Structural characterization and biological activity of exopolysaccharide from Lysinibacillus fusiformis

The exopolysaccharide (EPS) of Lysinibacillus fusiformis were evaluated for their antioxidant properties. The total antioxidant capacity of EPS extracts was found to be maximum in malt medium 80.13 ± 0.26%. The hydrogen peroxide radical scavenging activity was found to be maximum in malt medium 78.3 ± 0.26%. The DPPH radical scavenging activity was found to be maximum in malt medium 48.39 ± 2.15%. 3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) inhibition assay was found to be maximum in malt medium 50.75 ± 3.85%. This study suggested that the EPS from L. fusiformis could potentially be used for antioxidant activity. Agarose gel electrophoresis was used to find the structure of mobility using acetate buffer (pH 3.6) and citrate buffer (pH 9.0). The physiochemical characterization of EPS was studied and the structure was confirmed by the Fourier transform infrared spectroscopy (FTIR), gas chromatography–mass spectrometry (GC-MS) and high performance liquid chromatography (HPLC) analysis.


INTRODUCTION
The biosynthesis of exopolysaccharide (EPS) is believed to serve many functions concerning promotion of the initial attachment of cells to solid surfaces formation maintenance of microcolony and mature biofilm structure and enhanced biofilm resistance to environmental stress and disinfectants.In some cases EPS matrix also enables the bacteria to capture nutrients.The production of EPS by attached microorganisms is a very complicated process, which is affected by many unique parameters.It is also considered that the mechanisms of biofilm development process are vastly different from species to species (Dunne, 2002).Thus, it is essential to develop and utilize effective and natural antioxidants, so that can be protecting the human body from free radicals and retard the progress of many chronic diseases (Nandita and Rajini, 2004).Many natural resources have attracted attention in the search for bioactive compounds to develop new drugs and healthy foods.Some polysaccharides have been demonstrated to play an important role as free-radical scavengers in vitro and as antioxidants for the prevention of oxidative damage in living organisms (Zhang et al., 2004).The microbial biofilm formed on a biotic surface is an important area of research because of the wide range of possible affects and the disinfectant resistance of the cells.The colonization of solid surfaces by microorganisms is a very complicated process that depends mostly on extracellular molecule production.The biosynthesis of EPS reflected not only the attachment and aggregation process but also provided an optimal environment for the exchange of genetic material between the cells.The comparative and comprehensive analysis of all documented data concerning EPS production can enable the development and effective control strategies for biofilms (Czaczyk and Myszka, 2007).
The polysaccharides play important roles in many biological processes, and they can function as the virulence determinants in the pathogens.Occurrence in nature the biological activities of polysaccharides have attracted more and more attention in biochemistry and medicine (Peng et al., 2008).EPS producing strains are of commercial value for both their technological and putative probiotic properties.Microbial polysaccharides of economic interest are usually produced at the industrial level by fermentation.Problems associated with the reproducibility of fermentation during the industrial scaleup may result in inconsistent productivity, yield and quality, all of which can translate into financial losses.This can be solved by optimising the inoculation methodology and the fermentation conditions.A high EPS production could result in the formation of a highly viscous fermentation broth, making the recovery of cells and further downstream processing difficult (Champagne et al., 2007).
The exopolysaccharides (EPS) are high molecular weight polymers which are long chain composed of sugar residues and secreted by microorganisms into the surrounding environment.Bacterial EPS consist of a complex mixture of macro molecular poly electrolytes including polysaccharides, proteins and nucleic acids.Each comprises of variable molecular mass and struc-tural properties (Vijayabaskar et al., 2011).L. fusiformis produce polysaccharides respond to environmental factors directly and for some, the nutritional conditions determine the degree of exopolysaccharide formation.To evaluate the potential antioxidant activities and characterization of AGE, FTIR, HPLC and GC-MS for exopolysaccharides (EPS) produced L. fusiformis.

Isolation and identification of the bacteria
Exopolysaccharide producing bacteria were isolated from soil samples collected from Ayya Nadar Janaki Ammal College campus near the garden.Isolates were obtained by serial dilution plating on nutrient agar medium.A total of 100 colonies were isolated and the exopolysaccharide producing bacteria were screened for their ability to produce exopolysaccharide, based on colony morphology (mucous and ropy).A mucous colony was isolated and identified by biochemical characterization and 16S rRNA sequencing.

Genomic DNA isolation from isolates
The isolated bacterial strain was grown in 25 ml LB broth overnight at 35°C.The culture was spin at 5000 rpm for 5 min.The pellet was resuspended in 400 µl of sucrose TE buffer (Tris EDTA).Lysozyme was added to a final concentration of 8 mg/ml and incubated for 1 h at 35°C.To this tube, 100 µl of 0.5 M EDTA (pH 8.0), 60 µl of SDS and 3 µl of proteinase-K (20 mg/ml) were added and incubated at 55°C.After incubation, they were centrifuged at 7000 rpm for 3 min and then the supernatant were extracted twice with phenol: chloroform (1:10 and again with chloroform: isoamylalcohol (24:1).It was precipitated with ethanol.The DNA pellet was resuspended in sterile buffer.

Amplification of 16S rRNA gene sequence
Bacterial 16S rDNA was amplified from the extracted genomic DNA using the following universal eubacterial 16S rRNA primers: forward primer 5' AGAGTTTGATCCTGGCTCAG 3' and reverse primer 5'ACGGCTACCTTGTTACGACTT 3'.Polymerase chain reaction was performed in a typical reaction mixture was 2 µl of template DNA and 1.5 µl of forward primer, 1.5 µl of reverse primer, 10 μl of 2X PCR master mixes and 5 µl of nuclease free water for 20 µl reaction.The reaction was performed with an initial denaturation at 94°C for 2 min, 30 cycles of denaturation at 94°C for 45 s, annealing at 56°C for 1 min, extension at 72°C for 1 min 30 s followed by final extension at 72°C for 5 min and hold at 4°C.The amplification of 16S rRNA gene was confirmed by running the amplification product in 1% agarose gel electrophoresis.

Sequencing of 16S rRNA sequence
Partial sequencing of the rRNA gene (about 1210 bp) for the isolated bacteria was carried out in Royal life science Pvt Ltd., (Hyderabad).

Culture process and exopolysaccharide production from bacteria
Bacteria culture was maintained on nutrient agar plates.It was sub cultured and slants were inoculated and maintained at 28ºC for h.Experiments were done using 250 ml flask each containing ml of basal and malt medium inoculated with the bacterial culture.The basal medium contains Glucose 10 g, yeast extract 3 g, malt extracts 3 g, peptone 5 g, MgSo4.7H2O 1 g, KH2PO4 0.3 g and mg of vitamin B1 incorporated at 28ºC with initial pH 7 in 1000 ml distilled water.Malt medium, malt extracts 40 g, peptone 5 g, distilled water 1000 ml and pH 7. The flask was incubated at 28°C on an orbital shaker incubator at 110 rpm for 72 h.

Bacterial exopolysaccharide (EPS) quantification
After 72 h of incubation both basal and malt medium samples were centrifuged at 5000 rpm for 20 min.The EPS was then precipitated from the supernatant by addition of equal volume of methanol.The mixture were agitated with addition of methanol to prevent local high concentration of the precipitate and left over night at 4ºC and centrifuged at 7000 rpm for 20 min.After centrifugation the precipitate was collected in a Petri plate and dried at 60ºC.

Determination of total antioxidant capacity
Total antioxidant activity of exopolysaccharides extract was determined according to the method (Mitsuda et al., 1996).45 ml of sul-furic acid (0.6 M), 0.9942 g of sodium sulfate (28 mM) and 1.235 g of ammonium molybtate (4 mM) were mixed together in 250 ml with distilled water and labeled as total antioxidant capacity.0.1 ml of the EPS extract (50, 100, 250, 500 and 1000 µg) was dissolved in 1 ml of total antioxidant capacity and absorbance was read at 695 nm after 15 min.Ascorbic acid was used as standard.

Determination of reducing power
Reducing power of the exopolysaccharides extract was determined by the following method (Yamaguchi et al., 1998).0.75 ml of various concentrations EPS (200, 400, 600, 800 and 1000 µg) was mixed with 0.75 ml phosphate buffer (pH 6.6) and 0.75 ml of 1% of potassium ferriccyanide was added.The mixture was incubated at 50ºC for 20 min.0.75 ml of 10% trichloro acetic acid was added to the mixture and centrifuged at 3000 rpm for 10 min.1.5 ml of distilled water and 0.1 ml of 0.1% ferric chloride was added.After an incubation of 10 min the absorbance was read at 700 nm.

Hydrogen peroxide scavenging assay
The free radical scavenging activity of exopolysaccharides extract was determined by Hydrogen peroxide assay (Gulcin et al., 2004).Hydrogen peroxide (10 mM) solution was prepared in phosphate buffered saline (0.1 M, pH 7.4). 1 ml of exopolysaccharides extract (50, 100, 250, 500 and 1000 µg) was rapidly mixed with 2 ml of hydrogen peroxide solution.The absorbance was measured at 230 nm in the UV spectrophotometer after 10 min of incubation at 37ºC against a blank (without hydrogen peroxide).The percentage scavenging of Hydrogen peroxide was calculated using the formula,

Picrylhydrazyl free radical (DPPH) radical scavenging assay
The free radical scavenging activity of exopolysaccharides extract was measured by the 1-1-Diphemy1-2picryl-hydrazyl (DPPH) following the method (Blois, 1958).Used as a reagent, DPPH evidently offers a convenient and accurate method for titrating the oxidizable groups of natural or synthetic antioxidants.0.1 mM solution of DPPH in methanol was prepared and 1 ml of this solution was added to 3 ml of exopolysaccharides extract at different concentrations (50, 100, 250, 500 and 1000 µg).After 10 min, absorbance was read at 517 nm.The percentage scavenging activity values were calculated as below.

3-Ethylbenzothiazoline-6-sulfonic acid) (ABTS) inhibition assay
The free radical scavenging activity of exopolysaccharides extract was determined by ABTS (2,2 azino bis (3-etheylbenzothiazoline-6sulphonicacid) diammonium salt) radical cation decolourization assay (Re et al., 1999).ABTS was generated by mixing 5 ml of 7 mM ABTS with 88 µl of 140 mM potassium persulfate under darkness at room temperature for 16 hours.The solution was diluted with 50% ethanol and the absorbance at 734 nm was measured.The ABTS radical cation scavenging activity was assessed by mixing 5 ml ABTS solution (absorbance of 0.7 ± 0.05) with 0.1 ml exopolysaccharides extract (50, 100, 250, 500 and 1000 µg).The final absorbance was measured at 743 nm with spectrophotometer.The percentage scavenging of was calculated by the following formula

Infra-red spectroscopy analysis of exopolysaccharide (EPS)
The bacterial exopolysaccharides were also characterized using Fourier transform infrared spectrophotometer.IR spectroscopies of bacterial EPS along with a standard, dextran sulfate were tested using Perkin-Elmer FT-IR instrument, which helped to analyze different sulfate, carboxyl and hydroxyl groups of these sample molecules (Wang et al., 2004).One part of extract was mixed with ninety nine parts of dried potassium bromide (KBr) separately and then compressed to prepare a salt disc of 3 mm diameter.These discs were subjected to IR-spectrophotometer. The absorption was read between 400 and 4000 cm -1 .

Agarose gel electrophoresis
The EPS samples (10 µg) were subjected to electrophoresis on a 0.6% agarose gel (Mauro et al., 1998).This experiment helps to find out the nature of the isolated product.The presence of EPS was analyzed by electrophoresis, using two different buffer systems such as acetate buffer (pH 3.6) and citrate buffer (pH 9) for 1hour at 100 v.After the electrophoresis, the gel was then fixed with 0.1% N-Cetyl-N,N,N-trimethyl ammonium bromide for 12 h.The gel was then dried and stained with toluidine blue solution 0.1% toluidene in acetic acid, ethanol and water in the ratio of (0.1:5:5 v/v).After staining, the gel was washed in destaining solution acetic acid, ethanol and water in the ratio (0.1:5:5 v/v) and result was documented.

High performance liquid chromatography (HPLC) analysis of exopolysaccharide (EPS)
The EPS were analyzed with a high performance liquid chromatography (HPLC) C18 system column (LC-10VP Shimadzu) and eluted with distilled water at a flow rate of 1.0 ml/min at 20°C.The separated components were monitored by a refractive index detector.The EPS after being hydrolyzed and derivatives with methanol was analyzed for its sugar composition by HPLC.The column was calibrated with different molecular mass standard and a standard curve was then established.

Gas chromatography-mass spectrometry (GC-MS) analysis of exopolysaccharide (EPS) extract
0.1 g sample was mixed with 1.25 ml of 92% of sulfuric acid with a glass sticks and incubated for 60 min at 30ºC.The mixture were distilled with 13.5 ml of distilled water and incubated in a boiling water bath for 4 hours.After incubation, mixtures were cooled and 3.1 ml of 32% of NaOH (w/v) was added.At the end of hydrolysis, 0.2 ml of sample was taken in a separate tube and 2 ml of 2% sodium borohydride in dimethyl sulfoxide was added.The mixtures were then stirred for 90 min at 40ºC after which 0.2 ml of glacial acetic acid was added to decompose excess of sodium borohydrizde.After cooling, 4 ml acetic anhydride and 0.4 ml of 1-methyl immidazole were added to the solution.The mixture was then incubated for 10 mints at room temperature and then 20 ml of distilled water was added to decompose the excess of acetic anhydride.After cooling, 8 ml of dichloromethane was added and mixture was vigorously shaken for total alditol acetate extraction.
Alditol acetates were separated on a 30 m x 0.25 mm ID x 0.25 µm film thickness column DB 5 ms (agilent) attached to the GC-2010 (GCMS-QP 2010 SHIMADZU) chromatography equipment with a flame-ionization detector and a split injector.High purity hy- drogen was used as the carrier gas at a flow rate of 1.40 ml/min.the column temperature was maintained at 200 and 240 o C respectively, and 1µl sample in dichloromethane was injected through a glass-lined splitter, set at 1/90 ratio.The absorption was read between 40 m/z and 800 m/z.

Screening of exopolysaccharied producing bacteria
The bacterial isolate was identified by standard morphological and biochemical characterization in accordance with Bergey's manual of determinative bacteriology.The morphology of the isolate was Gram positive, rod shaped, spore forming and motile aerobes and biochemical characterization of the isolate was carried out and the results (Table 1).The 16S rRNA gene of the EPS strain was PCR amplified using the 16S rDNA universal primers and both the strands were sequenced.The sequences were compared with the 16S rDNA sequences available in the RDP database (http: //rdp.cme.msu.edu/).Sequence analysis revealed that the strains were phylogenetically closely related to the genus Lysinibacillus.BLAST analysis of the 16S rDNA sequence of EPS producing isolate revealed that it is more score bit with Lysinibacillus fusiformis.Though the isolates had a close similarity, the dendrogram was constructed based on their phylogenetic relationship revealed that all the isolates were distinctly placed under separate clusters.16S rRNA gene sequen-cing confirmed that the isolate was Lysinibacillus fusiformis (Figure 1).The Lysinibacillus fusiformis strain ME was submitted in the Genbank with Accession number JF906500.

Total antioxidant capacity
The total antioxidant capacity of EPS extracts of L. fusiformis along with standard ascorbic acid (Figure 2).The activity of extracts was calculated based on inhibition percentage.The total antioxidant capacity of EPS extracts of L. fusiformis was found to be maximum in malt medium (80.13 ± 0.26%).

Reducing power
The reducing capacities of various concentration of EPS extracts from L. fusiformis in basal and malt medium is compared with standard compound which implies that as the concentration increases the reducing power of the extracts also increases L. fusiformis EPS extracts (Figure 3).

Hydrogen peroxide scavenging assay
The activity observed in hydrogen peroxide radical assay was directly proportional to change and as found to be concentration gradients to change.The hydrogen peroxide inhibition activity for the L. fusiformis of basal and malt medium EPS extracts.Which indicate higher activity EPS extracts of L. fusiformis was found to be maximum in malt medium (78.3 ± 0.26%) (Figure 4).

Picrylhydrazyl free radical (DPPH) scavenging assy
The activity observed in DPPH radical scavenging assay of EPS extracts of L. fusiformis along with standard Gallic acid (Figure 5).Which indicate higher activity for EPS extracts of L. fusiformis was found to be maximum in malt medium (48.39 ± 2.15%).

3-Ethylbenzothiazoline-6-sulfonic acid) (ABTS) inhibition assay
The activity observed in ABTS inhibition assay of EPS extrats of L. fusiformis along with standard Gallic acid (Figure 6).Which indicate higher activity for crude EPS extracts of L. fusiformis was found to be maximum in malt medium (50.75 ± 3.85%).showed the presence of hydrogen bonded compound, possible acid or amine salt.The L. fusiformis EPS extracts revealed characteristic absorption bands of EPS as observed in the reference compound dextran sulphate.The spectram of polysaccharide sample showed the band at 606. 63, 929.72, 1036.77, 1077.28, 1404.22, 1456.30cm -1 in spectrum of L. fusiformis malt EPS (Figure 7).In addition the spectrum showed the band around 1000, 1200, 1400, 1500 and 1600 cm -1 revealed the (1,3) -βglucan linkages in addition to the bands in  the region of 2900 and 3400 cm -1 chemical bands were presented.

IR spectroscopy of intact exopolysaccharides (EPS)
The broad peak at 3442 cm -1 was OH stretching peak, the carbonyl (C=O) stretching was at 1647 cm -1 , the peak between 1900 and 2800 cm -1 was C-H stretching peak.N-H deformation was at 1404 cm form.In the anomeric region (1000-1600 cm -1 ) the polysaccharides exhibited the obvious characteristic absorption at 1037 cm -1 .

Agarose gel eletrophoresis
The mobility of the exopolysaccharides extracted from both the L. fusiformis was checked using agarose gel electrophoresis by applying various pH for two different buffer systems.The highest mobility was observed for higher pH values (pH 3.6 acetate buffer and pH 9.0 citrate buffer in the two buffer systems, especially the citrate buffer (pH 9.0) showed highest mobility for both the EPS extracts when compared with dextran sulfate (Figure 8

High performance liquid chromatography (HPLC)
The obtained fractions were analyzed with a high performance liquid chromatography (HPLC) system C18 column.HPLC was applied to elucidate the relative molecular mass of the exopolysaccharides.It has been confirmed with the previous reference.The EPS production has been quantified through HPLC, in which for EPS production from L. fusiformis from potential EPS malt medium with the retention time as the EPS production was found to be higher for the EPS malt medium.Here independent peaks were identified with retention time (Figure 9).

Gas chromatography-mass spectrometry (GC-MS) analysis of exopolysaccharide (EPS) extract
The electron impact fragmentation patterns of the mass spectra of derived alditol acetates were prepared from the hydrolyzed exopolysaccharides.In bacterial exopolysaccharide the peaks are corresponding to 1-methyl-2formylimidazole (47.95%) with various retention times.
The retention times and the percentage of monosaccharide concentration of the fractionated products of both the exopolysaccharide contained alditol acetate residues.
In this result the exhibited proper and various peaks compared to L. fusiformis (Malt) (Figure 10).

DISCUSSION
In present study the total antioxidant capacity of EPS extracts of L. fusiformis was found to be maximum in malt medium (80.13 ± 0.26%).In present study of L. fusiformis EPS extract from malt medium showed better reducing power when compared with L. fusiformis basal medium.(Czochra and Widensk, 2002) it can cross membranes and may slowly oxidize a number of compounds.Hydrogen peroxide itself is not very reactive but sometimes it can be toxic to cells because of rise in the hydroxyl radicals in the cells.In present study the which indicate higher activity EPS extracts of L. fusiformis was found to be maximum in malt medium (78.3 ± 0.26%).The ABTS radical reactions involve electron transfer and the process take place faster rate when compared to DPPH radicals.In the present study the ABTS radical scavenging activity was more in higher activity EPS extracts of L. fusiformis was found to be maximum in malt medium (50.75 ± 3.85%).The radical scavenging ability using the same per weight basis the antiradical performance of polysaccharide fractions with respect to DPPH radicals was measured and compared.The order of effectiveness of polysaccharide fractions in inhibiting free radicals was as follows: AP-III>AP-II>AP-I.AP-III had the highest radical scavenging activity, followed by AP-II.After 60 min incubation, 63.01% of DPPH radical were quenched by fraction AP-III, followed by fraction AP-II which was able to quench 45.3%.Surprisingly, inhibition of DPPH radicals was only 21.9% pure dextran (Sigma) was assayed (Mohsen et al., 2007).In present study of DPPH radicals which indicate higher activity EPS extracts of L. fusiformis was found to be maximum in malt medium (48.39 ± 2.15%).
The bacterial EPS extracts gave characteristics bands for EPS.Here, carbonyl (C= O) stretching peak and OH stretching peak was at broad and the maximum peak and the band at 1000-1500 showed the presence of polysaccharide.The cultivation temperature by 10˚C below optimal level inhibits the EPS biosynthesis by microbial cells.However, under low temperature of the growth, environment profiles of the high productivity of extracellular polysaccharide occur by bacterial cells (Sutherland 2002).The IR spectrum of the polymer proved the presence of carboxyl group, which may serve as binding sites for divalent cations.The carboxyl group may also work as functional moieties to generate new or modified polymer variants using different approaches like novel.The time dependent increase in the Si-O stretching vibration (1200 to 1000 cm -1 ) hindered our ability to extract IR data from this region of the EPS silica spectrum symmetric phosphate stretch of nucleic acid and C-O stretching modes of sugar/sugar phosphate (Omoike and Chorover, 2006).
The electrophoretic migration of exopolysaccharides in agarose gel, using various buffer systems at various pH depends on the structure of polysaccharide.The exopolysaccharides extracts of L. fusiformis in the gel depending upon the size of the molecules.These molecules were compared with standard dextran sulfate.The exopolysaccharides had different electrophoretic mobility for different buffer system depending on the structure of the polysaccharide (Dietrich et al., 1985).
The fully methylated products were hydrolyzed with as it, converted into the alditol acetate and analyzed by GC-MS.Methylation analysis of the polysaccharides glucan such as 2,3,4-tetra-Me.Glu.and 2,3,6-tri Me-Glu.When the oligosaccharide alditol contain hexoses.(Gal) or deoxyhexoses (Rha), the sequence of this saccharide may be determined on the basis of MS.The electron impact fragmentation patterns of the mass spectra of derived alditol acetates were prepared from the hydrolysed EPS (Vijayabaskar et al., 2011).In present study the EPS production has been quantified through HPLC, in which for EPS production from L. fusiformis from potential EPS malt medium with the retention time as the EPS production was found to be higher for the EPS malt medium.In present study the L. fusiformis exopolysaccharide the peaks are corresponding to 1-methyl-2-formylimidazole (47.95%) with various retention times.The retention times and the percentage of monosaccharide concentra-tion of the fractionated products of both the exopolysaccharide contained alditol acetate residues.In case of complex EPS neutral sugars are identified by their derivatives, alditol acetates by GC-MS (Hoebler et al., 1989).
a and b).

Table 1 .
Biochemical characteristics of the Lysinibacillus sp.
+ Positive Results.-Negative Results