Rishikesh Shukla

Structural and biocompatibility properties of dextran from Weissella cibaria JAG8 as food additive

Abstract Dextran produced from Weissella cibaria JAG8 was purified and characterized. The molecular mass of dextran as determined by the gel filtration and copper bicinchoninate method was approximately, 800u2009kDa. Monosaccharide analysis revealed that the polysaccharide comprised only glucose units. Dynamic light scattering study confirmed the mono-disperse nature of dextran with hydrodynamic radius of 900u2009nm. Surface morphology study of dextran by scanning electron microscopy showed the porous web like structure. Cytotoxicity studies on human cervical cancer (HeLa) cell line showed non-toxic and biocompatible nature of dextran. The relative browning for dextran from W. cibaria JAG8 was similar to commercial prebiotic Nutraflora P-95 and 3-fold lower than Raftilose P-95. Synthesis of dextran by dextransucrase treated, sucrose-supplemented skimmed milk revealed the promising potential of dextran as a food additive.

Novel dextran from Pediococcus pentosaceus CRAG3 isolated from fermented cucumber with anti-cancer properties

The porous branched dextran of molecular mass 2.93 × 10(5) Da with 50% overall yield (4.5mg/ml) was produced from Pediococcus pentosaceus CRAG3 (GenBank ID: JX679020), a novel isolate from fermented cucumber. It contained glucose monomers linked through 75% α-(1,6) linkage with 25% (α-1,3) branching as displayed by spectroscopic analysis. The peak analysis showed α-(1,3) branching after every five glucose units of main chain. In vitro cytotoxicity analysis of dextran displayed anti-cancer activity against cervical cancer (HeLa) and colon cancer (HT29) cell lines opening new horizons in its pharmaceutical applications. Dextran showed enhanced growth of macrophage lines revealing its biocompatible nature.

Recovery and Purification of Oligosaccharides from Copra Meal by Recombinant Endo-β-mannanase and Deciphering Molecular Mechanism Involved and Its Role as Potent Therapeutic Agent

Production of manno-oligosaccharides (MOSs) from pretreated and defatted copra meal (dFCO) hydrolysis was achieved by endo-mannanase. Structural characterization of dFCO by FT-IR and NMR exhibited resemblance with galactomannan. The time-dependent hydrolysis of dFCO by recombinant endo-β-(1xa0→xa04)-mannanase of Clostridium thermocellum by TLC and HPAEC displayed the release of mannose and MOSs mannobiose and mannotriose. Purified MOSs yielded 40xa0% mannobiose and 18xa0% mannotriose confirmed by mass spectroscopy which showed mannobiose (m/zxa0=xa0365) and mannotriose (m/zxa0=xa0527). The homology based structural analysis of catalytic endo-mannanase (CtManT) showed the catalytic core composed of Glu181 and Glu300 acting as acid/base and Glu288 as a nucleophile during galactomannan hydrolysis. Sub-site mapping of CtManT exhibited two aglycone and four glycone sites at cleavage sites existing on either side of β-(1xa0→xa04)-linkage of galactomannan. Isolated MOSs displayed potential prebiotic characteristics and supported higher growth of probiotic Lactobacillus acidophilus and Bifidobacterium infantis than with standard inulin. Moreover, MOSs displayed over 97xa0% tolerance to simulated gastric juice, intestinal fluid, and α-amylase proving its potential as a stable prebiotic over inulin. In vitro cytotoxicity assay of MOSs (500xa0µg/mL) on human epithelial colorectal adenocarcinoma cell line (HT-29) demonstrated 60xa0% decreased viability of cells after 48xa0h displaying anti-tumorigenic property.

Probiotic Potential of Pediococcus pentosaceus CRAG3: A New Isolate from Fermented Cucumber

Abstract A novel strain of lactic acid bacterium isolated from fermented cucumber was selected due to its high glucansucrase activity. It was identified on the basis of 16S rRNA sequence analysis as Pediococcus pentosaceus CRAG3 (GenBank accession number JX679020). The isolate was round shaped, Gram positive, and catalase negative displaying typical features of lactic acid bacterium. It produced 145xa0±xa03.27xa0mg lactic acid per ml of cell-free supernatant. It showed ability to ferment carbohydrates such as sucrose, dextrose, and arabinose; showed resistance to antibiotics such as ciprofloxacin, kanamycin, vancomycin; displayed acid production in triple sugar iron agar test and non-motile nature. Interestingly, the isolate also displayed potential probiotic properties such as hydrophobicity, autoaggregation, coaggregation, and in vitro cell adhesion ability. It exhibited resistance against lysozyme and simulated gastric juice at pH 3.0 with 75 and 58xa0% survival, respectively. It also showed tolerance toward 0.3xa0%, w/v bile salts with 73xa0% survival and ability to deconjugate bile salts. The isolate exhibited antibacterial activity and ability to utilize prebiotics such as inulin and raffinose. These results indicate both probiotic property and glucansucrase-producing ability of P. pentosaceus CRAG3.

Scale up of dextran production from a mutant of Pediococcus pentosaceus (SPAm) using optimized medium in a bioreactor

The mutant of Pediococcus pentosaceus (SPAm) produced earlier by UV-mutagenesis exhibiting higher dextransucrase activity as compared to wild-type was used. The generated mutant SPAm gave 12.2 mg/ml, a 20% higher dextran than wild-type. Response surface methodology was carried out for further enhancement of dextran production. To enhance dextran production by the mutant SPAm, Plackett-Burman Design and a 22 full factorial Central Composite Design was employed. After response optimization, the optimum concentration of sucrose and yeast extract was 5.115% (w/v) and 0.635% (w/v), respectively. The experimental values of dextran 36.0 mg/ml at flask level and 35.0 mg/ml at bioreactor level were in good agreement with the predicted value of 40.8 mg/ml. The increase in dextran production by the mutant SPAm using the optimized medium was 3 fold higher as compared to unoptimized medium.

PURIFICATION AND CHARACTERIZATION OF DEXTRANSUCRASE FROM LEUCONOSTOC MESENTEROIDES NRRL B-1149

ABSTRACT Leuconostoc mesenteroides NRRL B-1149 produces extracellular dextransucrase which in this study was purified using different concentrations of polyethylene glycol (PEG). The dextran produced by this enzyme is unique in that it contains α - (1→6) and α-(1→3) linkages which have clinical applications. The cell free supernatant with 0.9 U/mg enzyme specific activity was subjected to fractionation by PEG-400 and PEG-1500. The 33% PEG-400 gave dextransucrase with specific activity of 9.2 U/mg and 10 fold purification and the 15% PEG-1500 gave dextransucrase with maximum specific activity of 15 U/mg and 17 fold purification in a single step. The purified enzyme showed multiple molecular forms on denaturing SDS-PAGE with three prominent bands. The purified dextransucrase confirmed the presence of glucan, after in-situ activity detection by Periodic acid Schiffs staining after running under denaturing SDS-PAGE. The three bands that appeared on denaturing SDS-PAGE stained with silver nitrate solution, corresponded to the three activity bands.