Synthesis of octenyl succinic anhydride-modified levan and investigation of its microstructural, physicochemical, and emulsifying properties.

In this study, levan from Bacillus licheniformis NS032 was modified in an aqueous medium by octenyl succinic anhydride (OSA), and the properties of the obtained derivatives were studied. The maximum efficiency in the synthesis reaction was achieved at 40 °C and a polysaccharide slurry concentration of 30 %. Increasing the reagent concentration (2-10 %) led to an increase in the degree of substitution (0.016-0.048). Structures of derivatives were confirmed by FTIR and NMR. Scanning electronic microscopy, thermogravimetry, and dynamic light scattering analyses showed that the derivatives with degrees of substitution of 0.025 and 0.036 retained levan's porous structure and thermostability and showed better colloidal stability than the native polysaccharide. The intrinsic viscosity of derivatives increased upon modification, while the surface tension of the 1 % solution was lowered to 61 mN/m. Oil-in-water emulsions prepared with sunflower oil (10 % and 20 %) by mechanical homogenization and 2 and 10 % derivatives in the continuous phase showed mean oil droplet sizes of 106-195 µm, while the distribution curves exhibited bimodal character. The studied derivatives have a good capacity to stabilize emulsions, as they have a creaming index ranging from 73 % to 94 %. The OSA-modified levans could have potential applications in new formulations of emulsion-based systems.

Cytotoxic activity of the crude polysaccharides/exopolysaccharides of Coprinus comatus and Coprinellus truncorum.

Fungi are an important source of polysaccharides (PSH) and phenolic compounds (PC). Numerous studies have highlighted the beneficial effects of fungal consumption, but the impact of submerged cultivated mycelia (M) and filtrate (F) has not been fully investigated. We aimed to investigate the cytotoxic activity of isolated crude PSH and exopolysaccharides (ePSH) of submerged cultivated M and F of edible Coprinus comatus and Coprinellus truncorum species. Both PSH and ePSH exhibited significant cytotoxic activity towards HepG2 cancer cells of human origin (three-way ANOVA). The C. truncorum PSH/ePSH was more efficient inducing maximal reduction in cell viability (≈50% at 450 µg/mL) after 24 h while C. comatus PSH/ePSH needed 72 h to reach similar effect (≈60% at 450 µg/mL). Partial least square regression (PLSR) analysis indicated that specific phenolic composition of the PSH/ePSH could be responsible for the difference in their activity.

Comparison of Two Schizophyllum commune Strains in Production of Acetylcholinesterase Inhibitors and Antioxidants from Submerged Cultivation.

In recent years, fungi have been recognized as producers of acetylcholinesterase (AChE) inhibitors, agents important for the prevention of Alzheimer's disease (AD). This study aimed to examine the AChE inhibitory, the antioxidative and antibacterial activity of two different Schizophyllum commune strains that originated from Serbia (SRB) and Italy (IT). Submerged cultivation of grown mycelia (M) and fermentation broth (F) of ethanol (EtOH) and polysaccharide (PSH) extracts lasted for 7, 14, 21 and 28 days. For AChE activity Ellman method was performed, while for antioxidative activity, sevendifferent assays were conducted: DPPH, ABTS, FRAP, SOA, OH, NO together with total phenolic content. Antimicrobial screen, LC-MS/MS technique and FTIR measurements were performed. Different isolates exhibited different AChE activity, with PSH being the strongest (SRB, M, 28 days IC90 79.73 ± 26.34 µg/mL), while in EtOH extracts, IT stood out (F, 14 days, IC50 0.8 ± 0.6 µg/mL). PSH extracts (7 days) exhibit significant antioxidative activity (AO), opposite to EtOH extracts where 14 and 21days periods stood out. Only tw extracts showed antibacterial activity. Following LC-MS/MS analysis p-hydroxybenzoic and gallic acids were the most abundant phenolics. PSH extracts demonstrated remarkable results, making this study debut and introducing S. commune as a valuable resource of AChE inhibitors.

Bacillus licheniformis levan as a functional biopolymer in topical drug dosage forms: From basic colloidal considerations to actual pharmaceutical application.

Ongoing demand in sustainable and biocompatible drug dosage forms is reflected in the search for novel pharmaceutical excipients with equal properties. A group of microbial exopolysaccharides offers a variety of biopolymers with many alleged uses and effects. This study aims to assess applicative properties of levan obtained from Bacillus licheniformis NS032, focusing on its potential co-stabilizing and drug release-controlling functions in pertaining emulsion systems. Despite its high molecular weight and partial existence in globular nanometric structures (180-190 nm), levan was successfully incorporated into both tested colloidal systems: those stabilized with synthetic/anionic or natural-origin/non-ionic emulsifiers. In the tested levan concentrations range (0.2-3.0% w/w) the monitored flow and thermal parameters failed to show linear concentration dependence, which prompted us to revisit certain colloidal fundamentals of this biopolymer. Being a part of the external phase of the investigated emulsion systems, levan contributed to formation of a matrix-like environment, offering additional stabilization of the microstructure and rheology modifying properties (hysteresis loop elevation as high as 4167±98 to 20792±3166 Pa•s-1), especially in case of the samples where lamellar liquid crystalline formation occurred. Apart from its good water solubility and considerable conformational flexibility, the investigated homofructan easily saturated the external phase of the samples stabilized with a conventional anionic emulsifier, leading to similar properties of 0.2% and 3.0% levan-containing samples. After closer consideration of thermal and release behavior, this was considered as a favorable property for a novel excipient, offering tailored formulation characteristics even with lower levan concentrations, consequently not compromising the potential cost of the final drug dosage form.

Coprinus comatus filtrate extract, a novel neuroprotective agent of natural origin.

In vitro acetylholinesterase (AChE) inhibitory activity of an autochthonous sample of the mushroom Coprinus comatus (encompassing fruiting body FB, mycelia M and filtrate F from the submerged cultivation) was the subject of this study. C. comatus F extract exhibited rather potent anti-AChE activity (73.0 ± 1.5%) at in liquid conditions, comparable to those of the conventional drug donepezil (80.6 ± 1.4%). Also, the same extract exhibited high anti-AChE activity (1 µg) in solid. While its FTIR spectrum indicated the presence of phenolic compounds, quercetin (28.1 µg g-1 d.w.) was found to affect the observed bioactivity (59.8 ± 0.9%). This is the first report of profound anti-AChE activity of any C. comatus extract, a medicinal mushroom that has been successfully cultivated in P.R. China, due to the demanding needs of food industry.

Microbial levan and pullulan as potential protective agents for reducing adverse effects of copper on Daphnia magna and Vibrio fischeri.

Microbial polysaccharides, due to their unique physiochemical properties, have found application in the food industry, cosmetics, pharmacy and medicine. In the environment, microbes can use polysaccharides to alleviate the adverse effects of heavy metals in their close proximity. This adaptive property shows interesting potential for bioremediation. Herein, the effects of the exopolysaccharides (EPS) levan, produced by the bacterium Bacillus licheniformis NS032 and pullulan, produced by the fungus Aureobasidium pullulans CH-1 in the presence of copper (Cu2+) have been investigated for the first time on antioxidant enzyme activity, respiration and Cu2+ bioaccumulation of Daphnia magna as well as the bioluminescence of Vibrio fischeri. Both EPS decreased toxicity of Cu2+ in the acute test with D. magna. The activity of catalase (CAT) was significantly diminished after acute exposure to Cu2+ in comparison to treatments with Cu2+ and EPS, while in the prolonged acute exposure the CAT activity did not show statistically significant (P ≤ 0.05) differences between treatments with and without the EPS. According to ICP-MS results, during prolonged acute exposure of neonates, the bioaccumulation of Cu2+ in treatments without the EPS was 52.03 µg/g of biomass (wet), while in treatments with EPS, the bioaccumulation was lower by one order of magnitude. The respiration of neonates during acute exposure to Cu2+ with or without the EPS was monitored using the MicroOxymax respirometer, and the results show the EPS can positively effect the respiration. In the case of bacterial bioluminescence, the toxicity of Cu2+ decreased in treatments with EPS (30 min EC10) from 3.54 mg/L to 140.61 mg/L (levan) and 45.00 mg/L (pullulan). This study demonstrates protective effect of EPS against Cu2+ toxicity on D. magna and V. fischeri, and opens the door for further investigation of potential application of levan and pullulan in bioremediation of heavy metals and mitigation of their adverse effects in the aquatic environment.

The polysaccharide extracts from the fungi Coprinus comatus and Coprinellus truncorum do exhibit AChE inhibitory activity.

The polysaccharide (PSH) extracts from the edible mushroom species Coprinus comatus and Coprinellus truncorum were screened in liquid for their acetylcholinesterase inhibitory (AChE) activity. Both extracts were found to display inhibition of the aforementioned enzyme reaching similar IC50 values of 0.62 ± 0.07 and 0.61 ± 0.03 mg/mL, respectively. According to the means of FTIR spectroscopy, these PSH extracts mostly contained ß-glucans. However, the presence of some proteins and polyphenolics as minor ingredients were also detected. Compared with existing literature data for anti-AChE activity of the sugar samples, the findings within this study may be treated as a profound bioactivity. Consequently, this study puts some light on the possible use of the screened macrofungi in the palliative treatment of Alzheimer's disease.

Anti-hydroxyl radical activity, redox potential and anti-AChE activity of Amanita strobiliformis polysaccharide extract.

This study outlines antioxidant and anti-AChE activities of the polysaccharide (PSH) extract from the mushroom species Amanita strobiliformis. Both the presence of α and ß glucans within the aforementioned extract was recorded. PSH extract displayed a profound scavenging activity of OH radicals (IC50 value, 11.86 ± 0.59 µg/mL) and high potential for reduction of Fe3+ ions (174.11 ± 8.70 mg eq. AA/g d.w.) being almost 48- and 5-fold more effective than mannitol and butylated hydroxytoluene used as a positive control, respectively. Compared with galanthamine (0.001 µg), the same extract exhibited a moderate anti-AChE activity (10 µg) in solid. Since purified PSH extract exhibited higher bioactivity (IC50 value 7.27 ± 0.31 µg/mL, 197.68 ± 9.47 mg eq. AA/g d.w. and 0.1 µg, respectively), it can be predominantly ascribed to the polysaccharide compounds. A. strobiliformis PSH extract may be considered as a promising resource of potent bioactive polysaccharides of natural origin successfully addressing both oxidative stress and lack of acetylcholine.

Brachybacterium sp. CH-KOV3 isolated from an oil-polluted environment-a new producer of levan.

Various microorganisms isolated from polluted environments, such as Pseudomonas sp. and Micrococcus sp. can synthesize exopolysaccharides (EPSs) which are natural, non-toxic and biodegradable polymers. EPSs play a key role in protection of microbial cells under various external influences. For humans, these substances have potential use in many industries. EPSs can be applied as a flavor or a fragrance carrier, an emulsifier, a stabilizer, a prebiotic, an antioxidant or an antitumor agent. In this study, we characterized an environmental microorganism that produces EPS, optimized EPS production by this strain and characterized the EPS produced. Isolate CH-KOV3 was identified as Brachybacterium paraconglomeratum. The sucrose level in the growth medium greatly influenced EPS production, and the highest yield was when the microorganism was incubated in media with 500g/L of sucrose. The optimal temperature and pH were 28°C and 7.0, respectively. The nuclear magnetic resonance (NMR) results and GC-MS analysis confirmed that the residues were d-fructofuranosyl residues with ß-configuration, where fructose units are linked by ß-2,6-glycosidic bonds, with ß-2,1-linked branches. All these data indicate that the investigated EPS is a levan-type polysaccharide. Thus, it was concluded that Brachybacterium sp. CH-KOV3 could constitute a new source for production of the bioactive polysaccharide, levan.

The lignicolous fungus Trametes versicolor (L.) Lloyd (1920): a promising natural source of antiradical and AChE inhibitory agents.

This study aimed to determine antiradical (DPPH• and •OH) and acetylcholinesterase (AChE) inhibitory activities along with chemical composition of autochtonous fungal species Trametes versicolor (Serbia). A total of 38 phenolic compounds with notable presence of phenolic acids were identified using HPLC/MS-MS. Its water extract exhibited the highest antiradical activity against •OH (3.21 µg/mL), among the rest due to the presence of gallic, p-coumaric and caffeic acids. At the concentration of 100 µg/mL, the same extract displayed a profound AChE inhibitory activity (60.53%) in liquid, compared to donepezil (89.05%), a drug in clinical practice used as positive control. The flavonoids baicalein and quercetin may be responsible compounds for the AChE inhibitory activity observed. These findings have demonstrated considerable potential of T. versicolor water extract as a natural source of antioxidant(s) and/or AChE inhibitor(s) to be eventually used as drug-like compounds or food supplements in the treatment of Alzheimer's disease.

Synthesis and characterization of a new type of levan-graft-polystyrene copolymer.

Novel macromolecular graft copolymers were synthesized by reaction of the hydroxyl groups of the microbial polysaccharide levan, produced using Bacillus licheniformis, with polystyrene (Lev-g-PS). Synthesis was performed by the free radical reaction using potassium persulfate (PPS) as initiator. The prepared copolymer was characterized by FTIR, SEM, TG/DTA, XRD and (13)C NMR. The influence of the different conditions (reaction temperature, air or nitrogen atmosphere, reaction time, type of amines and ascorbic acid (AA) concentration) on the grafting reaction was investigated. Results showed that maximum percentage of grafting (58.1%) was achieved at a reaction temperature 70°C, in a nitrogen atmosphere and using dimethylethanolamine (DMEA) as the amine activator. On the basis of the obtained results, the likely reaction mechanism was proposed. Synthesized copolymers have better thermal stability in comparison with their initial components. Copolymers such as Lev-g-PS could potentially have many applications, such as compatibilizers and material for membranes.

Antioxidants of Edible Mushrooms.

Oxidative stress caused by an imbalanced metabolism and an excess of reactive oxygen species (ROS) lead to a range of health disorders in humans. Our endogenous antioxidant defense mechanisms and our dietary intake of antioxidants potentially regulate our oxidative homeostasis. Numerous synthetic antioxidants can effectively improve defense mechanisms, but because of their adverse toxic effects under certain conditions, preference is given to natural compounds. Consequently, the requirements for natural, alternative sources of antioxidant foods identified in edible mushrooms, as well as the mechanistic action involved in their antioxidant properties, have increased rapidly. Chemical composition and antioxidant potential of mushrooms have been intensively studied. Edible mushrooms might be used directly in enhancement of antioxidant defenses through dietary supplementation to reduce the level of oxidative stress. Wild or cultivated, they have been related to significant antioxidant properties due to their bioactive compounds, such as polyphenols, polysaccharides, vitamins, carotenoids and minerals. Antioxidant and health benefits, observed in edible mushrooms, seem an additional reason for their traditional use as a popular delicacy food. This review discusses the consumption of edible mushrooms as a powerful instrument in maintaining health, longevity and life quality.

Nutraceutical properties of the methanolic extract of edible mushroom Cantharellus cibarius (Fries): primary mechanisms.

The methanolic extract of the wild edible mushroom Cantharellus cibarius Fr. (chanterelle) was analyzed for in vitro antioxidative, cytotoxic, antihypertensive and antibacterial activities. Various primary and secondary metabolites were found. Phenols were the major antioxidant components found in the extract (49.8 mg g(-1)), followed by flavonoids, whose content was approximately 86% of the total phenol content. Antioxidant activity, measured by four different methods, was high for inhibition of lipid peroxidation (EC50 = 1.21 mg mL(-1)) and chelating ability (EC50 = 0.64 mg mL(-1)). The antioxidant activity of the C. cibarius methanol extract was achieved through chelating iron compared to hydrogen atom and/or electron transfer. The extract showed good selectivity in cytotoxicity on human cervix adenocarcinoma HeLa, breast carcinoma MDA-MB-453 and human myelogenous leukemia K562, compared to normal control human fetal lung fibroblasts MRC-5 and human lung bronchial epithelial cells BEAS-2B. The extract had inhibitory activity against angiotensin converting I enzyme (ACE) (IC50 = 0.063 mg mL(-1)). The extract revealed selective antimicrobial activity against Gram-positive bacteria with the highest potential against E. faecalis. The medicinal and health benefits, observed in wild C. cibarius mushroom, seem an additional reason for its traditional use as a popular delicacy food.

Dietary polysaccharide extracts of Agaricus brasiliensis fruiting bodies: chemical characterization and bioactivities at different levels of purification.

Polysaccharides of the European strain of A. brasiliensis were obtained by hot water extraction and ethanol precipitation (HWPE I) of fruiting bodies, and further purified by dialysis (HWPE II) and pronase incubation (PPE). These polysaccharides consisted mainly of (1→6)-ß-d-glucans. PPE was free of proteins and polyphenols as demonstrated by quantitative assays and NMR profiling. They showed a clear IFN-γ inducing activity in human PBMCs, which suggests these polysaccharides to have proinflammatory effects. Treatment by ß-glucosidase caused the polysaccharides to be degraded into smaller fragments and at the same time increased their IFN-γ inducing activity in PBMCs fourfold. In vitro, PPE showed a dose-dependent inhibition of the proliferation of the human leukemia Jurkat cell. At 100µg/mL the cells' viability was decreased by appr. 51% compared to the control. EPR spin trapping demonstrated a high antioxidative activity against •OH and •O2- radicals of HWPE I and PPE. Further, the results of the antioxidant assays indicated that antioxidant activity against •OH radicals in the Fenton system was achieved through scavenging or through chelating iron mechanisms. The good immunomodulating and antioxidative properties of A. brasiliensis polysaccharide extract obtained by hot water extraction and ethanol precipitation make it suitable for everyday use as an inexpensive dietary supplement.

The edible mushroom Laetiporus sulphureus as potential source of natural antioxidants.

Hot water extract (LN), partially purified polysaccharides (LP) and hot alkali extracted polysaccharides (LNa) obtained from fruiting bodies of the wild basidiomycete Laetiporus sulphureus were examined for their antioxidant activities. LNa was the most active antioxidant, as shown by the median effective concentrations (EC50 values) of 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging activity (0.5 ± 0.2 mg/ml), reducing power (4.0 ± 0.3 mg/ml) and ferrous ion-chelating ability (1.5 ± 0.1 mg/ml). LNa contained the highest level of α-glucan (17.3 ± 1.2 g/100 g dw), whereas LP contained mostly ß-glucans (66.8 ± 1.3 g/100 g dw). The prevalent monosaccharide in all extracts was glucose. The EC50 values of all three antioxidant activity assays were well-correlated with the α-glucan content. Strong and significant correlation was found between total phenolic compounds and DPPH scavenging ability and also reducing power. The three investigated extracts (at concentrations of 0.1-10 mg/ml) were not toxic to HTR-8/SVneo trophoblast cell line.

Simultaneous production of pullulan and biosorption of metals by Aureobasidium pullulans strain CH-1 on peat hydrolysate.

It was demonstrated that during the growth of Aureobasidium pullulans strain CH-1 on the acid hydrolysate of peat from the Vlasina Lake, the content of metals (Cu, Fe, Zn, Mn, Pb, Cd, Ni and Cr) decreased due to biosorption. The reduction in the metal content was found to be in the range (%): 38.2-62.2, 67.7-97.3, 0.02-62.05, 0.05-23.97, 0.16-4.24, 3.45-51.72, 1.18-35.82, 0.86-44.44, for Cu, Fe, Zn, Mn, Pb, Cd, Ni and Cr, respectively. During this process, the metals were accumulated in the biomass, while pullulan, an extracellular polysaccharide produced by Aureobasidium pullulans strain CH-1, was found not to bind the above-mentioned metals.

Natural and modified (1-->3)-beta-D-glucans in health promotion and disease alleviation.

A number of polysaccharides with beta-glycosidic linkage are widespread in nature in a variety of sources. All have a common structure and the (1-->3)-beta-D-glucan backbone is essential. They have attracted attention over the years because of their bioactive and medicinal properties. In many cases their functional role is a mystery, in others it is well established. Because of their insoluble chemical nature, particulate (1-->3)-beta-D-glucans are not suitable for many medical applications. Various methods of changing or modifying the beta-D-glucan chemical structure and transforming it to a soluble form have been published. The beta-D-glucan bioactive properties can be affected positively or negatively by such modifications. This review examines beta-glucan sources in nature, health effects and structure-activity relationships. It presents the current state of beta-D-glucan solubilization methods and discusses their effectiveness and application possibilities for the future.