Characterization of a new efficient low molecular weight Bacillus subtilis NRC 16 levansucrase and its levan.

Screening of 18 bacterial honey isolates revealed that all the isolates were levansucrase producers. The most potent isolate that achieved the highest activity (45.66 U/ml) was identified as Bacillus subtilis NRC based on morphological examination and 16S rRNA. The results recorded the necessity of starch (5 g/L), baker's yeast (12.5 g/L), and AlCl3 (5 mM) in improvement of the enzyme productivity. The Bacillus subtilis levansucrase was eluted as a single protein in one purification step. The enzyme molecular weight was (14 kDa). It showed its optimum activity at 45°C and could retain 60% of its activity after incubation at 50°C for 2 h. Its optimum activity was obtained at pH 8.2 and the enzyme showed great pH stability in both acidic and alkaline ranges. Unlike, most levansucrases all tested metals had an adverse effect in enzyme activity. The enzyme had antioxidant activities and were characterized as spherical micro- and nanoparticles by transmission electron microscopy. The effect of growth conditions and medium composition in levan structure and its fibrinolytic activity was evaluated.

Evaluation of lupine seeds (Lupinus albus L.) neutral extract as a texture improver in low-fat yogurt production.

Aqueous lupine seeds (Lupinus albus L.) extracts were evaluated as a natural fat substitute in low-fat yogurt production. Thus, the chemical composition, particle size, molecular weight, total phenolic (TPC), and total flavonoids (TFC) of the selected extract were estimated. Also, the antimicrobial activity and antioxidant capacity of selected extract were investigated. Yogurt with neutral lupine extract (NeLP) had the highest all sensorial attributes compared to other extracts. Also, the incorporation of NeLP during low-fat yogurt processing increased the solid content, and viscosity, as well as improved the textural profile and sensorial attributes without any negative effect on the yogurt's color. SEM micrographs of NeLP-yogurt microstructure showed a matrix characterized by large fused casein micelles clusters with comparatively lower porosity compared to control yogurt (without NeLP). The chemical composition of NeLP indicated that the major sugar constituents are glucose and galactose with different molar fractions. The molecular weight of NeLP is 460.5 kDa with a particle size of 1519.9 nm. Also, IC50 of NeLP is 0.589 mg/ml, while TPC and TFC are 7.17, and 0.0137 g/100 g sample, respectively. Hence, lupine neutral extract (0.25%) could be used as a fat replacer or texture improver ingredient in such low-fat yogurt which led to improved its characteristics without any negative defect during 7 days at 5 °C.

Enhancement of the quality attributes and health benefits synbiotic yoghurt from cow's milk.

The present work delighted on extraction of galactomannan polysaccharide from guar gum beans and microbial galactomannan source. Effect of replacing non-fat dry milk that used to fortify cow's milk in yoghurt industry with the two extracted galactomannans and commercial galactomannan as food additives was studied. Control yoghurt treatment was made from 3.0% fat cow's milk that was fortified with 1.5% non-fat dry milk. Another 6 yoghurt treatmentwas fortified with 0.15, 0.25% of commercial, guar and microbial galactomannan respectively. All treatments were cultured with the probiotic starter (1.0% Streptococcus thermophilus + 1.0% Lactobacillus delbrueckii subsp. Bulgaricus + 1.0% Bifidobacteriumbifidum). The obtained results indicated that yoghurt supplementation with the three types of galactomannans increased the acidity, curd tension, total solids content, decreased pH values and syneresis of yoghurt treatments. Control yoghurt and commercial galactomannan yoghurt were not significantly different from the corresponding batches those made with either guar galactomannan and microbial galactomannan in fat, protein and ash content. Yoghurt treatments which supplemented with the three types of galactomannans have higher bifidobacteria counts and organoleptic scores than the control treatment yoghurt.

Novel naringin tablet formulations of agro-resides based nano/micro crystalline cellulose with neuroprotective and Alzheimer ameliorative potentials.

This study aimed to prepare micro/nanocrystalline cellulose-loaded naringin (NAR) tablets and evaluate their neuro-protective/therapeutic potentials in Alzheimer's disease (AD) model. Micro/nanocellulose was prepared from different agro-wastes, and the different cellulose preparations were then used to formulate eight oral tablets of naringin micro/nanoparticles by direct compression. AD-like symptoms were induced in adult male Sprague Dawley rats by co-administration of 150 mg/kg AlCl3 and 300 mg/kg D-galactose (oral administration/one week), and NAR tablets were assessed for neuroprotective/therapeutic potentials in terms of behavioral changes, levels of neurodegenerative and inflammatory markers, brain redox status, neurotransmitter tones, and cortex/hippocampus histopathological alterations. NAR treatments have significantly reversed the neurotoxic effect of AlCl3 as demonstrated by improved spatial and cognitive memory functions and promoted antioxidant defense mechanisms in treated AD animals. Also, the neurodegeneration was markedly restrained as reflected by marked histopathological enhancement, and prevention/amelioration of neuropsychiatric disorders, besides the restorative effect on dysregulated neurotransmitters tone. Both NAR tablet forms showed an overall higher ameliorative effect compared to the DPZ reference drug. The formulated tablets represent promising neuroprotective/therapeutic agents for Alzheimer's disease.

Preliminary study on the possible impact of Thalassodendron ciliatum (Forss.) den Hartog acidic polysaccharide fractions against TAA induced liver failure.

The water-soluble acidic polysaccharide from Thalassodendron ciliatum (Forss.) den Hartog was successfully extracted, fractionated and purified. The phytochemical profile of the two water-soluble fractions (F1 and F2), were detected using different analytic techniques. GC-MS analysis revealed the presence of 22 saccharide. Acidic polysaccharide, galacturonic and glucuronic acid were the most abundant. Moreover, paper chromatography and electrophoresis also performed as a preliminary chemical characterization of the polymer. The hepatoprotective activity of the fractions against thioacetamide (TAA) induced liver failure; antioxidant potential and preliminary immunomodulatory activity were assigned in-vivo. The results revealed a potent competence to improve the liver function profile (ALT, AST, total bilirubin, total glyceride, etc.) and a remarkable improvement in liver architecture in comparison to the challenged intoxicated groups. Moreover, they showed high anti-oxidative properties and a promising immunomodulatory influence via Il6. These findings provide new insight into the possible role of polysaccharide purified two fractions in the treatment of acute liver injury.

Characterization of Aspergillus niger MK981235 xylanase with extraction of anti-hepatotoxic, antioxidant, hypocholesterolemic and prebiotic Corchorus olitorius stems xylooligosaccharides.

The object of this study was to utilize agro-industrial waste Corchorus olitorius stems (molokhia stems, MS) as substrate, for Aspergillus niger MK981235 xylanase production and as source of biologically active xylooligosaccharides (XOS). This study succeeded in utilization of Aspergillus niger MK981235 xylanase under different saccharification conditions designed by central composite design (CCD) for extraction of 15 biologically active XOS (anti-hepatotoxic, antioxidant, hypocholesterolemic and prebiotic) with different monosaccharides constituents composition and percent. A. niger MK981235 xylanase showed the highest activity 6.60 U·ml-1 at 50 °C with 1.5% xylan. The kinetics included Km and Vmax were determined to be 6.67 mg·ml-1 and 20 µmol·ml-1·min-1, respectively. Moreover, A. niger MK981235 xylanase thermodynamics Ea (activation energy) and Ed (activation energy of denaturation) were determined to be 21.95 and 39.51 KJ·mol-1, respectively. The highest prebiotic effect (growth promation) was exerted by the central MS XOS on Lactobacillus plantarum and Lactobacillus rhamnosus (125 and 135.3%, respectively). Also, the central MS XOS, exerted the highest cholesterol reduction and antioxidant activities 74.7 and 92%, respectively, showed remarkable in vivo protective role against the hepatic toxicity of lithium carbonate evaluated by changes in body weight, liver function markers (AST, ALT, Alb, total bilirubin) and tissue makers (MDA and GSH).

Role of levan extracted from bacterial honey isolates in curing peptic ulcer: In vivo.

Peptic ulcer is one of the worldwide diseases where 10% of adults are affected by peptic ulcers at least once in their lifetime. The goal of this study was to evaluate the effect of levan in treating peptic ulcer. The bacterial honey isolates called Bacillus sp. levan was utilized. Levan was chemically characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), 1H and 13C NMR analysis. Levan was used to treat gastric ulcers induced in rats by oral administration of 5 mL/kg ethanol. Microscopic examination of stomach sections indicated that treatment with 200 mg/kg levan effectively healed the ulcers. Levan had no antimicrobial activity against a common cause of ulcers such as Helicobacter pylori bacteria. Rather, we proposed that the high adhesion (manifested as a protective coating) and prebiotic activity of levan may account for the observed beneficial effects. The immunohistochemical examination showed that levan led to a noticeable Bacillus sp. levan reduction in NF-κB in the upper gastric mucosa. The results concluded that the role of levan was more protective rather than preventive and suggested that levan could play a fundamental role in solving the peptic ulcer problems.

Improvement of Aspergillus flavus saponin hydrolase thermal stability and productivity via immobilization on a novel carrier based on sugarcane bagasse.

Soyasapogenol B (SB) is known to have many biological activities such as hepatoprotective, anti-inflammatory, anti-mutagenic, antiviral and anticancer activities. Enzymatic conversion of soyasaponins to SB was carried out using saponin hydrolase (SH) extracted from Aspergillus flavus. The partially purified enzyme was immobilized on different carriers by physical adsorption, covalent binding or entrapment. Among the investigated carriers, Eupergit C and sugarcane bagasse (SCB) activated by DIC and NHS were the most suitable two carriers for immobilization (the immobilized forms recovered 46.5 and 37.1% of the loaded enzyme activity, respectively). Under optimized immobilization conditions, immobilized SH on Eupergit C and on activated SBC recovered 87.7 and 83.3% of its original activity, respectively. Compared to free SH, immobilized SH on Eupergit C and on activated SCB showed higher optimum pH, activation energy, half-lives and lower deactivation constant rate. Also, their SB productivities were improved by 2.3- and 2.2-folds compared to free SH (87.7 and 83.3 vs. 37.5%, respectively). Hence, being SCB more sustainable and an inexpensive material, it can be considered a good alternative to Eupergit C as a support for SH immobilization. SH immobilization on industrially applicable and inexpensive carrier is necessary to improve SB yield and reduce its production cost. The chemical structure of SCB and the resulting cellulose derivatives were studied by ATR-IR spectroscopy. The thermal analysis technique was used to study the chemical treatment of SCB and coupling with the enzyme. This technique confirmed the removal of lignin and hemicellulose by chemical treatment of SCB.

New applied pharmacological approach/trend on utilization of agro-industrial wastes.

This study aimed to transform the locally available lignocellulosic residual palm frond (PF) and rice straw (RS) wastes into multifunction added products like methylated cellulose and sulfated and phosphorylated hemicelluloses by simple processes. Hydrolysis with 2 N sulfuric acid was the most suitable reaction for microcrystalline cellulose production. The characteristics of the prepared products were studied to obtain the optimum reaction conditions. Palm frond hemicellulose (PFHC) recorded the highest antimicrobial activity against Staphylococcus aureus, Escherichia coli, and Candida albicans (22, 22, 26 mm), respectively, and phosphorylated palm frond hemicellulose (PPFHC) exhibited the highest potential antioxidant activity of approximately 60%, suggesting a possible correlation between the two bioactivities. Most of extracted celluloses and their derivatives had a variety of promising probiotic activities which are expected to reduce the side effects of the gastric mucosa and possibly play a role in curing the gastric ulcer. Accordingly, the determination of anti-inflammatory and gastroprotective activity results revealed that methylcellulose, sulfated and phosphorylated hemicelluloses showed anti-inflammatory and gastroprotective activities and the capability of all tested compounds to ameliorate the ethanol-induced gastric ulcer in rats' stomach. All results recommended PF and RS and their derivatives to be used as a medicinal food.

Chemically-modified polysaccharide extract derived from Leucaena leucocephala alters Raw 264.7 murine macrophage functions.

In this study, a chemical modification of the polysaccharides extract (E) derived from Leucaena leucocephala seeds was performed to prepare C-glycosidic 2-propanol derivative (PE), and its sulphated derivative (SPE). This study aimed to characterize immunomodulatory activities of the original extract and its derivatives by exploring their effects on Raw macrophage 264.7 functions and their antioxidant activity. Our results indicated that PE was an effective radical scavenger to hydroxyl, peroxyl, and superoxide anion radicals, and SPE was a peroxyl radical scavenger. PE and SPE were found to influence the macrophage functions. Both of PE and SPE enhanced the macrophage proliferation and phagocytosis of FITC-zymosan; PE inhibited nitric oxide (NO) generation and tumor necrosis factor-alpha (TNF-alpha) secretion in lipopolysaccharide (LPS)-stimulated Raw macrophage 264.7. In contrast, SPE over-induced NO generation and TNF-alpha secretion. Moreover, PE strongly inhibited the binding affinity of FITC-LPS to Raw 264.7, as indicated by flow cytometry analysis. These findings revealed that PE may act as a potent anti-inflammatory agent; however SPE may act as an inducer of macrophage functions against pathogens.

Cancer chemopreventive and anti-inflammatory activities of chemically modified guar gum.

Guar gum (G) is a simple characterized branched polysaccharide, which is frequently used in food industries. We prepared the gum C-glycosylated derivative (GG), and its sulphated derivative (SGG), aiming to characterize their cancer chemopreventive, and anti-inflammatory properties. Estimation of cancer chemopreventive activity, specifically anti-initiation, including the modulation of carcinogen metabolism and the antioxidant capacity, revealed that GG was a potent anti-initiator, where it inhibited not only the carcinogen activator enzyme, cytochrome P450 1A (CYP1A), but also induced the carcinogen detoxification enzymes glutathione-S-transferases (GSTs), while SGG inhibited both CYP1A and GSTs. SGG was an effective radical scavenger than GG against hydroxyl, peroxyl, and superoxide anion radicals. GG and SGG were found to modulate the macrophage functions into an anti-inflammatory pattern. Thus, both enhanced the macrophage proliferation and phagocytosis of fluorescein isothiocyanate (FITC)-zymosan; however, they also inhibited strongly the nitric oxide generation and tumor necrosis factor-alpha secretion in lipopolysaccharide (LPS)-stimulated RAW macrophage 264.7. Unexpectedly, both GG and SGG dramatically inhibited the binding affinity of FITC-LPS to RAW 264.7, as indicated by flow cytometry analysis. GG and SGG exhibited a significant anti-proliferative activity against human hepatocellular carcinoma cells (Hep G2), and only SGG was specifically cytotoxic for human breast carcinoma cells (MCF-7), but neither was significantly cytotoxic for human lymphoblastic leukemia cells (1301). SGG led to a major disturbance in cell cycle phases of Hep G2 cells as indicated by concomitant arrest in S- and G2/M-phases, a disturbance that was associated with an induced cell death as a result of necrosis, but not apoptosis in both GG- and SGG-treated cells. Taken together, the modified gums could be used as an alternative of G in health food industries to provide cancer prevention in risk populations.

Production optimization of invertase by Lactobacillus brevis Mm-6 and its immobilization on alginate beads.

A sequential optimization strategy, based on statistical experimental designs, was employed to enhance the production of invertase by Lactobacillus brevis Mm-6 isolated from breast milk. First, a 2-level Plackett-Burman design was applied to screen the bioprocess parameters that significantly influence the invertase production. The second optimization step was performed using fractional factorial design in order to optimize the amounts of variables have the highest positive significant effect on the invertase production. A maximal enzyme activity of 1399U/ml was more than five folds the activity obtained using the basal medium. Invertase was immobilized onto grafted alginate beads to improve the enzyme's stability. Immobilization process increased the operational temperature from 30 to 60°C compared to the free enzyme. The reusability test proved the durability of the grafted alginate beads for 15 cycles with retention of 100% of the immobilized enzyme activity to be more convenient for industrial uses.

Scaling up, characterization of levan and its inhibitory role in carcinogenesis initiation stage'.

Honey isolate Bacillus subtilis M was cultivated in shake flasks and in 16-l bioreactor cultures to investigate cell growth, bio-metabolites production kinetics and bioprocess scalability. The respective maximal levan and levansucrase productions of 59.5 g/l and 74.1 U/ml were achieved in bioreactor cultures under pH controlled condition (pH=7.0) after only 24 h. Crude levan (levE) was isolated, characterized and fractionated into F1, F2, and F3 with different molecular weight (21.8, 13.118, 9.53 kDa). (1)H NMR and (13)C NMR spectroscopy proved that LevE and their fractions were mainly ß-(2, 6)-linked levan-type polysaccharide. The cancer chemo-preventive activity indicated that the levE and its fraction 3 were promising inhibitors of cytochrome P-450 1A activity, inducers of glutathione-S-transferase activity in Murine hepatomaHepa1c1c7cells and possessed highest radical scavenging affinity to both ROO and OH. They inhibited the induced-DNA fragmentation. None of the tested samples triggered apoptosis or necrosis in splenocytes, except F2.

Levansucrase optimization using solid state fermentation and levan biological activities studies.

Bacillus subtilis NRC1aza produced levansucrase under solid state fermentation using starch as support. A sequential optimization strategy, based on statistical experimental designs is employed to enhance enzyme productivity. First, a 2-level Plackett-Burman design was applied for bioprocess parameters screen that significantly increase levansucrase production. Second optimization step was performed using fractional factorial design in order to optimize the amounts of highest positive variables that had significant effect on levansucrase productivity. Maximal enzyme productivity of 170 U/gds was achieved in presence of glucose, yeast extract, and pH 8. In vitro, experiments confirmed that LevCR and LevQT had an antitumor activity against different animal and human cancer cell lines by demonstrating inhibitory effects on growth of Ehrlich ascites carcinoma cell line, human MCF-7 breast and liver HepG2 cancer cell lines, in particular LevQT was found to be efficacious compared to anticancer drug, cisplatin. Result focused in LevCR as strong fibrinolytic agent.

Antitumor and antioxidant activities of levan and its derivative from the isolate Bacillus subtilis NRC1aza.

The novel levansucrase produced from Bacillus subtilis NRC1aza yielded two types of levan L1 and L2 with different molecular weights 85.23 kDa and 31.95 kDa, respectively. The levan identification was detected by paper chromatography and high-pressure liquid chromatography. The antioxidant activity of levan and their derivatives (SL1 and SL2) exhibited a strong free radical scavenging activity with DPPH. The antitumor activity of SL1 was tested against different human cancer cell lines. The cell death was explored mechanistically through evaluation of Apoptosis/necrosis ratio, DNA fragmentation, histone deacetylase activity, mitochondrial transmembrane potential (Δψm), cytochrome C release, total caspases, caspase-3, and caspase-9, and cell cycle. SL1 showed high selective cytotoxicity against HepG2 cells. SL1 led to DNA damaging and fragmentation that was associated with induced apoptosis via mitochondrial pathway, which initiated by the impairment of Δψm and then increased mitochondria, released cytochrome c, that in turn activated caspase-9 and caspase-3 and induced apoptosis.