Extraction of chitin-glucan complex from shiitake (Lentinula edodes) fruiting bodies using natural deep eutectic solvents and its prebiotic potential.

Chitin-glucan complex (CGC) is an emerging novel prebiotic with numerous physiological activities in amelioration of clinical manifestations. In the present work, natural deep eutectic solvent (NADES), ultrasonication, and submerged fermentation using probiotic microorganisms were deployed for the extraction of CGC from Shiitake fruiting bodies. CGC obtained through non-ultrasonication assisted fermentation employing Lactiplantibacillus plantarum exhibited maximum polysaccharide yield (27.86 ± 0.82 % w/w). However, based on antioxidant potential, NADES combination of urea: glycerol (1:1 M ratio) was selected for further characterization. The rheological behavior of CGC under optimized conditions showed shear thinning property in both 0.1 M NaCl and salt-free solution. FTIR, 1H-(1D), and 2D 1H1H Homonuclear NMR spectra displayed distinctive patterns associated with ß-glycosidic linkage and ß-d-glucopyranose sugar moiety. XRD profiles of CGC exhibited characteristic peaks at 2θ = 23°, 25°, and 28° with corresponding hkl values of (220), (101), and (130) lattice planes, respectively. Enhanced radical scavenging activities were noticed due to the triple helical structure and anionic nature of CGC. CGC exhibited potential prebiotic activity (prebiotic score 118-134 %) and short chain fatty acids liberation (maximum 9.99 ± 0.41 mM by Lactobacillus delbrueckii). Simulated static in-vitro digestion demonstrated that CGC withstands acidic environment of gastric phase, which indicated its suitability for use as a prebiotic in nutraceutical-enriched food products.

Evaluation of Prebiotic Properties of Galactooligosaccharides Produced by Transgalactosylation Using Partially Purified ß-Galactosidase from Enterobacter aerogenes KCTC2190.

Transgalactosylation reaction is the penultimate step in the production of galactooligosaccharides (GOSs) which has prominent applications in the treatment of disorders. In the present study, partially purified ß-galactosidase from Enterobacter aerogenes KCTC2190 was used for the synthesis of prebiotic GOSs. GOSs were produced using lactose as substrate. Structural elucidation of collected fractions of GOSs by liquid chromatography electrospray ionization mass spectrometry exhibited the appearance of major peaks of produced GOSs at m/z 241.20, 481.39, 365.11, 527.17, and 701.51 respectively. GOSs facilitated the growth of potential probiotic strains (Lactobacillus delbrueckii ssp. helveticus, Bifidobacterium bifidum, and Lactiplantibacillus plantarum) and liberated propionate and butyrate as principal short-chain fatty acids which established its prebiotic potency. Synbiotic combinations exhibited good antioxidant activities. Synbiotic combinations also exhibited antimicrobial activities against pathogenic microorganisms namely Staphylococcus aureus and Escherichia coli. Synbiotic combinations of GOSs and the respective probiotic microorganisms were able to decrease viable human bone cancer cells (MG-63).

Studies on production and evaluation of biopigment and synthetic dye decolorization capacity of laccase produced by A. oryzae cultivated on agro-waste.

The present study investigated the screening of mono and co-culture fungal cultivations for laccase production using extracted lignin as the substrate obtained from cauliflower wastes by two different pretreatment methods. Amongst mono and mixed culture fungal cultivations, monoculture of Aspergillus oryzae exhibited the highest enzymatic activity of 29.7 ± 0.6 U mL-1 under submerged conditions and using alkali extracted lignin as substrate. Under the optimal conditions (pH 4.5, 30 °C, 12 days, 1% (w/v) lignin and 0.5 mM Cu2+ concentration) the maximum laccase activity was estimated to be 41.3 ± 2.8 U mL-1 and production yield of 153.3 ± 2.4 mg L-1. Maximum decolorization of pigment extracted from Aspergillus heteromorphus CBS 117.55 cultivated culture media was achieved by administration of 40 U g-1 of crude enzyme concentration. Thermal and pH stability of crude laccase was observed over wide ranges. The dye decolorization efficiency of crude A. oryzae laccase was studied and Congo Red exhibited maximum decolorization percentage (64 ± 1.3%) at 15 µM, 50 °C and pH 4.5. The kinetic study of different dye (Congo Red) concentrations obtained Vmax and Km values of 0.123 × 10-3 M and 0.724 mol L-1 min-1, respectively.

Phytochemical screening and antioxidant and antimicrobial activities of crude extracts of different filamentous fungi.

Five filamentous fungal strains that grew in different whey-based media under submerged fermentation were investigated for antioxidant properties and phytochemicals. Phytochemical analysis revealed the presence of alkaloids, tannin, flavonoids, glycosides, phenols, saponins, and terpenes in the crude intra- and extracellular ethyl acetate extracts of different strains. All fungal extracts exhibited effective antioxidant activities in terms of TPC, TFC, DPPH, FRAP, ABTS, reducing power, and metal chelating capacity. The activities of intracellular extracts were higher than the extracellular metabolites. Fermentation media with sugar and salt supplementation significantly influenced antioxidant production. Aspergillus niger in glucose-supplemented whey medium was found to exhibit the highest antioxidant properties. The antimicrobial activity of A. niger and Penicillium expansum extracts by microtiter plate assay showed a promising result against some pathogenic bacterial strains. Chromatographic analysis of the fungal extracts revealed the presence of chlorogenic acid, trans-cinnamic acid, ferulic acid quercetin, myricetin, kaempferol, and catechin which are known for their antioxidant properties. Accumulation of nutrients in fungal biomass under constraint environment produces secondary metabolites which has demonstrated efficacy towards alleviation of several degenerative diseases. The antioxidative enriched phytochemicals present in these five different fungal strains will provide a breakthrough in the utilisation of whey as inexpensive source of substrate for the growth of these fungi. Moreover, phytochemicals could be utilized as therapeutic agents in a cost-effective and environmentally friendly manner.

Evaluation of nutraceutical application of xylooligosaccharide enzymatically produced from cauliflower stalk for its value addition through a sustainable approach.

There is increasing attention on the exploration of waste feedstocks as economically viable substrates for the production of prebiotic oligosaccharides, especially xylooligosaccharides, as excellent candidates for the maintenance and promotion of gut microbiota. XOS, an emerging prebiotic that has several functional attributes and beneficial health effects, is mainly produced by different processes, especially enzymatic hydrolysis through the valorisation of xylan enriched lignocellulosic materials. The present study deals with the enzymatic production of xylooligosaccharide (XOS) from xylan rich cauliflower stalk, a novel source. Delignification with alkali (NaOH) was found to be more efficient than acid and autohydrolysis, resulting in a higher extraction yield of xylan (18.42%). Alkaline extraction for 120 minutes at 1.25 M alkali concentration produced maximum xylan yield. FTIR analysis of xylan extracted from cauliflower stalk by an alkaline (NaOH) pretreatment method showed typical absorption bands at 1729 cm-1 that correspond to acetyl groups exhibiting the typical xylan specific band. Enzymatic hydrolysis was carried out with indigenously produced crude endoxylanase obtained from Aspergillus niger MTCC 9687 and the effects of substrate concentration, enzyme concentration, pH, time and temperature were investigated. High resolution MS analysis showed the presence of xylobiose as the major XOS. The major 1H spectral signals of XOS liberated from enzymatically hydrolysed alkali extracted cauliflower stalk xylan showed the presence of ß-anomeric protons in the spectral region of 4.0-4.7 ppm. Prebiotic efficacy of cauliflower stalk derived XOS alone and synbiotic combinations with known probiotic strains (Lactiplantibacillus plantarum, Bifidobacterium bifidum, Lactobacillus delbrueckii ssp. Helveticus) were evaluated. Butyrate was found to be the major short chain fatty acid produced by XOS supplemented fermentation media. All the synbiotic combinations showed significantly higher antioxidant and antimicrobial activities and reduced the viability of human bone cancer MG-63 cells. The individual profiles of antimicrobial components of XOS were identified as dihydroxy benzoic acid and aspartic acid by HPLC coupled to a photodiode array detector.

Effect of pretreatment with organic solvent on enzymatic digestibility of cauliflower wastes.

The present study investigated the operational conditions for different pretreatment approaches and subsequent enzymatic hydrolysis of cauliflower wastes (stalk and leaf) for better release of fermentable sugars. The structural analysis of raw and pretreated lignocellulosic biomasses was investigated using scanning electron microscopy (SEM), X-ray diffraction (XRD), and Fourier transforms infrared (FTIR) analysis. Results demonstrated that the highest cellulose conversion rate and removal of most of the hemicellulose and lignin were obtained with organosolvent pretreatment. Using methanol in presence of sodium (Na) acetate was most effective in delignification of cauliflower wastes. In the present study, methanol (100% v/v) in presence of 0.1 M Na-acetate at 121 °C for 45 and 60 min for stalk and leaf, respectively, gave maximum reducing sugar yield. Response surface methodology was used to optimize different process parameters for enzymatic saccharification using microbial cellulase and xylanase. The optimum operation condition of enzymatic hydrolysis of organosolvent pretreated cauliflower wastes were substrate loading (2.5% w/v for both stalk and leaf), enzyme loading (15 and 10 U/g for stalk and leaf, respectively), pH (4.46 and 5.48 for stalk and leaf, respectively), at 60 °C and for 180 min.