International Journal of Biological Macromolecules 43 (2008) 283–288
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International Journal of Biological Macromolecules
journal homepage: www.elsevier.com/locate/ijbiomac
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
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 keﬁr grain and was identiﬁed 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.
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
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 [1–3]. The exopolysaccharides produced by microorganisms 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]. Examples of industrially important microbial exopolysaccharides are dextrans, xanthan, gellan, pullulan, yeast glucans and bacterial alginates . Polysaccharides of microbial origin have been developed as food additive including xanthan from Xanthomonas campestris  and gellan from Pseudomona elodea . 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 . Lactic acid group of bacteria (LAB) which excretes polysaccharide of elevated molecular weight (MW) has been studied extensively during the last decade [6,10–12]. Their particular phys-
∗ Corresponding author. Tel.: +86 22 60601400; fax: +86 22 60601478. E-mail address: firstname.lastname@example.org (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 keﬁranofaciens, which was isolated from keﬁr grains and used as the starter Caucasian cultured milk, produces an exopolysaccharide called keﬁran . Various isolates have been reported and described as Lactobacillus keﬁr , L. keﬁranofaciens , Lactobacillus sp. KPB-167B , L. keﬁrgranum and L. parakeﬁr . This non-exhaustive listing indicates that the complex taxonomic relationships among the bacterial species of keﬁr have not been completely explored. In addition, the inﬂuence of the geographical 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 modulus of these gels. This phenomenon was strengthened by the previous heat treatment usually applied for yogurts manufacture . 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. keﬁranofaciens ZW3 isolated from Tibet keﬁr such as thermal stability, emulsifying capability, ﬂoc-
PCR product was checked with agarose gel electrophoresis.5% glucose and 1% lactose only. which was adjusted to pH 5. The contents of the dialysis bag were freeze-dried to provide EPS. The ﬁnal pH of each medium was adjusted to 6. Exopolysaccharide productions of different strains were then determined by phenol–sulphuric acid method  until no single sugar was detected in distilled water. heated for 30 min at 100 ◦ C.284
Y.7. 1 ml of homogenized and serially diluted in salt solution keﬁr grain was plated on whey agar medium.2. ribose. DNA fragments were ampliﬁed as follows: initial denaturation at 94 ◦ C for 10 min. / International Journal of Biological Macromolecules 43 (2008) 283–288
culating activity and FT-IR spectra which previously had not been reported yet. lactose. 2. tréhalose. Media Media used were supplemented MRS. at above given conditions. 2. at a resolution of 4 cm−1 and processed by Bruker OPUS software.gov/blast). galactose. The nucleotide sequences were used for the analysis of sequence similarity through BLAST (http://www. A primer pair.05 g cysteine monohydrochloride. 0. 0. and ﬁltering.000 × g for 15 min. Crude EPS was precipitated by the addition of an equal volume of chilled absolute ethanol to the supernatant ﬂuid. respectively was used to clone 16S rRNA genes of target isolate.ncbi. 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. 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. 37 and 45 ◦ C. cellobiose. maltose. After incubation at 30 ◦ C for 7–9 days in an anaerobic atmosphere with a GasPack ﬁlled with a gas mixture consisting of 80% N2 . and the pellet was retained. annealing at 58 ◦ C for 30 s. sorbitol. Experimental 2.5 g glucose. The precipitated protein was removed by centrifugation at 12. Ltd. Supplemented MRS broth medium was prepared by addition of following components in commercial MRS broth (Oxoid) medium: 5 mM CaCl2 . For FTIR spectrum of ZW3 EPS was obtained using KBr method. . China and was preserved by our laboratory and propagated at 25 ◦ C.6 with 2N HCl. corresponding to positions 8–28 and 542–549 of the 16S rDNA. The resulting supernatant was adjusted to pH 6.000 × g for 30 min.5 g tryptone. and d-rafﬁnose. Sugar composition For sugar composition determinations. The strain identiﬁcation was also conﬁrmed by partially sequencing 16S rRNA genes analysis. for 72 h at 4 ◦ C. 0. supplemented M17 and supplemented whey. The ZW3 EPS proved to be having good emulsion stability and ﬂocculating activity as compared to other commercially available gums. 10% CO2 and 10% H2 (v/v).5 g sodium acetate. 1 ml mineral solution. catalase reaction. Ampliﬁed products of about 550 bp in length after veriﬁcation were sequenced by using DNA sequencing kit (Shanghai Sangon Biological Engineering Technology & Services Co. The ﬂasks were taken out and heat at 100 ◦ C for 30 min to dissolve cell attached EPS and subsequently centrifuged at 12. Identiﬁcation of strain ZW3 The strain ZW3 was identiﬁed by using Gram stain reaction. ropy bacteria were isolated. Milk whey used in liquid whey media was deproteinized by adjusting skim milk to pH 4. heating for 30 min at 100 ◦ C. polysaccharides were hydrolyzed by treatment with 2 M TFA (120 ◦ C for 2 h). 2. The tightly capped bottles were incubated at 30 ◦ C for 24–72 h in anaerobic conditions.1 g/l NaCl.1 ml Tween 80. This EPS was named as partially puriﬁed EPS and was later on used to study physical characteristic. and ﬁltered to obtain deproteinized whey. mannitol. Keﬁr grain Keﬁr grain was taken from Tibet. supplemented MRS and supplemented M17 media were aimed at exopolysaccharide production. Following primitively screened isolates were inoculated into 50 ml liquid whey medium in the screw-capped bottles with an inoculation percentage of 2%.8 with 2N NaOH.nih. mélibiose. Whey medium were prepared as described by Yokoi et al. Wang et al. Total chromosomal DNA from MRS (Oxoid) 48 h broth culture was extracted as described by Forsman and Alatossava . 2. 0. EPS-producing LAB strains were ﬁrst screened according to the stickiness and ropiness characteristics of their colonies. 0. gas production from glucose. The pellet was dissolved in double distilled water and was lyphophilized. ability to grow at 15.). After the broth was centrifuged at 12. The mineral solution was composed of 0.1. d-fructose. The polysaccharide samples were pressed into KBr pellets at sample: KBr ratio 1:100. Isolation and puriﬁcation of EPS The EPS was puriﬁed by using method of García-Garibay and Marshall . Screening of the isolates for EPS production The keﬁr grains washed with sterile distilled water were homogenized with a Waring blender.5. and was subsequently autoclaved at 115 ◦ C for 20 min. 2. The sample was centrifuged at 25. the sample was centrifuged. P1 (5 -GAGTTTGATCCTGG CTCAG-3 ) and P2 (5 -TACCGCGGCTGCTGGCAC-3 ). and 0.2. 2. where as M17 (Oxoid) broth media was supplemented with 0. After overnight precipitation at 4 ◦ C. 2. and 2 g agar.3. 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. 0. arginine hydrolysis and by sugar
fermentation which included: l-arabinose.07% MgSO4 and % lactose monohydrate. against three changes of distilled water per day. For isolation. with some modiﬁcation. Supplemented whey medium contained 100 of milk whey.5 with lactic acid and heated for 30 min at 100 ◦ C.6. Milk whey used in the agar media was prepared by the ﬁltration of skim milk.000 rpm for 15 min. the released sugars were converted to their alditol acetates and analyzed by
. where as supplemented liquid whey. 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. 0. d-xylose. extension at 72 ◦ C for 1 min and a 10-min ﬁnal extension step at 72 ◦ C.4 g/l of MgSO4 ·7H2 O.15 g/l of MnSO4 ·4H2 O. saccharose.04% MnSO4 ·4H2 O.nlm. 0. The purpose of using agar whey media was to isolate ropy strains. The resulted supernatant was adjusted to pH 7 and EPS was precipitated by putting an equal volume of chilled ethanol at −21 ◦ C. 1 g lactose monohydrate. Study of infrared (FT-IR) spectroscopy The major structural groups of the puriﬁed EPS were detected using Fourier-transformed infrared spectroscopy.18 g/l of FeSO4 ·7H2 O.4. 0. Partially puriﬁed EPS was further puriﬁed by dissolving it in 14% trichloroacetic acid (TCA) and stirred over night. The Fourier transform-infrared spectra were recorded on a Bruker Vector 22 instrument (Germany) in the region of 4000–400 cm−1 .000 × g for 25 min at 4 ◦ C. followed by 30 cycles consisting of denaturation at 94 ◦ C for 30 s.
partial sequencing of variable regions of 16S rRNA genes was also performed. Keﬁranofaciens.8 mM). ribose. The absorbance at 540 nm was read immediately before and after vortexing (A0 ). keﬁranofaciens subsp. It did not grow at 15 ◦ C. 3.8.000 rpm at 4 ◦ C for 15 min.
screening and exopolysaccharide production. arabinose. catalase negative and rod shaped bacteria. Gram positive. was selected for present study. Results and discussion 3. 2. About 550 base pair (bp) variable regions of 16S rRNA genes was ampliﬁed and 500 bp were sequenced. 3. Flocculating activity The ﬂocculating activity was measured by using the method as described by Lim et al. glucose. Strain ZW3 produced a very high amount of EPS up to 1215 mg/l.5 ml deionized water by heating at 100 ◦ C for about 15–20 min and allowed to cool to room temperature (25 ◦ C). After incubation for 48–72 h.2. column ﬂow: 1 ml/min. The fall in absorbance was recorded after incubation at room temperature for 30 and 60 min (At ). The turbidities of the upper 1 ml phase were measured at 550 nm. supplemented M17 and supplemented whey media was used for screening of EPS-producing strains. 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. The nucleotide sequences were used for the analysis of sequence similarity through BLAST (http://www.ncbi.10. keﬁranofaciens subsp. were selected for next step. Initially the strains were screened on the basis of the morphology and colonies which have mucoid.10 m column under following conditions—injector temperature: 320 ◦ C. EPS production. France). the broth was centrifuged at 12.5 ml hexadecane. The emulsiﬁcation activity was expressed as the percentage retention of emulsion during incubation for time t: At /A0 × 100.25 mm × 0. keﬁranofaciens .1. B: turbidity of control. heating in a mixture of
A: turbidity of EPS-containing suspension. This strain did not produce gas from arginine.1 ml of CaCl2 solution (6. 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.35]. The volume was then made up to 2 ml using phosphate-buffered saline (PBS).Y. keﬁranofaciens species . After removing cell. To this mixture. Screening and identiﬁcation of ZW3 strain Keﬁr samples were taken from Tibet. heating at 60 ◦ C in saline solution. 2. Emulsion stability The emulsifying activity of EPS was assayed as described by Bramhachari et al. keﬁranofaciens subsp. Ropy behaviour of colony of L. keﬁrgranum and L. To conﬁrm the biochemical results. A control was run simultaneously with 2 ml PBS and 0. .5 mg) was dissolved in 0. for determining the melting point and enthalpy change. Charcoal-activated carbon that was used as a testing material was suspended in deionized water at a concentration of 5 g/l. xylose. 10 ml of a charcoal-activated carbon suspension was added and mixed with 0. mannose.nih. Strain ZW3 was. keﬁranofaciens ZW3 strain. keﬁranofaciens subsp. In a test tube. supplemented MRS. Isolation and quantiﬁcation Initially L. 1).9. 1. China. Heat treatment of the samples as a ﬁrst step in the polysaccharide isolation procedure is critical for complete recovery of the EPS. using empty pan as a reference. Analysis of thermal properties The thermal properties of EPS were analyzed by using a differential scanning calorimeter (DSC Model 141 SETARAM Scientiﬁc & Industrial Equipment Co Limited.2 mg of dried EPS sample in an aluminium pan. But differentiate physical test between two subspecies such as growth at 15 ◦ C. it was sealed and analyzed. Other drastic methods include boiling the cell suspension for 15 min in water. both for
. The ﬂocculating activity (%) was deﬁned and calculated according to the following equation: ﬂocculating activity = B−A × 100 × dilution rate B
Fig. The standard alditol acetates were generated by subjecting an intimate mixture of equal proportions of rhamnose. various amounts of EPS were added and vortexed for 30 s and allowed to stand for 10 min at room temperature.5 ml hexadecane. The ropy strain of ZW3 which produced the highest amount of EPS among screened strains. 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. slimy or ropy appearance. its convex colony with extremely sliminess appearance (Fig. In ﬁnal step capability of strains to produce EPS were tested by phenol sulphuric acid method. . 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. A control experiment without the EPS was also pursued in the same manner. Wang et al. The sample was vortexed for 1 min after the addition of 0. 2. the supernatant was dialysed and EPS amount was determined by phenol sulphuric acid method using glucose as standard. keﬁranofaciens ZW3 belonged to subsp. Keﬁranofaciens ZW3. This homofermentative proﬁle along with the combination of sugar fermentation pattern suggests that strain ZW3 might belong to the L. After placing 4. keﬁranofaciens ZW3 and other strains were grown in 50 ml liquid whey medium to screen the strains for EPS quantiﬁcation.gov/blast) and it gave 100% similarity with L. carrier gas: He 99. But we found the supplemented whey media as the best.nlm. split ratio: 10. keﬁranofaciens and named as L. Lyophilized EPS (0. / International Journal of Biological Macromolecules 43 (2008) 283–288
GC–MS. The heating rate was 10 ◦ C min−1 from 20 to 300 ◦ C. Analysis was performed by using a Varian GC/MS 4000 instrument (USA) equipped with VF-5ms 30 m × 0.999%. large amount of exopolysaccharide production and negative aesculin hydrolysis proved that strain L. Different media such as. So strain ZW3 was identiﬁed as L.
As for the exopolysaccharides obtained from a mutant of Bacillus polymyxa. the melting point was 183.9
. The absorption in that region (Fig. Similar biochemical compositions were observed in previous studies of the EPS from L. Emulsion stability Microbial and plant gums as well as some plant and animal proteins have been known to possess lipid emulsifying effects. 3. hydroxyl. Polysaccharides contain a signiﬁcant number of hydroxyl groups. which shows more complex pattern of peaks from 2950 to 1200 cm−1 .3 cal/g .286
Y. Autoclaving is the most frequently used treatment for releasing capsular polysaccharides from cells . 93. Further. The melting of ZW3 EPS. keﬁranofaciens may be has better capability of EPS production than previously reported strains. Analysis of thermal properties Besides chemical properties. applicability of polysaccharide is largely dependent on its thermal and rheological behaviour .4 ◦ C with an enthalpy of 1. The IR spectra of puriﬁed exopolysaccharide ZW3 is given in Fig. so in the ﬁngerprint region (region below 1500 cm−1 where bands characterise the molecule as a whole). and amine groups.5. 3.4. Especially. 2.2 and 192. keﬁranofaciens species isolated from keﬁr . Thus the ZW3 polysaccharide showed a different thermal behaviour than xanthan gum and guar gum. 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. 2) has the rounded trait typical of hydroxyl groups  which suggests that the substance is polysaccharide. the measurement of the thermal characteristics of levan synthesized with levansucrase showed the highest melting point to be 178. the strongest absorption band at 1067 cm−1 is attributed to that substance is polysaccharide . As for the thermal characteristics of exopolysaccharides. which exhibit a broad rounded absorption band above wave number 3000 cm−1 . xanthan gum with microorganism origin has been widely used in the food industry because of its high emulsifying
Table 1 Thermal properties of L. The IR spectra of L.9.3. respectively (Table 1). The presence of different sugar moieties suggests that the exopolymer is a heteropolysaccharide.1 ◦ C. Sugar analysis and infrared (FT-IR) spectroscopy Fourier transform infrared spectroscopy has been a useful tool in monitoring structural changes in biopolymers . and 490. 3). keﬁranofaciens ZW3 was high as compared to previously reported EPS production in the same medium which was up to 405 mg . The FT-IR spectra of the polymer evidenced the presence of carboxyl groups. 3. and a peak at 1378 cm−1 could be assigned to C O str of the COO− and C–O bond from COO− [41.7 93. xanthan gum and guar gum were 249.7. and enthalpy was 100. In an earlier report. keﬁranofaciens ZW3. respectively. 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 . xanthan gum and guar gum started at about 93. Fourier-transformed infrared (FT-IR) spectrum of the exopolysaccharide produced by L. and the endothermic enthalpy change ( H) required to melt 1 g of ZW3 EPS. In this contrast it did not differ from previously reported results. C–O at 1000–1200 cm−1 corresponds to the presence of carbohydrates . / International Journal of Biological Macromolecules 43 (2008) 283–288
phenol water at 65 ◦ C or sonicating the cell suspension. which may serve as binding sites for divalent cations .4 490. The sugar composition of the EPS. Also the amount of EPS produced by L.66 cal/g. Here only qualitative results are given which revealed that ZW3 exopolysaccharide is composed of glucose and
galactose only. the spectrum showed the presence of carboxyl.1 Enthalpy (J g−1 ) 249.4.2 192. Gas chromatogram of alditol acetate derivative of hydrolyzed exopolysaccharide from L.42]. Wang et al. similar to the thermal characteristics of the exopolysaccharides derived from legacy microorganisms . 2. So the strain L. 3.25 ◦ C. which are the preferred groups for the ﬂocculation process similar to that observed in polyelectrolyte . analyzed using MS–gas chromatography (Fig.38. 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 . A broad stretch of C–O–C. Strong absorption at 1643 cm−1 which corresponds to amide I > C O str and C–N bending of protein and peptide amines.
Fig. heat absorption and emission are accompanied with the physical change by deformation of polymer structure or melting of crystalline polysaccharides. 2940 cm−1 (C–H stretch) and 3400 cm−1 (–OH stretch) . keﬁranofaciens ZW3. keﬁranofaciens ZW3 exopolysaccharide (EPS) by differential scanning calorimetry (DSC) Peak temperature (◦ C) ZW3 EPS Xanthan gum Guar gum 97.38 153. 153. Carbohydrates such as xanthan components are recognized by peaks at wave numbers of 1040 cm−1 (C–O bond from the alcohol group).
A control was run with 2 ml PBS without EPS.3–0. respectively. 4 that the ﬂocculating capability initially increased with increasing concentration 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. where as xanthan gum produced 92. Flocculating capability test was performed at EPS concentration ranging from 0.34 ± 3. 3.5 mg/l.003 0.1 to 0.98 100 ± 0. polyethyleneimine) and natural ﬂocculants or bio ﬂocculants (gelatin. A large molecular weight ﬂocculant is usually long enough and has a sufﬁcient number of free functional groups that can act as bridges to bring many suspended particles together.302 ± 0.09% and 84.313 ± 0.31 17. locust gum. On the contrary.004 0.00 38. 0.41 100 ± 0.00 92.023
Emulsifying activity (%) 100 ± 0.6. xanthan gum and guar gum.34% and 65.0.421 ± 0. and gave the greatest ﬂocculating activity between concentration range of 0.
. 4. respectively.017 0. dredging.
activity . respectively.4 mg/ml.004 0. keﬁranofaciens ZW3 exopolysaccharide were compared with various commercial polysaccharides including xanthan gum. Flocculating capability A variety of ﬂocculants.8 mM CaCl2 ·H2 O (Fig. only particles around ﬂocculants participated in the ﬂocculating reaction at that moment. keﬁranofaciens ZW3 exopolysaccharide.10 ± 2. guar gum and locust gum and results are listed in Table 2.015 0. The other polysaccharides such as locust bean and guar gum showed relatively poor emulsifying activities as compared to L.249 ± 0.214 ± 0.63 100 ± 0. Wang et al.011 0.00 91. The emulsifying activity of EPS is determined by its strength in retaining the emulsion of the hydrocarbon in water. keﬁranofaciens ZW3 exopolysaccharide showed almost similar activity. This capability initially increased with increasing the concentration of EPS. keﬁranofaciens ZW3 EPS
Fig.15 ± 2.009 0.Y.571 ± 0.05 88. 86.91 ± 1.3 and 0. Recently. Flocculating capacity of L.00 72. chitosan guar gum) have been widely used in chemical and mineral industrial ﬁelds such as tap water producing. The puriﬁed fraction of the exopolymer produced by L.09 ± 2.12 ± 1. Results of partially puriﬁed fraction of the exopolymer.009 1.007 0.013 0. such as inorganic ﬂocculants (polyaluminium chloride and aluminium sulphate).30 84.5 mg/l and on word it had a decreasing trend as the EPS (ﬂocculant) concentration increased. keﬁranofaciens ZW3 EPS.25% and 88.518 ± 0. The absorbance reading after 30 and 60 min gives a fairly good indication of the stability of the emulsion . vortexed for 1 min and the absorbance monitored at 540 nm.343 ± 0.83
ZW3 EPS puriﬁed
ZW3 EPS partially puriﬁed
Hexadecane.415 ± 0.009 0. L.10% emulsion activity after 30 and 60 min. they have neurotoxic and carcinogenic monomers and their usage is restricted [1. which did not differ much from puriﬁed fraction.25 ± 2.029 1. keﬁranofaciens ZW3 is expected to have a great potential for use as an emulsiﬁer. which are industrially used [55–57]. Generally the emulsion breaks rapidly within an initial incubation of 30 min.47%. keﬁranofaciens ZW3 retained 91.22 65.066 ± 0.67 ± 2. was added to 0. / International Journal of Biological Macromolecules 43 (2008) 283–288 Table 2 Emulsifying activity of ZW3 exopolysaccharide (EPS) Emulsiﬁer 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. respectively. diluted to 2 ml with phosphate buffer saline (PBS). wastewater treatment.6 mg in 5 mg/l dispersion of charcoal-activated carbon containing 6. Although chemical ﬂocculants have been used widely due to their effective ﬂocculating activity and low cost. the polysaccharide produced by L.04 ± 2. Because of incomplete dispersion of excess ﬂocculants. The optimal ﬂocculant concentration in test solution was determined to be 0. 4).26 ± 0. and hence causes a larger ﬂoc size in the ﬂocculation reaction .215 ± 0.68 100 ± 0.163 ± 0.173 ± 0.014 0.032 1.53].12% after 30 and 60 min.006 0.04% of the emulsiﬁcation activity after 30 and 60 min.46%. Where as the optimal ﬂocculant concentration for xanthan gum and guar gum was 0.67% and 37.74 100 ± 0. So the puriﬁed and partially puriﬁed L.00 88. As shown in Fig. The guar gum retained 72. The emulsion stabilities of L.031 ± 0. fermentation and food industries [50–52]. where as puriﬁed exopolysaccharide showed better activity when compared with xanthan gum.43 37.46 ± 2. downstream processing.44 81. produced 88.47 ± 2.008 0.015 0. organic ﬂocculants (polyacrylamide.5 ml EPS (1 mg/ml).06 ± 0. bioﬂocculants produced by microorganisms during their growth are safe and biodegradable polymers .66 ± 2.00 86. From these results.66% and 81. The ﬂocculating capability of isolated exopolysaccharide was compared with that of xanthan gum and guar gum used as control.5 ml.480 ± 0. many studies have been reported on the ﬂocculating effect of microbial polysaccha-
rides to replace synthetic ﬂocculants.11 ± 0.
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