Production and Cross Linking Studies of Dextran from Leuconostoc Mesenteroides MTCC10508 for Biopolymer Preparation

 

V. Mohanasrinivasan1*, Taniya Ghosal, Thaslim J.B., Hurmat Jahan Zeba, E.Selvarajan1, V. Suganthi1, C. Subathra Devi2

1Environmental Biotechnology Division, School of Biosciences and Technology, Vellore Institute of Technology University, Vellore. Tamil Nadu, India.

2Industrial Biotechnology Division, School of Biosciences and Technology, Vellore Institute of Technology University, Vellore. Tamil Nadu, India

*Corresponding Author E-mail: v.mohan@vit.ac.in

 

 

ABSTRACT:

Dextran is easily soluble, biodegradable and biocompatible biopolymers comprised by glucose units. Due to these characteristics, high purity of dextran serve as important starting and intermediate reagents in a broad range of synthesis in biotechnological and food industries. In this study, Leuconostoc mesenteroides MTCC10508 were obtained from MTCC and were transferred in MRS broth medium at 30°C and pH 6.8 for 24 h. The produced dextran from Leuconostoc mesenteroides MTCC10508 was compared with standard dextran. Furthermore, FT-IR analysis results showed that there were similarities between produced dextran and standard dextran. Produced dextran was cross linked with epichlorohydrin to prepare biopolymer. FT-IR result showed that there was similar bands present between cross linked produced dextran and standard dextran.

 

KEYWORDS: Dextran, Leuconostoc mesenteroides MTCC10508, epichlorohydrin, Biopolymer, FT-IR.

 


INTRODUCTION:

Dextran is a straight chain of polysaccharide consisting of glucose molecules joined by α (1,6) glycosidic bonds. Polysaccharides are long carbohydrate molecules of monosaccharide units joined together by glycosidic bonds. Dextran is produced by the species of L. mesenteroides MTCC10508, Streptococcus and Acetobacter1. But commercially dextran is produced by L. mesenteroides MTCC10508 2. Sucrose acts as the main substrate for the production of dextran. The strain is grown in sucrose rich media producing an enzyme, dextransucrase which converts excess of sucrose to dextran and fructose3. Sucrose is the only major known substrate able to synthesize the dextran3. L. mesenteroides MTCC10508 is gram positive cocci in chains. It grows best at temperature between 5° to 30° C  and pH  range 6.0 to 6.9 4. Dextran is being used in microsurgery to decrease vascular thrombosis. It is also being used in pharmaceutical industry, food industry and in chemical industry as blood volume expander, emulsifiers, adjuvants, stabilizer and carrier5, 6, 7. Another use of dextran is the production of Biopolymer gel beads 8.

 

Cross-linked dextran with epichlorohydrin known as biopolymer. They are made up of monomeric units that are bonded to form larger structures by covalent bonds. Biopolymers are widely used for separation and purification of various products like protein in research and industry 9. It also maintains flavor, appearance of food stuffs, enhances moisture retention and prevents crystallization of sugars. Biopolymer is a trademark for cross-linked dextran gel and has wide application in gel filtration and purification process.  

 

The present aim of this study was to produce dextran from L. mesenteroides MTCC10508 and cross linking studies of dextran with epichlorohydrin.

 

MATERIALS AND METHODS:

Yeast extract, peptone, sucrose, MRS and all other chemicals were purchased from HiMedia, Mumbai, India.

 

Stock culture:

L. mesenteroides MTCC10508 were obtained from the Microbial Type Culture Collection (MTCC). The microorganisms were maintained in test tubes containing MRS broth medium at 30°C and pH 6.8. Pure cultures were maintained at 4°C.

Inoculum preparation:

A culture medium composed of Sucrose (1.5 g/L), Yeast extract (0.05 g/L), Peptone (0.05g/L), K2HPO4 (0.15 g/L), MgCl2 (0.00019 g/L), NaCl (0.00019 g/L), CaCl2 (0.0005 g/L) broth was used for preparation of inoculum. About 20 ml of this medium in 100 ml flasks was autoclaved at 121˚C for 15 minutes prior to inoculation.  After cooling, the flasks were inoculated with the stock culture and incubated for overnight at 37˚C, 100 rpm at pH 6.8. The organisms were collected by centrifugation at 8000rpm for 10 min and resuspended in liquid medium and the bacteria were inoculated into liquid fermentation media in flasks.

 

Production and extraction of Dextran:

Inoculum at O.D 0.6 was transferred to 90 ml sterile Dextran production broth containing Yeast extract, (13.3g/L), Bactopeptone (0.45g/L), K2HPO4 (1.35 g/L), MgCl2 (0.00171 g/L), NaCl (0.00171 g/L), CaCl2 (0.0045 g/L) at pH 7.0 and incubated at 37°C for 24 h in a shaker at 120 rpm 10. The turbidity and optical density value was observed at regular intervals till incubation period. After incubation the culture was centrifuged at 12,000 rpm for 10 min at 4°C. The pellet obtained was discarded and to the supernatant, twice the volume of absolute ethanol was added and incubated overnight at 4°C. Centrifugation at 12,000 rpm for 10 minutes at 4°C was performed to pellet out the slimy Dextran. The pellet was washed thrice with distilled water by repeated centrifugation at 12,000 rpm for 10 minutes at 4°C. The samples were kept in hot air oven at 80°C for 2 h to attain proper drying of the samples. Finally, dried sample were weighed and the total yield obtained was calculated.

 

Fourier transform infrared spectroscopy (FT-IR)

The FT-IR spectrum of the produced sample was obtained at a resolution of 4 cm-1. The sample was incorporated into KBr (spectroscopic grade) and pressed into a 2 mm pellet. IR spectra11 were recorded in the transmittance mode from 4000 to 400 cm-1

 

Cross linking of Dextran:

For cross linking process, 200µl of the epichlorohydrin was added to 0.25 g of the produced dextran obtained from L. mesenteroides MTCC10508. The whole process was carried out in a glass beaker and mixed using magnetic stirrer. The above procedure was followed again to crosslink standard dextran (0.25 g) with epichlorohydrin (200µl). The cross linked samples were examined using FT-IR spectra.

 

RESULTS AND DISCUSSION:

Production of Dextran: 

During the fermentation process, pH decreased from 7.0 to 4.2. Sucrose broth media consisting of produced dextran is showed in Figure 1. The total yield of produced dextran sample was . So, dextran from L. mesenteroides MTCC10508 was further carried out for cross linking process.

 

Figure.1 Dextran produced by L. mesenteroides MTCC10508

 

FT-IR analysis:

This technique was used to investigate the functional groups of Dextran produced. The FTIR spectrum of standard and produced Dextran is shown in Fig 2, 3 respectively. The band in the region of 3329.14 cm-1 is due to the hydroxyl stretching vibrations of polysaccharides. The band in the region of 2927 cm-1 is due to C-H stretching vibration and the band region of 1641 cm-1 is due to the carboxyl group.


 

Figure 2. FTIR result of Standard Dextran

 

Figure 3. FTIR results of produced Dextran from L. mesenteroides MTCC10508

 

Figure 4. FTIR result of produced Biopolymer from Standard dextran.

 


The main characteristic bands found in the spectra of Dextran at 1157and 1041cm-1  are due to the valent vibrations of C-O and C-C bonds and deformation vibrations of  -CCH and –HCO bonds. The band at 1157 cm-1 is assigned to valent vibrations of C-O-C bond and glycosidic bridges. It was also observed that absorption peaks at 916.19 cm-1 and 862.18 cm-1 are characteristics of (1-3)-α-D glucan. FTIR spectra analysis of dextran contains both α-(1-6) and α-(1-3) linkages.

 

Cross linking of Dextran  

Fig 4 shows the FT-IR results of biopolymer from the standard dextran that was compared with fig 5 of FT-IR results of biopolymer from produced dextran. The FT-IR spectra of biopolymer from produced dextran showed the presence of following peaks: 3223 cm-1, 1265 cm-1, 1062 cm-1, 1016 cm-1, 923 cm-1, 864 cm-1, 813 cm-1 and 623 cm-1. At 623 cm-1  it has C-Br stretch, 1016 cm-1  showed C-O stretch, 1265 cm-1  indicating the presence of ether C-O-C stretch and at 3223 cm-1  which has –OH stretch. –CH bend was observed in 923 cm-1, 864 cm-1 and 813 cm-1 range.  The FT-IR spectra of the standard sample showed stretch bands at 765 cm-1 , 858 cm-1 and 3115 cm-1 whereas bend bands were observed at 916 cm-1 , 1384 cm-1  and 1159 cm-1 . –CH bend was observed at 916 cm-1, 858 cm-1 and 765 cm-1. -N-H bend was observed at 1620 cm-1 , nitro group (-NO2) at 3184 cm-1  and –OH stretch at 3155 cm-1 . Ether group (C-O-C) stretch band was observed at 1159 cm-1.

 


 

Figure 5. FTIR result of Biopolymer from strain of L. mesenteroidesMTCC10508

.


There was similarity between  standard and produced dextran sample at 2926 cm-1  and 1402 cm-1  which indicates that there was no cross linking in this ranges. The FT-IR analysis of dextran cross linked with camptothecin (CPT) and Fa-DEX-CPT nanoparticles showed the presence of different peaks which had no difference with raw dextran as reported by yuangang et al, 2011. Likewise Hoffmann et al stated that cross linking of glutaraldehyde or oxidized dextran and chitosan showed both NH2 stretch band at 3352 cm-1, 3289 cm-1 and bend band were observed at 1586 cm-

 

Furthermore, a band belonging to imine binding at 1624 cm-1 and 1635 cm-1 respectively, recognized the crosslinking of glutaraldehyde or oxidised dextran and chitosan.

 

The functional ionic groups attached by ether linkage to glucose units of the polysaccharide chain is the main characteristic feature of cross linked dextran. So, presence of ether linkage at 1265 cm-1 of the dextran sample confirms the cross linking of dextran with epichlorohydrin.

 

CONCLUSION:

From the present study it is concluded that, FT-IR spectrum showed there were similarities between produced dextran from L. mesenteroides MTCC10508 and standard dextran. FT-IR analysis showed there were different ranges of bands present between cross linked produced dextran and standard dextran. Furthermore, dextran produced from L. mesenteroides MTCC10508 was successfully cross linked with epichlorohydrin to produce biopolymer.

 

ACKNOWLEDGEMENT:

The authors are grateful to the management of Vellore Institute of Technology University, Vellore, India for providing us the facilities to conduct the research work.

 

REFERENCES:

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Received on 07.11.2013       Modified on 25.11.2013

Accepted on 04.12.2013      © RJPT All right reserved

Research J. Pharm. and Tech. 7(1): Jan. 2014; Page   08-11