Phytochemical screening and antioxidative property evaluation of lipid-producing fungi.

The increased demands for natural bioactive compounds have induced the search for unusual sources. Microorganisms, especially fungi are a potent source of secondary metabolites, which could act mainly as antioxidant compounds to prevent oxidative stress. In the present study three soil-isolated fungi Aspergillus niger, Aspergillus heteromorphus and Aspergillus fumigatus, were screened for their oleaginous property as well as their potential for the production of bioactive compounds. Fungal biomasses were freeze dried and extracted with methanol using a cold percolation process for the production of intracellular metabolites and the fungal culture media after fermentation were examined for extracellular metabolites. Intracellular and extracellular extracts of the isolated fungi along with the single-cell oils extracted from those fungi were screened for phytochemicals, which showed the presence of alkaloids, flavonoides, glycosides, phenols, saponins and terpenoids. All strains showed potent antioxidant activity, determined using 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid (ABTS) and ferric reducing antioxidant power (FRAP) activity. Extracellular extract and single-cell oil of A. heteromorphus showed the highest antioxidant activity with maximum ABTS radical scavenging activity and reducing potential. Highest content of phenolic and flavonoid compounds within the isolated fungi was found to be 37.58 mg gallic acid equivalent (GAE)/g and 62.07 mg catechine equivalent (CE)/g, respectively. Chromatographic analysis of the intracellular and extracellular extracts of the fungi showed the presence of gallic acid, di-hydroxy benzoic acid, ferulic acid, quercetin, epigerin, kampferol, trans cinnamic acid, chlorogenic acid and rutin, which made them biologically important and beneficial for human health.

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.

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.

Emulsions stabilized by soy protein nanoparticles as potential functional non-dairy yogurts.

BACKGROUND: Rice bran oil and soy protein nanoparticles (SPNs) may be considered as novel functional food ingredients for soy yogurt production. Formulation of soy yogurt with SPNs and rice bran oil, which has significant physiological functions, will convert them into functional food products. This study was conducted to develop rice bran oil-based soy protein nanoparticles emulsion (SPNE) and to evaluate physical properties, antioxidant activities, oxidative stability and microbiological load as well as textural attributes of SPNs incorporated yogurt (SPNY) during storage at 4 °C for 45 days. RESULTS: SPNs were prepared from soy protein isolate of defatted soy flour. Solubilization, crystallization and ultrasonication was carried out six times. After the sixth cycle of repeated solubilizing, crystallization and ultrasonication, the size of nano protein was reduced to 72.42 ± 0.22 nm from 586.72 ± 0.75 nm (after first cycle). Viscosity, penetration values and water-holding capacity of SPNs added to yogurt were decreased with increase in reduction size of SPNs. SPNs added to yogurt exhibited greater antiradical scavenging ability and ferric reducing antioxidant property than control yogurt. Fortified soy yogurt had significant higher oxidative stability and proteolytic activity. CONCLUSION: Fortification of non-dairy food products with SPNs, which has significant physiological functions, convert conventional soy yogurt into functional food products. © 2019 Society of Chemical Industry.

Bioconversion of Jackfruit Seed Waste to Fungal Biomass Protein by Submerged Fermentation.

The bioconversion of jackfruit seed into a valuable product like Single-Cell Protein (SCP) was carried out using Generally Recognized As Safe (GRAS), five filamentous fungi such as Penicillium expansum, Aspergillus niger, Aspergillus oryzae, Rhizopus microsporus, and Candida intermedia by submerged fermentation. Data obtained from the study showed that supplementation of jackfruit seed extracts with inorganic nitrogen sources and glucose as a carbon source enhanced fungal biomass and SCP production. Among the various fungal strains, A. niger gave the highest biomass and protein yield of 4.01 ± 0.07 g/l and 1.82 ± 0.03 g/l, respectively, on the 9th day of fermentation followed by P. expansum (3.65 ± 0.04 g/l and 1.68 ± 0.03 g/l, respectively). C. intermedia growth was not recorded in all the experimental media. The present study revealed that fungal biomass has shown low crude fat, crude fiber, and total genomic DNA content ranged from 1.10 ± 0.20 to 4.95 ± 0.40%. A. niger and P. expansum were the most efficient in the conversion of sugar (55.83 ± 0.3% and 54.71 ± 0.4%, respectively) to yield biomass in sugar supplementation media. P. expansum and A. niger were the most promising fungal strains to produce fungal biomass protein using inexpensive agro-waste materials.

Characterization of a blue-green pigment extracted from Pseudomonas aeruginosa and its application in textile and paper dyeing.

Microorganisms are a promising source of colorants with large economic potential. Owing to better bio-degradability and higher eco-compatibility, microbial pigments propose promising avenues and can thus be a smart substitute for artificial pigments. The present work focused on the screening, isolation, and extraction of a blue-green pigment produced by soil microorganisms. The pigment-producing microorganism was identified as Pseudomonas aeruginosa on the basis of standard biochemical tests and by 16S rRNA sequencing. The purified blue pigment was characterized by high-performance liquid chromatography and gas chromatography-mass spectrometry. The antimicrobial activity of the microbial biocolor (3 × 108 CFU/ml) was studied, and the zone of inhibition was found to be 10 mm, 13 mm, 9 mm, and 7 mm for E. coli, S. aureus, B. subtilis, and S. typhi, respectively. The evaluation of the biocolor as a dye was executed on different types of textiles and paper. The dyed fabrics were checked for washing, rubbing, and light and temperature fastness. Standard fabric properties of the fabrics dyed with the extracted microbial pigment were also assessed. The dyed fabrics were finally subjected to a patch test to check for any kind of allergic or hypersensitivity on human skin. The extracted pigment from Pseudomonas aeruginosa exhibited remarkable dyeing properties, indicating the scope for utilization of the pigment as a colorant on different types of textile and paper materials. The present study highlights the application of a bacterial pigment as a dyeing agent, which may raise its market value and probably replace toxic synthetic dyes due to its nontoxic nature, compatibility with various textiles, and cost-effectiveness.

Effect of Fungal Fermentation on Enhancement of Nutritional Value and Antioxidant Activity of Defatted Oilseed Meals.

Agro-industrial residues contain high nutritive value. Nowadays, various advanced researches have been done for the production of various value-added products, using these wastes as substrates in the fermentation media. Flaxseed, mustard, and rice bran meal, residues of oil industry, were used as substrates for fermentation. Submerged fermentation with soil-isolated fungal species of the genus Aspergillus sp. was done for oil production by using these substrates in the fermentation media. Effect of fermentation by the oleaginous species of Aspergillus on the nutritive value and functional properties of flaxseed, mustard, and rice bran meal has been discussed for the first time in the present study. After fermentation, the seed meals showed substantial increase in the protein and ash content. The fungal strains utilized the carbohydrate present in the seed meals for the production of highly nutritional metabolites, which decrease the sugar contents of the meals. The fungi also showed extracellular amylase and cellulase activities which helped to hydrolyze the carbohydrates present in these meals, to utilize them for their metabolism. The enhancement was also observed in terms of antioxidant activity of the meals. Increase in the total phenolic and flavonoid contents was observed after fermentation along with radical scavenging activity of 1,1-diphenyl-2-picrylhydrazyl and 2,2-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid reagents and ferric reduction potential. These effects of fermentation modify these cheap waste materials into nutrient dense substrates, which could be further used in the formulation of value-added 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).

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.

Production and immobilization of ß-galactosidase isolated from Enterobacter aerogenes KCTC2190 by entrapment method using agar-agar organic matrix.

In the present study, Enterobacter aerogenes KCTC2190 was isolated from soil around a cattle shed area, which was capable of producing intracellular ß-galactosidase. Partially purified ß-galactosidase was immobilized by entrapment method in agar-agar gel matrix. Agar-agar entrapped beads were prepared by dropping the enzyme-agar solution to ice-cooled toluene-chloroform ((3:1 (v/v)). 45.88±0.11% activity of partially purified ß-galactosidase was retained after immobilization (bead shape). Maximum immobilization yield was observed in the presence of 2.5% agar-agar concentration. After immobilization, optimum temperature required for the enzyme-substrate reaction was shifted from 50 to 60 °C and the optimum reaction time was shifted from 15 to 25 min. The optimum pH for both free and immobilized ß-galactosidase was pH 7. Free enzyme showed lower activation energy in comparison with the immobilized one. For free as well as immobilized ß-galactosidase thermal deactivation, rate constant (kd) increased with increasing temperature while the values of decimal reduction time (D-values) and half-lives (t1/2) decreased. Immobilization process increased the t1/2 and D-values of ß-galactosidase while it decreased the kd. Thermostability of immobilized ß-galactosidase was higher as they showed higher enthalpy (ΔΗ0) and Gibb's free energy (ΔG0)value than those of the free ß-galactosidase. The negative entropy (ΔS0) of free and immobilized ß-galactosidase established that both were in a more ordered state within the temperature range (50 to 70 °C) studied. Immobilized ß-galactosidase was able to retain 51.65±1.61% of its initial activity after 7 batches of enzyme-substrate reaction. Immobilized ß-galactosidase showed 78.09±3.69% of its initial activity even after 40 days of storage at 4 °C.

Hepatoprotective effects of synbiotic soy yogurt on mice fed a high-cholesterol diet.

OBJECTIVES: The role of probiotic yogurt in the prevention and treatment of hypercholesterolemia has attracted global attention. Mounting evidence has indicated that probiotics and prebiotics improve lipid metabolism by lowering low-density lipoprotein cholesterol concentration in plasma of hypercholesterolemic patients. The present study aimed to develop synbiotic soy yogurt that had a greater cholesterol-lowering effect in hypercholesterolemic mice compared with control soy yogurt. METHODS: Synbiotic soy yogurt was prepared using soy milk and synbiotic capsule containing LactoBacil Plus (SCLBP) probiotic cultures and fructo-oligosaccharide. Synbiotic soy yogurt was analyzed for proximate composition and microbiological and antioxidative properties during storage periods of 28 d. To study hypocholesterolemic effect, hypercholesterolemia was induced in mice with administration of 1.25% cholesterol and 0.5% cholic acid for 4 wk. After that 24 male Balb/c mice were randomly divided into four groups and fed basic, high-cholesterol, high-cholesterol with soy yogurt, or high-cholesterol with synbiotic soy yogurt diet for 5 wk. Blood samples were collected to measure lipids concentration and oxidative and antioxidative status. RESULTS: Proximate composition of SCLBP-formulated soy yogurt exhibits a marked difference from control soy yogurt in terms of total solids, moisture, protein, fat, ash, carbohydrate, and energy content. Results indicated that the 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity (75.28%) in synbiotic yogurt containing 2% SCLBP was significantly greater (P ≤ 0.05) compared with control soy yogurt (52.98%). In mice with hypercholesterolemia that were fed synbiotic soy yogurts, the yogurts had a favorable effect in reducing blood cholesterol, triglycerides, and low-density lipoprotein cholesterol and lipid peroxidation in liver. These led to a significant decrease of the atherogenic index compared with soy yogurt (control) only. Treatment with synbiotic soy yogurt cultures ameliorates lipid peroxidation in liver. CONCLUSIONS: These results indicated that the synbiotic soy yogurts have beneficial effects against hypercholesteroemia and can be used as a therapeutic agent in hypercholesteremic patients.

Purification and molecular characterization of a sialic acid specific lectin from the phytopathogenic fungus Macrophomina phaseolina.

A lectin was isolated and purified from the culture filtrate of the plant pathogenic fungus Macrophomina phaseolina by a combination of ammonium sulfate precipitation, affinity chromatography on fetuin-Sepharose 4B and ion-exchange chromatography on DEAE-A 50. The lectin designated MPL was homogeneous by PAGE and HPLC and a monomeric protein with a molecular weight of approximately 34 kDa as demonstrated by SDS-PAGE. It is a glycoprotein and agglutinated human erythrocytes regardless of the human blood type. Neuraminidase treatment of erythrocytes reduced the agglutination activity of the lectin. It is thermally stable and exhibits maximum activity between pH 6 and 7.2. Its carbohydrate binding specificity was investigated both by hapten inhibition of hemagglutination and by enzyme-conjugated lectin inhibition assay. Although, M. phaseolina lectin bound sialic acid, it exhibited binding affinity towards neuraminyl oligosaccharides of N-linked glycoproteins, alpha-Neu5Ac-(2-->3)-beta-Gal-(1-->4)-GlcNAc being maximum.

Adsorption behavior of rhodamine B on Rhizopus oryzae biomass.

The removal of a carcinogenic dye rhodamine B (C. I. 45170) from wastewater by biomass of different moulds and yeasts is described. Among all of the fungal species tested, the biomass of Rhizopus oryzae MTCC 262 is found to be the most effective. Dye adsorption reaches maximum with the biomass harvested from the early stationary phase of growth. The optimum temperature and pH for adsorption are observed to be 40 degrees C and 7.0, respectively. The adsorption rate is very fast initially and attains equilibrium after 5 h. The adsorption isotherm follows the Langmuir isotherm model satisfactorily within the studied dye concentration range. Of the different metabolic inhibitors tested, 2,4-ditrophenol (DNP) and N,N'-dicyclohexylcarbodiimide (DCCD) decrease dye adsorption by approximately 30% suggesting the role of energy metabolism in the process. Spectrophotometric study indicates that the removal of rhodamine B by R. oryzae biomass involves an adsorption process. Scanning (SEM) and transmission (TEM) electron microscopic investigations have been carried out to understand the probable mechanism of the dye-biomass interaction.