Chapter 3. Purification, optimization of assay and stability studies of
5.3 Results and Discussion…
5.3.2 Determination of molecular mass of dextran from Weissella cibaria
The dextran from Weissella cibaria JAG8 was purified by gel filtration using Sephacryl S-500HR. The fractions (3 ml) were collected at a flow rate of 1 ml/min at room temperature. It was observed that dextran started eluting from fraction no. 16 and maximum concentration of dextran eluted at fraction no. 19, (i.e.) 57 ml (19 x 3 ml). The standard dextran 2000 kDa started eluting from fraction no. 12 and the maximum concentration was observed at fraction no. 15, (i.e.) at 45 ml (15 x 3 ml).
While, 200 kDa standard dextran started eluting from fraction no. 21 and the peak was observed at fraction no. 25, (i.e.) 75 ml (25 x 3 ml) and 70 kDa was eluted from fraction no. 27 and peak was observed at fraction no. 29, (i.e.) 87 ml (29 x 3 ml). The elution profile of dextran from Weissella cibaria JAG8 and standard dextrans are displayed in Fig. 5.3.1. The results clearly suggested that dextran produced by Weissella cibaria JAG8 is of high molecular weight. Molecular mass of dextran produced from Weissella cibaria JAG8 was eluted earlier than standard dextran 200 kDa and 70 kDa indicating that its molecular mass is more than 200 kDa and less than 2000 kDa. Based on elution profile of dextran from Weissella cibaria JAG8 that molecular weight was approximately, in the range of 500-1000 kDa. The average
molecular weight was confirmed further by bichinconinate method as described in Section 5.3.3.
Fraction no.
4 8 12 16 20 24 28 32 36 40
Dextran Concentration (mg/ml)
0.0 0.2 0.4 0.6 0.8
2000 kDa Dextran JAG8 200 kDa 70 kDa
Fig. 5.3.1 Gel filtration of dextran from Weissella cibaria JAG8 using Sephacryl S- 500HR matrix.
.
5.3.3 Determination of number average molecular weight (MWn) and degree of polymerization (Dpn) of dextran by Bichinconinate (BCA) method
The molecular weight of dextran from Weissella cibaria JAG8 was further confirmed by BCA method and it was found to be approximately, 800 kDa. The molecular mass determined by gel filtration and BCA method were in agreement.
Dextran eluted in the fractions from 16-26 were pooled after determining the total carbohydrate content by phenol sulphuric acid method and was subjected for freezing at -20°C for 3-4 h and finally lyophilized to powder.
5.3.4 Structure analysis of dextran Weissella cibaria JAG8 by spectroscopy 5.3.4.1 FT-IR Spectroscopic analysis of dextran
The FT-IR spectrum of dextran provided information on the functional groups, monomeric units and linkages present in dextran. Generally carbohydrates show high absorbance in the region of 1200-950 cm-1. The bands obtained in the FT-IR spectrum of dextran are shown in Table 5.3.1 and Fig. 5.3.2. The band in the region 2447 cm-1 indicated C-H stretching vibration and the band in the 1638 cm-1 was due to carboxyl group stretching and the results were in agreement with the earlier report of Liu et al., (2007). The absorption peaks at 865 and 920 cm-1 indicated the existence of α- glycosidic bond and the results were in accordance with the earlier report of Majumder et al., (2009). The absorption at 1084 and 988 cm-1 indicated C-O, C-C bond and deformational vibration of CCH, COH and HCO bonds. The band at 1084 cm-1 was assigned to be valent vibrations of C-O-C bond and glycosidic bond as reported earlier by Purama et al., (2009). The band at 988 cm-1 in polysaccharide indicated α(1→6) linkage and can be considered as the characteristic for the type of inter-unit link as also reported by Shingel (2002).The band obtained in the region of 3402 cm-1 was due to the hydroxyl stretching vibration of the polysaccharide as also reported by Purama et al., (2009).
Table 5.3.1 Characterization of functional groups present in dextran by FT-IR.
Wave length (cm-1) Functional Group
3402 Represents hydroxyl stretching vibration.
2447 C-H group stretching vibration.
1638 Carboxyl group.
1401, 1306 C=C (Aromatic/Cyclic) group.
1084, 988 C-O, C-C bond and deformational vibration of CCH, COH and HCO bonds.
920, 866 α-Glycosidic linkage.
540 C-Br stretching vibrations.
Fig. 5.3.2 FT-IR spectrum of dextran produced from the purified dextransucrase of isolate Weissella cibaria JAG8.
5.3.4.2 1H NMR spectroscopic analysis of dextran
The structural characterization of dextran was performed by NMR spectroscopic analysis. The 1H data is shown in Fig. 5.3.3 and Table 5.3.2. Based on the data obtained from 1H NMR, the anomeric α(1→6) proton appeared at 4.96 ppm and a low intensity peak at 5.3 ppm indicated the presence of α(1→3) branching (Maina et al., 2008). No peaks were detected in the range 4.9-5.3 ppm indicating the presence of no branching other than α(1→3) as shown in Fig. 5.3.3. It was reported earlier that the dextran produced from Weissella speciessuch as W. cibaria and W. confusa are linear in nature with lower percentage (2.4% to 3.4%) of α(1→3) branching (Maina et al., 2008; Bounaix et al., 2009 and Rifat et al., 2012). Based on integration analysis of 1H NMR, it was observed that Weissella cibaria JAG8 produces dextran with 93.0% of
α(1→6) and 7.0% of α(1→3) branching with average chain length of 14 glucose units between branched linkages. There are 69 branch linkages for every 1000 glucose units in case of dextran produced from Weissella cibaria JAG8 as analysed based on the report of Vettori et al., (2012). Dextran from Weissella cibaria JAG8 has 2-fold more α(1→3) branching than the dextran from W. cibaria CMGDEX3 (Rifat et al., 2012).
The branched dextrans are resistant to enzyme hydrolysis by exodextranases and glucosidases indicating its applications for the production of prebiotic oligosaccharides (Remaud-Simeon et al., 2000). This is the first report on Weissella cibaria JAG8 with 7.0% of α (1→3) branching thus giving a different structure and characteristic feature to the dextran.
Fig. 5.3.3 1H NMR (400 MHz, D2O) spectrum of dextran produced by the purified dextransucrase from Weissella cibaria JAG8.
5.3.4.3 13CNMR analysis of dextran
The structure of dextran was further confirmed by 100 MHz C13 NMR. The resonances at 97.832, 71.543, 73.541, 69.652, 70.315 and 65.655 ppm were obtained, which are the characteristic peaks of linear dextran as reported by Seymour (1979a) and Uzochukwu et al., (2002). The C13 NMR spectra displayed two prominent regions (a) the 95-105 ppm region, which is the anomeric region and (b) the 75 to 85 ppm for dextran branched at C-2, C-3 (or) C-4. C13 NMR resonances with in the 70-75 ppm region are associated with free positions at C-2, C-3 and C-4 residues. As no additional peaks were observed in the 75-85 ppm region indicated the absence of branched linkages (Seymour 1979a). The major resonance in the anomeric region occurs generally at 98.7 (97.8) showing the C-1 is linked. An equal intensity peak at 66.5 (65.6 ppm), indicated that the most of the C-6 are also linked as reported by Uzochukwu et al., (2002). The equal peak intensity at 97.8 and 65.6 ppm confirmed that glucose residues in dextran are linked by α(1→6) glycosidic bond and no peaks at 75-85 ppm confirmed the linear nature of dextran from Weissella cibaria JAG8 as reported in Weissella cibaria CMU (Kang et al., 2006). The resonance data of 13C NMR is represented in Table 5.3.2 and Fig 5.3.4.
Table 5.3.2 1H and 13C NMR chemical shift of dextran from W. cibaria JAG8
Atoms H1/C1 H2/C2 H3/C3 H4/C4 H5/C5 H6/C6
1H 4.96 3.55 3.70 3.51 3.89 3.97
13C 97.83 71.54 73.54 69.65 70.31 65.65
Fig. 5.3.4 13C NMR (100 MHz, D2O) spectrum of dextran produced by the purified dextransucrase from Weissella cibaria JAG8.