Low molecular weight chitosan from Pleurotus ostreatus waste and its prebiotic potential.

The booming mushroom industry envisages economic merits, and massive unutilized waste production (∼ 20 %) creates an opportunity for valorization. Chitosan, a bioactive polysaccharide, has drawn immense attention for its invaluable therapeutic potential. Thus, the present study was conducted to extract chitosan from mushroom waste (MCH) for its prebiotic potential. The structural characterization of MCH was carried out using NMR, FTIR, and XRD. The CP/MAS-13CNMR spectrum of MCH appeared at δ 57.67 (C2), 61.19 (C6), 75.39 (C3/C5), 83.53 (C4), 105.13 (C1), 23.69 (CH3), and 174.19 (C = O) ppm. The FTIR showed characteristic peaks at 3361 cm-1, 1582 cm-1, and 1262 cm-1 attributed to -NH stretching, amide II, and amide III bands of MCH. XRD interpretation of MCH exhibited a single strong reflection at 2θ =20.19, which may correspond to the "form-II" polymorph. The extracted MCH (∼ 47 kDa) exhibited varying degrees of deacetylation from 79 to 84 %. The prebiotic activity score of 0.73 to 0.82 was observed for MCH (1 %) when supplemented with probiotic strains (Lactobacillus casei, L. helveticus, L. plantarum, and L. rhamnosus). MCH enhanced the growth of Lactobacillus strains and SCFA's levels, particularly in L. rhamnosus. The MCH also inhibited the growth of pathogenic strains (MIC of 0.125 and 0.25 mg/mL against E. coli and S. aureus, respectively) and enhanced the adhesion efficiency of probiotics (3 to 8 % at 1 % MCH supplementation). L. rhamnosus efficiency was higher against pathogens in the presence of MCH, as indicated by anti-adhesion assays. These findings suggested that extracted polysaccharides from mushroom waste can be used as a prebiotic for ameliorating intestinal dysbiosis.

Structural characteristics and functional properties of a fucose containing prebiotic exopolysaccharide from Bifidobacterium breve NCIM 5671.

AIM: To evaluate the structure and functions of capsular exopolysaccharide (CPS) from Bifidobacterium breve NCIM 5671. METHODS AND RESULTS: A CPS produced by the probiotic bacteria B. breve NCIM 5671 was isolated and subjected to characterization through GC analysis, which indicated the presence of rhamnose, fucose, galactose, and glucose in a molar ratio of 3:1:5:3. The average molecular weight of the CPS was determined to be ∼8.5 × 105 Da. Further, NMR analysis revealed the probable CPS structure to be composed of major branched tetra- and penta-saccharide units alternately repeating and having both α- and ß-configuration sugar residues. CPS displayed an encouraging prebiotic score for some of the studied probiotic bacteria. Compared to standard inulin, CPS showed better resistance to digestibility against human GI tract in vitro. DPPH, total antioxidant, and ferric reducing assays carried out for CPS displayed decent antioxidant activity too. CONCLUSION: This study indicates that the CPS from B. breve NCIM 5671 has the potential to be utilized as a prebiotic food supplement. It is a high-molecular-weight (∼8.5 × 105 Da) capsular heteropolysaccharide containing rhamnose, fucose, galactose, and glucose.

Rotenone mediated developmental toxicity in Drosophila melanogaster.

Rotenone (ROT) is a widely used natural pesticide, and its effect on growth and developmental toxicity remain unclear. In the present study, the effects of ROT exposure on the reproductive structure and function of the female Drosophila melanogaster and third instar larvae were investigated. ROT exposure on female Drosophila melanogaster resulted in developmental inhibition and ovarian abnormality, which were evident from the disruptive growth of border cells as well as morphological changes in the orientation of nurse cells during the 9th-10th stage of developing egg chamber of in the Drosophila ovary. Other abnormalities, such as, altered developmental gene expression (Osk, Grk, Nos, Bic-d), inhibition in the kinesin motor protein level (KIF-5B), increased caspases activities (Caspase 3, 8, & 9) and apoptosis were also observed. Subsequently, ROT treated larvae exhibited behavioral deficits and delay in developmental time. The above findings demonstrate that the exposure of ROT causes developmental toxicity in Drosophila melanogaster.

Self-assembled chitosan derived microparticles inhibit tumor angiogenesis and induce apoptosis in Ehrlich-ascites-tumor bearing mice.

Self-assembled microparticles from chitosan (SAMC) was prepared by depolymerization induced by potassium persulfate. Particle size distribution data showed averaged around 5 µm size and SEM indicated the sequential formation of "RBC" shaped particles. Soluble SAMC consists of 'deacetylated' residues as revealed by 13C NMR. SAMC showed antitumor efficacy in human breast cancer cell lines through mitigation in cell proliferation, colony formation and cell migration. Anti-tumor and anti-angiogenic properties of SAMC was found in vivo Ehrlich ascites tumor (EAT) bearing mice model resulting in tumor growth inhibition (EAT control, 17.4 ml; SAMC treated, 6.8 ml) and improved survival potency (15 days). Moreover, the decrease in ascites VEGF secretion (EAT control, 1354 ng; SAMC treated, 351 ng) accompanied with reduction in neovessel formation. Apoptosis induction by SAMC was confirmed by DNA fragmentation, caspase activities and fluorescence staining methods respectively. SAMC may be a safe candidate for anti-tumor dietary supplement production in food industry.

Physicochemical Composition and Characterization of Bioactive Compounds of Mulberry (Morus indica L.) Fruit During Ontogeny.

Mulberry fruit is well recognized as one of the richest sources of bioactive compounds. We investigated the physicochemical composition and characterized the bioactive compounds during different ripening stages of mulberry (Morus indica) fruit and evaluated their anti-quorum sensing activity on Chromobacterium violaceum. The proximate components such as carbohydrates, proteins and lipids were found to be high in the ripe fruit compared to unripe and mid-ripe fruit. The ripe fruit contained higher content of total phenolics and flavonoids (336.05 and 282.55 mg/100 g fresh weight (FW), respectively). Epicatechin and resveratrol were the major polyphenols detected in the fruit with the range 5.13-19.46 and 4.07-14.45 mg/100 g FW, respectively. Chlorogenic acid and myricetin were predominant in the unripe and mid-ripe fruit (7.14 and 1.84 mg/100 g FW, respectively). The fruit was found to be an excellent source of anti-diabetic compound 1-deoxynojirimycin. The highest content of 1-deoxynojirimycin was present in the mid-ripe fruit, with a content of 2.91 mg/100 g FW. Furthermore, fruit extracts exhibited anti-quorum sensing activity against Chromobacterium violaceum by effectively inhibiting violacein production. Ripe fruit extracts showed the highest activity of 76.30% at 1 mg/mL and thus, could be used as a potent anti-quorum sensing agent. The results could be promising in the selection of appropriate developmental stages for M. indica fruit commercial exploitation in the food formulations rich in potential health components.

GH36 α-galactosidase from Lactobacillus plantarum WCFS1 synthesize Gal-α-1,6 linked prebiotic α-galactooligosaccharide by transglycosylation.

α-Galactosidases are potent industrial glycoside hydrolases which are relatively less explored for their transglycosylation potential, especially from Lactobacillus genera. A GH36 α-galactosidase from Lactobacillus plantarum WCFS1 was cloned and over expressed in Hi-control Escherichia coli BL21(DE3). Ni-NTA affinity gel chromatography resulted in purified α-galactosidase (LpαG; specific activity 3077.35 U mg-1) having a monomeric weight of ~80 kDa with 29.3% yield. Size exclusion chromatography of LpαG showed native molecular mass of ~240.5 kDa. LpαG displayed optimum activity at pH 6 and 37 °C. The Km,Vmax and kcat/Km of LpαG towards pNPαGal were found to be 0.93 mM and 714.3 µmol ml-1 min-1 and 12,075 s-1 mM-1, respectively. LpαG displayed maximum transglycosylation activity towards melibiose substrate (as both donor and acceptor) and synthesized majorly a trisaccharide with 0.26 mg ml-1 yield. Nuclear magnetic resonance (NMR) characterization revealed that trisaccharide consist of only single species of α-linked galactooligosaccharide (manninotriose; α-d-Galp-(1 â†’ 6)-α-d-Galp-(1 â†’ 6)-d-Glcp) with α-(1 â†’ 6) regioselectivity. Manninotriose displayed prebiotic property by supporting the growth of probiotic L. plantarum WCFS1 and Bifidobacteria adolescentis DSM 20083.

Structural diversity and prebiotic potential of short chain ß-manno-oligosaccharides generated from guar gum by endo-ß-mannanase (ManB-1601).

Size exclusion chromatography of short chain ß-manno-oligosaccharides (GG-ß-MOS) produced after endo-mannanase (ManB-1601) hydrolysis of guar gum resulted in seven (P1-P7) peaks. Electron spray ionization mass-spectrometry (ESI-MS) revealed P3, P4, P5 and P6 peaks as pentasaccharide (DP5), tetrasaccharide (DP4), trisaccharide (DP3) and disaccharide (DP2), respectively. DP2 and DP3 GG-ß-MOS were structurally characterized by NMR (1H and 13C), FTIR and XRD. DP2 GG-ß-MOS was composed of two species (A) mannopyranose ß-1,4 mannopyranose and (B) α-1,6-galactosyl-mannopyranose while, DP3 oligosaccharide showed presence of three species i.e. (A) α-d-galactosyl-ß-d-mannobiose (galactosyl residue at reducing end), (B) α-d-galactosyl-ß-d-mannobiose (galactosyl residue at non-reducing end) and (C) mannopyranose ß-1,4 mannose ß-1,4 mannopyranose. In batch fermentation, DP2 GG-ß-MOS was preferred over DP3 by all Lactobacillus sp. except Lactobacillus casei var rhamnosus. DP2/DP3 and GG-ß-MOS mixture inhibited the growth of enteropathogens in monoculture and co-culture fermentations, respectively. Fermentation of GG-ß-MOS mixture by Lactobacillus sp. produced short chain fatty acids.

In Vitro Antidiabetic Effects of Isolated Triterpene Glycoside Fraction from Gymnema sylvestre.

A triterpene glycoside (TG) fraction isolated and purified from ethanolic extract of Gymnema sylvestre (EEGS) was investigated for blood glucose control benefit using in vitro methods. The HPLC purified active fraction TG was characterized using FTIR, LC-MS, and NMR. The purified fraction (TG) exhibited effective inhibition of yeast α-glucosidase, sucrase, maltase, and pancreatic α-amylase with IC50 values 3.16 ± 0.05 µg/mL, 74.07 ± 0.51, 5.69 ± 0.02, and 1.17 ± 0.24 µg/mL, respectively, compared to control. TG was characterized to be a mixture of triterpene glycosides: gymnemic acids I, IV, and VII and gymnemagenin. In vitro studies were performed using mouse pancreatic ß-cell lines (MIN6). TG did not exhibit any toxic effects on ß-cell viability and showed protection against H2O2 induced ROS generation. There was up to 1.34-fold increase in glucose stimulated insulin secretion (p<0.05) in a dose-dependent manner relative to standard antidiabetic drug glibenclamide. Also, there was further one-fold enhancement in the expression of GLUT2 compared to commercial standard DAG (deacylgymnemic acid). Thus, the present study highlights the effective isolation and therapeutic potential of TG, making it a functional food ingredient and a safe nutraceutical candidate for management of diabetes.

Structural Characterization and in Vitro Fermentation of ß-Mannooligosaccharides Produced from Locust Bean Gum by GH-26 endo-ß-1,4-Mannanase (ManB-1601).

Size exclusion chromatography of ß-mannooligosaccharides (ß-MOS) mixtures, obtained from ManB-1601 hydrolysis of locust bean gum, resulted in separation of oligosaccharides with various degrees of polymerization (DP 2, 3, and 5). The oligosaccharides were structurally [ESI-MS, FTIR, XRD, TGA, and NMR (1H and 13C)] and functionally (in vitro fermentation) characterized. DP2 oligosaccharide was composed of two species, (A) mannopyranose ß-1,4 mannopyranose and (B) α-1,6-galactosyl-mannopyranose, while DP3 oligosaccharide showed the presence of only one species, i.e. α-d-galactosyl-ß-d-mannobiose. ManB-1601 was capable of cleaving near the branch points in the substrate, resulting in oligosaccharides with galactose at the terminal position apart from attacking unsubstituted ß-1,4-glycosidic linkages. DP2 and DP3 improved the growth of three out of seven species of Lactobacillus while DP5 resulted in poor growth of all Lactobacillus spp. under in vitro conditions. DP2, DP3, and DP5 were found to inhibit the growth of Escherichia coli, Listeria monocytogenes and Salmonella typhi.

Structural analyses and immunomodulatory properties of fructo-oligosaccharides from onion (Allium cepa).

Onion (Allium cepa) is an immune-boosting food rich in fructans. The major aim of this study is to characterize and investigate the immunomodulatory properties of onion fructo-oligosaccharides (FOS). FOS was isolated from onion bulbs by hot 80% ethanol extraction (yield: ∼4.5 g/100 g fw) followed by gel permeation chromatography. NMR of onion FOS revealed unusual ß-D-Glc terminal residue at the non-reducing end. TLC and ESI-MS analyses showed that onion FOS ranged from trisaccharides to hexasaccharides. Onion FOS (50 µg/mL) significantly increased (∼3-fold) the proliferation of mouse splenocytes/thymocytes vs. control. Further, onion FOS enhanced (∼2.5-fold) the production of nitric oxide by peritoneal exudates cells (PECs) from Wistar rats; intracellular free radicals production and phagocytic activity of isolated murine PECs were also augmented. Our structural and in vitro results indicate that onion FOS comprising of tri- to hexasaccharide units belongs to inulin-type fructans, and possess immunostimulatory activities towards murine lymphocytes and macrophages.

alpha-Lipoic acid ameliorates altered colonic contractility and intestinal transit in STZ-diabetic rats.

alpha-Lipoic acid treatment (100 mg/kg/day for 2 weeks after 6 weeks of untreated diabetes) of streptozotocin diabetic rats partially but significantly reversed both reduced contractile response of distal colon to acetylcholine and delayed transit of charcoal meal in small intestine compared to diabetic control. These effects of alpha-Lipoic acid were associated with complete reversal of diabetes induced increased plasma lipid peroxidation level. alpha-Lipoic acid had no effect on any of the parameters measured in non-diabetic rats. These findings demonstrate contribution of oxidative stress in the development of physiological changes of gut in diabetes.