Nutritional Composition and Pharmacological Activity of Musa balbisiana Colla Seed: An Insight into Phytochemical and Cellular Bioenergetic Profiling.

Musa balbisiana Colla belongs to the family Musaceae which is well-known for its nutritional and pharmacological properties. Here, we have analysed the phytochemical content and evaluated the nutritional, antioxidant, anti-glycation, α-amylase, and α-glucosidase inhibition potential. Moreover, for the first time, we have studied the bioenergetic profiles of the bioactive fractions of M. balbisiana seeds extract against oxidative stress-related mitochondrial and cellular dysfunction using XFe24 extracellular flux analyzer. M. balbisiana seeds have high nutritional values with significant levels of carbohydrates, starch, protein, and minerals (Ca, Na, Mg, Cu, Fe, and Zn). Bioactivity-guided fractionation of the methanolic extract of M. balbisiana seeds revealed that the ethyl acetate fraction (EAF) showed the highest antioxidant, anti-glycation, and phytochemical content as compared to other fractions. Moreover, the EAF showed a lower α-amylase inhibition and a higher α-glucosidase inhibitory activity. Most importantly, our GC-MS analyses of EAF revealed the presence of unique and previously unreported 14 phytochemical compounds. A strong correlation between the biological activities and total phenolic/tannin content was observed. In addition, the bioactive fraction of M. balbisiana seeds (EAF) improved the bioenergetic profiles of free fatty acid-induced oxidative stress with a concomitant increase in ATP production, and respiratory and glycolytic capacity. Altogether, our findings suggest that M. balbisiana seeds can be used as a natural supplement to boost antioxidant levels and combat oxidative stress and non-enzymatic glycation.

Ultrasound-Assisted Extraction of Verbascoside from Clerodendrum glandulosum Leaves for Analysis of Antioxidant and Antidiabetic Activities.

Verbascoside (VER) is a phenylethanoid glycoside compound found in Clerodendrum species and is an important part of traditional medicine. It is found in the leaves of Clerodendrum glandulosum, which is taken as a soup or vegetable and also utilized in traditional medicine by the people of Northeast India, especially against hypertension and diabetes. In the present study, VER was extracted from C. glandulosum leaves using ultrasound-assisted extraction through the solvent extraction method (ethanol-water, ethanol, and water). The ethanol extract had the highest phenolic and flavonoid contents, viz., 110.55 mg GAE/g and 87.60 mg QE/g, respectively. HPLC and LC-MS were used to identify the active phenolic compound, and VER was found to be the main component present in the extraction with a molecular weight of 624.59 g/mol. NMR (1H, 2D-COSY) analysis showed the presence of hydroxytyrosol, caffeic acid, glucose, and rhamnose in the VER backbone. Further, different antioxidant activities and antidiabetic and antihyperlipidemia enzyme markers' inhibition against VER-enriched ethanol extract were evaluated. The results showed that ultrasound extraction of polyphenols using ethanol from C. glandulosum could be a promising technique for the extraction of bioactive compounds.

Molecular insight into Aspergillus oryzae ß-mannanase interacting with mannotriose revealed by molecular dynamic simulation study.

Fungal ß-mannanases hydrolyze ß-1, 4-glycosidic bonds of mannans and find application in the generation of mannose and prebiotic mannooligosaccharides (MOS). Previously, a MOS generating ß-mannanase from Aspergillus oryzae MTCC 1846 (ßManAo) was characterized and its structural and functional properties were unraveled through homology modeling and molecular dynamics in this study. The ßManAo model was validated with 92.9% and 6.5% of the residues found to be distributed in the most favorable and allowed regions of the Ramachandran plot. Glu244 was found to play a key role in the interaction with mannotriose, indicating conserved amino acids for the catalytic reaction. A detailed metadynamic analysis of the principal components revealed the presence of an α8-helix in the C-terminus which was very flexible in nature and energy landscapes suggested high conformation sub-states and the complex dynamic behavior of the protein. The binding of the M3 substrate stabilized the ß-mannanase and resulted in a reduction in the intermediate conformational sub-states evident from the free energy landscapes. The active site of the ß-mannanase is mostly hydrophilic in nature which is accordance with our results, where the major contribution in the binding energy of the substrate with the active site is from electrostatic interactions. Define Secondary Structure of Proteins (DSSP) analysis revealed a major transition of the protein from helix to ß-turn for binding with the mannotriose. The molecular dynamics of the ßManAo-mannotriose model, and the role and interactions of catalytic residues with ligand were also described. The substrate binding pocket of ßManAo was found to be highly dynamic and showed large, concerted movements. The outcomes of the present study can be exploited in further understanding the structural properties and functional dynamics of ßManAo.

Heterologous expression and molecular modelling of L-asparaginase from Bacillus subtilis ETMC-2.

Bacterial L-asparaginase is the key therapeutic enzyme in cancer therapy and is also witnessing demand as a food processing aid. In this study, L-asparaginase of newly isolated Bacillus subtilis ETMC-2 was cloned and over-expressed in Escherichia coli as an active soluble protein using ligation independent cloning strategy. The molecular mass was estimated to be 40 kDa and was optimally active at 50 °C. Zymography revealed that the enzyme was active in homo-tetramer state (~160 KDa). The encoded protein after BLASTp analysis on NCBI showed 99.73% similarity with L-ASNase that of Bacillus sp. Physico-chemical properties were predicted using Protparam leading to categorization of the enzyme as a stable protein with an instability index (II) of 19.02. The calculated aliphatic index (85.44) indicated the high thermal stability of the protein with GRAVY value of -0.317. Protein-Ligand docking revealed that the residues Thr89, Thr121, and Asp122 were fundamental in protein-ligand complexation. After homology modelling, model validation was performed using Ramachandran plot, VERIFY3D, and RMSD. The paper describes cloning, heterologous expression, catalytic characteristics and physico-chemical properties of the type II B. subtilis L-ASNase.

Hemicellulose-Derived Oligosaccharides: Emerging Prebiotics in Disease Alleviation.

The gut microbiota in the human body is an important component that plays a pivotal role in the ability of the host to prevent diseases and recover from these diseases. If the human microbiome changes for any reason, it affects the overall functioning of the host. Healthy and vigorous gut microbiota require dietary fiber supplementation. Recently, oligosaccharides have been found to play a significant role in the modulation of microbiota. Several such oligosaccharides, i.e., xylooligosaccharides (XOS), mannooligosaccharides (MOS), and arabino-xylooligosaccharides (AXOS), are derived from hemicellulosic macromolecules such as xylan, mannan, and arabino-xylan, respectively. These oligosaccharides serve as substrates for the probiotic production of health-promoting substances (short-chain fatty acids, branched chain amino acids etc.), which confer a variety of health benefits, including the prevention of some dreaded diseases. Among hemicellulose-derived oligosaccharides (HDOs), XOS have been largely explored, whereas, studies on MOS and AXOS are currently underway. HDOs, upon ingestion, help reduce morbidities by lowering populations of harmful or pathogenic bacteria. The ATP-binding cassette (ABC) transporters are mainly utilized for the uptake of oligosaccharides in probiotics. Butyrate generated by the selective fermentation of oligosaccharides, along with other short-chain fatty acids, reduces gut inflammation. Overall, oligosaccharides derived from hemicelluloses show a similar potential as conventional prebiotics and can be supplemented as functional foods. This review summarizes the role of HDOs in the alleviation of autoimmune diseases (inflammatory bowel disease, Crohn's disease), diabetes, urinary tract infection, cardiovascular diseases, and antimicrobial resistance (AMR) through the modulation of the gut microbiota. The mechanism of oligosaccharide utilization and disease mitigation is also explained.

Prebiotic mannooligosaccharides: Synthesis, characterization and bioactive properties.

Functional oligosaccharides are non-digestible food ingredients that confer numerous health benefits. Among these, mannooligosaccharides (MOS) are emerging prebiotics that have characteristic potential bio-active properties. Microbial mannanases can be used to break down mannan rich agro-residues to yield MOS. Various applications of MOS as health promoting functional food ingredient may open up newer opportunities in food and feed industry. Enzymatic hydrolysis is the widely preferred method over chemical hydrolysis for MOS production. Presently, commercial MOS is being derived from yeast cell wall mannan and is widely used as prebiotic in feed supplements for poultry and aquaculture. Apart from stimulating the growth of probiotic microflora, MOS impart anticancer and immunomodulatory effects by inducing different gene markers in colon cells. This review summarizes recent developments and future prospects of enzymatic synthesis of MOS from various mannans, their structural characteristics and their potential health benefits.

Characteristics and bioactive properties of mannooligosaccharides derived from agro-waste mannans.

Mannooligosaccharides (MOS) were derived using Aspergillus oryzae ß-mannanase (ManAo) from different mannan-rich agro-wastes, palm kernel cake (PKC), guar gum and copra meal (CM). Guar gum (GG) released higher amount of MOS (56.31% w/w) from which purification of mannobiose (0.68 mg) and mannotriose (1.26 mg) was demonstrated using size-exclusion chromatography. FTIR analysis of mannan hydrolysates showed characteristic peaks in 1200-900 cm-1 region indicating the presence of MOS. 1H &13C NMR spectra showed presence of anomeric sugar forms of MOS in different mannan hydrolysates. MOS from locust bean gum and guar gum had both α- and ß-anomers while PKC and CM had only α-anomer. Growth promotional activities of different MOS were demonstrated using two probiotic Lactobacilli. Besides, enzymatically derived MOS also showed metal (Fe2+) chelating and anti-oxidant activities, wherein best anti-glycating agent was evaluated as MOS from PKC. PKC derived MOS showed highest cytotoxicity (74.19%) against human colon adenocarcinoma cell line (Caco-2). This study demonstrated the prebiotic potential of agro-waste derived MOS and possibility of their utilization as a functional food ingredient.

Immobilization of Aspergillus quadrilineatus RSNK-1 multi-enzymatic system for fruit juice treatment and mannooligosaccharide generation.

Aspergillus quadrilineatus RSNK-1 produced a multi-enzymatic system containing (U/gds) ß-mannanase (1021), endo-xylanase (1 9 1), α-galactosidase (3.42), ß-xylosidase (0.07) and ß-glucosidase (0.28) on low-cost copra meal (CM) in SSF. The enzyme preparation was covalently immobilized on aluminum oxide pellets (3 mm) under statistically optimized conditions leading to 73.17% immobilization yield. The immobilized enzyme (Man-AOP) displayed enhanced thermal and pH stability. Man-AOP was characterized by FTIR, SEM and PXRD revealing a covalent interaction. The bio-conjugate was successfully recycled for mannooligosaccharide (MOS) generation from locust bean gum (LBG) up to 10 cycles, yielding an average of 0.95 mg MOS/cycle. Man-AOP was also effective in clarification of apple, kiwi, orange and peach juices and enhanced their reducing sugar content. The bio-conjugate was useful in generation of MOS from mannan and enrichment of fruit juices.

Production optimization and characterization of mannooligosaccharide generating ß-mannanase from Aspergillus oryzae.

A multi-tolerant ß-mannanase (ManAo) was produced by Aspergillus oryzae on copra meal, a low-cost agro waste. Under statistically optimized conditions, 4.3-fold increase in ß-mannanase production (434 U/gds) was obtained. Purified ManAo had MW ∼34 kDa and specific activity of 335.85 U/mg with optimum activity at 60 °C and at pH 5.0. Activity of ManAo was enhanced by most metal ions and modulators while maximum enhancement was noticed with Ag+ and Triton X-100. Km and Vmax were 2.7 mg/mL and 1388.8 µmol/min/mg for locust bean gum while the enzyme showed lower affinity towards konjac gum (8.8 mg/mL, 555.5 µmol/min/mg). Evaluation of various thermodynamic parameters indicated high-efficiency of the ManAo with activation energy 12.42 KJ/mol and 23.31 KJ/mol towards LBG and konjac gum, respectively. End product analysis of ß-mannanase action by fluorescence assisted carbohydrate electrophoresis (FACE) revealed the generation of sugars from DP 1-4 with some higher DP MOS from different mannans.