectrum of the exopolysaccharide produced by L. keranofaciens ZW3.

Fig. 3. Gas chromatogram of alditol acetate derivative of hydrolyzed exopolysaccharide from L. keranofaciens ZW3.

phenol water at 65 C or sonicating the cell suspension. Autoclaving is the most frequently used treatment for releasing capsular polysaccharides from cells [35]. Also the amount of EPS produced by L. keranofaciens 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. keranofaciens 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]. Carbohydrates such as xanthan components are recognized by peaks at wave numbers of 1040 cm1 (CO bond from the alcohol group), 2940 cm1 (CH stretch) and 3400 cm1 (OH stretch) [37]. The IR spectra of puried exopolysaccharide ZW3 is given in Fig. 2, which shows more complex pattern of peaks from 2950 to 1200 cm1 . Polysaccharides contain a signicant number of hydroxyl groups, which exhibit a broad rounded absorption band above wave number 3000 cm1 . 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. keranofaciens ZW3 exopolysaccharide revealed characteristic functional such as a broad-stretching hydroxyl group at 3405 cm1 and a weak CH stretching peak of methyl group at 2924 cm1 [31]. A broad stretch of COC, CO at 10001200 cm1 corresponds to the presence of carbohydrates [39], so in the ngerprint region (region below 1500 cm1 where bands characterise the molecule as a whole), the strongest absorption band at 1067 cm1 is attributed to that substance is polysaccharide [40]. Strong absorption at 1643 cm1 which corresponds to amide I > C O str and CN bending of protein and peptide amines, and a peak at 1378 cm1 could be assigned to C O str of the COO and CO 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 occulation process similar to that observed in polyelectrolyte [43]. Noticeably the exopolysaccharide differ from the algal polysaccharide by having an additional peak at around 1240 cm1 region due to the presence of o-acetyl ester [44]. The sugar composition of the EPS, analyzed using MSgas chromatography (Fig. 3). Here only qualitative results are given which revealed that ZW3 exopolysaccharide is composed of glucose and

galactose only. The presence of different sugar moieties suggests that the exopolymer is a heteropolysaccharide. Similar biochemical compositions were observed in previous studies of the EPS from L. keranofaciens species isolated from ker [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 absorption and emission are accompanied with the physical change by deformation of polymer structure or melting of crystalline polysaccharides. 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 measurement 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. keranofaciens ZW3 exopolysaccharide (EPS) by differential scanning calorimetry (DSC) Peak temperature ( C) ZW3 EPS Xanthan gum Guar gum 97.38 153.4 490.1 Enthalpy (J g1 ) 249.7 93.2 192.9

Y. Wang et al. / International Journal of Biological Macromolecules 43 (2008) 283288 Table 2 Emulsifying activity of ZW3 exopolysaccharide (EPS) Emulsier Standard Incubation time (min) 0 30 60 0 30 60 0 30 60 0 30 60 0 30 60 0 30 60 Sample OD at A540 nm 0.173 0.0.009 0.066 0.004 0.031 0.003 0.518 0.011 0.480 0.015 0.421 0.013 0.571 0.015 0.415 0.014 0.214 0.017 0.249 0.004 0.215 0.008 0.163 0.006 0.343 0.009 0.313 0.007 0.302 0.009 1.26 0.032 1.11 0.029 1.06 0.023

287

Emulsifying activity (%) 100 0.00 38.15 2.31 17.91 1.74 100 0.00 92.66 2.44 81.10 2.68 100 0.00 72.67 2.43 37.47 2.98 100 0.00 86.34 3.22 65.46 2.41 100 0.00 91.25 2.05 88.04 2.63 100 0.00 88.09 2.30 84.12 1.83

Xanthan gum

Guar gum

Locust gum

ZW3 EPS puried

ZW3 EPS partially puried

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. keranofaciens ZW3 exopolysaccharide were compared with various commercial polysaccharides including xanthan gum, guar gum and locust gum and results are listed in Table 2. The puried fraction of the exopolymer produced by L. keranofaciens ZW3 retained 91.25% and 88.04% of the emulsication activity after 30 and 60 min, respectively. Results of partially puried fraction of the exopolymer, which did not differ much from puried 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. keranofaciens ZW3 exopolysaccharide. The guar gum retained 72.67% and 37.47%, locust gum, 86.34% and 65.46%, where as xanthan gum produced 92.66% and 81.10% emulsion activity after 30 and 60 min, respectively. So the puried and partially puried L. keranofaciens ZW3 exopolysaccharide showed almost similar activity, where as puried exopolysaccharide showed better activity when compared with xanthan gum. From these results, the polysaccharide produced by L. keranofaciens ZW3 is expected to have a great potential for use as an emulsier. 3.6. Flocculating capability A variety of occulants, such as inorganic occulants (polyaluminium chloride and aluminium sulphate), organic occulants (polyacrylamide, polyethyleneimine) and natural occulants or bio occulants (gelatin, chitosan guar gum) have been widely used in chemical and mineral industrial elds such as tap water producing, wastewater treatment, dredging, downstream processing, fermentation and food industries [5052]. Although chemical occulants have been used widely due to their effective occulating activity and low cost, they have neurotoxic and carcinogenic monomers and their usage is restricted [1,53]. On the contrary, bioocculants produced by microorganisms during their growth are safe and biodegradable polymers [54]. Recently, many studies have been reported on the occulating effect of microbial polysaccha-

rides to replace synthetic occulants, which are industrially used [5557]. 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 CaCl2 H2 O (Fig. 4). The occulating capability of isolated exopolysaccharide was compared with that of xanthan gum and guar gum used as control. This capability initially increased with increasing the concentration of EPS, and gave the greatest occulating activity between concentration range of 0.30.5 mg/l and on word it had a decreasing trend as the EPS (occulant) concentration increased. The optimal occulant concentration in test solution was determined to be 0.4 mg/ml. Where as the optimal occulant concentration for xanthan gum and guar gum was 0.3 and 0.5 mg/l, respectively. As shown in Fig. 4 that the occulating capability initially increased with increasing concentration and then started to decrease after attaining a highest and puried point and this may be due to that the adsorption of excess occulants destabilized the particles. Because of incomplete dispersion of excess occulants, only particles around occulants participated in the occulating reaction at that moment. A large molecular weight occulant is usually long enough and has a sufcient number of free functional groups that can act as bridges to bring many suspended particles together, and hence causes a larger oc size in the occulation reaction [32]. L. keranofaciens ZW3 EPS

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

288

Y. Wang et al. / International Journal of Biological Macromolecules 43 (2008) 283288 [23] H. Maeda, X. Zhu, K. Omura, S. Suzuki, S. Kitamura, J. Agric. Food Chem. 52 (2004) 55335538. [24] K.L. Rodrigues, L.R. Gaudino Caputo, J.C. Tavares Carvalho, J. Evangelista, J.M. Schneedorf, Int. J. Antimicrob. Agents 25 (2005) 404408. [25] G. Vinderola, G. Perdign, J. Duarte, E. Farnworth, C. Matar, Cytokine 36 (2006) 254260. [26] M. Medrano, P.F. Prez, A.G. Abraham, Int. J. Food Microbiol. 122 (2008) 17. [27] P.S. Rimada, A.G. Abraham, Int. Dairy J. 16 (2006) 3339. [28] H. Yokoi, T. Watanabe, Y. Fujii, T. Toba, S. Adachi, J. Dairy Sci. 73 (1990) 16841689. [29] P. Forsman, T. Alatossava, Arch. Virol. 137 (1994) 4354. [30] M. Dubois, K.A. Gilles, J.K. Hamilton, P.A. Rebers, F. Smith, Anal. Chem. 28 (1956) 350356. [31] P.V. Bramhachari, P.B. Kavi kishor, R. Ramadevi, R. Kumar, B.R. Rao, S.K. Dubey, J. Microbiol. Biotechnol. 17 (2007) 1451. [32] D.J. Lim, J.D. Kim, M.Y. Kim, S.H. Yoo, J.U. Kong, J. Microbiol. Biotechnol. 17 (2007) 979984. [33] I. Mainville, N. Robert, B. Lee, E.R. Farnworth, Syst. Appl. Microbiol. 29 (2006) 5968. [34] P.S. Rimada, A.G. Abraham, Lait 83 (2003) 7987. [35] A.S. Kumar, K. Mody, B. Jha, J. Basic Microbiol. 47 (2007) 103117. [36] R.H. Wilson, B.J. Goodfellow, P. Belton, Food Hydrocolloids 2 (1988) 169178. [37] S. Cetin, A. Erdincler, Water Sci. Technol. 5 (2004) 4956. [38] K.J. Howe, K.P. Ishida, M.M. Clark, Desalination 147 (2002) 251255. [39] P.J. Bremer, G.G. Geesey, Biofouling 3 (1991) 89100. [40] S. Nataraj, R. Schomacker, M. Kraume, M.I. Mishra, A. Drews, J. Membr. Sci. 308 (2008) 152161. [41] D. Helm, D. Naumann, FEMS Microbiol. Lett. 126 (1995) 7580. [42] K. Haxaire, Y. Marechal, M. Milas, M. Rinaudo, Biopolymers 72 (2003) 1020. [43] J.E. Zajic, E. Knetting, Development in Industrial Microbiology, American Institute of Biological Science, Washington, D.C., 1970, pp. 8798. [44] S.K. Kazy, P. Sar, S.P. Singh, A.K. Sen, S.F. DSouza, World J. Microbiol. Biotechnol. 18 (2002) 583588. [45] E. Marinho-Soriano, E. Bourret, Bioresour. Technol. 96 (2005) 379382. [46] G.S. Kwon, H.K. Joo, T.K. Oh, Kor. J. Appl. Microbiol. Biotechnol. 20 (1992) 34 39. [47] S.J. Jung, K.B. Song, B.Y. Kim, U.H. Chun, S.K. Rhee, Food Eng. Prog. 3 (1999) 176180. [48] M.C. Cirigliano, G.M. Carman, Appl. Environ. Microbiol. 51 (1985) 846850. [49] S. Royan, C. Parulekar, S. Mavinkurve, Lett. Appl. Microbiol. 29 (1999) 342 346. [50] H. Salehizadeh, M. Vossoughi, I. Alemzadeh, Biochem. Eng. J. 5 (2000) 3944. [51] H. Salehizadeh, S.A. Shojaosadati, Biotechnol. Adv. 19 (2001) 371385. [52] Z.Q. Zhang, B. Lin, S.Q. Xia, X.J. Wang, A.M. Yang, J. Environ. Sci. China 19 (2007) 660666. [53] C. Ruden, Food Chem. Toxicol. 42 (2004) 335349. [54] S.B. Deng, R.B. Bai, X.M. Hu, Q. Luo, Appl. Microbiol. Biotechnol. 60 (2003) 588593. [55] K. Toeda, R. Kurane, Agric. Biol. Chem. 55 (1991) 27932799. [56] J. Zhang, Z. Liu, S. Wang, P. Jiang, Appl. Microbiol. Biotechnol. 59 (2002) 517 522. [57] X.W. Huang, W. Cheng, Y.Y. Hu, J. Environ. Sci. China 17 (2005) 494498.

showed a better occulating activity than guar gum and almost similar to xanthan gum. Moreover the occulating behaviour of ZW3 is also supported by its IR analysis. So ZW3 EPS is expected to be useful occulating agents in the areas of wastewater treatment, drinking water processing, and downstream processing in the food industry because of their biodegradability and harmlessness towards humans and the environment. Acknowledgments This work was supported by grant from China the Fifteenth National Scientic Support Grant (No. 2006BAD 04A 06) and by Tianjin Municipal Science and Technology Commission Grant (No. 08JCYBJC01900). References
[1] C. Arezoo, Nephrol. Dial. Transplant. 16 (2002) 1720. [2] I.Y. Lee, W.T. Seo, G.J. Kim, M.K. Kim, S.G. Ahn, G.S. Kwon, Bioprocess Biosyst. Eng. 16 (1997) 7175. [3] L. De Vuyst, F. Vanderveken, S. van den Ven, B. Degeest, J. Appl. Microbiol. 84 (1998) 10591068. [4] J. Bender, S.R. Eaton, U.M. Ekanemesang, P. Phillips, Appl. Environ. Microbiol. 60 (1994) 23112315. [5] I.W. Sutherland, Trends Biotechnol. 16 (1998) 4146. [6] L. De Vuyst, B. Degeest, FEMS Microbiol. Rev. 23 (1999) 153177. [7] M. Garciagaribay, V.M.E. Marshall, J. Appl. Bacteriol. 70 (1991) 325328. [8] S. Matsukawa, T. Watanabe, Food Hydrocolloids 21 (2007) 13551361. [9] A. Laws, Y. Gu, V. Marshall, Biotechnol. Adv. 19 (2001) 597625. [10] L. De Vuyst, F. De Vin, F. Vaningelgem, B. Degeest, Int. Dairy J. 11 (2001) 687708. [11] A.D. Welman, I.S. Maddox, Trends Biotechnol. 21 (2003) 269274. [12] A. Laws, V. Marshall, Int. Dairy J. 11 (2001) 709721. [13] L. Jolly, S.J.F. Vincent, P. Duboc, J.R. Neeser, Antonie Leeuwenhoek 82 (2002) 367374. [14] O. Kandler, P. Kunath, Syst. Appl. Microbiol. 4 (1983) 286294. [15] P. Kooiman, Carbohyd. Res. 7 (1968) 220221. [16] T. Fujisawa, S. Adachi, T. Toba, K. Arimara, T. Mitsuoka, Int. J. Syst. Bacteriol. 38 (1988) 1214. [17] H. Yokoi, Y. Fujii, T. Mukai, T. Toba, S. Adachi, Int. J. Food Microbiol. 13 (1991) 257264. [18] S. Takizawa, S. Kojima, S. Tamura, S. Fujinaga, Y. Benno, T. Nakase, Int. J. Syst. Bacteriol. 44 (1994) 435438. [19] G. Ottogalli, A. Galli, P. Resmini, G. Volonterio, Annu. Microbiol. 23 (1973) 109121. [20] L. Angulo, E. Lopez, C. Lema, J. Dairy Res. 60 (1993) 263267. [21] M.E. Pintado, J.A. Lopes Da Silva, P.B. Fernandes, F. Xavier Malcata, T.A. Hogg, Int. J. Food Sci. Technol. 31 (1996) 1526. [22] M. Murofushi, M. Shiomi, K. Aibara, Jpn. J. Med. Sci. Biol. 36 (1983) 4953.