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

Abstract

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-(1 → 4)-mannanase of Clostridium thermocellum by TLC and HPAEC displayed the release of mannose and MOSs mannobiose and mannotriose. Purified MOSs yielded 40 % mannobiose and 18 % mannotriose confirmed by mass spectroscopy which showed mannobiose (m/z = 365) and mannotriose (m/z = 527). 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-(1 → 4)-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 97 % 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 (500 µg/mL) on human epithelial colorectal adenocarcinoma cell line (HT-29) demonstrated 60 % decreased viability of cells after 48 h displaying anti-tumorigenic property.

Exploration of endo-xylanase from novel strain ofBacillus velezensisAG20 isolated from the cave of Meghalaya

“…Apparently, 90% of the XOS will remain intact under the systemic factors influence for better performance as prebiotics. This result corroborated with the earlier report (34). The discrimination in growth promotion by mixed xylo-oligosaccharides of probiotic Lactobacillus and Bifidobacterium was due to the generation of short chain fatty acids and lactate from anaerobic fermentation in the presence of prebiotics that involved in supply energy for colonic epithelium and suppression of pathogenic intestinal bacteria by lowering of pH (35).…”

“…At low concentrations (300 μg/ml) of mixed XOS suppressed the proliferation of HT-29 and Caco-2 cells 90% and 75%, respectively, after 48 h. Such a finding can initiate the advancement of application of XOS of this kind in anticancer therapy and may pave the applications of XOS as nutraceutical and theragonostic implications as well. Earlier studies suggested that the fructooligosaccharides and manno-oligosaccharides from apple and coconut were effective against human colon cancer cell line due to cell cycle arrest in S phase leading to apoptosis (34, 54). Similar mechanisms might have enlighten in the present study.…”

“…To become a potential prebiotic agent, oligosaccharides must bear some significant characteristics such as 1) promotion of growth of human gut probiotic microbiota, 2) should be able to withstand gastric juice, intestinal fluid and oral enzymes (34). In order to perpetuate as growth promoter, in vitro prebiotic potential of the mixed oligosaccharides were investigated on the growth of probiotc strains.…”

“…The purified XOS were pooled and a mixture was prepared for the investigation. Probiotic and human gut non probiotic gut microbes were cultured using enriched and basal medium as described elsewhere (34, 35). Here each growth medium for specific culture of microorganism supplemented with 1% (w/v) inulin, considered as positive control and mixed XOS as test prebiotics.…”

“…Effect of gastric juice on the mixed XOS was examined and compared with the commercial prebiotic inulin for reference. The simulated gastric juice has been prepared (pH 1-4) following the method described elsewhere (34, 36). Freeze-dried XOS mixture was diluted in MlliQ water (pH 5.3) to prepare 1% (w/v) test sample and similarly the reference inulin.…”